# Patent

## Title
Genes and uses for plant improvement

## Abstract

Transgenic seed for crops with improved traits are provided by trait-improving recombinant DNA where plants grown from such transgenic seed exhibit one or more improved traits as compared to a control plant. Of particular interest are transgenic plants that have increased yield. The invention also provides recombinant DNA molecules for expression of a protein.

## Background
CROSS REFERENCE TO RELATED APPLICATIONS
This application is a divisional of and claims the benefit of priority of U.S. application Ser. No. 11/188,298, filed Jul. 22, 2005, which application claims the benefit of priority under 35USC §119(e) of U.S. provisional application Ser. No. 60/592,978, filed Jul. 31, 2004, herein incorporated by reference in its entirety.


INCORPORATION OF SEQUENCE LISTING
Two copies of the sequence listing (Copy 1 and Copy 2) and a computer readable form (CRF) of the sequence listing, all on CD-ROMs, each containing the file named “pa—01184—53452B.rpt”, which is 67,108,864 bytes (measured in MS-WINDOWS, MEDIUM TYPE: CD-ROM (ASC11 TEXT) COMPUTER: IBM PC/XT/AT, IBM PS/2 OR COMPATIBLES. OPERATING SYSTEM: DOS/WINDOWS 2000/NT) and was created on Jul. 18, 2005, are herein incorporated by reference.
FIELD OF THE INVENTION
Disclosed herein are inventions in the field of plant genetics and developmental biology. More specifically, this invention provides transgenic seeds for crops, wherein the genome of said seed comprises recombinant DNA, the expression of which results in the production of transgenic plants that have improved trait(s).
BACKGROUND OF THE INVENTION
Transgenic plants with improved traits such as improved yield, environmental stress tolerance, pest resistance, herbicide tolerance, modified seed compositions, and the like are desired by both farmers and consumers. Although considerable efforts in plant breeding have provided significant gains in desired traits, the ability to introduce specific DNA into plant genomes provides further opportunities for generation of plants with improved and/or unique traits. The ability to develop transgenic plants with improved traits depends in part on the identification of genes that are useful in recombinant DNA constructs for production of transformed plants with improved properties.
SUMMARY OF THE INVENTION
This invention provides transgenic seeds, transgenic plants and DNA constructs with trait-improving recombinant DNA from a gene for a protein having an amino acid sequence with at least 90% identity to a consensus amino acid sequence in the group consisting of SEQ ID NO: 270 and its homologs through SEQ ID NO: 538, where the respective homolog proteins have amino acid sequences SEQ ID NO: 539 through SEQ ID NO: 22568, as indicated in Table 17. In some cases of trait improvement, the recombinant DNA encodes a protein; in other cases, the recombinant DNA suppresses endogenous protein expression. In a broad aspect this invention provides transgenic seeds for growing crop plants with improved traits, such crop plants with improved traits and the plant parts including transgenic seed produced by such crop plants. The improved traits provided by the recombinant DNA in the transgenic crop plant of this invention are identified by comparison to a control plant, i.e., a plant without the trait-improving recombinant DNA. In one aspect of the invention, transgenic crop plant grown from the transgenic seed has improved yield, as compared to the yield of a control plant, e.g., a plant without the recombinant DNA that produces the increased yield. Some plants of this invention exhibit increased yield by producing a yield increase under non-stress conditions. Other plants of this invention exhibit increased yield by producing a yield increase under one or more environmental stress conditions including, but not limited to, water deficit stress, cold stress, heat stress, high salinity stress, shade stress, and low nitrogen availability stress. Still other plants of this invention have other improved phenotypes, such as improved plant development, plant morphology, plant physiology or seed composition as compared to a corresponding trait of a control plant. The various aspects of this invention are especially useful for transgenic seed and transgenic plants having improved traits in corn (maize), soybean, cotton, canola (rape), wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
The invention also comprises recombinant DNA constructs. In one aspect, such recombinant DNA constructs useful for the transgenic seed and transgenic plants of this invention comprise a promoter functional in a plant cell operably linked to a DNA segment for expressing a protein associated with a trait in a model plant or a homologue. In another aspect the recombinant DNA constructs useful for the transgenic seed and transgenic plants of this invention comprise a promoter functional in a plant cell operably linked to a DNA segment for suppressing the level of an endogenous plant protein which is a homologue to a model-plant protein, the suppression of which is associated with an improved trait. Suppression can be effected by any of a variety of methods known in the art, e.g., post transcriptional suppression by anti-sense, sense, dsRNA and the like or by transcriptional suppression.
This invention also provides a method of producing a transgenic crop plant having at least one improved trait, wherein the method comprises providing to a grower of transgenic seeds comprising recombinant DNA for expression or suppression of a trait-improving gene provided herein, and growing transgenic plant from said transgenic seed. Such methods are used to generate transgenic crop plants having at least one improved trait under one or more environmental stress conditions including, but not limited to, water deficit stress, cold stress, heat stress, high salinity stress, shade stress, and low nitrogen availability stress. In another aspect, such methods are used to generate transgenic crop plants having improved plant development, plant morphology, plant physiology or seed component phenotype as compared to a corresponding phenotype of a control plant. Of particular interest are uses of such methods to generate transgenic crop plants having increased yield under non-stress condition, or under one or more stress conditions.


DETAILED DESCRIPTION OF THE INVENTION
This invention provides transgenic plant seed having in its genome trait-improving recombinant DNA and transgenic plants grown from such seed which exhibit an improved trait as compared a control plant. In one aspect, the invention provides transgenic plants where the improved trait is one or more of improved drought stress tolerance, improved heat stress tolerance, improved cold stress tolerance, improved high salinity stress tolerance, improved low nitrogen availability stress tolerance, improved shade stress tolerance, improved plant growth and development at the stages of seed imbibition through early vegetative phase, and improved plant growth and development at the stages of leaf development, flower production and seed maturity. Particular transgenic plants grown from transgenic seeds of this invention exhibit increased seed yield. Recombinant DNA constructs used in this invention comprise recombinant DNA disclosed herein which produces mRNA to modulate gene expression imparting improved traits to plants.
“Gene” means all or part of the DNA that encodes a protein or mRNA, e.g., chromosomal DNA, plasmid DNA, cDNA, or synthetic DNA, and includes DNA regions flanking the coding sequences, e.g., introns, 5′UTR, 3′UTR, promoters and other DNA involved in the regulation of expression.
“Transgenic seed” means plant seed having a genome altered by the incorporation of recombinant DNA, e.g., by transformation. “Transgenic plant” means a plant produced from an original transformation event, or progeny from later generations or crosses of a plant to a transformed plant, so long as the progeny contains the recombinant DNA in its genome. “Recombinant DNA” means a DNA molecule having a genetically engineered modification introduced through a combination of endogenous and/or exogenous DNA elements in a transcription unit, manipulation via mutagenesis, restriction enzymes, and the like or simply by inserting multiple copies of a native transcription unit. Recombinant DNA may comprise DNA segments obtained from different sources, or DNA segments obtained from the same source, but which have been manipulated to join DNA segments which do not naturally exist in the joined form. Recombinant DNA can exist outside of a cell, e.g., as a PCR fragment or in a plasmid, or can be integrated into a genome such as a plant genome.
“Trait” means a physiological, morphological, biochemical, or physical characteristic of a plant or particular plant material or cell. In some instances the characteristic is visible to the human eye, e.g., seed or plant size, or can be measured by biochemical techniques, e.g., detecting the protein, starch, or oil content of seed or leaves, or by observation of a metabolic or physiological process, e.g., by measuring uptake of carbon dioxide, or by the observation of the expression level of a gene or genes, e.g., by employing Northern analysis, RT-PCR, microarray gene expression assays, or reporter gene expression systems, or by agricultural observations such as stress tolerance, yield, or pathogen tolerance.
“Control plant” is a plant without trait-improving recombinant DNA. A control plant is used to measure and compare trait improvement in a transgenic plant with such trait-improving recombinant DNA. One suitable control plant is a non-transgenic plant of the parental line that was used to generate a transgenic plant. Another suitable control plant is a transgenic plant that comprises recombinant DNA without the specific trait producing DNA, e.g., simply a marker gene. Another suitable control plant is a negative segregant progeny of hemizygous transgenic plant. In certain demonstrations of trait improvement, e.g., in field conditions, the use of a limited number of control plants can cause a wide variation in the control dataset. To minimize the effect of the variation within the control dataset, a “reference” is used, i.e., a trimmed mean of all data from both transgenic and control plants grown under the same conditions and at the same developmental stage. The trimmed mean is calculated by eliminating a specific percentage, i.e., 20%, of the smallest and largest observation from the data set and then calculating the average of the remaining observation.
“Trait improvement” means a detectable and desirable difference in a characteristic in a transgenic plant relative to a control plant or a reference. In some cases, the trait improvement is measured quantitatively. For example, the trait improvement can entail at least a 2% desirable difference in an observed trait, at least a 5% desirable difference, at least about a 10% desirable difference, at least about a 20% desirable difference, at least about a 30% desirable difference, at least about a 50% desirable difference, at least about a 70% desirable difference, or at least about a 100% difference, or an even greater desirable difference. In other cases, the trait improvement is only measured qualitatively. It is known that there are natural variations in a trait. Therefore, the trait improvement observed entails a change of the normal distribution of the trait in the transgenic plant compared with the trait distribution observed in a control plant or a reference, which is evaluated by statistical methods provided herein. Trait improvement includes, but not limited to, yield increase, including increased yield under non-stress conditions and increased yield under environmental stress conditions. Stress conditions may include, for example, drought, shade, fungal disease, viral disease, bacterial disease, insect infestation, nematode infestation, cold temperature exposure, heat exposure, osmotic stress, reduced nitrogen nutrient availability, reduced phosphorus nutrient availability and high plant density. Many agronomic traits can affect “yield”, including without limitation, plant height, pod number, pod position on the plant, number of internodes, incidence of pod shatter, grain size, efficiency of nodulation and nitrogen fixation, efficiency of nutrient assimilation, resistance to biotic and abiotic stress, carbon assimilation, plant architecture, resistance to lodging, percent seed germination, seedling vigor, and juvenile traits. Other traits that can affect yield include, efficiency of germination (including germination in stressed conditions), growth rate (including growth rate in stressed conditions), ear number, seed number per ear, seed size, composition of seed (starch, oil, protein) and characteristics of seed fill. Also of interest is the generation of transgenic plants that demonstrate desirable phenotypic properties that may or may not confer an increase in overall plant yield. Such properties include improved plant morphology, plant physiology or improved components of the mature seed harvested from the transgenic plant.
“Yield-limiting environment” means a condition under which a plant would have the limitation on yield including environmental stress conditions.
“Stress condition” means a condition unfavorable for a plant, which adversely affects plant metabolism, growth and/or development. A plant under the stress condition typically shows reduced germination rate, retarded growth and development, reduced photosynthesis rate, and eventually leading to reduction in yield. Specifically, “water deficit stress” means sub-optimal conditions for water and humidity needed for normal growth of natural plants. Relative water content (RWC) is one physiological measure of plant water deficit. RWC measures the effect of osmotic adjustment in plant water status, when a plant is under stressed conditions. RWC can result from heat, drought, high salinity and induced osmotic stress.
“Cold stress” means exposure of a plant to temperatures below, e.g., at least two or more degrees Celsius below, those temperatures that are normal for a particular species or particular strain of plant.
“Sufficient nitrogen growth condition” means a growth condition where the soil or growth medium contains or receives enough amounts of nitrogen nutrient to sustain a healthy plant growth and/or for a plant to reach its typical yield for a particular plant species or a particular strain. “Nitrogen nutrient” means any one or any mix of the nitrate salts commonly used as plant nitrogen fertilizer, including, but not limited to, potassium nitrate, calcium nitrate, sodium nitrate, ammonium nitrate. “Ammonium” means any one or any mix of the ammonium salts commonly used as plant nitrogen fertilizer, e.g., ammonium nitrate, ammonium chloride, ammonium sulfate, etc. Those skilled in the art know what constitutes such soil, media and fertilizer inputs for most plant species. “Low nitrogen availability stress” means a plant growth condition that does not contain sufficient nitrogen nutrient to maintain a healthy plant growth and/or for a plant to reach its typical yield under a sufficient nitrogen growth condition; a useful low nitrogen availability stress is a growth condition with 50% or less of the conventional nitrogen inputs.
“Shade stress” means a limited light availability that triggers the shade avoidance response in plant. Plants are subject to shade stress when localized at lower part of the canopy, or in close proximity of neighboring vegetation. Shade stress is exacerbated when the planting density exceeds the average prevailing density for a particular plant species. The average prevailing densities per acre of a few other examples of crop plants in the USA in the year 2000 were: wheat 1,000,000-1,500,000; rice 650,000-900,000; soybean 150,000-200,000, canola 260,000-350,000, sunflower 17,000-23,000 and cotton 28,000-55,000 plants per acre.
“Increased yield” of a transgenic plant of this invention is evidenced and measured in a number of ways, including test weight, seed number per plant, seed weight, seed number per unit area (i.e., seeds, or weight of seeds, per acre), bushels per acre, tons per acre, tons per acre, kilo per hectare. For example, corn yield is measured as production of shelled corn kernels per unit of production area, e.g., in bushels per acre or metric tons per hectare, often reported on a moisture adjusted basis, e.g., at 15.5% moisture. Increased yield is often achieved from improved utilization of key biochemical compounds, such as nitrogen, phosphorous and carbohydrate, or from improved responses to environmental stresses, such as cold, heat, drought, salt, and attack by pests or pathogens. Trait-improving recombinant DNA is used to provide transgenic plants having improved growth and development, and ultimately increased yield, as the result of modified expression of plant growth regulators or modification of cell cycle or photosynthesis pathways.
“Expression” means transcription of DNA to produce RNA. The resulting RNA includes mRNA encoding a protein, antisense RNA that is complementary to an mRNA encoding a protein, or an RNA transcript comprising a combination of sense and antisense gene regions, such as for use in RNAi gene suppression. Expression also means production of encoded protein from mRNA.
“Promoter” means a region of DNA upstream from the start of transcription and involved in recognition and binding of RNA polymerase and other proteins to initiate transcription. A “plant promoter” is a promoter capable of initiating transcription in plant cells whether or not its origin is a plant cell. Exemplary plant promoters include, but are not limited to, those that are obtained from plants, plant viruses, and bacteria which comprise genes expressed in plant cells such as Agrobacterium or Rhizobium. “Tissue preferred” promoters preferentially regulate expression in certain tissues, such as leaves, roots, or seeds. “Tissue specific” promoters predominately regulate expression only in certain tissues. “Cell type” specific promoter primarily regulate expression in certain cell types in one or more organs, for example, vascular cells in roots or leaves. “Inducible” and “repressible” promoters regulate expression under environmental influences, under the effect of anaerobic conditions, certain chemicals, or the presence of light. Tissue specific, tissue preferred, cell type specific, and inducible promoters constitute a class of “non-constitutive” promoters. “Constitutive” promoters are promoters which are active under most conditions. “Anti-sense orientation” refers to a DNA sequence that is operably linked to a promoter in an orientation where the anti-sense strand is transcribed. “Operably linked” refers to an association of two or more DNA elements in a single construct so that the function of one is affected by the other. For example, a promoter is operably linked with transcribable DNA when it is capable of affecting the expression of that DNA; that is, the coding DNA is under the transcriptional control of the promoter.
“Consensus sequence” means an artificial, amino acid sequence of conserved parts of the proteins encoded by homologous genes, e.g., as determined by a CLUSTALW alignment of amino acid sequence of homolog proteins.
“Homologs” means genes that produce functionally similar proteins, e.g., in the same organism or in different organisms. A gene can be related to a homolog gene by descent from a common ancestral DNA. Homologs include genes where the relationship is by speciation, e.g., often called orthologs, or by genetic duplication, e.g., often called paralogs. More specifically, “orthologs” include homologs in different species that evolved from a common ancestral gene by specification. Normally orthologs retain the same function in the course of evolution. “Paralogs” include homologs in the same species that have diverged from each other as a consequence of genetic duplication.
“Percent identity” means the extent to which two optimally aligned DNA or protein segments are invariant throughout a window of alignment of components, e.g., nucleotide sequence or amino acid sequence. An “identity fraction” for aligned segments of sequences is the number of identical components which are shared divided by the total number of sequence components in the segment used as a reference over a window of alignment which is the smaller of the sequences. “Percent identity” (“% identity”) is the identity fraction times 100. “% identity” to a consensus amino acid sequence” is 100 times the identity fraction in a window of alignment of an amino acid sequence of a test protein optimally aligned to consensus amino acid sequence of this invention.
“Arabidopsis” means plants of Arabidopsis thaliana. 
Recombinant DNA Constructs
This invention provides recombinant DNA constructs comprising DNA elements for imparting one or more improved traits to transgenic plant. Such constructs typically comprise a promoter operatively linked to DNA to provide for expression of a protein or RNA for gene suppression in a target plant. Recombinant DNA constructs can also include additional regulatory elements, such as 5′ or 3′ untranslated regions (UTRs) such as polyadenylation sites, introns, and transit or signal peptides. Such recombinant DNA constructs are assembled using methods known to those of ordinary skill in the art.
In certain embodiments, recombinant DNA constructs comprise sense-oriented, trait-imparting DNA operably linked to a promoter that is functional in a plant to provide for expression of the trait-imparting DNA in the sense orientation such that a desired protein is produced. In other embodiments at least a part of the trait-imparting DNA is in an anti-sense orientation for gene suppression activity.
Recombinant DNA constructs, especially for expressing proteins are typically prepared with a 3′ UTR that a polyadenylation site and signal. Recombinant DNA constructs can also include a transit peptide for targeting of a gene target to a plant organelle, particularly to a chloroplast, leucoplast or other plastid organelle. For descriptions of the use of chloroplast transit peptides, see U.S. Pat. No. 5,188,642 and U.S. Pat. No. 5,728,925, incorporated herein by reference.
Table 1 provides a list of genes that can provide trait-imparting DNA for recombinant DNA constructs. DNA from each gene was used in a model plant (Arabidopsis) to discover associations with improved traits. The DNA was also used to identify homologs from which a consensus amino acid sequence is defined for characterizing the aspects of the invention where recombinant DNA is incorporated in the transgenic seeds, transgenic plants, DNA constructs and methods of this invention. With reference to Table 1:

    
    
        “NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing.
        “PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA
        “construct_id” refers to arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.
        “gene” refers to an arbitrary name used to identify the particular DNA.
        “orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relative to the promoter.
        “species” refers to the organism from which the particular DNA was derived.
    
    














TABLE 1





Nuc SEQ ID
Pep SEQ ID
construct_id
Gene
orientation
Species




















1
270
14324
CGPG1560
SENSE
Arabidopsis thaliana


2
271
17484
CGPG2630
SENSE
Arabidopsis thaliana


3
272
19109
CGPG1381
ANTI-SENSE
Arabidopsis thaliana


4
273
70423
CGPG3165
SENSE
Arabidopsis thaliana


5
274
70424
CGPG3180
SENSE
Arabidopsis thaliana


6
275
70480
CGPG3833
SENSE
Arabidopsis thaliana


7
276
70509
CGPG2420
SENSE
Arabidopsis thaliana


8
277
70647
CGPG4334
SENSE
Arabidopsis thaliana


9
278
70675
CGPG4519
SENSE
Arabidopsis thaliana


10
279
70829
CGPG518
SENSE
Arabidopsis thaliana


11
280
70849
CGPG596
SENSE
Arabidopsis thaliana


12
281
71627
CGPG1270
SENSE
Arabidopsis thaliana


13
282
71934
CGPG2294
SENSE
Arabidopsis thaliana


14
283
72615
CGPG4829
SENSE
Arabidopsis thaliana


15
284
72927
CGPG1477
SENSE
Arabidopsis thaliana


16
285
73014
CGPG5692
SENSE
Xenorhabdus nematophilus 85816


17
286
73559
CGPG6535
SENSE
Bacillus subtilis 168


18
287
74251
CGPG5489
SENSE
Arabidopsis thaliana


19
288
19631
CGPG3627
SENSE
Arabidopsis thaliana


20
289
70121
CGPG2380
SENSE
Saccharomyces cerevisiae


21
290
70654
CGPG4352
SENSE
Arabidopsis thaliana


22
291
70696
CGPG4590
SENSE
Arabidopsis thaliana


23
292
70713
CGPG1462
ANTI-SENSE
Arabidopsis thaliana


24
293
70740
CGPG3700
SENSE
Arabidopsis thaliana


25
294
71321
CGPG4418
SENSE
Arabidopsis thaliana


26
295
71835
CGPG4634
SENSE
Arabidopsis thaliana


27
296
72934
CGPG5798
SENSE
Saccharomyces cerevisiae


28
297
72945
CGPG5787
SENSE
Saccharomyces cerevisiae


29
298
72980
CGPG5773
SENSE
Saccharomyces cerevisiae


30
299
73504
CGPG6480
SENSE
Synechocystis sp. PCC 6803


31
300
73507
CGPG6504
SENSE
Bacillus subtilis 168


32
301
73573
CGPG6462
SENSE
Agrobacterium tumefacians C58


33
302
73586
CGPG6471
SENSE
Bacillus subtilis 168


34
303
73770
CGPG5435
SENSE
Arabidopsis thaliana


35
304
74105
CGPG6574
SENSE
Xenorhabdus nematophilus 86068


36
305
74111
CGPG6622
SENSE
Escherichia coli K-12


37
306
74136
CGPG6632
SENSE
Synechocystis


38
307
74139
CGPG6561
SENSE
Escherichia coli K-12


39
308
74267
CGPG5364
SENSE
Arabidopsis thaliana


40
309
74291
CGPG5363
SENSE
Arabidopsis thaliana


41
310
74318
CGPG5826
SENSE
Arabidopsis thaliana


42
311
74319
CGPG5831
SENSE
Arabidopsis thaliana


43
312
74324
CGPG5885
SENSE
Arabidopsis thaliana


44
313
74512
CGPG32
SENSE
Arabidopsis thaliana


45
314
74583
CGPG6649
SENSE
Ralstonia metallidurans CH34


46
315
70427
CGPG3067
SENSE
Arabidopsis thaliana


47
316
71811
CGPG4426
SENSE
Arabidopsis thaliana


48
317
73463
CGPG6384
SENSE
Ralstonia metallidurans CH34


49
318
72081
CGPG5279
SENSE
Glycine max


50
319
10139
CGPG101
ANTI-SENSE
Arabidopsis thaliana


51
320
11410
CGPG103
SENSE
Arabidopsis thaliana


52
321
11604
CGPG48
ANTI-SENSE
Arabidopsis thaliana


53
322
12368
CGPG1006
SENSE
Arabidopsis thaliana


54
323
13502
CGPG1354
SENSE
Arabidopsis thaliana


55
324
13745
CGPG1576
ANTI-SENSE
Arabidopsis thaliana


56
325
13821
CGPG1569
SENSE
Arabidopsis thaliana


57
326
14240
CGPG1697
SENSE
Arabidopsis thaliana


58
327
14718
CGPG1082
SENSE
Arabidopsis thaliana


59
328
17022
CGPG1774
SENSE
Arabidopsis thaliana


60
329
17924
CGPG2882
SENSE
Arabidopsis thaliana


61
330
18259
CGPG3368
SENSE
Arabidopsis thaliana


62
331
19171
CGPG2952
SENSE
Saccharomyces cerevisiae


63
332
19201
CGPG2332
SENSE
Arabidopsis thaliana


64
333
19317
CGPG3662
SENSE
Xanthomonas


65
334
70417
CGPG3427
SENSE
Arabidopsis thaliana


66
335
70467
CGPG3785
SENSE
Arabidopsis thaliana


67
336
70806
CGPG712
SENSE
Arabidopsis thaliana


68
337
70818
CGPG479
SENSE
Arabidopsis thaliana


69
338
70820
CGPG655
SENSE
Arabidopsis thaliana


70
339
70919
CGPG4029
SENSE
Glycine max


71
340
71623
CGPG4696
SENSE
Arabidopsis thaliana


72
341
71662
CGPG4679
SENSE
Glycine max


73
342
71693
CGPG4652
SENSE
Glycine max


74
343
72384
CGPG4639
SENSE
Saccharomyces cerevisiae


75
344
72439
CGPG5075
SENSE
Arabidopsis thaliana


76
345
72619
CGPG4835
SENSE
Arabidopsis thaliana


77
346
72624
CGPG4842
SENSE
Arabidopsis thaliana


78
347
72715
CGPG5521
SENSE
Saccharomyces cerevisiae


79
348
72754
CGPG5548
SENSE
Saccharomyces cerevisiae


80
349
72819
CGPG4989
SENSE
Arabidopsis thaliana


81
350
75516
CGPG7689
SENSE
Glycine max


82
351
75701
CGPG7856
SENSE
Glycine max


83
352
73515
CGPG6473
SENSE
Bacillus subtilis 168


84
353
74684
CGPG6360
SENSE
Arabidopsis thaliana


85
354
19542
CGPG3069
SENSE
Arabidopsis thaliana


86
355
19618
CGPG3574
SENSE
Arabidopsis thaliana


87
356
19649
CGPG3140
SENSE
Arabidopsis thaliana


88
357
19745
CGPG3973
SENSE
Glycine max


89
358
19768
CGPG4096
SENSE
Glycine max


90
359
19772
CGPG3939
SENSE
Glycine max


91
360
19779
CGPG4113
SENSE
Glycine max


92
361
19833
CGPG4074
SENSE
Glycine max


93
362
19862
CGPG3961
SENSE
Glycine max


94
363
19879
CGPG4009
SENSE
Glycine max


95
364
70445
CGPG3728
SENSE
Arabidopsis thaliana


96
365
70738
CGPG3195
SENSE
Arabidopsis thaliana


97
366
71437
CGPG4043
SENSE
Glycine max


98
367
71572
CGPG4520
SENSE
Arabidopsis thaliana


99
368
71617
CGPG1227
SENSE
Arabidopsis thaliana


100
369
72532
CGPG4780
SENSE
Arabidopsis thaliana


101
370
72757
CGPG5572
SENSE
Arabidopsis thaliana


102
371
73412
CGPG6448
SENSE
Pseudomonas syringae var tomato DC3000


103
372
74102
CGPG6550
SENSE
Bacillus halodurans C-125


104
373
72633
CGPG4853
SENSE
Arabidopsis thaliana


105
374
72456
CGPG4745
SENSE
Arabidopsis thaliana


106
375
72963
CGPG1746
SENSE
Arabidopsis thaliana


107
376
70426
CGPG3199
SENSE
Arabidopsis thaliana


108
377
70772
CGPG4627
SENSE
Arabidopsis thaliana


109
378
71137
CGPG125
SENSE
Arabidopsis thaliana


110
379
71529
CGPG2808
SENSE
Arabidopsis thaliana


111
380
71601
CGPG1858
SENSE
Arabidopsis thaliana


112
381
72362
CGPG983
SENSE
Arabidopsis thaliana


113
382
72466
CGPG4767
SENSE
Arabidopsis thaliana


114
383
72524
CGPG4770
SENSE
Arabidopsis thaliana


115
384
73085
CGPG5689
SENSE
Synechocystis sp. PCC 6803


116
385
74241
CGPG5457
SENSE
Arabidopsis thaliana


117
386
74247
CGPG5475
SENSE
Arabidopsis thaliana


118
387
74284
CGPG5413
SENSE
Arabidopsis thaliana


119
388
74652
CGPG6168
SENSE
Arabidopsis thaliana


120
389
70437
CGPG3706
SENSE
Arabidopsis thaliana


121
390
71633
CGPG857
SENSE
Arabidopsis thaliana


122
391
72948
CGPG5617
SENSE
Arabidopsis thaliana


123
392
72519
CGPG4749
SENSE
Arabidopsis thaliana


124
393
10475
CGPG399
SENSE
Arabidopsis thaliana


125
394
11120
CGPG459
ANTI-SENSE
Arabidopsis thaliana


126
395
19736
CGPG4129
SENSE
Glycine max


127
396
71606
CGPG4715
SENSE
Arabidopsis thaliana


128
397
71840
CGPG4353
SENSE
Arabidopsis thaliana


129
398
74240
CGPG5454
SENSE
Arabidopsis thaliana


130
399
74331
CGPG5834
SENSE
Arabidopsis thaliana


131
400
74610
CGPG6048
SENSE
Arabidopsis thaliana


132
401
75527
CGPG7682
SENSE
Glycine max


133
402
70681
CGPG4584
SENSE
Arabidopsis thaliana


134
403
71663
CGPG4638
SENSE
Xanthomonas


135
404
72769
CGPG5573
SENSE
Arabidopsis thaliana


136
405
71508
CGPG1541
SENSE
Arabidopsis thaliana


137
406
74248
CGPG5476
SENSE
Arabidopsis thaliana


138
407
72771
CGPG2166
SENSE
Arabidopsis thaliana


139
408
72085
CGPG5228
SENSE
Arabidopsis thaliana


140
409
72744
CGPG5563
SENSE
Saccharomyces cerevisiae


141
410
73039
CGPG810
SENSE
Arabidopsis thaliana


142
411
73054
CGPG5754
SENSE
Saccharomyces cerevisiae


143
412
73501
CGPG6456
SENSE
Agrobacterium tumefacians C58


144
413
19707
CGPG4179
SENSE
Glycine max


145
414
19951
CGPG3941
SENSE
Glycine max


146
415
19967
CGPG4032
SENSE
Glycine max


147
416
70543
CGPG3815
SENSE
Arabidopsis thaliana


148
417
70707
CGPG1273
ANTI-SENSE
Arabidopsis thaliana


149
418
70719
CGPG1712
ANTI-SENSE
Arabidopsis thaliana


150
419
71134
CGPG817
SENSE
Arabidopsis thaliana


151
420
71146
CGPG2928
SENSE
Arabidopsis thaliana


152
421
71660
CGPG4690
SENSE
Arabidopsis thaliana


153
422
72086
CGPG5236
SENSE
Arabidopsis thaliana


154
423
72632
CGPG4852
SENSE
Arabidopsis thaliana


155
424
72716
CGPG5529
SENSE
Saccharomyces cerevisiae


156
425
72723
CGPG1848
SENSE
Arabidopsis thaliana


157
426
72987
CGPG1787
SENSE
Arabidopsis thaliana


158
427
74109
CGPG6606
SENSE
Xenorhabdus nematophilus 86068


159
428
74140
CGPG6569
SENSE
Bacillus halodurans C-125


160
429
74191
CGPG6597
SENSE
Rhodobacter sphaeroides 2.4.1


161
430
74265
CGPG5356
SENSE
Arabidopsis thaliana


162
431
74369
CGPG6076
SENSE
Arabidopsis thaliana


163
432
70217
CGPG6
SENSE
Arabidopsis thaliana


164
433
72711
CGPG1846
SENSE
Arabidopsis thaliana


165
434
70932
CGPG4089
SENSE
Glycine max


166
435
73518
CGPG6497
SENSE
Pseudomonas fluorescens PfO-1


167
436
19771
CGPG4011
SENSE
Glycine max


168
437
73549
CGPG6460
SENSE
Xenorhabdus nematophilus 85816


169
438
72994
CGPG5803
SENSE
Saccharomyces cerevisiae


170
439
71928
CGPG1617
SENSE
Arabidopsis thaliana


171
440
72903
CGPG5584
SENSE
Arabidopsis thaliana


172
441
73017
CGPG5733
SENSE
Saccharomyces cerevisiae


173
442
74587
CGPG6774
SENSE
Agrobacterium tumefacians C58


174
443
72453
CGPG4735
SENSE
Arabidopsis thaliana


175
444
72967
CGPG5742
SENSE
Saccharomyces cerevisiae


176
445
72961
CGPG5591
SENSE
Arabidopsis thaliana


177
446
73070
CGPG5627
SENSE
Arabidopsis thaliana


178
447
73475
CGPG6385
SENSE
Rhodopseudomonas palustris CGA009


179
448
72916
CGPG1814
SENSE
Arabidopsis thaliana


180
449
72969
CGPG5789
SENSE
Saccharomyces cerevisiae


181
450
74449
CGPG6659
SENSE
Agrobacterium tumefaciens


182
451
16615
CGPG2539
SENSE
Agrobacterium


183
452
19187
CGPG3310
SENSE
Arabidopsis thaliana


184
453
19648
CGPG3134
SENSE
Arabidopsis thaliana


185
454
70354
CGPG3995
SENSE
Glycine max


186
455
70421
CGPG2942
SENSE
Arabidopsis thaliana


187
456
70459
CGPG3758
SENSE
Arabidopsis thaliana


188
457
70465
CGPG3775
SENSE
Arabidopsis thaliana


189
458
70683
CGPG4587
SENSE
Arabidopsis thaliana


190
459
70725
CGPG2097
ANTI-SENSE
Arabidopsis thaliana


191
460
70852
CGPG1465
SENSE
Arabidopsis thaliana


192
461
71112
CGPG934
SENSE
Arabidopsis thaliana


193
462
71127
CGPG945
SENSE
Arabidopsis thaliana


194
463
71132
CGPG1561
SENSE
Arabidopsis thaliana


195
464
71217
CGPG95
SENSE
Arabidopsis thaliana


196
465
71645
CGPG4688
SENSE
Arabidopsis thaliana


197
466
71726
CGPG3894
SENSE
Arabidopsis thaliana


198
467
72432
CGPG4562
SENSE
Arabidopsis thaliana


199
468
72450
CGPG4732
SENSE
Arabidopsis thaliana


200
469
72455
CGPG4742
SENSE
Arabidopsis thaliana


201
470
72727
CGPG5522
SENSE
Saccharomyces cerevisiae


202
471
72817
CGPG4987
SENSE
Arabidopsis thaliana


203
472
72992
CGPG5777
SENSE
Saccharomyces cerevisiae


204
473
73007
CGPG5760
SENSE
Saccharomyces cerevisiae


205
474
73073
CGPG5688
SENSE
Synechocystis sp. PCC 6803


206
475
73506
CGPG6496
SENSE
Pseudomonas fluorescens PfO-1


207
476
74107
CGPG6590
SENSE
Sinorhizobium meliloti 1021


208
477
74117
CGPG6575
SENSE
Xenorhabdus nematophilus 86068


209
478
74131
CGPG6592
SENSE
Synechocystis sp. PCC 6803


210
479
74344
CGPG5929
SENSE
Arabidopsis thaliana


211
480
14320
CGPG1229
SENSE
Arabidopsis thaliana


212
481
16756
CGPG2117
SENSE
Arabidopsis thaliana


213
482
17448
CGPG2673
SENSE
Arabidopsis thaliana


214
483
17633
CGPG2839
SENSE
Arabidopsis thaliana


215
484
18876
CGPG3096
SENSE
Arabidopsis thaliana


216
485
19120
CGPG1976
ANTI-SENSE
Arabidopsis thaliana


217
486
19221
CGPG2958
SENSE
Arabidopsis thaliana


218
487
70206
CGPG4116
SENSE
Glycine max


219
488
70223
CGPG53
SENSE
Arabidopsis thaliana


220
489
70347
CGPG3147
SENSE
Arabidopsis thaliana


221
490
70406
CGPG1687
SENSE
Arabidopsis thaliana


222
491
70469
CGPG3791
SENSE
Arabidopsis thaliana


223
492
70564
CGPG1864
SENSE
Arabidopsis thaliana


224
493
70601
CGPG2917
SENSE
Arabidopsis thaliana


225
494
70612
CGPG3721
SENSE
Arabidopsis thaliana


226
495
70720
CGPG1358
ANTI-SENSE
Arabidopsis thaliana


227
496
70735
CGPG2661
SENSE
Arabidopsis thaliana


228
497
70846
CGPG377
SENSE
Arabidopsis thaliana


229
498
70923
CGPG4020
SENSE
Glycine max


230
499
71149
CGPG3457
SENSE
Arabidopsis thaliana


231
500
71608
CGPG4687
SENSE
Arabidopsis thaliana


232
501
71739
CGPG4345
SENSE
Arabidopsis thaliana


233
502
72014
CGPG5230
SENSE
Arabidopsis thaliana


234
503
72051
CGPG5241
SENSE
Arabidopsis thaliana


235
504
74259
CGPG5343
SENSE
Arabidopsis thaliana


236
505
72463
CGPG4760
SENSE
Arabidopsis thaliana


237
506
72902
CGPG5597
SENSE
Arabidopsis thaliana


238
507
74572
CGPG6640
SENSE
Synechocystis


239
508
73055
CGPG5768
SENSE
Saccharomyces cerevisiae


240
509
74103
CGPG6558
SENSE
Escherichia coli K-12


241
510
72921
CGPG5781
SENSE
Saccharomyces cerevisiae


242
511
72968
CGPG5772
SENSE
Saccharomyces cerevisiae


243
512
19703
CGPG4172
SENSE
Glycine max


244
513
19946
CGPG4097
SENSE
Glycine max


245
514
19980
CGPG3914
SENSE
Glycine max


246
515
70435
CGPG3701
SENSE
Arabidopsis thaliana


247
516
71114
CGPG1657
SENSE
Arabidopsis thaliana


248
517
72451
CGPG4733
SENSE
Arabidopsis thaliana


249
518
72947
CGPG5607
SENSE
Glycine max


250
519
73012
CGPG5786
SENSE
Saccharomyces cerevisiae


251
520
73022
CGPG5622
SENSE
Arabidopsis thaliana


252
521
73488
CGPG6394
SENSE
Bacillus subtilis 168


253
522
73901
CGPG5237
SENSE
Arabidopsis thaliana


254
523
73964
CGPG5804
SENSE
Saccharomyces cerevisiae


255
524
74019
CGPG5706
SENSE
Bacillus subtilis 168


256
525
74022
CGPG5724
SENSE
Arabidopsis thaliana


257
526
74114
CGPG6551
SENSE
Agrobacterium tumefacians C58


258
527
74262
CGPG5353
SENSE
Arabidopsis thaliana


259
528
74292
CGPG5367
SENSE
Arabidopsis thaliana


260
529
74302
CGPG5384
SENSE
Arabidopsis thaliana


261
530
74325
CGPG5898
SENSE
Arabidopsis thaliana


262
531
74429
CGPG6689
SENSE
Bacillus subtilis 168


263
532
74440
CGPG6682
SENSE
Bacillus halodurans C-125


264
533
74462
CGPG6668
SENSE
Synechocystis


265
534
74465
CGPG6692
SENSE
Bacillus subtilis 168


266
535
74474
CGPG6669
SENSE
Synechocystis


267
536
74505
CGPG6783
SENSE
Escherichia coli K-12


268
537
74507
CGPG6799
SENSE
Xenorhabdus nematophilus 85816


269
538
74562
CGPG6764
SENSE
Bacillus subtilis 168





“NUC SEQ ID NO” refers to a SEQ ID NO. for particular DNA sequence in the Sequence Listing .


“PEP SEQ ID NO” refers to a SEQ ID NO. in the Sequence Listing for the amino acid sequence of a protein cognate to a particular DNA


“construct_id” refers to an arbitrary number used to identify a particular recombinant DNA construct comprising the particular DNA.


“gene” refers to an arbitrary name used to identify the particular DNA.


“orientation” refers to the orientation of the particular DNA in a recombinant DNA construct relativeto the promoter.


“species” refers to the organism from which the particular DNA was derived.






Recombinant DNA

Trait-imparting DNA for use in this invention for improved traits in plants is disclosed herein as having a DNA sequence of SEQ ID NO:1 through SEQ ID NO:269 and any of the respective homologs. A subset of the trait-imparting DNA includes fragments with less than the full DNA sequence, e.g., consisting of oligonucleotides of at least about 15 to 20 or more consecutive nucleotides from one of the disclosed sequences. Such oligonucleotides are fragments of the larger molecules having a sequence selected from the group consisting of SEQ ID NO: 1 through SEQ ID NO: 269, and find use, for example as probes and primers for detection of the polynucleotides of the invention or for cloning DNA for use in this invention.
Useful DNA includes variants of the disclosed DNA. Such variants include naturally occurring, including homologous DNA from genes of the same or a different species, or non-natural variants, for example DNA synthesized using chemical synthesis methods, or generated using recombinant DNA techniques. Degeneracy of the genetic code provides the possibility to substitute at least one nucleotide of a disclosed DNA without causing the amino acid sequence of the protein produced to be changed. Hence, useful DNA can have any base sequence that has been changed from the sequences provided herein by substitution in accordance with degeneracy of the genetic code.
Homologs of the trait-imparting DNA generally demonstrate significant identity with the DNA provided herein. Homologous DNA is substantially identical to a trait-imparting DNA if, when the nucleotide sequences are optimally aligned there is at least about 60% nucleotide identity, or higher, e.g., at least 70% or 80% or 85% or even 90% identity or higher, such as 95% or 98% identity over a comparison window of at least 50 to 100 nucleotides, and up to the entire length of the trait-imparting DNA. Optimal alignment of sequences for aligning a comparison window can be conducted by algorithms including computerized implementations of the algorithms (for example, the Wisconsin Genetics Software Package Release 7.0-10.0, Genetics Computer Group, 575 Science Dr., Madison, Wis.). The reference DNA sequence can represent a full-length coding sequence or a portion.
Proteins useful for imparting improved traits are entire proteins or at least a sufficient portion of the entire protein to impart the relevant biological activity of the protein. Proteins useful for generation of transgenic plants having improved traits include the proteins with an amino acid sequence provided herein as SEQ ID NO: 270 through SEQ ID NO: 538, as well as homologs of such proteins.
One method to identify homologs of the proteins useful in this invention is by comparison of the amino acid sequence of the trait-imparting protein to amino acid sequences of proteins from the same or different organisms, e.g., manually or by using known homology-based search algorithms such as those commonly known and referred to as BLAST, FASTA, and Smith-Waterman. In one method a local sequence alignment program, e.g., BLAST, is used to search a database of sequences to find similar sequences, and the summary Expectation value (E-value) is used to measure the sequence base similarity. As a protein hit with the best E-value for a particular organism may not necessarily be an ortholog or the only ortholog, a reciprocal BLAST search is used to filter hit sequences with significant E-values for ortholog identification. The reciprocal BLAST entails search of the significant hits against a database of amino acid sequences from the base organism that are similar to the sequence of the query protein. A hit is a likely ortholog, when the reciprocal BLAST's best hit is the query protein itself or a protein encoded by a duplicated gene after speciation. Thus, homolog is used herein to described proteins that are assumed to have functional similarity by inference from sequence base similarity. The relationship of homologs with amino acid sequences of SEQ ID NO: 539 through SEQ ID NO: 22568 to the proteins with amino acid sequences of SEQ ID NO: 270 through SEQ ID NO: 538 is found is found in Table 17.
Aspects of the invention also use DNA encoding functional homolog proteins which differ in one or more amino acids from those of protein encoded by disclosed trait-imparting DNA as the result of one or more of the well-known conservative amino acid substitutions, e.g., valine is a conservative substitute for alanine and threonine is a conservative substitute for serine. Conservative substitutions for an amino acid within the native sequence are selected from other members of a class to which the naturally occurring amino acid belongs. Representative amino acids within these various classes include, but are not limited to: (1) acidic (negatively charged) amino acids such as aspartic acid and glutamic acid; (2) basic (positively charged) amino acids such as arginine, histidine, and lysine; (3) neutral polar amino acids such as glycine, serine, threonine, cysteine, tyrosine, asparagine, and glutamine; and (4) neutral nonpolar (hydrophobic) amino acids such as alanine, leucine, isoleucine, valine, proline, phenylalanine, tryptophan, and methionine. Conserved substitutes for an amino acid within a native amino acid sequence are selected from other members of the group to which the naturally occurring amino acid belongs. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide-containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulfur-containing side chains is cysteine and methionine. Naturally conservative amino acids substitution groups are: valine-leucine, valine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, aspartic acid-glutamic acid, and asparagine-glutamine. A further aspect of the invention uses DNA encoding proteins that differ in one or more amino acids from those of protein encoded from a described trait-imparting DNA as the result of deletion or insertion of one or more amino acids in a native sequence.
Homologs of the proteins encoded by disclosed trait-improving DNA will generally demonstrate significant sequence identity, e.g., at least 50% amino acid sequence identity or higher such as at least 70% identity or at least 80% or at least 90% identity with an amino acid sequence of SEQ ID NO:270 through SEQ ID NO:538. Identity of protein homologs is determined by optimally aligning the amino acid sequence of a putative protein homolog with a defined amino acid sequence of a protein encoded by a disclosed trait-imparting DNA and by calculating the percentage of identical and conservatively substituted amino acids over the window of comparison. The window of comparison for determining identity can be the entire amino acid sequence disclosed herein, e.g., the full sequence of any of SEQ ID NO:270 through SEQ ID NO:538.
Genes that are homologs to each other can be grouped into families and included in multiple sequence alignments to allow a consensus sequence to be derived. This analysis enables the derivation of conserved and class- (family) specific residues or motifs that are functionally important. These conserved residues and motifs can be further validated with 3D protein structure if available. A consensus sequence is used to define the full scope of the invention, e.g., to identify proteins with a homolog relationship and the corresponding trait-imparting DNA. Thus, this invention contemplates that protein homologs include proteins with an amino acid sequence that has at least 90% identity to such a consensus amino acid sequence.
Promoters
Numerous promoters that are active in plant cells have been described in the literature. These include promoters present in plant genomes as well as promoters from other sources, including nopaline synthase (NOS) promoter and octopine synthase (OCS) promoters carried on tumor-inducing plasmids of Agrobacterium tumefaciens, caulimovirus promoters such as the cauliflower mosaic virus or figwort mosaic virus promoters. For instance, see U.S. Pat. Nos. 5,858,742 and 5,322,938 which disclose versions of the constitutive promoter derived from cauliflower mosaic virus (CaMV35S), U.S. Pat. No. 5,378,619 which discloses a Figwort Mosaic Virus (FMV) 35S promoter, U.S. Pat. No. 6,437,217 which discloses a maize RS81 promoter, U.S. Pat. No. 5,641,876 which discloses a rice actin promoter, U.S. Pat. No. 6,426,446 which discloses a maize RS324 promoter, U.S. Pat. No. 6,429,362 which discloses a maize PR-1 promoter, U.S. Pat. No. 6,232,526 which discloses a maize A3 promoter, U.S. Pat. No. 6,177,611 which discloses constitutive maize promoters, U.S. Pat. No. 6,433,252 which discloses a maize L3 oleosin promoter, U.S. Pat. No. 6,429,357 which discloses a rice actin 2 promoter and intron, U.S. Pat. No. 5,837,848 which discloses a root specific promoter, U.S. Pat. No. 6,084,089 which discloses cold inducible promoters, U.S. Pat. No. 6,294,714 which discloses light inducible promoters, U.S. Pat. No. 6,140,078 which discloses salt inducible promoters, U.S. Pat. No. 6,252,138 which discloses pathogen inducible promoters, U.S. Pat. No. 6,175,060 which discloses phosphorus deficiency inducible promoters, U.S. Patent Application Publication 2002/0192813A1 which discloses 5′, 3′ and intron elements useful in the design of effective plant expression vectors, U.S. patent application Ser. No. 09/078,972 which discloses a coixin promoter, U.S. patent application Ser. No. 09/757,089 which discloses a maize chloroplast aldolase promoter, and U.S. patent application Ser. No. 10/739,565 which discloses water-deficit inducible promoters, all of which are incorporated herein by reference. These and numerous other promoters that function in plant cells are known to those skilled in the art and available for use in recombinant DNA to provide for expression of desired genes in transgenic plant cells.
It is well known in the art that promoters are usefully altered to contain multiple “enhancer sequences” to assist in elevating gene expression. By including an enhancer sequence with such constructs, expression is generally enhanced. These enhancers often are found 5′ to the start of transcription in a promoter that functions in eukaryotic cells, and can also be inserted in the forward or reverse orientation 5′ or 3′ to the coding sequence. In some instances, 5′ enhancing elements are introns. Particularly useful enhancers are the 5′ introns of the rice actin 1 gene and the rice actin 2 gene. Other enhancers include elements from the CaMV 35S promoter, octopine synthase genes, the maize alcohol dehydrogenase gene, the maize shrunken 1 gene and promoters from non-plant eukaryotes.
In some aspects of the invention it is preferred that the promoter element in the DNA construct be capable of causing sufficient expression in water deficit conditions. Such promoters can be identified and isolated from the regulatory region of plant genes that are over expressed in water deficit conditions. Specific water-deficit-inducible promoters for use in this invention are derived from the 5′ regulatory region of genes identified as a heat shock protein 17.5 gene (HSP17.5), an HVA22 gene (HVA22), a Rab17 gene and a cinnamic acid 4-hydroxylase (CA4H) gene (CA 4H) of Zea maize. Such water-deficit-inducible promoters are disclosed in U.S. 2004-0123347 A1, incorporated herein by reference.
In other aspects of the invention, sufficient expression in plant seed tissues is desired to effect improvements in seed composition. Exemplary promoters for use for seed composition modification include promoters from seed genes such as napin (U.S. Pat. No. 5,420,034), maize L3 oleosin (U.S. Pat. No. 6,433,252), zein Z27 (Russell, et al., (1997) Transgenic Res. 6(2):157-166), globulin 1 (Belanger, et al., (1991) Genetics 129:863-872), glutelin 1 (Russell (1997) supra), and peroxiredoxin antioxidant (Per1) (Stacy, et al., (1996) Plant Mol. Biol. 31(6):1205-1216).
In still other aspects of the invention, preferential expression in plant green tissues is desired. Promoters of interest for such uses include those from genes such as SSU (Fischhoff, et al., (1992) Plant Mol. Biol. 20:81-93), aldolase and pyruvate orthophosphate dikinase (PPDK) (Taniguchi, et al., (2000) Plant Cell Physiol. 41(1):42-48).
Gene Overexpression
“Gene overexpression” means expression, e.g., of a gene at a level in its native host that exceeds levels of expression in a non-trans genic host. In many embodiments of the invention, a recombinant DNA construct provides gene overexpression, e.g., as identified in Table 1.
Gene Suppression
Gene suppression includes any of the well-known methods for suppressing expression, typically indicated by reduced levels of protein. Posttranscriptional gene suppression is mediated by transcription of integrated recombinant DNA to form double-stranded RNA (dsRNA) having homology to a gene targeted for suppression. This formation of dsRNA most commonly results from transcription of an integrated inverted repeat of an element of a target gene, and is a common feature of gene suppression methods known as anti-sense suppression, co-suppression and RNA interference (RNAi). Transcriptional suppression can be mediated by a transcribed dsRNA having homology to a promoter DNA sequence to effect what is called promoter trans suppression.
More particularly, posttranscriptional gene suppression by inserting a recombinant DNA construct with one or more copies of anti-sense oriented DNA to regulate gene expression in plant cells is disclosed in U.S. Pat. No. 5,107,065 (Shewmaker, et al.,) and U.S. Pat. No. 5,759,829 (Shewmaker, et al.,). Transgenic plants transformed using such anti-sense oriented DNA constructs for gene suppression can comprise integrated DNA arranged as an inverted repeats that result from insertion of the DNA construct into plants by Agrobacterium-mediated transformation, as disclosed by Redenbaugh, et al., in “Safety Assessment of Genetically Engineered Flavr SavrTM Tomato, CRC Press, Inc. (1992). Inverted repeat insertions can comprises a part or all of the T-DNA construct, e.g., an inverted repeat of a complete transcription unit or an inverted repeat of transcription terminator sequence. Screening for inserted DNA comprising inverted repeat elements can improve the efficiency of identifying transformation events effective for gene silencing whether the transformation construct is a simple anti-sense DNA construct which must be inserted in multiple copies or a complex inverted repeat DNA construct (e.g., an RNAi construct) which can be inserted as a single copy.
Posttranscriptional gene suppression by inserting a recombinant DNA construct with sense-oriented DNA to regulate gene expression in plants is disclosed in U.S. Pat. No. 5,283,184 (Jorgensen, et al.) and U.S. Pat. No. 5,231,020 (Jorgensen, et al.). Inserted T-DNA providing gene suppression in plants transformed with such sense constructs by Agrobacterium is organized predominately in inverted repeat structures, as disclosed by Jorgensen, et al., Mol. Gen. Genet., 207:471-477 (1987). See also Stam, et al., The Plant Journal, 12(1), 63-82 (1997) who used segregation studies to support Jorgensen's finding that gene silencing is mediated by multimeric transgene T-DNA loci in which the T-DNAs are arranged in inverted repeats. Screening for inserted DNA comprising inverted repeat elements can improve the gene silencing efficiency when transforming with simple sense-orientated DNA constructs. Gene silencing efficiency can also be improved by screening for single insertion events when transforming with an RNAi construct containing inverted repeat elements
As disclosed by Redenbaugh, et al., gene suppression can be achieved by inserting into a plant genome recombinant DNA that transcribes dsRNA. Such a DNA insert can be transcribed to an RNA element having the 3′ region as a double stranded RNA. RNAi constructs are also disclosed in EP 0426195 A1 (Goldbach, et al.,—1991) where recombinant DNA constructs for transcription into hairpin dsRNA for providing transgenic plants with resistance to tobacco spotted wilt virus. Double-stranded RNAs were also disclosed in WO 94/01550 (Agrawal, et al.,) where anti-sense RNA was stabilized with a self-complementary 3′ segment. Agrawal, et al., referred to U.S. Pat. No. 5,107,065 for using such self-stabilized anti-sense RNAs for regulating gene expression in plant cells; see International Publication No. 94/01550. Other double-stranded hairpin-forming elements in transcribed RNA are disclosed in International Publication No. 98/05770 (Werner, et al.,) where the anti-sense RNA is stabilized by hairpin forming repeats of poly(CG) nucleotides. See also U.S. Patent Application Publication No. 2003/0175965 A1 (Lowe, et al.,) which discloses gene suppression using and RNAi construct comprising a gene coding sequence preceded by inverted repeats of 5′UTR. See also U.S. Patent Application Publication No. 2002/0048814 A1 (Oeller) where RNAi constructs are transcribed to sense or anti-sense RNA which is stabilized by a poly(T)-poly(A) tail. See also U.S. Patent Application Publication No. 2003/0018993 A1 (Gutterson, et al.,) where sense or anti-sense RNA is stabilized by an inverted repeat of the 3′ untranslated region of the NOS gene. See also U.S. Patent Application Publication No. 2003/0036197 A1 (Glassman, et al.,) where RNA having homology to a target is stabilized by two complementary RNA regions.
Gene silencing can also be affected by transcribing RNA from both a sense and an anti-sense oriented DNA, e.g., as disclosed by Shewmaker, et al., in U.S. Pat. No. 5,107,065 where in Example 1 a binary vector was prepared with both sense and anti-sense aroA genes. See also U.S. Pat. No. 6,326,193 where gene targeted DNA is operably linked to opposing promoters.
Gene silencing can also be affected by transcribing from contiguous sense and anti-sense DNA. In this regard see Sijen, et al., The Plant Cell, Vol. 8, 2277-2294 (1996) discloses the use of constructs carrying inverted repeats of a cowpea mosaic virus gene in transgenic plants to mediate virus resistance. Such constructs for posttranscriptional gene suppression in plants by double-stranded RNA are also disclosed in International Publication No. WO 99/53050 (Waterhouse, et al.,), International Publication No. WO 99/49029 (Graham, et al.), U.S. 2004-0029283 A1 (Fillatti), U.S. Pat. No. 6,506,559 (Fire, et al.,). See also U.S. 2004-0006792 A1 (Shewmaker, et al.,) that discloses constructs and methods for simultaneously expressing one or more recombinant genes while simultaneously suppressing one or more native genes in a transgenic plant. See also U.S. Pat. No. 6,448,473 (Mitsky, et al.,) that discloses multi-gene suppression vectors for use in plants. All of the above-described patents, applications and international publications disclosing materials and methods for posttranscriptional gene suppression in plants are incorporated herein by reference. Transcriptional suppression such as promoter trans suppression can be affected by a expressing a DNA construct comprising a promoter operably linked to inverted repeats of promoter DNA for a target gene. Constructs useful for such gene suppression mediated by promoter trans suppression are disclosed by Mette, et al., The EMBO Journal, Vol. 18, No. 1, pp. 241-148, 1999 and by Mette, et al., The EMBO Journal, Vol. 19, No. 19, pp. 5194-5201-148, 2000, both of which are incorporated herein by reference.
Suppression can also be achieved by insertion mutations created by transposable elements may also prevent gene function. For example, in many dicot plants, transformations with the T-DNA of Agrobacterium are readily achieved and large numbers of transformants can be rapidly obtained. Also, some species have lines with active transposable elements that are efficiently be used for the generation of large numbers of insertion mutations, while some other species lack such options. Mutant plants produced by Agrobacterium or transposon mutagenesis and having altered expression of a polypeptide of interest are identified using the polynucleotides of this invention. For example, a large population of mutated plants are screened to detect mutated plants having an insertion in the gene encoding the polypeptide of interest.
Gene Stacking
This invention also contemplates that the trait-improving recombinant DNA is used in combination with other recombinant DNA to create plants with a multiple desired traits. The combinations generated include multiple copies of any one or more of the recombinant DNA constructs. These stacked combinations are created by any method, including but not limited to cross breeding of transgenic plants, or multiple genetic transformation.
Plant Transformation Methods
Numerous methods for transforming plant cells with recombinant DNA are known in the art and are useful in producing the transgenic seeds of this invention. Two commonly used methods for plant transformation are Agrobacterium-mediated transformation and microprojectile bombardment. Microprojectile bombardment methods are illustrated in U.S. Pat. No. 5,015,580 (soybean); U.S. Pat. No. 5,550,318 (corn); U.S. Pat. No. 5,538,880 (corn); U.S. Pat. No. 5,914,451 (soybean); U.S. Pat. No. 6,160,208 (corn); U.S. Pat. No. 6,399,861 (corn) and U.S. Pat. No. 6,153,812 (wheat) and Agrobacterium-mediated transformation is described in U.S. Pat. No. 5,159,135 (cotton); U.S. Pat. No. 5,824,877 (soybean); U.S. Pat. No. 5,591,616 (corn); and U.S. Pat. No. 6,384,301 (soybean), all of which are incorporated herein by reference. For Agrobacterium tumefaciens based plant transformation system, additional elements present on transformation constructs include T-DNA left and right border sequences to facilitate incorporation of the recombinant polynucleotide into the plant genome.
In general it is preferred to introduce heterologous DNA randomly, i.e., at a non-specific location, in the genome of a target plant line. In special cases it is useful to target heterologous DNA insertion in order to achieve site-specific integration, e.g., to replace an existing gene in the genome, to use an existing promoter in the plant genome, or to insert a recombinant polynucleotide at a predetermined site known to be active for gene expression. Several site specific recombination systems exist which are known to function implants include cre-lox as disclosed in U.S. Pat. No. 4,959,317 and FLP-FRT as disclosed in U.S. Pat. No. 5,527,695, both incorporated herein by reference.
Transformation methods of this invention are preferably practiced in tissue culture on media and in a controlled environment. “Media” means any of the numerous nutrient mixtures that are used to grow cells in vitro, that is, outside of the intact living organism. Recipient cell targets include, but are not limited to, meristem cells, callus, immature embryos and gametic cells such as microspores, pollen, sperm and egg cells. It is contemplated that any cell from which a fertile plant is regenerated is useful as a recipient cell. Callus is initiated from tissue sources including, but not limited to, immature embryos, seedling apical meristems, microspores and the like. Cells capable of proliferating as callus are also recipient cells for genetic transformation. Practical transformation methods and materials for making transgenic plants of this invention, e.g., various media and recipient target cells, transformation of immature embryos and subsequent regeneration of fertile transgenic plants are disclosed in U.S. Pat. Nos. 6,194,636 and 6,232,526 and U.S. 2004-0216189 A1, which are incorporated herein by reference.
In practice DNA is introduced into only a small percentage of target cells in any one experiment. Marker genes are used to provide an efficient system for identification of those cells that are stably transformed by receiving and integrating a transgenic DNA construct into their genomes. Preferred marker genes provide selective markers that confer resistance to a selective agent, such as an antibiotic or herbicide. Potentially transformed cells are exposed to the selective agent. In the population of surviving cells are those cells where, generally, the resistance-conferring gene has been integrated and expressed at sufficient levels to permit cell survival. Cells are tested further to confirm stable integration of the exogenous DNA. Useful selective marker genes include those conferring resistance to antibiotics such as kanamycin (nptII), hygromycin B (aph IV) and gentamycin (aac3 and aacC4) or resistance to herbicides such as glufosinate (bar or pat) and glyphosate (EPSPS). Examples of such selectable are illustrated in U.S. Pat. Nos. 5,550,318; 5,633,435; 5,780,708 and 6,118,047, all of which are incorporated herein by reference. Screenable markers which provide an ability to visually identify transformants are also often employed, e.g., a gene expressing a colored or fluorescent protein such as a luciferase or green fluorescent protein (GFP) or a gene expressing a beta-glucuronidase or uidA gene (GUS) for which various chromogenic substrates are known. It is also contemplated that combinations of screenable and selectable markers will be useful for identification of transformed cells. See PCT publication WO 99/61129 which discloses use of a gene fusion between a selectable marker gene and a screenable marker gene, e.g., an NPTII gene and a GFP gene.
Cells that survive exposure to the selective agent, or cells that have been scored positive in a screening assay, are cultured in regeneration media and allowed to mature into plants. Developing plantlets are transferred to soil less plant growth mix, and hardened off, e.g., in an environmentally controlled chamber at about 85% relative humidity, 600 ppm CO2, and 25-250 microeinsteins m−2s−1 of light, prior to transfer to a greenhouse or growth chamber for maturation. Plants are preferably matured either in a growth chamber or greenhouse. Plants are regenerated from about 6 wk to 10 months after a transformant is identified, depending on the initial tissue. During regeneration, cells are grown to plants on solid media at about 19 to 28 degrees C. After regenerating plants have reached the stage of shoot and root development, they are transferred to a greenhouse for further growth and testing. Plants are pollinated using conventional plant breeding methods known to those of skill in the art and seed produced.
Progeny are recovered from transformed plants and tested for expression of the exogenous recombinant polynucleotide. Useful assays include, for example, “molecular biological” assays, such as Southern and Northern blotting and PCR; “biochemical” assays, such as detecting the presence of RNA, e.g., double stranded RNA, or a protein product, e.g., by immunological means (ELISAs and Western blots) or by enzymatic function; plant part assays, such as leaf or root assays; and also, by analyzing the phenotype of the whole regenerated plant.
Discovery of Trait-Improving Recombinant DNA
To identify recombinant DNA that confer improved traits to plants, Arabidopsis plants were transformed with a large population of recombinant DNA constructs for expressing a large variety of distinct DNA. Transgenic plants were produced and screened to identify those plants having recombinant DNA constructs expressing trait-imparting DNA. A two-step screening process was employed which comprised two passes of trait characterization to ensure that the trait modification was dependent on expression of the recombinant DNA, but not due to the chromosomal location of the integration of the transgene. Twelve independent transgenic lines for each recombinant DNA construct were established and assayed for the transgene expression levels. Five transgenic lines with high transgene expression levels were used in the first pass screen to evaluate the transgene's function in T2 transgenic plants. Subsequently, three transgenic events, which had been shown to have one or more improved traits, were further evaluated in the second pass screen to confirm the transgene's ability to impart an improved trait. The following Table 2 summarizes the improved traits that have been confirmed as provided by a recombinant DNA construct.
In particular, Table 2 reports
“PEP Seq ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.


    
    
        “construct_id” is an arbitrary name for the recombinant DNA describe more particularly in Table 1.
        “annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.
        “description” refers to the description of the top BLAST hit.
        “e-value” provides the expectation value for the BLAST hit.
        “identity” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.
        “traits” identified by two letters codes the confirmed improvement in a transgenic plant provided by the recombinant DNA. The codes for improved traits are:
        “CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;
        “CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;
        “DS” which indicates drought tolerance improvement identified by a PEG induced osmotic stress tolerance screen;
        “PEG” which idicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;
        “HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;
        “SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;
        “LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen;
        “LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;
        “PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen;
        “SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.
    
    












TABLE 2







Pep

annotation















SEQ
construct

e
%





Id
id
gene
value
identity
ncbi id
description
traits






















270
14324
CGPG1560
1.00E−127
86
gi|15232185|
ref|NP_191546.1|expressed
CK
SS












protein [Arabidopsis














thaliana]]








271
17484
CGPG2630
0
93
gi|15220912|
ref|NP_173239.1|zinc finger
CK













(C3HC4-type RING finger)














family protein [Arabidopsis














thaliana]








272
19109
CGPG1381
9.00E−31
81
gi|18404521|
ref|NP_565870.1|expressed
CK













protein [Arabidopsis














thaliana]








273
70423
CGPG3165
1.00E−134
96
gi|30688808|
ref|NP_850953.1|MADS-
CK
CS
CK
HS
PP









box protein (AGL9)














[Arabidopsis thaliana]














gb|AAM65812.1| putative














floral homeotic protein,














AGL9 [Arabidopsis














thaliana]








274
70424
CGPG3180
1.00E−142
81
gi|25405039|
pir||H96827protein
CK













F20B17.12 [imported]-














Arabidopsis thaliana














gb|AAF68121.1| F20B17.12














[Arabidopsis thaliana]








275
70480
CGPG3833
1.00E−122
99
gi|18411867|
ref|NP_565174.1|14-3-3
CK













protein GF14 pi (GRF13)














[Arabidopsis thaliana]








276
70509
CGPG2420
1.00E−113
82
gi|15225186|
ref|NP_180770.1|ovate
CK













protein-related [Arabidopsis














thaliana]








277
70647
CGPG4334
1.00E−171
94
gi|15237269|
ref|NP_200093.1|ornithine
CK













cyclodeaminase/mu-














crystallin family protein














[Arabidopsis thaliana]














dbj|BAB10429.1]








278
70675
CGPG4519
0
100
gi|15224730|
ref|NP_180115.1|2-
CK













oxoglutarate-dependent














dioxygenase, putative














[Arabidopsis thaliana]














pir||E84648 probable














dioxygenase]








279
70829
CGPG518
0
92
gi|15232841|
ref|NP_186854.1|potassium
CK













transporter (KUP3)














[Arabidopsis thaliana]








280
70849
CGPG596
1.00E−166
96
gi|15224801|
ref|NP_179547.1|cytidine
CK













deaminase (CDD)/cytidine














aminohydrolase














[Arabidopsis thaliana]








281
71627
CGPG1270
0
99
gi|18398032|
ref|NP_566315.1|ABC1
CK













family protein [Arabidopsis














thaliana]








282
71934
CGPG2294
1.00E−154
79
gi|15233973|
ref|NP_195575.1|26S
CK













proteasome regulatory














subunit S5A (RPN10)














[Arabidopsis thaliana]














sp|P55034|PSD4_ARATH














26S proteasome non-














ATPase regulatory subunit














4 (26S proteasome














regulatory








283
72615
CGPG4829
2.00E−49
88
gi|18422886|
ref|NP_568693.1|expressed
CK













protein [Arabidopsis














thaliana]








284
72927
CGPG1477
1.00E−114
81
gi|15234815|
ref|NP_194797.1|MA3
CK













domain-containing protein














[Arabidopsis thaliana]














pir||A85359 translation














initiation factor-like protein








285
73014
CGPG5692
1.00E−180
93
gi|37528369|
ref|NP_931714.1|Fructose-
CK
PP












1,6-bisphosphatase (D-














fructose-1,6-bisphosphate














1-phosphohydrolase)














(FBPase) [Photorhabdus














luminescens subsp.














laumondii TTO1]








286
73559
CGPG6535
0
93
gi|16078422|
ref|NP_389241.1|similar to
CK













aspartate aminotransferase














[Bacillus subtilis]














sp|O31665|MTNE_BACSU














Transaminase mtnE














pir||F69863 probable














transaminase (EC 2.6.1.—)














ykrV








287
74251
CGPG5489
1.00E−171
87
gi|15238437|
ref|NP_200760.1|zinc
CK
SP












transporter (ZIP2)














[Arabidopsis thaliana]














sp|Q9LTH9|ZIP2_ARATH














Zinc transporter 2 precursor














(ZRT/IRT-like protein 2)








288
19631
CGPG3627
1.00E−94
90
gi|18410249|
ref|NP_565053.1|SNF7
CS













family protein [Arabidopsis














thaliana] pir||G96755














developmental protein














homolog DG1118














[imported]-Arabidopsis














thaliana








289
70121
CGPG2380
1.00E−111
100
gi|6323765|
ref|NP_013836.1|Hypothetical
CS













ORF; Ymr118cp














[Saccharomyces














cerevisiae]














sp|Q04487|YM07_YEAST














Putative succinate














dehydrogenase cytochrome














B subunit, mitochondrial














precursor








290
70654
CGPG4352
2.00E−63
88
gi|18400941|
ref|NP_566531.1|expressed
CS













protein [Arabidopsis














thaliana]








291
70696
CGPG4590
8.00E−92
87
gi|25408379|
pir||E84768hypothetical
CS
PP












protein At2g35430








292
70713
CGPG1462
0
95
gi|30678679|
ref|NP_191966.2|malate
CS













oxidoreductase, putative








293
70740
CGPG3700
0
94
gi|18402759|
ref|NP_566667.1|transcription
CS
LL
PP











factor jumonji (jmjC)














domain-containing protein








294
71321
CGPG4418
0
91
gi|13878402|
sp|Q9STL0|C71N_ARATH
CS













Cytochrome P450 71A23














pir||T06712 probable














cytochrome P450














T29H11.180








295
71835
CGPG4634
0
100
gi|15234361|
ref|NP_192100.1|DC1
CS
SP












domain-containing protein














[Arabidopsis thaliana]














pir||E85024 probable CHP-














rich zinc finger protein








296
72934
CGPG5798
0
98
gi|6323512|
ref|NP_013583.1|High-
CS













affinity inorganic phosphate














(Pi) transporter and low-














affinity manganese














transporter; regulated by














Pho4p and Spt7p; mutation














confers resistance to














arsenate; exit from the ER














during maturation requires














Pho86p; Pho84p














[Saccharomyces














cerevisiae]








297
72945
CGPG5787
0
94
gi|6319991|
ref|NP_010071.1|GABA-
CS













specific transport protein;














Uga4p [Saccharomyces














cerevisiae]














sp|P32837|UGA4_YEAST














GABA-specific permease














(GABA-specific transport














protein)








298
72980
CGPG5773
0
89
gi|6321960|
ref|NP_012036.1|Subunit of
CS













the anaphase-promoting














complex/cyclosome














(APC/C), which is a














ubiquitin-protein ligase














required for degradation of














anaphase inhibitors,














including mitotic cyclins,














during the














metaphase/anaphase














transition; Cdc23p








299
73504
CGPG6480
1.00E−178
100
gi|16330153|
ref|NP_440881.1|fructokinase
CS
PP












[Synechocystis sp. PCC














6803] pir||S77227














fructokinase (EC 2.7.1.4)-














Synechocystis sp. (strain














PCC 6803)








300
73507
CGPG6504
1.00E−173
100
gi|16078547|
ref|NP_389366.1|similar to
CS
LL
PP
PEG










glutaminase [Bacillus














subtilis]








301
73573
CGPG6462
0
100
gi|15890038|
ref|NP_355719.1|AGR_C_5067p
CS
PP












[Agrobacterium














tumefaciens str. C58]














ref|NP_533456.1|3-














isopropylmalate














dehydrogenase








302
73586
CGPG6471
1.00E−177
100
gi|160776841
ref|NP_388498.1|similar to
CS
DS
PP











fructokinase [Bacillus














subtilis]








303
73770
CGPG5435
2.00E−34
68
gi|15235771|
ref|NP_193383.1|cysteine
CS
PP












protease inhibitor family














protein/cystatin family














protein [Arabidopsis














thaliana]








304
74105
CGPG6574
0
73
gi|23059330|
ref|ZP_00084307.1|COG1012:
CS













NAD-dependent














aldehyde dehydrogenases














[Pseudomonas fluorescens














PfO-1]








305
74111
CGPG6622
0
99
gi|16131442|
ref|NP_418028.1|alpha-
CS













amylase [Escherichia coli














K12]








306
74136
CGPG6632
9.00E−81
99
gi|16330993|
ref|NP_441721.1|unknown
CS
CK
HS











protein [Synechocystis sp.














PCC 6803]








307
74139
CGPG6561
1.00E−180
95
gi|24112825|
ref|NP_707335.1|glyceraldehyde-
CS
LL












3-phosphate














dehydrogenase A [Shigella














flexneri 2a str. 301]








308
74267
CGPG5364
0
97
gi|18399375|
ref|NP_566402.1|U-box
CS













domain-containing protein














[Arabidopsis thaliana]








309
74291
CGPG5363
0
94
gi|18401867|
ref|NP_565676.1|armadillo/
CS













beta-catenin repeat family














protein/U-box domain-














containing protein














[Arabidopsis thaliana]








310
74318
CGPG5826
0
100
gi|15219730|
ref|NP_176847.1|cell
CS
HS












division protein kinase,














putative [Arabidopsis














thaliana]








311
74319
CGPG5831
0
96
gi|15224359|
ref|NP_181907.1|mitogen-
CS













activated protein kinase,














putative/MAPK, putative














(MPK6) [Arabidopsis














thaliana]








312
74324
CGPG5885
1.00E−174
95
gi|42569304|
ref|NP_180094.2|protein
CS













kinase family protein














[Arabidopsis thaliana]








313
74512
CGPG32
0
96
gi|15217945|
ref|NP_176132.1|amino
CS
HS
SP











acid permease I (AAP1)














[Arabidopsis thaliana]








314
74583
CGPG6649
1.00E−151
83
gi|22978283|
ref|ZP_00024043.1|COG0252:
CS
PP












L-














asparaginase/archaeal Glu-














tRNAGln amidotransferase














subunit D [Ralstonia














metallidurans]








315
70427
CGPG3067
0
100
gi|42572771|
ref|NP_974481.1|kelch
CS
DS












repeat-containing F-box














family protein [Arabidopsis














thaliana]








316
71811
CGPG4426
0
97
gi|15223341|
ref|NP_171627.1|cytochrome
CS
DS
LL
LN










P450, putative














[Arabidopsis thaliana]








317
73463
CGPG6384
0
100
gi|22977164|
ref|ZP_00022985.1|COG0538:
DS













Isocitrate














dehydrogenases [Ralstonia














metallidurans]








318
72081
CGPG5279
7.00E−66
77
gi|42570373|
ref|NP_850277.2|CCAAT-
DS
PEG












box binding transcription














factor, putative [Arabidopsis














thaliana]








319
10139
CGPG101
0
89
gi|15229877|
ref|NP_187154.1|sodium
DS













proton exchanger, putative














(NHX2) [Arabidopsis














thaliana]








320
11410
CGPG103
0
82
gi|15236418|
ref|NP_192555.1|homeobox
DS













protein knotted-1 like 1














(KNAT1) [Arabidopsis














thaliana]








321
11604
CGPG48
0
92
gi|15233457|
ref|NP_194642.1|hexokinase
DS













1 (HXK1) [Arabidopsis














thaliana]








322
12368
CGPG1006
1.00E−146
85
gi|15231451|
ref|NP_190238.1|epsin N-
DS













terminal homology (ENTH)














domain-containing protein/














clathrin assembly protein-














related [Arabidopsis














thaliana]








323
13502
CGPG1354
0
95
gi|15224557|
ref|NP_180632.1|serine/threonine
DS
PP












protein kinase,














putative [Arabidopsis














thaliana]








324
13745
CGPG1576
1.00E−112
84
gi|15222987|
ref|NP_177749.1|hypothetical
DS













protein [Arabidopsis














thaliana] gb|AAF17642.1|














T23E18.15 [Arabidopsis














thaliana]








325
13821
CGPG1569
1.00E−155
85
gi|18416499|
ref|NP_567716.1|expressed
DS













protein [Arabidopsis














thaliana]








326
14240
CGPG1697
0
94
gi|15241302|
ref|NP_197527.1|expressed
DS













protein [Arabidopsis














thaliana]








327
14718
CGPG1082
0
86
gi|18407200|
ref|NP_566090.1|expressed
DS













protein [Arabidopsis














thaliana]








328
17022
CGPG1774
1.00E−159
100
gi|15237803|
ref|NP_197755.1|nodulin
DS













MtN3 family protein














[Arabidopsis thaliana]








329
17924
CGPG2882
3.00E−92
100
gi|15233350|
ref|NP_192875.1|zinc finger
DS













(C3HC4-type RING finger)














family protein (RHA1b)














[Arabidopsis thaliana]








330
18259
CGPG3368
2.00E−94
88
gi|30685085|
ref|NP_849549.1|zinc finger
DS
PP












protein (LSD1) [Arabidopsis














thaliana]








331
19171
CGPG2952
0
91
gi|6320063|
ref|NP_010143.1|plasma
DS













membrane glucose sensor;














Rgt2p [Saccharomyces














cerevisiae]








332
19201
CGPG2332
0
96
gi|15233948|
ref|NP_194205.1|protein
DS













kinase (AFC2) [Arabidopsis














thaliana]














sp|P51567|AFC2_ARATH














Protein kinase








333
19317
CGPG3662
1.00E−151
91
gi|21232858|
ref|NP_638775.1|conserved
DS













hypothetical protein














[Xanthomonas campestris














pv. campestris str. ATCC














33913]








334
70417
CGPG3427
0
81
gi|18396278|
ref|NP_566180.1|integral
DS
PP
SP











membrane family protein














[Arabidopsis thaliana]








335
70467
CGPG3785
0
100
gi|15241416|
ref|NP_196953.1|no apical
DS













meristem (NAM) family














protein [Arabidopsis














thaliana]








336
70806
CGPG712
0
100
gi|15218225|
ref|NP_173010.1|cyclin,
DS













putative [Arabidopsis














thaliana]








337
70818
CGPG479
1.00E−157
92
gi|30691978|
ref|NP_568508.2|bZIP
DS













transcription factor family














protein [Arabidopsis














thaliana]








338
70820
CGPG655
0
93
gi|15224342|
ref|NP_181899.1|acyl-[acyl-
DS













carrier-protein] desaturase/














stearoyl-ACP desaturase














(SSI2) [Arabidopsis














thaliana]








339
70919
CGPG4029
1.00E−169
73
gi|6996560|
emb|CAB75429.1|oligouridylate
DS













binding protein














[Nicotiana plumbaginifolia]








340
71623
CGPG4696
1.00E−150
95
gi|15236511|
ref|NP_192588.1|mitogen-
DS













activated protein kinase,














putative [Arabidopsis














thaliana] pir||T01835














serine/threonine-specific














protein kinase ARA.KIN














homolog T15F16.3-














Arabidopsis thaliana








341
71662
CGPG4679
1.00E−173
91
gi|5929964|
gb|AAD56659.1|malate
DS













dehydrogenase [Glycine














max]








342
71693
CGPG4652
6.00E−86
56
gi|21553460|
gb|AAM62553.1|snap25a
DS













[Arabidopsis thaliana]








343
72384
CGPG4639
0
98
gi|1169548|
sp|P38604|
DS













ERG7_YEASTLanosterol














synthase














(Oxidosqualene--lanosterol














cyclase) (2,3-














epoxysqualene--lanosterol














cyclase) (OSC)














gb|AAA64377.1|2,3-














oxidosqualene-lanosterol














cyclase








344
72439
CGPG5075
8.00E−57
90
gi|22331337|
ref|NP_683594.1|NPR1/NIM1-
DS













interacting protein 2














(NIMIN-2)








345
72619
CGPG4835
6.00E−83
88
gi|15237317|
ref|NP_200108.1|expressed
DS













protein [Arabidopsis














thaliana]








346
72624
CGPG4842
0
100
gi|15242148|
ref|NP_200558.1|expressed
DS













protein [Arabidopsis














thaliana]








347
72715
CGPG5521
0
91
gi|6323933|
ref|NP_014004.1|Carboxy-
DS
SS












terminal domain (CTD)














phosphatase, essential for














dephosphorylation of the














repeated C-terminal














domain of the RNA














polymerase II large subunit














(Rpo21p); Fcp1p














[Saccharomyces














cerevisiae]








348
72754
CGPG5548
1.00E−169
100
gi|728961|
sp|Q00618|
DS













BET4_YEASTGeranylgeranyl














transferase














type II alpha subunit (Type














II protein geranyl-








349
72819
CGPG4989
0
100
gi|18417026|
ref|NP_567780.1|pfkB-type
DS













carbohydrate kinase family














protein [Arabidopsis














thaliana]








350
75516
CGPG7689
1.00E−138
70
gi|42568081|
ref|NP_197938.2|zinc finger
DS













(C3HC4-type RING finger)














family protein [Arabidopsis














thaliana]








351
75701
CGPG7856
8.00E−41
37
gi|15225413|
ref|NP_182037.1|zinc finger
DS
LN












(C2H2 type) family protein














[Arabidopsis thaliana]








352
73515
CGPG6473
1.00E−162
100
gi|16079626|
ref|NP_390450.1|similar to
HS
CS
PEG











6-phosphogluconate














dehydrogenase (pentose














phosphate) [Bacillus














subtilis]








353
74684
CGPG6360
1.00E−64
100
gi|18390735|
ref|NP_563782.1|expressed
CS
HS












protein [Arabidopsis














thaliana]








354
19542
CGPG3069
0
90
gi|18403574|
ref|NP_564592.1|F-box
HS













family protein [Arabidopsis














thaliana]








355
19618
CGPG3574
1.00E−121
100
gi|15218423|
ref|NP_177373.1|trypsin
HS













and protease inhibitor














family protein/Kunitz














family protein [Arabidopsis














thaliana] pir||F96746














probable drought induced














protein








356
19649
CGPG3140
1.00E−141
87
gi|18412787|
ref|NP_567287.1|vesicle-
HS













associated membrane














family protein/VAMP














family protein








357
19745
CGPG3973
2.00E−60
46
gi|15239303|
ref|NP_201424.1|expressed
HS













protein [Arabidopsis














thaliana]








358
19768
CGPG4096
1.00E−179
81
gi|25052804|
gb|AAN65180.1|mitogen-
HS
SS












activated protein kinase 4














[Petroselinum crispum]








359
19772
CGPG3939
3.00E−82
79
gi|7488744|
pir||T09700MADS-box
HS













protein —alfalfa (fragment)














gb|AAB51377.1| MADS-box














protein [Medicago sativa]








360
19779
CGPG4113
1.00E−153
89
gi|30681126|
ref|NP_196201.2|phosphate
CS
HS












translocator-related














[Arabidopsis thaliana]








361
19833
CGPG4074
1.00E−107
79
gi|6683777|
gb|AAF23363.1|CAGL2
CS
HS
PP











[Cucumis sativus]








362
19862
CGPG3961
2.00E−89
56
gi|15229637|
ref|NP_188469.1|no apical
HS













meristem (NAM) family














protein [Arabidopsis














thaliana] dbj|BAB01106.1|














unnamed protein product














[Arabidopsis thaliana]








363
19879
CGPG4009
0
75
gi|18401703|
ref|NP_564504.1|protein
HS
CS

SS










phosphatase 2C-related/














PP2C-related [Arabidopsis














thaliana]








364
70445
CGPG3728
2.00E−51
88
gi|30696602|
ref|NP_200357.2|protease
HS













inhibitor/seed storage/lipid














transfer protein (LTP)














family protein [Arabidopsis














thaliana]








365
70738
CGPG3195
1.00E−96
100
gi|15234797|
ref|NP_194791.1|expressed
HS
PP












protein [Arabidopsis














thaliana]








366
71437
CGPG4043
1.00E−164
81
gi|15241535|
ref|NP_196433.1|serine/threonine
HS













protein kinase,














putative [Arabidopsis














thaliana]








367
71572
CGPG4520
3.00E−83
92
gi|18403850|
ref|NP_565804.1|expressed
HS













protein [Arabidopsis














thaliana]








368
71617
CGPG1227
0
100
gi|15236219|
ref|NP_195218.1|1-
HS
CK












phosphatidylinositol














phosphodiesterase-related














[Arabidopsis thaliana]








369
72532
CGPG4780
1.00E−118
90
gi|15236659|
ref|NP_194120.1|expressed
HS













protein [Arabidopsis














thaliana]








370
72757
CGPG5572
0
89
gi|15242402|
ref|NP_197088.1|zinc finger
HS
LL
PEG











protein CONSTANS (CO)














[Arabidopsis thaliana]








371
73412
CGPG6448
0
99
gi|28867589|
ref|NP_790208.1|glutamine
HS













synthetase, type I














[Pseudomonas syringae pv.














tomato str. DC3000]








372
74102
CGPG6550
1.00E−167
94
gi|15614187|
ref|NP_242490.1|L-
HS













asparaginase [Bacillus














halodurans C-125]








373
72633
CGPG4853
1.00E−145
86
gi|15238013|
ref|NP_199519.1|casein
CS
LL
PEG











kinase II beta chain,














putative [Arabidopsis














thaliana]








374
72456
CGPG4745
1.00E−75
92
gi|15239846|
ref|NP_196763.1|17.6 kDa
DS
LL












class II heat shock protein














(HSP17.6-CII) [Arabidopsis














thaliana]








375
72963
CGPG1746
1.00E−151
87
gi|15222239|
ref|NP_172174.1|ovate
LL
LN












family protein [Arabidopsis














thaliana]








376
70426
CGPG3199
8.00E−54
88
gi|18397268|
ref|NP_564336.1|double-
LL













stranded DNA-binding














family protein [Arabidopsis














thaliana]








377
70772
CGPG4627
1.00E−92
80
gi|15220084|
ref|NP_173175.1|MADS-
LL













box protein (AGL100)














[Arabidopsis thaliana] P








378
71137
CGPG125
1.00E−111
90
gi|15218957|
ref|NP_176202.1|two-
LL













component responsive














regulator/response














regulator 3 (ARR3)














[Arabidopsis thaliana]








379
71529
CGPG2808
1.00E−131
72
gi|42562375|
ref|NP_174152.3|Dof-type
LL













zinc finger domain-














containing protein














[Arabidopsis thaliana]








380
71601
CGPG1858
1.00E−168
92
gi|15231425|
ref|NP_187378.1|transcriptional
LL













activator, putative














[Arabidopsis thaliana]








381
72362
CGPG983
1.00E−163
95
gi|15242779|
ref|NP_200562.1|xylogluca
LL













n: xyloglucosyl transferase,














putative/xyloglucan














endotransglycosylase,














putative/endo-xyloglucan














transferase, putative














[Arabidopsis thaliana]








382
72466
CGPG4767
3.00E−60
83
gi|15234046|
ref|NP_195030.1|glutaredo
CK
LL
PEG











xin family protein














[Arabidopsis thaliana]








383
72524
CGPG4770
1.00E−134
91
gi|18412649|
ref|NP_567140.1|expressed
CK
LL












protein [Arabidopsis














thaliana]








384
73085
CGPG5689
1.00E−134
100
gi|16331347|
ref|NP_442075.1|triosephosphate
LL













isomerase














[Synechocystis sp. PCC














6803]








385
74241
CGPG5457
0
89
gi|444790|
prf||1908224Anucleotide
LL













translocator








386
74247
CGPG5475
1.00E−159
100
gi|18411863|
ref|NP_565172.1|protein
LL













phosphatase 2C, putative/














PP2C, putative














[Arabidopsis thaliana]








387
74284
CGPG5413
0
97
gi|15230577|
ref|NP_190087.1|serine
LL













carboxypeptidase III,














putative [Arabidopsis














thaliana]








388
74652
CGPG6168
1.00E−90
80
gi|15235970|
ref|NP_194879.1|expressed
LL
DS












protein [Arabidopsis














thaliana]








389
70437
CGPG3706
1.00E−172
100
gi|30678824|
ref|NP_186983.2|short-
CK
LN












chain














dehydrogenase/reductase














(SDR) family protein














[Arabidopsis thaliana]








390
71633
CGPG857
1.00E−100
86
gi|6690274|
gb|AAF24061.1|v-SNARE
DS
LN












AtVTI1a [Arabidopsis














thaliana]








391
72948
CGPG5617
0
94
gi|15225456|
ref|NP_182059.1|leucine-
LN
PEG












rich repeat transmembrane














protein kinase, putative














[Arabidopsis thaliana]








392
72519
CGPG4749




LN
SS






393
10475
CGPG399
1.00E−164
96
gi|15240972|
ref|NP_195761.1|stress-
LN













responsive protein, putative














[Arabidopsis thaliana]








394
11120
CGPG459
0
100
gi|15227169|
ref|NP_179812.1|inositol-3-
LN













phosphate synthase














isozyme 2/myo-inositol-1-














phosphate synthase 2/MI-














1-P synthase 2/IPS 2














[Arabidopsis thaliana]








395
19736
CGPG4129
2.00E−94
67
gi|13346194|
gb|AAK19619.1|GHMYB9
LN













[Gossypium hirsutum]








396
71606
CGPG4715
0
91
gi|15218674|
ref|NP_171800.1|phototropic-
LN













responsive NPH3 family














protein [Arabidopsis














thaliana]








397
71840
CGPG4353
0
96
gi|18401087|
ref|NP_566542.1|mitotic
DS
LL
LN











phosphoprotein N′ end














(MPPN) family protein














[Arabidopsis thaliana]








398
74240
CGPG5454
1.00E−155
90
gi|15233884|
ref|NP_194188.1|mitochondrial
CK
LN












substrate carrier family














protein [Arabidopsis














thaliana] pir||T05577














uncoupling protein homolog














F22K18.230-Arabidopsis














thaliana








399
74331
CGPG5834
0
94
gi|15220416|
ref|NP_172003.1|protein
LN













kinase family protein














[Arabidopsis thaliana]








400
74610
CGPG6048
1.00E−117
100
gi|15217568|
ref|NP_172434.1|Ras-
LL
LN












related GTP-binding














protein, putative














[Arabidopsis thaliana]














sp|O04486|RB1C_ARATH














Ras-related protein














Rab11C








401
75527
CGPG7682
1.00E−55
65
gi|15240946|
ref|NP_195750.1|phosphati
LN













dylethanolamine-binding














family protein [Arabidopsis














thaliana]








402
70681
CGPG4584
9.00E−64
93
gi|18411465|
ref|NP_567196.1|auxin-
CK
PEG












responsive family protein














[Arabidopsis thaliana]








403
71663
CGPG4638
0
93
gi|21230153|
ref|NP_636070.1|conserved
CK
PEG












hypothetical protein














[Xanthomonas campestris














pv. campestris str. ATCC














33913]








404
72769
CGPG5573
0
100
gi|15225499|
ref|NP_182075.1|cytochrome
CK
PEG












P450, putative














[Arabidopsis thaliana]








405
71508
CGPG1541
2.00E−24
100
gi|15241504|
sp|Q9SD80|OM05_ARATH
PEG
CS
SP
PEG










Mitochondrial import














receptor subunit TOM5














homolog (Translocase of














outer membrane 5 kDa














subunit homolog)








406
74248
CGPG5476
0
100
gi|15226152|
ref|NP_180926.1|protein
PEG
CS












phosphatase 2C, putative/














PP2C, putative














[Arabidopsis thaliana] p








407
72771
CGPG2166
5.00E−44
100
gi|18395032|
ref|NP_564151.1|expressed
PEG
CK
HS
SS










protein [Arabidopsis














thaliana]








408
72085
CGPG5228
0
94
gi|15241541|
ref|NP_199275.1|cytochrome
PEG
HS












P450 family protein














[Arabidopsis thaliana]














dbj|BAA98115.1| flavonoid














3′,5′-hydroxylase-like;














cytochrome P450














[Arabidopsis thaliana]








409
72744
CGPG5563
1.00E−136
96
gi|6321574|
ref|NP_011651.1|20S
HS
PEG
CK











proteasome beta-type














subunit; the only














nonessential 20S subunit;














Pre9p [Saccharomyces














cerevisiae]








410
73039
CGPG810
0
96
gi|15242124|
ref|NP_197599.1|molybdopterin
HS
PEG












biosynthesis CNX1














protein/molybdenum














cofactor biosynthesis














enzyme CNX1 (CNX1)














[Arabidopsis thaliana]








411
73054
CGPG5754
2.00E−98
100
gi|6324827|
ref|NP_014896.1|Nat5p
PEG
HS
SS
PEG










[Saccharomyces














cerevisiae] pir||S67150














hypothetical protein














YOR253w-yeast














(Saccharomyces














cerevisiae)








412
73501
CGPG6456
0
97
gi|15888752|
ref|NP_354433.1|AGR_C_2631p
HS
PEG












[Agrobacterium














tumefaciens str. C58]














sp|Q8UFH1|ENO_AGRT5














Enolase (2-














phosphoglycerate














dehydratase) (2-phospho-














D-glycerate hydro-lyase)








413
19707
CGPG4179
1.00E−86
49
gi|15236282|
ref|NP_195242.1|O-
PEG
CS












methyltransferase family 2














protein [Arabidopsis














thaliana]








414
19951
CGPG3941
5.00E−91
54
gi|15221582|
ref|NP_177064.1|basic
PEG
CK












helix-loop-helix (bHLH)














family protein [Arabidopsis














thaliana]








415
19967
CGPG4032
1.00E−127
67
gi|4760710|
dbj|BAA77395.1|SLL2-S9-
PEG













protein [Brassica rapa]








416
70543
CGPG3815
0
95
gi|15220994|
ref|NP_175222.1|E2F
PEG
CK












transcription factor-2














(E2F2)/transcription factor














E2Fc (E2Fc) [Arabidopsis














thaliana]








417
70707
CGPG1273
1.00E−108
100
gi|15219558|
ref|NP_177523.1|Ssu72-
PEG













like family protein














[Arabidopsis thaliana]














pir||F96765 unknown














protein F








418
70719
CGPG1712
0
86
gi|18394560|
ref|NP_564043.1|expressed
PEG













protein [Arabidopsis














thaliana]








419
71134
CGPG817
8.00E−55
100
gi|15240471|
ref|NP_200327.1|small
PEG
HS
PP











ubiquitin-like modifier 2














(SUMO) [Arabidopsis














thaliana]








420
71146
CGPG2928
1.00E−86
92
gi|29165403|
gb|AAO65311.1|MADS
PEG













affecting flowering 3 variant














II [Arabidopsis thaliana]








421
71660
CGPG4690
3.00E−80
100
gi|18415773|
ref|NP_567637.1|methionine
PEG













sulfoxide reductase














domain-containing protein/














SeIR domain-containing














protein [Arabidopsis














thaliana]








422
72086
CGPG5236
0
100
gi|15232215|
ref|NP_191556.1|
PEG
PP
PEG











methylenetetrahydrofolate














reductase














1 (MTHFR1) [Arabidopsis














thaliana]-








423
72632
CGPG4852
4.00E−99
90
gi|18425032|
ref|NP_569028.1|expressed
PEG













protein [Arabidopsis














thaliana]








424
72716
CGPG5529
2.00E−85
89
gi|6320196|
ref|NP_010276.1|subunit of
PEG













the Anaphase Promoting














Complex; all known APC














subunits co-














immunoprecipitate with














epitope-tagged Apc11p;














Apc11p [Saccharomyces














cerevisiae]








425
72723
CGPG1848
0
97
gi|15237500|
ref|NP_199487.1|human
PEG













Rev interacting-like family














protein/hRIP family protein














[Arabidopsis thaliana]














dbj|BAB08919.1| zinc finger














protein Glo3-like














[Arabidopsis thaliana]








426
72987
CGPG1787
5.00E−81
77
gi|15231568|
ref|NP_189282.1|octicosapeptide/
PEG
SP












Phox/Bem1p (PB1)














domain-containing protein














[Arabidopsis thaliana]








427
74109
CGPG6606
0
77
gi|37524479|
ref|NP_927823.1|maltodextrin
PEG













phosphorylase














[Photorhabdus luminescens














subsp. laumondii TTO1]








428
74140
CGPG6569
0
99
gi|15613102|
ref|NP_241405.1|NADP-
PEG
PP
PEG











dependent aldehyde














dehydrogenase [Bacillus














halodurans C-125]








429
74191
CGPG6597
0
96
gi|22960294|
ref|ZP_00007935.1|COG1850:
PEG













Ribulose 1,5-














bisphosphate carboxylase,














large subunit [Rhodobacter














sphaeroides]








430
74265
CGPG5356
1.00E−117
100
gi|15237288|
ref|NP_197727.1|GRAM
PEG
PP
PEG











domain-containing protein/














ABA-responsive protein-














related [Arabidopsis














thaliana]








431
74369
CGPG6076
2.00E−86
96
gi|18409647|
ref|NP_564994.1|ubiquitin-
PEG
CK
PP
PEG










conjugating enzyme family














protein [Arabidopsis














thaliana]








432
70217
CGPG6
0
97
gi|15231536|
ref|NP_189259.1|cytochrome
CK
PP
SP











P450 family protein














[Arabidopsis thaliana]








433
72711
CGPG1846
4.00E−75
79
gi|15221048|
ref|NP_175816.1|transcription
CK
PP
SP











initiation factor IID














(TFIID) 31 kDa subunit














(TAFII-31) family protein














[Arabidopsis thaliana]








434
70932
CGPG4089
1.00E−129
56
gi|15223134|
ref|NP_177792.1|expressed
CS
HS
PP











protein [Arabidopsis














thaliana]








435
73518
CGPG6497
1.00E−177
63
gi|22981996|
ref|ZP_00027327.1|COG1012:
CS
CK
PP











NAD-dependent














aldehyde dehydrogenases














[Burkholderia fungorum]








436
19771
CGPG4011
3.00E−90
80
gi|18418200|
ref|NP_568342.1|rubredoxin
PP
HS
SS











family protein














[Arabidopsis thaliana]














dbj|BAB10504.1|














gene_id: MKP11.2~unknown














protein [Arabidopsis














thaliana] g








437
73549
CGPG6460
0
90
gi|37524978|
ref|NP_928322.1|5-
HS
DS
PP











carboxymethyl-2-














hydroxymuconate














semialdehyde














dehydrogenase














[Photorhabdus luminescens














subsp. laumondii TTO1]








438
72994
CGPG5803
0
83
gi|6322702|
ref|NP_012776.1|Vacuolar
CK
PEG
CS
PP
PEG









transporter, exports large














neutral amino acids from














the vacuole; member of a














family of seven S. cerevisiae














genes (AVT1-7)














related to vesicular GABA-














glycine transporters; Avt3p














[Saccharomyces














cerevisiae]








439
71928
CGPG1617
0
100
gi|18394888|
ref|NP_564120.1|catalase 3
CS
PEG
CK
PP
PEG









(SEN2) [Arabidopsis














thaliana]








440
72903
CGPG5584
0
91
gi|6322293|
ref|NP_012367.1|Histone
PEG
PP
SS











methyltransferase with a














role in transcriptional














elongation, methylates a














lysine residue of histone














H3; associates with the C-














terminal domain of Rpo21p;














histone methylation activity














is regulated by














phosphorylation status of














Rpo21p; Set2p














[Saccharomyces














cerevisiae]














sp|P46995|SET2_YEAST














SET domain protein 2








441
73017
CGPG5733
0
94
gi|6325368|
ref|NP_015436.1|kinase
PEG
PP
PEG











required for late nuclear














division; Dbf20p














[Saccharomyces














cerevisiae]








442
74587
CGPG6774
0
94
gi|17938451|
ref|NP_535240.1|succinate
PEG
DS
HS
PP
SS









semialdehyde














dehydrogenase














[Agrobacterium














tumefaciens str. C58]








443
72453
CGPG4735
6.00E−67
91
gi|15218924|
ref|NP_174236.1|auxin-
CK
PP
SP
SS










responsive family protein














[Arabidopsis thaliana]














pir||A86417 probable auxin-














induced protein, 45653-45228








444
72967
CGPG5742
0
99
gi|6321525|
ref|NP_011602.1|Cytosolic
CS
CK
HS
LL
PP
SS








catalase T, has a role in














protection from oxidative














damage by hydrogen














peroxide, Ctt1p














[Saccharomyces














cerevisiae]








445
72961
CGPG5591
0
95
gi|15228498|
ref|NP_186975.1|UTP--
PEG
SS
HS
PP










glucose-1-phosphate














uridylyltransferase, putative/














UDP-glucose














pyrophosphorylase,














putative/UGPase, putative














[Arabidopsis thaliana]








446
73070
CGPG5627
0
90
gi|15225044|
ref|NP_181451.1|protein
PEG
PP
SS











kinase family protein














[Arabidopsis thaliana]








447
73475
CGPG6385
0
100
gi|39934021|
ref|NP_946297.1|glyceraldehyde-
PEG
PP
SS











3-phosphate














dehydrogenase(GAPDH)














[Rhodopseudomonas














palustris CGA009]








448
72916
CGPG1814
0
97
gi|15228871|
ref|NP_188303.1|protein
PP
SS












phosphatase 2C, putative/














PP2C, putative














[Arabidopsis thaliana]








449
72969
CGPG5789
0
94
gi|6321886|
ref|NP_011962.1|Low-
PP
SP
SS











affinity glucose transporter














of the major facilitator














superfamily, expression is














induced by Hxk2p in the














presence of glucose and














repressed by Rgt1p when














glucose is limiting; Hxt1p














[Saccharomyces














cerevisiae]








450
74449
CGPG6659
0
96
gi|15890426|
ref|NP_356098.1|AGR_L_619p
PP
SS












[Agrobacterium














tumefaciens str. C58]














pir||A98170 hypothetical














protein AGR_L_619














[imported]-Agrobacterium








451
16615
CGPG2539
0
98
gi|15890896|
ref|NP_356568.1|AGR_L_1560p
PP













[Agrobacterium














tumefaciens str. C58]














ref|NP_534561.1| glucose-














1-phosphate














adenylyltransferase














[Agrobacterium














tumefaciens str. C58]








452
19187
CGPG3310
0
91
gi|18423163|
ref|NP_568731.1|squamos
PP













a promoter-binding protein,














putative [Arabidopsis














thaliana]








453
19648
CGPG3134
1.00E−179
96
gi|18413950|
ref|NP_568102.1|short-
PP













chain














dehydrogenase/reductase














(SDR) family protein














[Arabidopsis thaliana]








454
70354
CGPG3995
0
64
gi|15241312|
ref|NP_196916.1|nodulin
DS
PP
SP











family protein [Arabidopsis














thaliana]








455
70421
CGPG2942
0
88
gi|30677977|
ref|NP_178317.2|zinc finger
PP













(C2H2 type) family protein














[Arabidopsis thaliana]








456
70459
CGPG3758
0
95
gi|15233315|
ref|NP_188242.1|F-box
PP













family protein [Arabidopsis














thaliana] dbj|BAB01261.1|














unnamed protein product














[Arabidopsis thaliana]








457
70465
CGPG3775
1.00E−155
90
gi|15236937|
ref|NP_195254.1|zinc finger
PP
SP












(C2H2 type) family protein














[Arabidopsis thaliana]








458
70683
CGPG4587
3.00E−65
64
gi|18423239|
ref|NP_568751.1|polyadenylate-
PP













binding protein,














putative/PABP, putative














[Arabidopsis thaliana]








459
70725
CGPG2097
0
91
gi|18420505|
ref|NP_568066.1|expressed
CS
PP












protein [Arabidopsis














thaliana]








460
70852
CGPG1465
0
93
gi|15237075|
ref|NP_195290.1|isocitrate
PP
SP












dehydrogenase, putative/














NAD+ isocitrate














dehydrogenase, putative














[Arabidopsis thaliana]








461
71112
CGPG934
1.00E−130
94
gi|15218701|
ref|NP_171806.1|expressed
CS
PP












protein [Arabidopsis














thaliana] pir||E86161














F10O3.11 protein-














Arabidopsis thaliana














gb|AAD25802.1| Belongs to














the PF|01027














Uncharacterized protein














family UPF0005 with 7














transmembrane domains.














[Arabidopsis thaliana]








462
71127
CGPG945
0
97
gi|15225307|
ref|NP_179604.1|26S
PP













protease regulatory














complex subunit 4, putative














[Arabidopsis thaliana]














pir||E84585 26S














proteasome subunit 4














[imported]-Arabidopsis














thaliana








463
71132
CGPG1561
0
98
gi|15232209|
ref|NP_191550.1|expressed
PP













protein [Arabidopsis














thaliana]








464
71217
CGPG95
0
100
gi|15221476|
ref|NP_172127.1|shaggy-
PP













related protein kinase iota/














ASK-iota (ASK9) (GSK1)














[Arabidopsis thaliana] (EC














2.7.1.—)








465
71645
CGPG4688
3.00E−69
100
gi|18401105|
ref|NP_566544.1|phosphotransfer
PP













family protein














[Arabidopsis thaliana]








466
71726
CGPG3894
0
93
gi|15217677|
ref|NP_171725.1|no apical
HS
PP












meristem (NAM) family














protein [Arabidopsis














thaliana]








467
72432
CGPG4562
1.00E−144
92
gi|20152540|
emb|CAD29662.1|putative
PP
SP












auxin response factor 23














[Arabidopsis thaliana]








468
72450
CGPG4732
1.00E−170
100
gi|15238890|
ref|NP_197366.1|zinc finger
LL
PP












(C3HC4-type RING finger)














family protein [Arabidopsis














thaliana]








469
72455
CGPG4742
1.00E−144
93
gi|15242893|
ref|NP_200597.1|anthranilate
PP
PEG












synthase beta subunit,














putative [Arabidopsis














thaliana]








470
72727
CGPG5522
1.00E−118
100
gi|6324107|
ref|NP_014177.1|functionally
PP













related to TFIIB, affects














start site selection in vivo;














Ssu72p [Saccharomyces














cerevisiae]








471
72817
CGPG4987
0
96
gi|30679158|
ref|NP_567238.2|AAA-type
PP













ATPase family protein














[Arabidopsis








472
72992
CGPG5777
0
90
gi|6324981|
ref|NP_015049.1|S-
PP
PEG












adenosylMethionine














Permease; Sam3p














[Saccharomyces














cerevisiae]








473
73007
CGPG5760
0
93
gi|6320865|
ref|NP_010944.1|One of
PP













three possible beta-














subunits of the Snf1 kinase














complex, allows nuclear














localization of the Snf1














kinase complex in the














presence of a














nonfermentable carbon














source; contains glycogen-














binding domain; Gal83p














[Saccharomyces














cerevisiae]








474
73073
CGPG5688
0
95
gi|16331010|
ref|NP_441738.1|fructose
PP













1,6-bisphosphatase














[Synechocystis sp. PCC














6803]








475
73506
CGPG6496
0
96
gi|23062569|
ref|ZP_00087347.1|COG10
PP













12: NAD-dependent














aldehyde dehydrogenases














[Pseudomonas fluorescens














PfO-1]








476
74107
CGPG6590
0
95
gi|15965198|
ref|NP_385551.1|PYRUVATE
PP
SS












DEHYDROGENASE














ALPHA2 SUBUNIT














PROTEIN [Sinorhizobium














meliloti 1021]








477
74117
CGPG6575
0
81
gi|37528116|
ref|NP_931461.1|
CS
PP












Phenylacetaldehyde














dehydrogenase














(PAD) [Photorhabdus














luminescens subsp.














laumondii TTO1]








478
74131
CGPG6592
0
96
gi|16329404|
ref|NP_440132.1|transaldolase
PP
SS












[Synechocystis sp.














PCC 6803] B-








479
74344
CGPG5929
1.00E−111
100
gi|15236410|
ref|NP_193147.1|COP9
HS
PP












signalosome subunit,














putative/CSN subunit,














putative (CSN8)














[Arabidopsis thaliana]








480
14320
CGPG1229
0
100
gi|18418018|
ref|NP_567894.1|expressed
SP













protein [Arabidopsis














thaliana]








481
16756
CGPG2117
1.00E−142
85
gi|18391249|
ref|NP_563885.1|expressed
SP













protein [Arabidopsis














thaliana]








482
17448
CGPG2673
1.00E−102
72
gi|15239624|
ref|NP_197993.1|PHD
SP













finger family protein














[Arabidopsis thaliana]














gb|AAM64729.1| nucleic














acid binding protein-like














[Arabidopsis thaliana]








483
17633
CGPG2839
1.00E−145
85
gi|18395124|
ref|NP_564171.1|basic
SP













helix-loop-helix (bHLH)














family protein [Arabidopsis














thaliana]








484
18876
CGPG3096
1.00E−172
89
gi|18394949|
ref|NP_564133.1|transporter-
SP













related [Arabidopsis














thaliana] pir||G86343














hypothetical protein














T22I11.10








485
19120
CGPG1976
0
100
gi|15232345|
ref|NP_188710.1|fertilization-
SP













independent endosperm














protein (FIE) [Arabidopsis














thaliana]








486
19221
CGPG2958
1.00E−159
78
gi|30690446|
ref|NP_182182.2|Dof zinc
SP













finger protein DAG2/Dof














affecting germination 2














(DAG2) [Arabidopsis














thaliana]








487
70206
CGPG4116
1.00E−139
64
gi|18412918|
ref|NP_565249.1|phospholipid/
SP













glycerol acyltransferase














family protein [Arabidopsis








488
70223
CGPG53
0
93
gi|15240313|
ref|NP_198006.1|hexose
SP













transporter, putative














[Arabidopsis thaliana]








489
70347
CGPG3147
1.00E−121
66
gi|18416267|
ref|NP_567693.1|Dof-type
SP













zinc finger domain-














containing protein














[Arabidopsis thaliana]








490
70406
CGPG1687
0
93
gi|18397470|
ref|NP_564354.1|early-
SP













responsive to dehydration














stress protein (ERD4)














[Arabidopsis thaliana]








491
70469
CGPG3791
1.00E−171
89
gi|15237581|
ref|NP_198936.1|MADS-
SP
HS












box family protein














[Arabidopsis thaliana]








492
70564
CGPG1864
0
89
gi|15219067|
ref|NP_173589.1|SWIRM
SP













domain-containing protein/














DNA-binding family protein














gb|AAD41423.1| Contains














similarity to gb|AF033823














moira protein from














Drosophila melanogaster














and contains a PF|00249














Myb-like DNA-binding














domain.








493
70601
CGPG2917
0
91
gi|15235140|
ref|NP_193702.1|zinc finger
SP
PP












(C3HC4-type RING finger)














family protein [Arabidopsis














thaliana] pir||T04748














hypothetical protein














T16H5.30-Arabidopsis














thaliana








494
70612
CGPG3721
0
96
gi|18416732|
ref|NP_568256.1|conserved
SP













oligomeric Golgi complex














component-related/COG














complex component-related














[Arabidopsis thaliana]








495
70720
CGPG1358
0
93
gi|15238483|
ref|NP_198387.1|lectin
SP













protein kinase family














protein [Arabidopsis














thaliana]








496
70735
CGPG2661
1.00E−109
100
gi|15231241|
ref|NP_187953.1|transcription
SP













initiation factor IID-1














(TFIID-1)/TATA-box factor














1/TATA sequence-binding














protein 1 (TBP1)














[Arabidopsis thaliana]








497
70846
CGPG377
1.00E−151
100
gi|15221223|
ref|NP_177577.1|zinc finger
SP













(C3HC4-type RING finger)














family protein [Arabidopsis














thaliana] pir||D96772














probable RING zinc finger














protein








498
70923
CGPG4020
0
87
gi|8132347|
gb|AAF73257.1|MAP
SP













kinase PsMAPK2 [Pisum














sativum]








499
71149
CGPG3457
0
83
gi|20141566|
sp|P48001|
SP













HKL4_ARATHHomeobox














protein knotted-1














like 4 (KNAT4) pir||T51795














HOMEOBOX PROTEIN














KNOTTED-1 LIKE 4














(KNAT4)-








500
71608
CGPG4687
0
100
gi|15220438|
ref|NP_72008.1|ent-
SP













kaurenoic acid hydroxylase














(KAO1)/cytochrome P450














88A3, putative (CYP88A3)














[Arabidopsis thaliana]








501
71739
CGPG4345
7.00E−89
80
gi|18406944|
ref|NP_566061.1|expressed
SP













protein [Arabidopsis














thaliana]








502
72014
CGPG5230
0
100
gi|25410898|
pir||D84423probable WD-
SP













40-repeat protein [imported]-














Arabidopsis thaliana














gb|AAD14533.1| putative














stress protein [Arabidopsis














thaliana]








503
72051
CGPG5241
0
93
gi|18401606|
ref|NP_566585.1|cyclic
SP













nucleotide-binding














transporter 1/CNBT1














(CNGC20) [Arabidopsis














thaliana]














sp|Q9LD37|CG20_ARATH














Probable cyclic nucleotide-














gated ion channel 20,














chloroplast precursor














(Cyclic nucleotide-binding














transporter 1)








504
74259
CGPG5343
0
96
gi|15222882|
ref|NP_175431.1|branched-
CS
HS
SS











chain amino acid














aminotransferase 6/














branched-chain amino acid














transaminase 6 (BCAT6)














[Arabidopsis thaliana] s








505
72463
CGPG4760
8.00E−48
100
gi|15236351|
ref|NP_193115.1|auxin-
CS
SS
HS
LN
PP









responsive protein, putative














[Arabidopsis thaliana]








506
72902
CGPG5597
0
88
gi|15240576|
ref|NP_199800.1|chloride
SS
CS
DS











channel protein (CLC-c)














[Arabidopsis thaliana]














sp|Q96282|CLCC_ARATH














Chloride channel protein














CLC-c (AtCLC-c)








507
74572
CGPG6640
1.00E−109
93
gi|16331001|
ref|NP_441729.1|unknown
CS
PP
SS











protein [Synechocystis sp.














PCC 6803]








508
73055
CGPG5768
0
97
gi|6321588|
ref|NP_011665.1|Hypothetical
SS
CS
HS











ORF; Ygr149wp














[Saccharomyces














cerevisiae]








509
74103
CGPG6558
0
99
gi|15833050|
ref|NP_311823.1|fructose-
HS
PP
SS











bisphosphate aldolase














class II [Escherichia coli














O157:H7] r








510
72921
CGPG5781
0
93
gi|6322892|
ref|NP_012965.1|general
CK
PEG
SS











amino acid permease;














Gap1p [Saccharomyces














cerevisiae]








511
72968
CGPG5772
0
99
gi|6321546|
ref|NP_011623.1|role in
PEG
LL
SS











DNA replication during S














phase; Clb6p














[Saccharomyces














cerevisiae]








512
19703
CGPG4172
0
83
gi|7488676|
pir||T07150G-box binding
HS
SS












factor 2A-soybean














(fragment) gb|AAB00097.1|














G-box binding factor








513
19946
CGPG4097
1.00E−46
34
gi|15219099|
ref|NP_175691.1|2-
SS













oxoglutarate-dependent














dioxygenase, putative














[Arabidopsis thaliana]








514
19980
CGPG3914
2.00E−63
49
gi|28629811|
gb|AAO45179.1|transcription
CS
SS












factor Myb1 [Malus














xiaojinensis]








515
70435
CGPG3701
1.00E−150
91
gi|15236597|
ref|NP_193499.1|casein
SS
PP
SP











kinase II beta chain,














putative [Arabidopsis














thaliana]








516
71114
CGPG1657
0
88
gi|30680729|
ref|NP_849990.1|K+ efflux
SS













antiporter, putative (KEA4)














[Arabidopsis thaliana]








517
72451
CGPG4733
0
94
gi|15239622|
ref|NP_197992.1|mitochondrial
SS













substrate carrier family














protein [Arabidopsis














thaliana]








518
72947
CGPG5607
3.00E−62
53
gi|1483230|
emb|CAA67968.1|MADS4
SS













protein [Betula pendula]








519
73012
CGPG5786
0
97
gi|6324187|
ref|NP_014257.1|belongs
SS













to a ubiquitous family of














cytoplasmic membrane














proteins that transport only














ammonium (NH(4)(+) + NH(3)).;














Mep2p














[Saccharomyces














cerevisiae]








520
73022
CGPG5622
0
86
gi|15225518|
ref|NP_182083.1|protein
SS













kinase family protein














[Arabidopsis thaliana]








521
73488
CGPG6394
1.00E−154
94
gi|16080620|
ref|NP_391447.1|UTP-
SS
CS
PP











glucose-1-phosphate














uridylyltransferase [Bacillus














subtilis]








522
73901
CGPG5237
0
92
gi|18400284|
ref|NP_565553.1|extra-
SS













large guanine nucleotide














binding protein/G-protein














(XLG)








523
73964
CGPG5804
0
88
gi|6319773|
ref|NP_009855.1|Na+/Pi
SS













cotransporter, active in














early growth phase; similar














to phosphate transporters














of Neurospora crassa;














transcription regulated by














inorganic phosphate














concentrations and Pho4p;














Pho89p [








524
74019
CGPG5706
2.00E−92
100
gi|16079815|
ref|NP_390639.1|adenine
SS













phosphoribosyltransferase














[Bacillus subtilis]








525
74022
CGPG5724
0
97
gi|18378991|
ref|NP_563659.1|glycosyl
SS
SP












hydrolase family 3 protein














[Arabidopsis thaliana]








526
74114
CGPG6551
0
99
gi|15888903|
ref|NP_354584.1|AGR_C_2
SS













921p [Agrobacterium














tumefaciens str. C58]














pir||H97551 probable














aminotransferase aatc








527
74262
CGPG5353
0
100
gi|18416245|
ref|NP_568226.1|histidinol-
SS
PP












phosphate














aminotransferase, putative














[Arabidopsis thaliana]








528
74292
CGPG5367
0
96
gi|15239204|
ref|NP_201393.1|U-box
SS













domain-containing protein














[Arabidopsis thaliana]








529
74302
CGPG5384
1.00E−59
82
gi|25313155|
pir||A96787protein F10A5.6
PP
SS












[imported]-Arabidopsis














thaliana








530
74325
CGPG5898
1.00E−174
86
gi|15230382|
ref|NP_188576.1|cinnamyl-
SS













alcohol dehydrogenase














(CAD) [Arabidopsis














thaliana]








531
74429
CGPG6689
0
96
gi|16077873|
ref|NP_388687.1|acetoin
SS













dehydrogenase E1














component (TPP-














dependent alpha subunit)














[Bacillus subtilis]








532
74440
CGPG6682
6.00E−90
82
gi|15613838|
ref|NP_242141.1|uridine
SS













kinase [Bacillus halodurans














C-125]








533
74462
CGPG6668
5.00E−67
99
gi|16332127|
ref|NP_442855.1|unknown
HS
SS












protein [Synechocystis sp.














PCC 6803]








534
74465
CGPG6692
1.00E−119
99
gi|16078642|
ref|NP_389461.1|similar to
PP
SS












ribulose-5-phosphate 3-














epimerase [Bacillus subtilis]








535
74474
CGPG6669
2.00E−85
82
gi|16331209|
ref|NP_441937.1|unknown
LL
SS












protein [Synechocystis sp.














PCC 6803]








536
74505
CGPG6783
0
100
gi|16129426|
ref|NP_415984.1|cryptic
SS













nitrate reductase 2 beta














subunit [Escherichia coli














K12]








537
74507
CGPG6799
0
82
gi|27479656|
gb|AAO17183.1|Orf17
SP
SS












[Photorhabdus














luminescens]








538
74562
CGPG6764
0
95
gi|16077501|
ref|NP_388315.1|similar to
SS













pyruvate oxidase [Bacillus














subtilis]





“PEP SEQ ID” which is the amino acid sequence of the protein cognate to the DNA in the recombinant DNA construct corresponding to a protein sequence of a SEQ ID NO. in the Sequence Listing.


“constructed” is an arbitrary name for the recombinant DNA describe more particularly in Table I.


“annotation” refers to a description of the top hit protein obtained from an amino acid sequence query of each PEP SEQ ID NO to GenBank database of the National Center for Biotechnology Information (ncbi). More particularly, “gi” is the GenBank ID number for the top BLAST hit.


“description” refers to the description of the top BLAST hit.


“e-value” provides the expectation value for the BLAST hit.


“identity” refers to the percentage of identically matched amino acid residues along the length of the portion of the sequences which is aligned by BLAST between the sequence of interest provided herein and the hit sequence in GenBank.


“traits” identified by two letters codes the confirmed improvement in a transgenic plant provided by the recombinant DNA . The codes for improved traits are:


“CK” which indicates cold tolerance improvement identified under a cold shock tolerance screen;


“CS” which indicates cold tolerance improvement identified by a cold germination tolerance screen;


“DS” which indicates drought tolerance improvement identified by a soil drought stress tolerance screen;


“PEG” which indicates osmotic stress tolerance improvement identified by a PEG induced osmotic stress tolerance screen;


“HS” which indicates heat stress tolerance improvement identified by a heat stress tolerance screen;


“SS” which indicates high salinity stress tolerance improvement identified by a salt stress tolerance screen;


“LN” which indicates nitrogen use efficiency improvement identified by a limited nitrogen tolerance screen;


“LL” which indicates attenuated shade avoidance response identified by a shade tolerance screen under a low light condition;


“PP” which indicates improved growth and development at early stages identified by an early plant growth and development screen;


“SP” which indicates improved growth and development at late stages identified by a late plant growth and development screen provided herein.






Screens for Identifying Trait Improving Genes

DS—Improvement of Drought Tolerance Identified by a Soil Drought Stress Tolerance Screen:
Drought is a water deficit condition that imposes osmotic stress on plants. Plants are particularly vulnerable to drought during the flowering stage. The drought condition in the screening process disclosed in Example 1B started from the flowering time and was sustained to the end of harvesting. The drought tolerance-imparting DNA defined for this invention are used in recombinant DNA constructs that improve plant survival rate under drought conditions. Exemplary recombinant DNA which has been identified for conferring such drought tolerance is identified as such in Table 2. Such identified recombinant DNA is useful in generating transgenic plants that are tolerant to the drought condition imposed during flowering time and in other stages of the plant life cycle. As demonstrated from the model plant screen, in some embodiments of transgenic plants with trait-improving recombinant DNA grown under such sustained drought condition also have increased total seed weight per plant in addition to the increased survival rate within a transgenic population, providing a higher yield potential as compared to control plants.
PEG-Improvement of Drought Tolerance Identified by PEG Induced Osmotic Stress Tolerance Screen:
Various drought levels can be artificially induced by using various concentrations of polyethylene glycol (PEG) to produce different osmotic potentials (Pilon-Smits et al., (1995) Plant Physiol. 107:125-130). Several physiological characteristics have been reported as being reliable indications for selection of plants possessing drought tolerance. These characteristics include the rate of seed germination and seedling growth. The traits can be assayed relatively easily by measuring the growth rate of seedling in PEG solution. Thus, a PEG-induced osmotic stress tolerance screen is a useful surrogate for drought tolerance screen. Certain embodiments of transgenic plants with trait-improving recombinant DNA identified in the PEG-induced osmotic stress tolerance screen survive drought conditions providing a higher yield potential as compared to control plants.
SS-Improvement of Drought Tolerance Identified by High Salinity Stress Tolerance Screen:
Three different factors are responsible for salt damages: (1) osmotic effects, (2) disturbances in the mineralization process, (3) toxic effects caused by the salt ions, e.g., inactivation of enzymes. While the first factor of salt stress results in the wilting of the plants that is similar to drought effect, the ionic aspect of salt stress is clearly distinct from drought. Exemplary recombinant DNA which has been identified to help plants maintain biomass, root growth and/or plant development in high salinity conditions are identified as such in Table 2. Since osmotic effect is one of the major components of salt stress, which is common to the drought stress, embodiments of trait-improving recombinant DNA identified in a high salinity stress tolerance screen also provide transgenic crops with improved drought tolerance. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a high salinity stress tolerance screen survive drought conditions and/or high salinity conditions providing a higher yield potential as compared to control plants.
HS-Improvement of Drought Tolerance Identified by Heat Stress Tolerance Screen:
Heat and drought stress often occur simultaneously, limiting plant growth. Heat stress can cause the reduction in photosynthesis rate, inhibition of leaf growth and osmotic potential in plants. Thus, genes identified as heat stress tolerance conferring genes may also impart improved drought tolerance to plants. As demonstrated from the model plant screen, embodiments of transgenic plants with trait-improving recombinant DNA identified in a heat stress tolerance screen can survive better heat stress conditions and/or drought conditions providing a higher yield potential as compared to control plants.
CK and CS-Improvement of Tolerance to Cold Stress:
Low temperature may immediately result in mechanical constraints, changes in activities of macromolecules, and reduced osmotic potential. Two screening conditions, i.e., cold shock tolerance screen (CK) and cold germination tolerance screen (CS), were set up to look for transgenic plants that display visual growth advantage at lower temperature. In cold germination tolerance screen, the transgenic Arabidopsis plants were exposed to a constant temperature of 8 degrees C. from planting until day 28 post planting. The trait-improving recombinant DNA identified by such screen are particular useful for the production of transgenic plant that can germinate more robustly in a cold temperature as compared to the wild type plants. In cold shock tolerance screen, the transgenic plants were first grown under the normal growth temperature of 22 degrees C. until day 8 post planting, and subsequently were placed under 8 degrees C. until day 28 post planting. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a cold shock stress tolerance screen and/or a cold germination stress tolerance screen survive cold conditions providing a higher yield potential as compared to control plants.
Improvement of Tolerance to Multiple Stresses:
Different kinds of stresses often lead to identical or similar reaction in the plants. Genes that are activated or inactivated as a reaction to stress can either act directly in a way the genetic product reduces a specific stress, or they can act indirectly by activating other specific stress genes. By manipulating the activity of such regulatory genes, i.e., multiple stress tolerance genes, plants are enabled to react to different kinds of stresses. For examples, DNA for expressing proteins of SEQ ID NO:352 and SEQ ID NO:353 is useful to improve both heat stress tolerance and cold stress tolerance in plants. Plants transformed with DNA for expressing protein of SEQ ID NO:508 resist heat stress, salt stress and cold stress. Thus, the disclosed stress tolerance conferring genes are useful in combinations to generate transgenic plants that resist multiple stress conditions.
PP-Improvement of Early Plant Growth and Development:
It is known in the art that to minimize the impact of disease on crop profitability, it is important to start the season with healthy vigorous plants. This means avoiding seed and seedling diseases, leading to increased nutrient uptake and increased yield potential. Traditionally early planting and applying fertilizer are the methods used for promoting early seedling vigor. In early development stage, plant embryos establish only the basic root-shoot axis, a cotyledon storage organ(s), and stem cell populations, called the root and shoot apical meristems, that continuously generate new organs throughout post-embryonic development. “Early growth and development” encompasses the stages of seed imbibition through the early vegetative phase. Certain DNA is identified as useful to produce transgenic plants that have advantages in one or more processes including, but not limited to, germination, seedling vigor, root growth and root morphology under non-stressed conditions. The transgenic plants starting from a more robust seedling are less susceptible to the fungal and bacterial pathogens that attach germinating seeds and seedling. Furthermore, seedlings with advantage in root growth are more resistant to drought stress due to extensive and deeper root architecture. Therefore, genes conferring the growth advantage in early stages to plants are used to generate transgenic plants that are more resistant to various stress conditions due to improved early plant development. Exemplary recombinant DNA that confers both stress tolerance and growth advantages to plants, is identified as such in Table 2, e.g., DNA encoding a protein of SEQ ID NO:444 can improve the plant early growth and development, and impart heat and cold tolerance to plants. Embodiments of transgenic plants with trait-improving recombinant DNA identified in the early plant development screen grow better under non-stress conditions and/or stress conditions providing a higher yield potential as compared to control plants.
SP-Improvement of Late Plant Growth and Development:
“Late growth and development” encompasses the stages of leaf development, flower production, and seed maturity. Transgenic plants with late growth and development advantages express DNA that is identified as such in Table 2. Such plants exhibit at least one phenotypic characteristics including, but not limited to, increased rosette radius, increased rosette dry weight, seed dry weight, silique dry weight, and silique length. For example, the rosette radius and rosette dry weight are used as the indexes of photosynthesis capacity, and thereby plant source strength and yield potential of a plant. Seed dry weight, silique dry weight and silique length are used as the indexes for plant sink strength, which are considered as the direct determinants of yield. Embodiments of transgenic plants with trait-improving recombinant DNA identified in the late development screen grow better and/or have improved development during leaf development and seed maturation providing a higher yield potential as compared to control plants.
LL-Improvement of Tolerance to Shade Stress Identified in a Low Light Screen:
The effects of light on plant development are especially prominent at the seedling stage. Under normal light conditions with unobstructed direct light, a plant seeding develops according to a characteristic photomorphogenic pattern, in which plants have open and expanded cotyledons and short hypocotyls. Then the plant's energy is devoted to cotyledon and leaf development while longitudinal extension growth is minimized. Under low light condition where light quality and intensity are reduced by shading, obstruction or high population density, a seedling displays a shade-avoidance pattern, in which the seedling displays a reduced cotyledon expansion, and hypocotyls extension is greatly increased. As the result, a plant under low light condition increases significantly its stem length at the expanse of leaf, seed or fruit and storage organ development, thereby adversely affecting of yield. Recombinant DNA that enables plants to have an attenuated shade avoidance response so that the source of plant can be contributed to reproductive growth efficiently provides embodiments of those plants with higher yield as compared to the wild type plants. Embodiments of transgenic plants with trait-improving recombinant DNA identified in a shade stress tolerance screen have attenuated shade response under shade conditions providing a higher yield potential as compared to control plants. The transgenic plants generated by this invention are suitable for a higher density planting, thereby resulting increased yield per unit area.
LN-Improvement of Tolerance to Low Nitrogen Availability Stress
Nitrogen is a key factor in plant growth and crop yield. The metabolism, growth and development of plants are profoundly affected by their nitrogen supply. Restricted nitrogen supply alters shoot to root ratio, root development, activity of enzymes of primary metabolism and the rate of senescence (death) of older leaves. All field crops have a fundamental dependence on inorganic nitrogenous fertilizer. Since fertilizer is rapidly depleted from most soil types, it must be supplied to growing crops two or three times during the growing season. Enhanced nitrogen use efficiency by plants should enable crops cultivated under low nitrogen availability stress condition resulted from low fertilizer input or poor soil quality.
Recombinant DNA that imparts enhanced nitrogen use efficiency in transgenic plants is identified in Table 2. Such plants exhibit one or more desirable traits including, but not limited to, increased seedling weight, increased number of green leaves, increased number of rosette leaves, altered root length and advanced flower bud formation. Such plants can also have altered amino acid or protein compositions, increased yield and/or better seed quality. Embodiments of such transgenic plants are productively cultivated under nitrogen nutrient deficient conditions, i.e., nitrogen-poor soils and low nitrogen fertilizer inputs that cause the growth of wild type plants to cease or to be so diminished as to make the wild type plants practically useless under such conditions. The transgenic plants also are advantageously used to achieve earlier maturing, faster growing, and/or higher yielding crops and/or produce more nutritious foods and animal feedstocks when cultivated using nitrogen non-limiting growth conditions.
Stacked Traits:
This invention also provides transgenic plants with stacked engineered traits, e.g., a crop having an improved phenotype resulting from expression of a trait-improving recombinant DNA, in combination with herbicide and/or pest resistance traits. For example, genes of the current invention can be stacked with other traits of agronomic interest, such as a trait providing herbicide resistance, for example a glyphosate resistance trait, or insect resistance, such as using a gene from Bacillus thuringiensis to provide resistance against lepidopteran, coliopteran, homopteran, hemiopteran, and other insects. Herbicides for which resistance is useful in a plant include glyphosate herbicides, phosphinothricin herbicides, oxynil herbicides, imidazolinone herbicides, dinitroaniline herbicides, pyridine herbicides, sulfonylurea herbicides, bialaphos herbicides, sulfonamide herbicides and gluphosinate herbicides. To illustrate that the production of transgenic plants with herbicide resistance is a capability of those of ordinary skill in the art, reference is made to U.S. 2003-0106096 A1 and 2002-0112260 A1 and U.S. Pat. Nos. 5,034,322; 5,776,760, 6,107,549 and 6,376,754, all of which are incorporated herein by reference. To illustrate that the production of transgenic plants with pest resistance is a capability of those of ordinary skill in the art reference is made to U.S. Pat. Nos. 5,250,515 and 5,880,275 which disclose plants expressing an endotoxin of Bacillus thuringiensis bacteria, to U.S. Pat. No. 6,506,599 which discloses control of invertebrates which feed on transgenic plants which express dsRNA for suppressing a target gene in the invertebrate, to U.S. Pat. No. 5,986,175 which discloses the control of viral pests by transgenic plants which express viral replicase, and to U.S. Patent Application Publication 2003/0150017 A1 which discloses control of pests by a transgenic plant which express a dsRNA targeted to suppressing a gene in the pest, all of which are incorporated herein by reference.
Once one recombinant DNA has been identified as conferring an improved trait of interest in transgenic Arabidopsis plants, several methods are available for using the sequence of that recombinant trait-imparting DNA and knowledge about the protein it encodes to identify homologs of that sequence from the same plant and different plant species or other organisms, e.g., bacteria and yeast. Thus, in one aspect, this invention provides methods for identifying a homologous gene with a DNA sequence homologous to any of SEQ ID NO:1 through SEQ ID NO:269, or a homologous protein with an amino acid sequence homologous to any of SEQ ID NO:270 through SEQ ID NO:538. In another aspect, this invention provides a consensus amino acid sequence for respective homologs for each of SEQ ID NO:270 through SEQ ID NO:538. In yet another aspect, this invention also includes linking or associating one or more desired traits, or gene function with a homolog sequence disclosed herein.
The trait-improving recombinant DNA and methods of using such trait-improving recombinant DNA for generating transgenic plants with improved traits provided by this invention are not limited to any particular plant species. Indeed, the plants of this invention encompass many species of monocots and dicots and include agriculturally useful plants which are cultivated for purposes of food production or industrial applications, e.g., corn and soybean plants and cotton plants. Recombinant DNA constructs optimized for soybean transformation and recombinant DNA constructs optimized for corn transformation are disclosed in the following examples. Other plants of this invention include canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
Thus, embodiments of this invention include the use of both DNA identified in Table 3 and homologs in recombinant DNA for transgenic crop plants with improved traits. Transgenic crop plants with improved traits are identified from populations of plants grown from transgenic events by screening to segregate the plants of this invention from plants without the improved traits. Preferred screens for transgenic crop plants identify plants with improved responses to stress conditions, e.g., assays using imposed stress conditions to detect improved responses to drought stress, nitrogen deficiency, cold growing conditions, or alternatively, under naturally present stress conditions, for example under field conditions. Biomass measures are made on greenhouse or field grown plants and include such measurements as plant height, stem diameter, root and shoot dry weights, and, for corn plants, ear length and diameter.
Trait data on morphological changes is collected by visual observation during the process of plant regeneration as well as in regenerated plants transferred to soil. Such trait data includes characteristics such as normal plants, bushy plants, taller plants, thicker stalks, narrow leaves, striped leaves, knotted phenotype, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other enhanced traits are identified by measurements taken under field conditions, such as days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barrenness/prolificacy, green snap, and pest resistance. In addition, trait characteristics of harvested grain are confirmed, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
To confirm hybrid yield in transgenic corn plants expressing trait-imparting DNA of this invention, it is useful to test hybrid plants over multiple years at multiple locations in a geographical location where corn is conventionally grown, e.g., in Iowa, Illinois and Kansas, under “normal” field conditions as well as under stress conditions, e.g., under drought or population density stress.
Transgenic crop plants are used to provide other aspects of this invention such as transgenic seeds of crop plants. Seeds of transgenic plants are used to propagate more progeny plants which contain the trait-improving recombinant DNA constructs of this invention. These progeny plants are within the scope of this invention when they contain a trait-improving recombinant DNA construct of this invention, whether or not these plants are selfed or crossed with different varieties of plants.
Screening Methods for Crop Transgenic Plants with Enhanced Agronomic Trait
Due to variability in transformation many transgenic events which survive to fertile transgenic plants that produce seeds and progeny plants do not exhibit an enhanced agronomic trait. Thus, screening is necessary to identify the transgenic events that produce the transgenic plants and seeds of this invention. Transgenic crop plants having enhanced traits are identified from populations of plants transformed as described herein by evaluating the trait in a variety of assays to detect an enhanced agronomic trait. Useful assays include analyses to detect changes in the chemical composition, biomass, physiological properties and morphology of the plant. Changes in chemical compositions such as nutritional composition of grain are detected by analysis of the seed composition and content of protein, free amino acids, oil, free fatty acids, starch or tocopherols. Changes in biomass characteristics are detected in greenhouse or field grown plants and include plant height, stem diameter, root and shoot dry weights; and, for corn plants, ear length and diameter. Changes in physiological properties are identified by evaluating responses to stress conditions, e.g., assays using imposed stress conditions such as water deficit, nitrogen deficiency, cold growing conditions, pathogen or insect attack or light deficiency, or increased plant density. Changes in morphology are measured by visual observation of tendency of a transformed plant with an enhanced agronomic trait to also appear to be a normal plant as compared to changes toward bushy, taller, thicker, narrower leaves, striped leaves, knotted trait, chlorosis, albino, anthocyanin production, or altered tassels, ears or roots. Other screening properties include days to pollen shed, days to silking, leaf extension rate, chlorophyll content, leaf temperature, stand, seedling vigor, internode length, plant height, leaf number, leaf area, tillering, brace roots, stay green, stalk lodging, root lodging, plant health, barrenness/prolificacy, green snap, and pest resistance. In addition, phenotypic characteristics of harvested grain are evaluated, including number of kernels per row on the ear, number of rows of kernels on the ear, kernel abortion, kernel weight, kernel size, kernel density and physical grain quality.
Seeds for transgenic crop plants with enhanced agronomic traits of this invention are corn, soybean and cotton seeds, as well as seeds for canola, wheat, sunflower, sorghum, alfalfa, barley, millet, rice, tobacco, fruit and vegetable crops, and turfgrass.
A. Screening for Nitrogen Use Efficiency
Many transgenic crop plants of this invention exhibit enhanced nitrogen use efficiency as compared to control plants. Higher nitrogen soil applications increase seed protein and starch accumulation, and lead to larger seed weight and larger kernel number per ear. Recent improvements in elite high yielding corn hybrid genotypes include the ability to utilize nitrogen efficiently. DNA causing the enhanced nitrogen use efficiency in crop plants are especially useful, e.g., for improving yield. Enhanced nitrogen use efficiency is assessed by measuring changes in plant growth such as leaf area production, shoot biomass, chlorophyll content in plants grown in nitrogen limiting conditions and/or nitrogen sufficient conditions. It is useful to conduct a first screen in nitrogen limiting conditions and confirm replicate transgenic events in both nitrogen limiting and nitrogen sufficient conditions. Table 3 shows an amount of nutrients in the nutrient solution for nitrogen limiting conditions (low N); and Table 4 shows an amount of nutrients in nutrient solution for nitrogen sufficient conditions (high N). These nutrient solutions when adjusted to pH 5.6, e.g. with HCl or KOH, are useful for nitrogen use efficiency screening. For example in a greenhouse screen pots of transgenic plants and control plants are treated with 100 ml of nutrient solution three times a week on alternate days starting at 8 and 10 days after planting for high N and low N screening, respectively.










TABLE 3








2 mM NH4NO3



Nutrient stock
Low nitrogen









1M NH4N03
2 mL/L



1M KH2PO4
0.5



1M MgSO4•7H2O
2



1M CaCl2
2.5



1M K2SO4
1




















TABLE 4








20 mM NH4NO3



Nutrient stock
High nitrogen









1M NH4N03
20 mL/L



1M KH2PO4
0.5



1M MgSO4•7H2O
2



1M CaCl2
2.5



1M K2SO4
1










After 28 days of plant growth for low N screening and 23 days for high N screening, measurements are taken for total shoot fresh mass, leaf chlorophyll, leaf area, leaf fresh mass and leaf dry mass.
B. Screening for Increased Yield
Many transgenic plants of this invention exhibit improved yield as compared to a control plant. Improved yield can result from a variety or other traits such as enhanced seed sink potential, e.g., the number and size of endosperm cells or kernels, and/or enhanced sink strength, e.g., the rate of starch biosynthesis. Sink potential is established very early during kernel development, as endosperm cell number and cell size are determined within the first few days after pollination.
Much of the increase in corn yield of the past several decades has resulted from an increase in planting density. During that period, corn yield has been increasing at a rate of 2.1 bushels/acre/year, but the planting density has increased at a rate of 250 plants/acre/year. A characteristic of modern hybrid corn is the ability of these varieties to be planted at high density. Many studies have shown that a higher than current planting density should result in more biomass production, but current germplasm does not perform well at these higher densities. One approach to increasing yield is to increase harvest index (HI), the proportion of biomass that is allocated to the kernel compared to total biomass, in high density plantings.
Effective yield screening of transgenic corn uses hybrid progeny of the transgenic event over multiple locations with plants grown under optimal production management practices, and maximum pest control. A useful target for improved yield is a 5% to 10% increase in yield as compared to yield produced by plants grown from seed for a control plant. Useful screening in multiple and diverse geographic locations, e.g., up to 16 or more locations, over one or more planting seasons, e.g., at least two planting seasons, is useful to statistically distinguish yield improvement from natural environmental effects. Useful hybrid screening includes planting multiple transgenic plants, positive and negative control plants, and pollinator plants in standard plots, e.g., 2 row plots, 20 feet long by 5 feet wide with 30 inches distance between rows and a 3 foot alley between ranges. Plants from separate transgenic events can be grouped by recombinant DNA constructs with groups randomly placed in the field. A pollinator plot of a high quality corn line is planted for every two plots to allow open pollination when using male sterile transgenic events. A useful planting density is about 30,000 plants/acre.
Surrogate indicators for screening for yield improvement include source capacity (biomass), source output (sucrose and photosynthesis), sink components (kernel size, ear size, starch in the seed), development (light response, height, density tolerance), maturity, early flowering trait and physiological responses to high density planting, e.g., at 45,000 plants per acre.
When screening for yield improvement a useful statistical measurement approach comprises three components, i.e., modeling spatial autocorrelation of the test field separately for each location, adjusting traits of recombinant DNA events for spatial dependence for each location, and conducting an across location analysis.
A first step in modeling spatial autocorrelation is estimating the covariance parameters of the semivariogram. A spherical covariance model is assumed to model the spatial autocorrelation. Because of the size and nature of the trial, it is likely that the spatial autocorrelation may change. Therefore, anisotropy is also assumed along with spherical covariance structure. The following set of equations describes the statistical form of the anisotropic spherical covariance model.




  
    
      C
      ⁡
      
        (
        
          h
          ;
          θ
        
        )
      
    
    =
    
      
        v
        ⁢
        
          
        
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          I
          ⁡
          
            (
            
              h
              =
              0
            
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      +
      
        
          
            σ
            2
          
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              1
              -
              
                
                  3
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                h
              
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where I(•) is the indicator function, h=√{square root over ({dot over (x)}2+{dot over (y)}2)}, and

{dot over (x)}=[cos(ρπ/180)(x1−x2)−sin(ρπ/180)(y1−y2)]/ωx 

{dot over (y)}=[sin(ρπ/180)(x1−x2)+cos(ρπ/180)(y1−y2)]/ωy 

where s1=(x1, y1) are the spatial coordinates of one location and s2=(x2, y2) are the spatial coordinates of the second location. There are 5 covariance parameters, θ=(ν, σ2, ρ, ωn, ωj) where ν is the nugget effect, σ2 is the partial sill, ρ is a rotation in degrees clockwise from north, ωn is a scaling parameter for the minor axis and ωj is a scaling parameter for the major axis of an anisotropical ellipse of equal covariance. The five covariance parameters that define the spatial trend will then be estimated by using data from heavily replicated pollinator plots via restricted maximum likelihood approach. In a multi-location field trial, spatial trend are modeled separately for each location.

After obtaining the variance parameters of the model, a variance-covariance structure is generated for the data set to be analyzed. This variance-covariance structure contains spatial information required to adjust yield data for spatial dependence. In this case, a nested model that best represents the treatment and experimental design of the study is used along with the variance-covariance structure to adjust the yield data. During this process the nursery or the seed batch effects can also be modeled and estimated to adjust the yields for any yield parity caused by seed batch differences.
After spatially adjusted data from different locations are generated, all adjusted data is combined and analyzed assuming locations as replications. In this analysis, intra and inter-location variances are combined to estimate the standard error of yield from transgenic plants and control plants. Relative mean comparisons are used to indicate statistically significant yield improvements.
C. Screening for Water Use Efficiency
Many transgenic crop plants of this invention exhibit improved yield resulting from improved water use efficiency and/or drought tolerance.
A greenhouse screen for transgenic corn plants for water use efficiency measures changes in plant growth rate, e.g., at least a 10% improvement, in height and biomass during a vegetative drought treatment, as compared to control plants. The hydration status of the shoot tissues following the drought is also measured. Shoot Initial Height (SIH) is plant height after 3 weeks of growth under optimum conditions. Shoot Wilt Height (SWH) is plant height at the end of a 6 day drought. Time course experiments have shown that at about 3 days of drought, wild type plants basically stop growing and begin to wilt. Thus a transgenic plant with improved water use efficiency will continue to grow (probably to a lesser extent than with water) and thereby end up as a significantly taller plant at the end of a drought experiment. Shoot Wilt Mass (SWM) is the amount of wet and dry matter in the shoot (plant separated from root ball at the soil line) at the end of the drought; SDM is measure after 2 to 3 weeks in a drying chamber. Shoot Turgid mass (STM) is the SWM plus the mass of the water that is transported into plant tissues in 3 days of soaking in 40 degree C. water in the dark. Experiments show that most of the water is pulled up in 24 hours but it takes 2 more days before additional increase becomes insignificant. STM-SWM is indicative of water use efficiency in plants where recovery from stress is more important than stress tolerance per se. Relative water content (RWC) is a measurement of how much (%) of the plant is water at harvest. RWC=(SWM−SDM)/(STM−SDM)*100. Fully watered corn plants are about 98% RWC. Typically, in a wilt screen the plants are about 60% RWC. Plants with higher RWC at the end of a drought are considered to be healthier plants and more fit for post-drought recovery and growth.
Relative Growth Rate (RGR) is calculated for each shoot using the formula RGR=(SWH−SIH)/((SWH+SIH)/2)*100
D. Screening for Growth Under Cold Stress
Many transgenic crop plants of this invention exhibit improved growth under cold stress, e.g., in a cold germination assay, in a cold shock assay, in an early seedling growth assay and in root-shoot biomass assay.
In a cold germination assay transgenic seeds from transgenic plants, e.g., R2 inbred seeds or F1 hybrid seeds, seeds of two types of control plants, e.g., negative segregants from the transgenic event or wild type, non-transgenic seeds of the transformed genotype, are treated with fungicide. A useful fungicide such as Captan fungicide (available from Arvesta Corp as MAESTRO® 80DF Fungicide) is applied at the rate of 0.43 mL Captan per 45 g of corn seeds which are dried to provide fungicide-coated seeds.
In a useful cold screen for transgenic corn seeds ten seeds per transgenic event are placed on filter paper (e.g., Whatman No. 1) in the lid of a Petri dish with 5 ml of water. A closed Petri dish is placed in a growth chamber set at 11 degrees C. for inbred corn seed or 9.5 degrees C. for hybrid corn seed. 2 ml of water is added on day 3 and day 10. Seeds are considered germinated if the emerged radicle size is 1 cm. Cold seeds are scored every 2 days from day 10 up to day 30. Tissue samples are collected at random on the last day of the experiment for confirmation of RNA expression. A germination index (GI) is calculated as

GI=(Σ([T+1−ni]*[Pi−Pi-1]))/T 

where “T” is the number of days for the experiment, “n” is the number of days after start, “i” is number of times germination is counted including the current day, “P” is the percentage of seed germinated during any given rating. Statistical differences are calculated between positive and wild type control.

In a cold shock assay, seeds are planted in potting media and placed in a growth chamber set at 23 degrees C., relative humidity of 65% with 12 hour day and night photoperiod (300 uE/m2-min). Planted seeds are watered for 20 minute every other day by sub-irrigation and flats are rotated every third day. On day 10 after planting the transgenic positive and wild type control plants are positioned in flats in an alternating pattern. Chlorophyll fluorescence of plants is measured on the tenth day during the dark period of growth by using a Walz PAM-2000 portable fluorometer following manufacturer's instructions. After chlorophyll measurements, leaf samples from each event are collected for confirming the expression of recombinant DNA. The plants are then exposed to temperatures of 5 degrees C. for 4 days. On the fourth day chlorophyll fluorescence is measured and plants are restored to a 23 degrees C. environment for recovery over 3 days. During the recovery period the length of the V3 leaf is measured on the first and third days. After two days of recovery V2 leaf damage is determined visually by estimating percent of green V2 leaf. Statistical differences in V3 leaf growth, V2 leaf necrosis and fluorescence during pre-shock and cold shock can be used for estimation of cold shock damage on corn plants.
In an early seedling growth assay three sets of seeds are assayed. The first set is a group of transgenic seeds from transgenic plants; the second set is negative segregants of the transgenic seed; and the third seed set is seed from two cold tolerant and two cold sensitive wild-type controls. All seeds are treated with a fungicide as indicated above. Seeds are grown in germination paper (12 inch×18 inch pieces of Anchor Paper #SD7606), wetted in a solution of 0.5% KNO3 and 0.1% Thyram. For each paper fifteen seeds are placed on the line evenly spaced such that the radicles will grow toward the same edge. The wet paper is rolled up evenly and tight enough to hold the seeds in place. The roll is secured into place with two large paper clips, one at the top and one at the bottom. The rolls are incubated in a growth chamber at 23 degrees C. for three days in a randomized complete block design within an appropriate container. The chamber is set for 65% humidity with no light cycle. For the cold stress treatment the rolls are then incubated in a growth chamber at 12 degrees C. for fourteen days. The chamber is set for 65% humidity with no light cycle. For the warm treatment the rolls are incubated at 23 degrees C. for an additional two days. After the treatment the germination papers are unrolled and the seeds that did not germinate are discarded. The lengths of the radicle and coleoptile for each seed are measured. A coleoptile sample is collected from six individual kernels of each entry for confirming the expression of recombinant DNA. Statistical differences in the length of radicle and shoot during pre-shock and cold shock are used for an estimation of the effect of the cold treatment on corn plants. The analysis is conducted independently for the warm and cold treatments.
In a root-shoot biomass assay two sets of seeds are used. The first set is transgenic seeds with recombinant DNA, e.g., R2 inbred seeds or F1 hybrid seeds; the second seed set is non-transgenic, wild type negative control made from the same genotypes as the transgenic seeds. All seeds are treated with a fungicide as indicated above. The seeds are planted in potting media in pots arranged in a randomized complete block design with 6 replications. Pots are watered as and when needed by filling water up to the brim of the pot. Plants are grown in a greenhouse to a V6 stage or approximately for 28 days. Greenhouse lights are turned on after emergence of seedlings with 14 hours of light 10 hours of dark. Plants are fertilized twice each week with water-soluble fertilizer containing 200-ppm nitrogen. For measurement of root and shoot dry weight, two pots are separated carefully to remove adhering sand by washing with water. Washed roots are cut at the first node. The roots are placed in a paper bag after squeezing excess water, folded once and stapled. The shoots are then folded up to a convenient size (approximately 15 cm), placed in a paper bag. Bags are placed over a wire shelve to facilitate drying in a ventilated room maintained at 120 degrees F. to a moisture content of about 13% then weighed to determine dried root and shoot biomass.
E. Screen for Enhanced Oil, Starch, or Protein Levels in Plant Seeds
Oil concentrations are determined in kernels by Near Infrared Transmittance (NIT) from inbred and from hybrid lines. Data are also obtained for protein and starch content from this measurement.
Inbred Kernel Oil Screen
The primary transformants are selfed to produce R1 seed which is planted to segregating seed. An untransformed control line is planted every sixth row. All plants are self-pollinated. A molecular assay is conducted to determine zygosity of the transgene in each plant. Ears are harvested at maturity, and well-filled ears are chosen for proximate analysis. Proximate analysis is conducted on up to 5 homozygous ears. If 5 good homozygous ears are not available, then hemizygous ears will be used to obtain 5 good transgene-positive ears. Statistical analysis is conducted to determine whether proximate values for transgenic events are different from controls. Events with an increase in oil with a p-value of less than or equal to 0.1 are termed “putative leads.” Kernel composition is confirmed in an inbred confirmation nursery which is conducted with selected events, and is run with a design similar to that of the Gen2 nursery. A “confirmed lead event” demonstrates an increase in oil with a p-value of less than or equal to 0.1 in two nurseries.
Hybrid Kernel Oil Screen
Grain samples from the multilocation hybrid yield trials are collected at the time of harvest and are analyzed by NIT. Controls are negative segregants, untransformed controls, or pollinators. Data from 3 to 12 locations are pooled for the statistical analysis. Putative leads have increased oil with a p-value of less than or equal to 0.1.
The various aspects of the invention are illustrated by means of the following examples which are in no way intended to limit the full breath and scope of claims.
Example 1
Identification of Recombinant DNA that Confers Improved Trait(s) to Plants
A. Expression Constructs for Arabidopsis Plant Transformation
Each gene of interest was amplified from a genomic or cDNA library using primers specific to sequences upstream and downstream of the coding region. Transformation vectors were prepared to constitutively transcribe DNA in either sense orientation (for enhanced protein expression) or anti-sense orientation (for endogenous gene suppression) under the control of an enhanced Cauliflower Mosaic Virus 35S promoter (U.S. Pat. No. 5,359,142) directly or indirectly (Moore, et al., PNAS 95:376-381, 1998; Guyer, et al., Genetics 149: 633-639, 1998; International patent application NO. PCT/EP98/07577). The transformation vectors also contain a bar gene as a selectable marker for resistance to glufosinate herbicide. The transformation of Arabidopsis plants was carried out using the vacuum infiltration method known in the art (Bethtold, et al., Methods Mol. Biol. 82:259-66, 1998). Seeds harvested from the plants, named as T1 seeds, were subsequently grown in a glufosinate-containing selective medium to select for plants which were actually transformed and which produced T2 transgenic seed.
B. Soil Drought Tolerance Screen
This example describes a soil drought tolerance screen to identify Arabidopsis plants transformed with recombinant DNA that wilt less rapidly and/or produce higher seed yield when grown in soil under drought conditions
T2 seeds were sown in flats filled with Metro/Mix® 200 (The Scotts® Company, USA). Humidity domes were added to each flat and flats were assigned locations and placed in climate-controlled growth chambers. Plants were grown under a temperature regime of 22° C. at day and 20° C. at night, with a photoperiod of 16 hours and average light intensity of 170 μmol/m2/s. After the first true leaves appeared, humidity domes were removed. The plants were sprayed with glufosinate herbicide and put back in the growth chamber for 3 additional days. Flats were watered for 1 hour the week following the herbicide treatment. Watering was continued every seven days until the flower bud primordia became apparent, at which time plants were watered for the last time.
To identify drought tolerant plants, plants were evaluated for wilting response and seed yield. Beginning ten days after the last watering, plants were examined daily until 4 plants/line had wilted. In the next six days, plants were monitored for wilting response. Five drought scores were assigned according to the visual inspection of the phenotypes: 1 for healthy, 2 for dark green, 3 for wilting, 4 severe wilting, and 5 for dead. A score of 3 or higher was considered as wilted.
At the end of this assay, seed yield measured as seed weight per plant under the drought condition was characterized for the transgenic plants and their controls and analyzed as a quantitative response according to example 1M.
Two approaches were used for statistical analysis on the wilting response. First, the risk score was analyzed for wilting phenotype and treated as a qualitative response according to the example 1L. Alternatively, the survival analysis was carried out in which the proportions of wilted and non-wilted transgenic and control plants were compared over each of the six days under scoring and an overall log rank test was performed to compare the two survival curves using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). Table 5 provides a list of recombinant DNA constructs that improve drought tolerance in transgenic plants.









TABLE 5









Survival Anaysis of



wilt response














Wilt Response Risk

diff



Pep

score
Seed Weight/plant
time



















SEQ



RS
p-


p-

to
p-



ID
Construct_id
Gene
Orientation
mean
value
c
delta
value
c
wilting
value
c






















319
10139
CGPG101
ANTI-SENSE
0.115
0.024
S
−0.123
0.804
/
−0.01
0.469
/


320
11410
CGPG103
SENSE
0.226
0.003
S
−0.366
0.926
/
0
1
/


321
11604
CGPG48
ANTI-SENSE
0.25
0.034
S
0.257
0.01
S
−0.24
0.38
/


322
12368
CGPG1006
SENSE
0.148
0.044
S
0.359
0.02
S
−0.06
0.764
/


323
13502
CGPG1354
SENSE
0.431
0
S
0.624
0
S
1.34
0.366
/


324
13745
CGPG1576
ANTI-SENSE
−0.021
0.711
/
0.664
0
S
−0.29
0.453
/


325
13821
CGPG1569
SENSE
0.135
0.025
S
0.128
0.402
/
0.24
0.972
/


326
14240
CGPG1697
SENSE
0.377
0.002
S
−1.305
0.991
/
0
1
/


327
14718
CGPG1082
SENSE
0.168
0.001
S
0.135
0.351
/
0.25
0.208
/


328
17022
CGPG1774
SENSE
0.06
0.124
T
0.563
0.043
S
0
0.961
/


329
17924
CGPG2882
SENSE
−0.093
0.914
/
0.288
0.021
S
0.09
0.935
/


330
18259
CGPG3368
SENSE
0.07
0.28
/
0.391
0.058
T
−0.27
0.591
/


331
19171
CGPG2952
SENSE
0.227
0.005
S
0.846
0.001
S
0.12
0.543
/


332
19201
CGPG2332
SENSE
0.124
0.027
S
−0.435
0.785
/
0.35
0.256
/


333
19317
CGPG3662
SENSE
0.338
0
S
−0.071
0.61
/
0.63
0.106
T


334
70417
CGPG3427
SENSE
0.253
0.016
S
−1.424
0.984
/
0
1
/


315
70427
CGPG3067
SENSE
−0.033
0.818
/
1.004
0.002
S
0.01
0.977
/


335
70467
CGPG3785
SENSE
0.127
0.023
S
−0.448
0.946
/
0.71
0.046
S


336
70806
CGPG712
SENSE
0.246
0.046
S
0.174
0.276
/
0.14
0.07
T


337
70818
CGPG479
SENSE
0.07
0.115
T
0.558
0.009
S
0.61
0.283
/


338
70820
CGPG655
SENSE
0.172
0.048
S
0.036
0.441
/
0.26
0.554
/


339
70919
CGPG4029
SENSE
0.167
0.009
S
−0.565
0.904
/
0.31
0.508
/


340
71623
CGPG4696
SENSE
0.158
0.047
S
0.421
0.04
S
0
1
/


390
71633
CGPG857
SENSE
0.121
0.017
S
−0.823
0.967
/
0.45
0.139
T


341
71662
CGPG4679
SENSE
0.063
0.013
S
−0.037
0.631
/
0.16
0.957
/


342
71693
CGPG4652
SENSE
0.074
0.042
S
0.246
0.06
T
0.34
0.616
/


316
71811
CGPG4426
SENSE
0.359
0.015
S
−0.729
0.903
/
0.15
0.822
/


318
72081
CGPG5279
SENSE
0.269
0.005
S
−0.372
0.987
/
0.17
0.404
/


343
72384
CGPG4639
SENSE
0.133
0.018
S
0.62
0.002
S
0.17
0.359
/


344
72439
CGPG5075
SENSE
0.175
0.013
S
−0.035
0.604
/
0.23
0.244
/


374
72456
CGPG4745
SENSE
0.53
0.01
S
−0.737
0.979
/
−0.08
0.823
/


345
72619
CGPG4835
SENSE
0.178
0.039
S
0.219
0.072
T
0.96
0.691
/


346
72624
CGPG4842
SENSE
0.163
0.021
S
0.356
0.051
T
0.3
0.375
/


347
72715
CGPG5521
SENSE
0.082
0.1
T
0.767
0.002
S
0.12
0.628
/


348
72754
CGPG5548
SENSE
0.131
0.031
S
−0.365
0.974
/
0
0.923
/


349
72819
CGPG4989
SENSE
0.094
0.026
S
0.362
0.069
T
0.03
0.83
/


350
75516
CGPG7689
SENSE
0.067
0.066
T
0.965
0.001
S
0.24
0.464
/


351
75701
CGPG7856
SENSE
0.147
0.006
S
0.986
0.015
S
0.15
0.448
/


317
73463
CGPG6384
SENSE
0.174
0.048
S
−1.359
0.959
/
0.09
0.984
/


454
70354
CGPG3995
SENSE
−0.005
0.563
/
0.444
0.002
S
10.29
0.9
/


397
71840
CGPG4353
SENSE
0.142
0.007
S
−0.212
0.859
/
9.29
0.99
/


506
72902
CGPG5597
SENSE
0.009
0.162
T
0.034
0.2
T
5
1
/


437
73549
CGPG6460
SENSE
0.119
0.037
S
−0.774
0.949
/
6.26
0.25
/


302
73586
CGPG6471
SENSE
0.003
0.451
/
0.588
0.002
S
6.34
0.723
/


442
74587
CGPG6774
SENSE
0.262
0.001
S
−0.117
0.574
/
7.49
0.041
S


388
74652
CGPG6168
SENSE
0.475
0
S
−0.766
0.92
/
7.48
0
S





S: represents that the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05, p value, of the delta of a quantitative response or of the risk score of a qualitative response, is the probability that the observed difference between the transgenic plants and the reference occur by chance)


T: represents that the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2


/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset.






C. Stress Tolerance Screen

Under high temperatures, Arabidopsis seedlings become chlorotic and root growth is inhibited. This example sets forth the heat stress tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are more resistant to heat stress based on primarily their seedling weight and root growth under high temperature.
T2 seeds were plated on 1⁄2×MS salts, 1% phytagel, with 10 μg/ml BASTA (7 per plate with 2 control seeds; 9 seeds total per plate). Plates were placed at 4° C. for 3 days to stratify seeds. Plates were then incubated at room temperature for 3 hours and then held vertically for 11 additional days at temperature of 34° C. at day and 20° C. at night. Photoperiod was 16 h. Average light intensity was  ̃140 μmol/m2/s. After 14 days of growth, plants were scored for glufosinate resistance, root length, final growth stage, visual color, and seedling fresh weight. A photograph of the whole plate was taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final grow stage at day 14 was scored as success if 50% of the plants had reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, et al., (2001) The Plant Cell 13, 1499-1510). The growth stage data was analyzed as a qualitative response according to example 1L. Table 6 provides a list of recombinant DNA constructs that improve heat tolerance in transgenic plants.











TABLE 6







Pep

Growth stage
Root Length
Seedling Weight



















SEQ



RS
p-


p-


p-



ID
Construct_id
Gene
Orientation
mean
value
c
delta
value
c
delta
value
c






















354
19542
CGPG3069
SENSE
0.842
0.021
S
0.296
0.023
S
1.616
0
S


355
19618
CGPG3574
SENSE
1.072
0.005
S
0.328
0.006
S
1.569
0
S


356
19649
CGPG3140
SENSE
0.72
0.042
S
0.243
0.054
T
1.665
0
S


357
19745
CGPG3973
SENSE
0.65
0.016
S
0.19
0.021
S
1.505
0
S


358
19768
CGPG4096
SENSE
1.06
0.035
S
0.17
0.034
S
1.171
0
S


436
19771
CGPG4011
SENSE
0.822
0.023
S
0.201
0.001
S
1.43
0
S


359
19772
CGPG3939
SENSE
0.247
0.014
S
0.151
0.014
S
1.344
0
S


360
19779
CGPG4113
SENSE
0.605
0.051
T
0.181
0.003
S
1.436
0
S


361
19833
CGPG4074
SENSE
0.965
0.026
S
0.266
0.007
S
1.01
0
S


362
19862
CGPG3961
SENSE
0.341
0.119
T
0.132
0.007
S
1.22
0
S


363
19879
CGPG4009
SENSE
0.734
0.002
S
0.22
0.001
S
1.499
0
S


364
70445
CGPG3728
SENSE
0.413
0.062
T
0.148
0.114
T
1.249
0
S


365
70738
CGPG3195
SENSE
0.687
0.055
T
0.205
0.041
S
1.261
0
S


366
71437
CGPG4043
SENSE
0.094
0.198
T
0.092
0.064
T
1.301
0
S


367
71572
CGPG4520
SENSE
0.938
0.052
T
0.441
0
S
1.633
0
S


368
71617
CGPG1227
SENSE
0.809
0.012
S
0.143
0.029
S
1.05
0.003
S


408
72085
CGPG5228
SENSE
1.234
0.02
S
0.192
0.043
S
1.162
0
S


369
72532
CGPG4780
SENSE
1.028
0.022
S
0.198
0.052
T
1.043
0.001
S


409
72744
CGPG5563
SENSE
0.17
0.146
T
0.04
0.359
/
0.827
0.004
S


370
72757
CGPG5572
SENSE
1.82
0.004
S
0.14
0.091
T
1.121
0
S


407
72771
CGPG2166
SENSE
1.776
0.001
S
0.36
0
S
1.428
0
S


444
72967
CGPG5742
SENSE
0.273
0.063
T
0.147
0.102
T
1.03
0
S


410
73039
CGPG810
SENSE
−0.048
0.774
/
−0.135
0.957
/
0.59
0.022
S


411
73054
CGPG5754
SENSE
0.055
0.312
/
0.236
0.001
S
1.434
0
S


508
73055
CGPG5768
SENSE
0.154
0.123
T
0.269
0
S
1.524
0
S


371
73412
CGPG6448
SENSE
0.187
0.118
T
0.134
0.06
T
1.181
0
S


412
73501
CGPG6456
SENSE
1.6
0.003
S
0.081
0.136
T
1.119
0
S


352
73515
CGPG6473
SENSE
−0.037
0.758
/
−0.024
0.604
/
0.694
0.008
S


437
73549
CGPG6460
SENSE
2.612
0
S
0.199
0.017
S
1.432
0
S


372
74102
CGPG6550
SENSE
0.34
0.035
S
0.268
0.002
S
1.355
0
S


509
74103
CGPG6558
SENSE
−0.013
1
/
−0.021
0.608
/
0.86
0
S


353
74684
CGPG6360
SENSE
0.44
0.018
S
0.254
0.002
S
1.383
0
S


512
19703
CGPG4172
SENSE
0.211
0.079
T
0.059
0.301
/
1.262
0
S


273
70423
CGPG3165
SENSE
1.456
0
S
0.418
0
S
1.912
0
S


491
70469
CGPG3791
SENSE
0.143
0.28
/
0.028
0.349
/
1.001
0.001
S


434
70932
CGPG4089
SENSE
0.354
0.1
T
0.03
0.419
/
1.254
0
S


419
71134
CGPG817
SENSE
0.522
0.106
T
0.077
0.248
/
1.225
0
S


466
71726
CGPG3894
SENSE
0.196
0.225
/
0.082
0.2
T
1.243
0
S


505
72463
CGPG4760
SENSE
1.182
0.003
S
0.202
0.026
/
1.702
0
S


445
72961
CGPG5591
SENSE
1.195
0.013
S
0.106
0.105
T
1.084
0
S


306
74136
CGPG6632
SENSE
0.886
0.012
S
0.306
0.009
S
1.42
0
S


504
74259
CGPG5343
SENSE
1.044
0.023
S
0.081
0.187
T
1.274
0
S


310
74318
CGPG5826
SENSE
0.407
0.075
T
0.116
0.052
T
1.118
0
S


479
74344
CGPG5929
SENSE
1.256
0.018
S
0.118
0.125
T
1.275
0
S


533
74462
CGPG6668
SENSE
0.846
0.022
S
0.26
0.011
S
1.425
0
S


313
74512
CGPG32
SENSE
0.241
0.118
T
0.146
0.044
S
1.062
0
S


442
74587
CGPG6774
SENSE
0
/
/
0.084
0.054
T
1.046
0
S





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2


/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset






D. Salt Stress Tolerance Screen

This example sets forth the high salinity stress screen to identify Arabidopsis plants transformed with the gene of interest that are tolerant to high levels of salt based on their rate of development, root growth and chlorophyll accumulation under high salt conditions.
T2 seeds were plated on glufosinate selection plates containing 90 mM NaCl and grown under standard light and temperature conditions. All seedlings used in the experiment were grown at a temperature of 22° C. at day and 20° C. at night, a 16-hour photoperiod, an average light intensity of approximately 120 umol/m2. On day 11, plants were measured for primary root length. After 3 more days of growth (day 14), plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was also taken on day 14.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success if 50% of the plants reached 3 rosette leaves and size of leaves are greater than 1 mm (Boyes, D. C., et al., (2001), The Plant Cell 13, 1499/1510). The growth stage data was analyzed as a qualitative response according to example 1L. Table 7 provides a list of recombinant DNA constructs that improve high salinity tolerance in transgenic plants












TABLE 7










Root Length at
Root Length at
Seedling Weight


Pep

Growth Stage
day 11
day 14
at day 14






















SEQ
Construct


RS
p-


p-


p-


p-



ID
id
Gene
Orientation
mean
value
c
delta
value
c
delta
value
c
delta
value
c

























512
19703
CGPG4172
SENSE
1.124
0.139
T
0.198
0.021
S
0.072
0.116
T
0.582
0.023
S


513
19946
CGPG4097
SENSE
1.201
0.072
T
0.02
0.89
/
0.069
0.573
/
0.443
0.266
/


514
19980
CGPG3914
SENSE
0.904
0.146
T
0.101
0.259
/
0.144
0.058
T
0.706
0.016
S


515
70435
CGPG3701
SENSE
1.363
0.031
S
−0.118
0.228
/
0.161
0.038
S
0.053
0.697
/


516
71114
CGPG1657
SENSE
0.138
0.399
/
0.245
0.009
S
0.187
0.025
S
0.472
0.069
T


517
72451
CGPG4733
SENSE
2.226
0.02
S
0.186
0.006
S
0.069
0.23
/
0.466
0.011
S


443
72453
CGPG4735
SENSE
1.539
0.031
S
0.232
0.002
S
0.216
0
S
0.737
0.001
S


505
72463
CGPG4760
SENSE
3.026
0.002
S
0.119
0.269
/
0.202
0.073
T
1.26
0
S


392
72519
CGPG4749
SENSE
0.598
0.041
S
0.126
0.055
T
0.172
0.003
S
0.635
0.008
S


506
72902
CGPG5597
SENSE
1.418
0.039
S
0.114
0.286
/
0.226
0.078
T
0.426
0.091
T


448
72916
CGPG1814
SENSE
0.682
0.181
T
0.07
0.532
/
0.201
0.046
S
0.387
0.079
T


510
72921
CGPG5781
SENSE
1.977
0.029
S
0.163
0.176
T
0.211
0.073
T
0.663
0.006
S


518
72947
CGPG5607
SENSE
1.505
0.028
S
0.024
0.899
/
0.204
0.022
S
0.466
0.216
/


445
72961
CGPG5591
SENSE
1.879
0.007
S
0.228
0.122
T
0.229
0.003
S
0.817
0.04
S


444
72967
CGPG5742
SENSE
2.427
0.004
S
0.386
0.009
S
0.369
0.001
S
1.254
0
S


511
72968
CGPG5772
SENSE
1.531
0.055
T
0.302
0.06
T
0.209
0.073
T
0.761
0.003
S


449
72969
CGPG5789
SENSE
0.67
0.078
T
0.029
0.789
/
0.239
0.008
S
0.603
0.013
S


519
73012
CGPG5786
SENSE
2.371
0.001
S
0.366
0
S
0.342
0
S
1.08
0
S


520
73022
CGPG5622
SENSE
1.408
0.036
S
0.22
0.056
T
0.347
0
S
0.492
0.03
S


508
73055
CGPG5768
SENSE
3.291
0.001
S
0.369
0.087
T
0.417
0.005
S
1.096
0.005
S


446
73070
CGPG5627
SENSE
2.755
0.005
S
0.188
0.392
/
0.275
0.011
S
0.685
0.025
S


447
73475
CGPG6385
SENSE
1.11
0.059
T
0.127
0.361
/
0.216
0.001
S
0.474
0.06
T


521
73488
CGPG6394
SENSE
2.373
0.013
S
0.314
0.006
S
0.262
0.002
S
1.126
0.005
S


522
73901
CGPG5237
SENSE
1.141
0.078
T
0.207
0.197
T
0.202
0.097
T
0.639
0.03
S


523
73964
CGPG5804
SENSE
1.235
0.043
S
0.428
0.008
S
0.317
0.022
S
0.955
0.002
S


524
74019
CGPG5706
SENSE
0.105
0.168
T
0.074
0.649
/
0.171
0.106
T
0.773
0.011
S


525
74022
CGPG5724
SENSE
0.033
0.327
/
−0.065
0.616
/
0.172
0.032
S
0.484
0.068
T


509
74103
CGPG6558
SENSE
1.225
0.074
T
0.26
0.042
S
0.267
0.004
S
0.543
0.042
S


526
74114
CGPG6551
SENSE
3.627
0
S
0.265
0.119
T
0.26
0
S
0.561
0.063
T


504
74259
CGPG5343
SENSE
2.802
0.003
S
0.249
0.098
T
0.256
0.037
S
0.995
0
S


527
74262
CGPG5353
SENSE
0.225
0.319
/
0.238
0.062
T
0.247
0
S
0.629
0.006
S


528
74292
CGPG5367
SENSE
0.327
0.199
T
0.16
0.067
T
0.105
0.166
T
0.565
0.013
S


529
74302
CGPG5384
SENSE
1.246
0.016
S
0.296
0.004
S
0.25
0
S
0.705
0.004
S


530
74325
CGPG5898
SENSE
1.596
0.021
S
−0.035
0.76
/
0.094
0.106
T
0.685
0.004
S


531
74429
CGPG6689
SENSE
1.796
0.008
S
0.298
0.037
S
0.207
0.006
S
0.496
0.029
S


532
74440
CGPG6682
SENSE
0.223
0.334
/
0.43
0.007
S
0.272
0.01
S
0.744
0.017
S


450
74449
CGPG6659
SENSE
0.693
0.19
T
0.204
0.104
T
0.205
0.022
S
0.451
0.095
T


533
74462
CGPG6668
SENSE
2.14
0.028
S
0.244
0.038
S
0.239
0.001
S
0.64
0.013
S


534
74465
CGPG6692
SENSE
1.245
0.016
S
0.35
0.01
S
0.215
0.001
S
0.575
0.043
S


535
74474
CGPG6669
SENSE
3.312
0.002
S
0.233
0.083
T
0.338
0.003
S
0.589
0.044
S


536
74505
CGPG6783
SENSE
1.731
0.043
S
0.272
0.007
S
0.208
0.01
S
0.493
0.009
S


537
74507
CGPG6799
SENSE
2.32
0.009
S
0.056
0.567
/
0.227
0.035
S
0.476
0.126
T


538
74562
CGPG6764
SENSE
1.405
0.038
S
−0.052
0.776
/
0.215
0.014
S
0.104
0.766
/


507
74572
CGPG6640
SENSE
1.425
0.025
S
0.201
0.009
S
0.267
0.001
S
1.184
0
S


270
14324
CGPG1560
SENSE
1.708
0.017
S
0.307
0.01
S
0.408
0
S
0.995
0.002
S


358
19768
CGPG4096
SENSE
1.496
0.061
T
0.163
0.187
T
0.129
0.013
S
0.66
0.002
S


436
19771
CGPG4011
SENSE
1.666
0.051
T
0.319
0.005
S
0.201
0.028
S
0.68
0.031
S


363
19879
CGPG4009
SENSE
2.117
0.029
S
0.16
0.139
T
0.091
0.202
/
0.545
0.028
S


347
72715
CGPG5521
SENSE
1.078
0.002
S
0.207
0.141
T
0.176
0.014
S
0.519
0.015
S


407
72771
CGPG2166
SENSE
0.564
0.256
/
−0.019
0.857
/
0.206
0.013
S
0.306
0.05
S


440
72903
CGPG5584
SENSE
0.645
0.196
T
−0.031
0.569
/
0.097
0.389
/
0.482
0.015
S


411
73054
CGPG5754
SENSE
1.52
0.035
S
0.198
0.089
T
0.098
0.187
T
0.696
0.004
S


476
74107
CGPG6590
SENSE
0.772
0.036
S
0.411
0
S
0.431
0.001
S
1.579
0
S


478
74131
CGPG6592
SENSE
1.747
0.044
S
0.316
0.004
S
0.115
0.01
S
0.663
0.001
S


442
74587
CGPG6774
SENSE
1.754
0.01
S
0.121
0.243
/
0.194
0.001
S
0.681
0
S





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2


/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset






E. Polyethylene Glycol (PEG) Induced Osmotic Stress Tolerance Screen

There are numerous factors, which can influence seed germination and subsequent seedling growth, one being the availability of water. Genes, which can directly affect the success rate of germination and early seedling growth, are potentially useful agronomic traits for improving the germination and growth of crop plants under drought stress. In this assay, PEG was used to induce osmotic stress on germinating transgenic lines of Arabidopsis thaliana seeds in order to screen for osmotically resistant seed lines.
T2 seeds were plated on BASTA selection plates containing 3% PEG and grown under standard light and temperature conditions. Seeds were plated on each plate containing 3% PEG, 1⁄2×MS salts, 1% phytagel, and 10 μg/ml glufosinate. Plates were placed at 4° C. for 3 days to stratify seeds. On day 11, plants were measured for primary root length. After 3 more days of growth, i.e., at day 14, plants were scored for transgenic status, primary root length, growth stage, visual color, and the seedlings were pooled for fresh weight measurement. A photograph of the whole plate was taken on day 14.
Seedling weight and root length were analyzed as quantitative responses according to example 1M. The final growth stage at day 14 was scored as success or failure based on whether the plants reached 3 rosette leaves and size of leaves are greater than 1 mm. The growth stage data was analyzed as a qualitative response according to example 1L. Table 8 provides a list of recombinant DNA constructs that improve osmotic stress tolerance in transgenic plants.












TABLE 8










Root Length at
Root Length at
Seedling Weight at


Pep

Growth Stage
day 11
day 14
day 14






















SEQ



RS
p-


p-


p-


p-



ID
Gene
Construct_id
Orientation
mean
value
c
delta
value
c
delta
value
c
delta
value
c

























413
19707
CGPG4179
SENSE
2.653
0.019
S
0.074
0.58
T
−0.031
0.81
/
0.427
0.063
T


414
19951
CGPG3941
SENSE
1.432
0.134
T
0.017
0.864
T
−0.08
0.28
/
0.476
0.054
T


415
19967
CGPG4032
SENSE
2.691
0.014
S
0.01
0.934
T
0.19
0.039
S
0.537
0.056
T


416
70543
CGPG3815
SENSE
1.735
0.077
T
0.084
0.561
T
0.323
0.007
S
0.676
0.006
S


402
70681
CGPG4584
SENSE
2.528
0.006
S
−0.065
0.682
/
−0.179
0.05
/
0.374
0.146
T


417
70707
CGPG1273
ANTI-
1.42
0.095
T
0.248
0.006
S
0.3
0.002
S
0.331
0.007
S





SENSE


418
70719
CGPG1712
ANTI-
1.43
0.106
T
−0.007
0.968
/
0.101
0.255
T
0.155
0.349
T





SENSE


419
71134
CGPG817
SENSE
1.478
0.13
T
0.119
0.15
T
0.035
0.684
T
0.198
0.277
T


420
71146
CGPG2928
SENSE
2.624
0.016
S
0.227
0.101
T
0.242
0.1
T
0.278
0.296
T


405
71508
CGPG1541
SENSE
3.153
0.001
S
0.446
0.054
T
0.384
0.048
S
0.782
0.003
S


421
71660
CGPG4690
SENSE
2.893
0.005
S
−0.024
0.795
/
0.225
0.074
T
0.165
0.329
T


403
71663
CGPG4638
SENSE
0.116
0.444
T
−0.061
0.536
/
0.358
0.041
S
0.058
0.702
T


439
71928
CGPG1617
SENSE
2.076
0.041
S
0.353
0.013
S
0.289
0.035
S
0.531
0.035
S


318
72081
CGPG5279
SENSE
1.262
0.138
T
0.174
0.243
T
0.2
0.197
T
−0.105
0.714
/


408
72085
CGPG5228
SENSE
4
0
S
0.22
0.046
S
0.371
0.004
S
0.929
0
S


422
72086
CGPG5236
SENSE
2.589
0.022
S
0.281
0.033
S
0.136
0.314
T
0.32
0.043
S


423
72632
CGPG4852
SENSE
1.663
0.053
T
0.254
0.069
T
0.094
0.548
T
0.471
0.015
S


424
72716
CGPG5529
SENSE
2.914
0.004
S
0.146
0.058
T
0.007
0.925
T
0.582
0.016
S


425
72723
CGPG1848
SENSE
2.138
0.066
T
0.043
0.728
T
0.2
0.068
T
−0.326
0.225
/


409
72744
CGPG5563
SENSE
1.636
0.05
/
0.195
0.151
T
0.059
0.751
T
0.539
0.005
S


404
72769
CGPG5573
SENSE
2.207
0.055
T
0.086
0.464
T
−0.134
0.162
/
0.389
0.086
T


407
72771
CGPG2166
SENSE
2.569
0.021
S
0.169
0.221
T
0.192
0.038
S
0.56
0.018
S


440
72903
CGPG5584
SENSE
2.161
0.061
T
0.035
0.856
T
0.245
0.162
T
0.06
0.871
T


510
72921
CGPG5781
SENSE
2.249
0.025
S
0.034
0.788
T
0.225
0.077
T
0.306
0.28
T


391
72948
CGPG5617
SENSE
2.267
0.005
S
0.054
0.495
T
0.117
0.019
S
0.289
0.077
T


445
72961
CGPG5591
SENSE
1.19
0.14
T
0.037
0.76
T
0.136
0.339
T
0.588
0.027
S


511
72968
CGPG5772
SENSE
3.142
0.007
S
0.178
0.072
T
−0.031
0.781
/
0.698
0.016
S


426
72987
CGPG1787
SENSE
2.055
0.078
T
−0.058
0.64
/
0.097
0.144
T
0.325
0.054
T


438
72994
CGPG5803
SENSE
2.674
0.013
S
0.245
0.124
T
0.07
0.657
T
0.599
0.035
S


441
73017
CGPG5733
SENSE
4
0
S
0.343
0.09
T
0.462
0.002
S
0.996
0
S


410
73039
CGPG810
SENSE
4
0
S
0.319
0.019
S
0.237
0.05
/
0.426
0.011
S


411
73054
CGPG5754
SENSE
3.048
0.002
S
0.556
0.003
S
0.26
0.063
T
1.12
0.002
S


446
73070
CGPG5627
SENSE
3.439
0.001
S
0.139
0.49
T
0.19
0.135
T
0.24
0.365
T


447
73475
CGPG6385
SENSE
1.933
0.033
S
0.04
0.476
T
0.009
0.897
T
0.459
0.073
T


412
73501
CGPG6456
SENSE
1.29
0.121
T
0.143
0.118
T
0.043
0.564
T
0.433
0.013
S


427
74109
CGPG6606
SENSE
3.517
0
S
0.159
0.136
T
0.249
0.004
S
0.333
0.025
S


428
74140
CGPG6569
SENSE
2.05
0.039
S
0.168
0.138
T
0.196
0.086
T
0.71
0.012
S


429
74191
CGPG6597
SENSE
2.565
0.019
S
0.336
0.092
T
0.199
0.112
T
0.54
0.02
S


406
74248
CGPG5476
SENSE
3.158
0.007
S
0.14
0.192
T
0.204
0.051
T
0.377
0.037
S


430
74265
CGPG5356
SENSE
2.208
0.023
S
0.317
0.034
S
0.419
0.006
S
0.577
0.008
S


431
74369
CGPG6076
SENSE
3.522
0
S
0.347
0.045
S
0.272
0.107
T
0.624
0.02
S


442
74587
CGPG6774
SENSE
3.325
0.002
S
0.073
0.468
T
0.414
0.002
S
0.577
0.016
S


405
71508
CGPG1541
SENSE
3.153
0.001
S
0.446
0.054
T
0.384
0.048
S
0.782
0.003
S


439
71928
CGPG1617
SENSE
2.076
0.041
S
0.353
0.013
S
0.289
0.035
S
0.531
0.035
S


422
72086
CGPG5236
SENSE
2.589
0.022
S
0.281
0.033
S
0.136
0.314
/
0.32
0.043
S


469
72455
CGPG4742
SENSE
1.367
0.15
T
0.026
0.764
/
0.024
0.786
/
0.255
0.04
S


382
72466
CGPG4767
SENSE
0.735
0.101
T
0.068
0.333
/
0.249
0.006
S
−0.341
0.278
/


373
72633
CGPG4853
SENSE
1.179
0.122
T
0.227
0.009
S
0.097
0.065
T
0.442
0.013
S


370
72757
CGPG5572
SENSE
2.272
0.017
S
0.125
0.361
/
0.11
0.317
/
0.425
0.047
S


472
72992
CGPG5777
SENSE
2.233
0.056
T
0.176
0.261
/
0.116
0.34
/
0.511
0.019
S


438
72994
CGPG5803
SENSE
2.674
0.013
S
0.245
0.124
T
0.07
0.657
/
0.599
0.035
S


441
73017
CGPG5733
SENSE
4
0
S
0.343
0.09
T
0.462
0.002
S
0.996
0
S


411
73054
CGPG5754
SENSE
3.048
0.002
S
0.556
0.003
S
0.26
0.063
T
1.12
0.002
S


300
73507
CGPG6504
SENSE
0.343
0.334
/
0.347
0.007
S
0.261
0.032
S
0.3
0.122
T


352
73515
CGPG6473
SENSE
3.336
0.002
S
0.279
0.009
S
0.241
0.003
S
0.328
0.05
S


428
74140
CGPG6569
SENSE
2.05
0.039
S
0.168
0.138
T
0.196
0.086
T
0.71
0.012
S


430
74265
CGPG5356
SENSE
2.208
0.023
S
0.317
0.034
S
0.419
0.006
S
0.577
0.008
S


431
74369
CGPG6076
SENSE
3.522
0
S
0.347
0.045
S
0.272
0.107
T
0.624
0.02
S


422
74587
CGPG6774
SENSE
3.325
0.002
S
0.073
0.468
/
0.414
0.002
S
0.577
0.016
S





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2


/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset






F. Cold Shock Tolerance Screen

This example set forth a screen to identify Arabidopsis plants transformed with the genes of interest that are more tolerant to cold stress subjected during day 8 to day 28 after seed planting. During these crucial early stages, seedling growth and leaf area increase were measured to assess tolerance when Arabidopsis seedlings were exposed to low temperatures. Using this screen, genetic alterations can be found that enable plants to germinate and grow better than wild type plants under sudden exposure to low temperatures.
Eleven seedlings from T2 seeds of each transgenic line plus one control line were plated together on a plate containing 1⁄2× Gamborg Salts with 0.8 PhytagelTM, 1% Phytagel, and 0.3% Sucrose. Plates were then oriented horizontally and stratified for three days at 4° C. At day three, plates were removed from stratification and exposed to standard conditions (16 hr photoperiod, 22° C. at day and 20° C. at night) until day 8. At day eight, plates were removed from standard conditions and exposed to cold shock conditions (24 hr photoperiod, 8° C. at both day and night) until the final day of the assay, i.e., day 28. Rosette areas were measured at day 8 and day 28, which were analyzed as quantitative responses according to example 1M. Table 9 provides a list of recombinant nucleotides that improve cold shock stress tolerance in plants.











TABLE 9











difference in rosette



rosette area
rosette area
area between day 28



at day 8
at day 28
and day 8



















Pep




p-


p-


p-



SEQ ID
Construct_id
Gene
Orientation
delta
value
c
delta
value
c
delta
value
c






















270
14324
CGPG1560
SENSE
0.054
0.429
/
0.258
0.017
S
−0.071
0.631
/


271
17484
CGPG2630
SENSE
−0.189
0.759
/
0.544
0.025
S
0.275
0.121
T


272
19109
CGPG1381
ANTI-SENSE
−0.016
0.523
/
0.541
0.008
S
0.818
0.014
S


273
70423
CGPG3165
SENSE
0.316
0.012
S
0.521
0.022
S
0.89
0.018
S


274
70424
CGPG3180
SENSE
0.474
0.003
S
0.695
0.003
S
0.693
0.043
S


275
70480
CGPG3833
SENSE
−0.066
0.591
/
0.175
0.159
T
0.474
0.059
T


276
70509
CGPG2420
SENSE
0.023
0.438
/
0.117
0.216
/
0.609
0.032
S


277
70647
CGPG4334
SENSE
−0.508
0.894
/
0.604
0.049
S
0.895
0.047
S


278
70675
CGPG4519
SENSE
0.2
0.2
/
0.303
0.153
T
0.507
0.034
S


279
70829
CGPG518
SENSE
−0.319
0.823
/
0.804
0.002
S
1.082
0.002
S


280
70849
CGPG596
SENSE
−0.039
0.564
/
0.698
0.001
S
0.707
0.001
S


281
71627
CGPG1270
SENSE
−0.146
0.748
/
0.349
0.05
T
0.3
0.12
T


282
71934
CGPG2294
SENSE
−0.068
0.796
/
0.757
0
S
0.922
0
S


283
72615
CGPG4829
SENSE
0.477
0.007
S
0.834
0
S
0.979
0.001
S


286
73559
CGPG6535
SENSE
0.143
0.093
T
−0.265
0.878
/
−0.344
0.821
/


287
74251
CGPG5489
SENSE
0.377
0.021
S
0.439
0.045
S
0.45
0.07
T


389
70437
CGPG3706
SENSE
−0.273
0.916
/
0.147
0.165
T
0.682
0.034
S


402
70681
CGPG4584
SENSE
0.352
0.155
T
0.252
0.261
/
0.269
0.328
/


403
71663
CGPG4638
SENSE
0.358
0.013
S
0.032
0.423
/
−0.031
0.585
/


404
72769
CGPG5573
SENSE
0.381
0.049
S
0.881
0.006
S
1.102
0.005
S


407
72771
CGPG2166
SENSE
0.993
0
S
1.381
0.003
S
1.536
0.003
S


432
70217
CGPG6
SENSE
0.275
0.067
T
0.126
0.289
/
0.362
0.215
/


433
72711
CGPG1846
SENSE
0.774
0.001
S
0.579
0.004
S
0.429
0.038
S


438
72994
CGPG5803
SENSE
0.116
0.381
/
0.708
0.068
T
0.744
0.069
T


510
72921
CGPG5781
SENSE
0.265
0.057
T
0.31
0.162
T
0.367
0.11
T


414
19951
CGPG3941
SENSE
0.729
0.006
S
0.473
0.017
s
0.846
0.006
S


273
70423
CGPG3165
SENSE
0.316
0.012
S
0.521
0.022
S
0.89
0.018
S


416
70543
CGPG3815
SENSE
1.584
0
S
0.86
0
S
0.82
0.002
S


368
71617
CGPG1227
SENSE
0.204
0.136
T
0.408
0.025
S
0.458
0.057
T


439
71928
CGPG1617
SENSE
0.104
0.265
/
0.786
0
S
0.836
0.001
S


382
72466
CGPG4767
SENSE
0.497
0.017
S
0.565
0.017
S
0.963
0.002
S


383
72524
CGPG4770
SENSE
0.438
0.02
S
0.377
0.025
S
0.385
0.043
S


409
72744
CGPG5563
SENSE
0.52
0.058
T
0.859
0.026
S
0.454
0.189
T


444
72967
CGPG5742
SENSE
0.955
0
S
0.629
0.009
S
0.403
0.189
T


435
73518
CGPG6497
SENSE
0.114
0.278
/
0.319
0.01
S
0.195
0.114
T


306
74136
CGPG6632
SENSE
0.606
0.007
S
0.523
0.036
S
0.598
0.025
S


398
74240
CGPG5454
SENSE
−0.099
0.644
/
1.277
0.003
S
1.498
0.006
S


431
74369
CGPG6076
SENSE
0.623
0.002
S
0.62
0.04
S
0.737
0.096
T





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2


/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset.






G. Cold Germination Tolerance Screen

This example sets forth a screen to identify Arabidopsis plants transformed with the genes of interests are resistant to cold stress based on their rate of development, root growth and chlorophyll accumulation under low temperature conditions.
T2 seeds were plated and all seedlings used in the experiment were grown at 8° C. Seeds were first surface disinfested using chlorine gas and then seeded on assay plates containing an aqueous solution of 1⁄2× Gamborg's B/5 Basal Salt Mixture (Sigma/Aldrich Corp., St. Louis, Mo., USA G/5788), 1% PhytagelTM (Sigma-Aldrich, P-8169), and 10 ug/ml glufosinate with the final pH adjusted to 5.8 using KOH. Test plates were held vertically for 28 days at a constant temperature of 8° C., a photoperiod of 16 hr, and average light intensity of approximately 100 umol/m2/s. At 28 days post planting, root length was measured, growth stage was observed, the visual color was assessed, and a whole plate photograph was taken.
The root length at day 28 was analyzed as a quantitative response according to example 1M. The growth stage at day 7 was analyzed as a qualitative response according to example 1L. Table 10 provides a list of recombinant DNA constructs that improve cold stress tolerance in transgenic plants.










TABLE 10









Growth stage
Root Length at



at day 28
day 28
















Pep SEQ



RS
p-


p-



ID
Construct_id
Gene
Orientation
mean
value
c
delta
value
c



















288
19631
CGPG3627
SENSE
2.229
0.052
T
0.094
0.252
/


289
70121
CGPG2380
SENSE
2.732
0.042
S
0.126
0.238
/


290
70654
CGPG4352
SENSE
2.474
0.026
S
0.263
0.019
S


291
70696
CGPG4590
SENSE
3.092
0.01
S
0.086
0.145
T


292
70713
CGPG1462
ANTI-SENSE
4
0
S
0.268
0.014
S


293
70740
CGPG3700
SENSE
2.485
0.024
S
0.244
0.012
S


294
71321
CGPG4418
SENSE
1.837
0.126
T
0.014
0.474
/


295
71835
CGPG4634
SENSE
3.349
0.002
S
0.353
0.036
S


296
72934
CGPG5798
SENSE
2.222
0.023
S
0.243
0.101
T


297
72945
CGPG5787
SENSE
3.478
0.001
S
0.236
0.011
S


298
72980
CGPG5773
SENSE
3.265
0.003
S
0.239
0.006
S


299
73504
CGPG6480
SENSE
4
0
S
0.521
0
S


300
73507
CGPG6504
SENSE
4
0
S
0.404
0.001
S


301
73573
CGPG6462
SENSE
4
0
S
0.268
0.004
S


302
73586
CGPG6471
SENSE
4
0
S
0.314
0.064
T


303
73770
CGPG5435
SENSE
3.091
0.01
S
0.303
0.085
T


304
74105
CGPG6574
SENSE
3.329
0.002
S
0.109
0.116
T


305
74111
CGPG6622
SENSE
2.226
0.021
S
0.445
0.006
S


306
74136
CGPG6632
SENSE
3.192
0.005
S
0.328
0.002
S


307
74139
CGPG6561
SENSE
4
0
S
0.254
0.092
T


308
74267
CGPG5364
SENSE
3.054
0.002
S
0.3
0
S


309
74291
CGPG5363
SENSE
4
0
S
0.142
0.142
T


310
74318
CGPG5826
SENSE
4
0
S
0.272
0.008
S


311
74319
CGPG5831
SENSE
3.207
0.005
S
0.201
0.002
S


312
74324
CGPG5885
SENSE
3.144
0.007
S
0.232
0.017
S


313
74512
CGPG32
SENSE
4
0
S
0.332
0.011
S


314
74583
CGPG6649
SENSE
3.249
0.004
S
0.28
0.001
S


315
70427
CGPG3067
SENSE
1.567
0.108
T
0.222
0.044
S


352
73515
CGPG6473
SENSE
4
0
S
0.324
0.003
S


353
74684
CGPG6360
SENSE
2.927
0.004
S
0.426
0.003
S


373
72633
CGPG4853
SENSE
2.121
0.027
S
0.289
0.048
S


405
71508
CGPG1541
SENSE
1.99
0.039
S
0.263
0.033
S


406
74248
CGPG5476
SENSE
2.385
0.011
S
0.217
0.017
S


434
70932
CGPG4089
SENSE
3.268
0.003
S
0.146
0.067
T


435
73518
CGPG6497
SENSE
3.373
0.002
S
0.352
0.032
S


439
71928
CGPG1617
SENSE
3.062
0.011
S
0.18
0.002
S


504
74259
CGPG5343
SENSE
3.511
0
S
0.308
0.024
S


505
72463
CGPG4760
SENSE
2.736
0.01
S
0.032
0.386
/


506
72902
CGPG5597
SENSE
3.105
0.009
S
0.278
0.037
S


507
74572
CGPG6640
SENSE
4
0
S
0.125
0.155
T


508
73055
CGPG5768
SENSE
3.173
0.006
S
0.407
0.004
S


413
19707
CGPG4179
SENSE
1.829
0.061
T
0.169
0.018
S


360
19779
CGPG4113
SENSE
4
0
S
0.213
0.017
S


361
19833
CGPG4074
SENSE
/
/
/
0.292
0.022
S


363
19879
CGPG4009
SENSE
4
0
S
0.34
0.001
S


514
19980
CGPG3914
SENSE
0.798
0.122
T
0.278
0.011
S


273
70423
CGPG3165
SENSE
2.906
0.004
S
0.105
0.114
T


459
70725
CGPG2097
ANTI-SENSE
1.949
0.044
S
0.122
0.148
T


461
71112
CGPG934
SENSE
2.579
0.018
S
0.185
0.17
T


444
72967
CGPG5742
SENSE
4
0
S
0.287
0.007
S


438
72994
CGPG5803
SENSE
1.072
0.098
T
0.161
0.04
S


521
73488
CGPG6394
SENSE
4
0
S
0.211
0.012
S


477
74117
CGPG6575
SENSE
2.567
0.02
S
0.123
0.04
S





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2


/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined compared to the reference in the current dataset






H. Shade Tolerance Screen

Plants undergo a characteristic morphological response in shade that includes the elongation of the petiole, a change in the leaf angle, and a reduction in chlorophyll content. While these changes may confer a competitive advantage to individuals, in a monoculture the shade avoidance response is thought to reduce the overall biomass of the population. Thus, genetic alterations that prevent the shade avoidance response are associated with higher yields. Genes that favor growth under low light conditions may also promote yield, as inadequate light levels frequently limit yield. This protocol describes a screen to look for Arabidopsis plants that show an attenuated shade avoidance response and/or grow better than control plants under low light intensity. Of particular interest, we were looking for plants that didn't extend their petiole length, had an increase in seedling weight relative to the reference and had leaves that were more close to parallel with the plate surface.
T2 seeds were plated on glufosinate selection plates with 1⁄2 MS medium. Seeds were sown on 1⁄2×MS salts, 1% Phytagel, 10 ug/ml BASTA. Plants were grown on vertical plates at a temperature of 22° C. at day, 20° C. at night and under low light (approximately 30 uE/m2/s, far/red ratio (655/665/725/735)-0.35 using PLAQ lights with GAM color filter #680). Twenty-three days after seedlings were sown, measurements were recorded including seedling status, number of rosette leaves, status of flower bud, petiole leaf angle, petiole length, and pooled fresh weights. A digital image of the whole plate was taken on the measurement day. Seedling weight and petiole length were analyzed as quantitative responses according to example 1M. The number of rosette leaves, flowering bud formation and leaf angel were analyzed as qualitative responses according to example 1L.
Table 11 provides a list of recombinant DNA constructs that improve shade tolerance in plants













TABLE 11









flowerbud


Number
seedling




formation at
Leaf Angle at
Petiole length at
of rosette
weight at


Pep

day 23
day 23
day 23
leaves at day 23
day 23
























SEQ


RS
p-

RS
p-

RS
p-

RS
p-


p-



ID
Construct_id
Orientation
mean
value
c
mean
value
c
mean
value
c
mean
value
c
delta
value
c



























376
70426
SENSE
0.719
0.09
T
0.163
0.244
/
−0.206
0.185
T
−0.225
0.984
/
0.484
0.003
S


377
70772
SENSE
−0.501
0.858
/
−0.066
0.724
/
−0.692
0.004
S
−0.626
0.983
/
−0.693
0.023
/


378
71137
SENSE
0.26
0.384
/
0.11
0.168
T
−0.869
0.029
S
1.064
0.073
T
−0.453
0.288
/


379
71529
SENSE
0.89
0.108
T
−0.014
1
/
−0.44
0.006
S
1.817
0.024
S
−0.022
0.914
/


380
71601
SENSE
1.79
0.066
T
0.195
0.172
T
−0.057
0.59
/
−0.483
0.961
/
0.264
0.308
/


381
72362
SENSE
1.763
0.072
T
0.218
0.262
/
−0.109
0.242
/
0.003
0.484
/
0.311
0.119
T


374
72456
SENSE
0.563
0.313
/
0.751
0.186
T
−0.848
0.001
S
−0.624
0.999
/
−0.85
0.025
/


382
72466
SENSE
−1.382
1
/
−0.092
0.645
/
−0.894
0
S
1.212
0.122
T
−0.879
0.027
/


383
72524
SENSE
−0.153
1
/
0.152
0.09
T
−0.952
0.002
S
−0.57
1
/
−1.269
0.01
/


373
72633
SENSE
3.513
0
S
−0.431
0.977
/
−0.426
0.001
S
−0.735
0.994
/
0.269
0.129
T


375
72963
SENSE
−0.911
0.988
/
0.236
0.396
/
−0.753
0.003
S
−0.083
0.665
/
−0.485
0.015
/


384
73085
SENSE
−0.073
0.756
/
1.212
0.122
T
0.175
0.097
/
4
0
S
0.51
0.027
S


385
74241
SENSE
−0.195
0.935
/
−0.098
0.64
/
−1.077
0.01
S
−0.821
0.999
/
−1.024
0.002
/


386
74247
SENSE
0.43
0.16
T
0.203
0.095
T
−0.22
0.197
T
1.166
0.07
T
0.483
0.018
S


387
74284
SENSE
0.062
0.22
/
2.077
0.038
S
−0.014
0.943
/
2.348
0.04
S
0.03
0.938
/


388
74652
SENSE
0.015
0.442
/
0.093
0.455
/
−0.073
0.621
/
0.967
0.184
T
0.173
0.569
/


363
19879
SENSE
0.585
/
/
0
/
/
0.152
/
/
0
/
/
0.708
/
/


293
70740
SENSE
0.44
0.137
T
0.609
0.103
T
0.161
0.186
/
/
/
/
0.45
0.001
S


316
71811
SENSE
−0.025
0.637
/
0.725
0.113
T
−0.205
0.01
S
/
/
/
−0.517
0.037
/


397
71840
SENSE
1.498
0.12
T
0.712
0.178
T
−0.379
0.001
S
−0.188
0.765
/
−0.95
0.124
/


468
72450
SENSE
0.068
0.349
/
−0.042
1
/
0.166
0.016
/
1.62
0.049
S
0.501
0.038
S


370
72757
SENSE
3.595
0
S
0.872
0.079
T
0.064
0.138
/
−0.767
0.997
/
0.546
0.001
S


444
72967
SENSE
1.829
0.063
T
1.123
0.065
T
0.133
0.196
/
−0.18
0.794
/
0.509
0.008
S


511
72968
SENSE
0.185
0.005
S
0.48
0.179
T
0.22
0.162
/
1.698
0.085
T
0.382
0.031
S


300
73507
SENSE
0.248
0.086
T
0.307
0.287
/
−0.304
0.031
S
/
/
/
0.05
0.86
/


307
74139
SENSE
−0.011
0.556
/
−0.071
1
/
−0.124
0.068
T
−0.511
1
/
−0.413
0.124
/


535
74474
SENSE
0.755
0.154
T
−0.145
0.857
/
0.142
0.396
/
/
/
/
0.765
0.005
S


400
74610
SENSE
0.572
0.222
/
0.177
0.22
/
−0.18
0.071
T
/
/
/
−0.341
0.173
/





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2


/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined compared to the reference in the current dataset.






I. Early Plant Growth and Development Screen

This example sets forth a plate based phenotypic analysis platform for the rapid detection of phenotypes that are evident during the first two weeks of growth. In this screen, we were looking for genes that confer advantages in the processes of germination, seedling vigor, root growth and root morphology under non-stressed growth conditions to plants. The transgenic plants with advantages in seedling growth and development were determined by the seedling weight and root length at day 14 after seed planting.
T2 seeds were plated on glufosinate selection plates and grown under standard conditions ( ̃100 □E/m2/s, 16 h photoperiod, 22° C. at day, 20° C. at night). Seeds were stratified for 3 days at 4° C. Seedlings were grown vertically (at a temperature of 22° C. at day 20° C. at night). Observations were taken on day 10 and day 14. Both seedling weight and root length at day 14 were analyzed as quantitative responses according to example 1M.
Table 12 provides a list recombinant DNA constructs that improve early plant growth and development.










TABLE 12









Root Length
Seedling Weight
















Pep SEQ ID
Construct_id
gene
Orientation
delta
p-value
c
delta
p-value
c



















432
70217
CGPG6
SENSE
0.038
0.469
/
0.375
0.047
S


433
72711
CGPG1846
SENSE
0.132
0.021
S
0.601
0.001
S


434
70932
CGPG4089
SENSE
0.328
0.005
S
0.473
0.017
S


435
73518
CGPG6497
SENSE
0.287
0
S
0.634
0.036
S


436
19771
CGPG4011
SENSE
0.218
0.076
T
0.581
0.018
S


437
73549
CGPG6460
SENSE
0.139
0.003
S
0.349
0.03
S


438
72994
CGPG5803
SENSE
0.44
0.004
S
0.791
0.001
S


439
71928
CGPG1617
SENSE
0.073
0.427
/
0.494
0.005
S


440
72903
CGPG5584
SENSE
0.298
0.002
S
0.399
0.044
S


441
73017
CGPG5733
SENSE
0.284
0.004
S
0.199
0.488
/


442
74587
CGPG6774
SENSE
0.111
0.075
T
0.538
0.006
S


443
72453
CGPG4735
SENSE
0.215
0.005
S
0.416
0.069
T


444
72967
CGPG5742
SENSE
0.103
0.212
/
0.568
0.008
S


445
72961
CGPG5591
SENSE
0.177
0.046
S
0.548
0.006
S


446
73070
CGPG5627
SENSE
0.221
0.007
S
0.652
0.005
S


447
73475
CGPG6385
SENSE
0.084
0.014
S
0.336
0.014
S


448
72916
CGPG1814
SENSE
0.182
0.067
T
0.353
0.012
S


449
72969
CGPG5789
SENSE
0.133
0.245
/
0.45
0.069
T


450
74449
CGPG6659
SENSE
0.314
0.002
S
0.579
0.027
S


451
16615
CGPG2539
SENSE
0.223
0.023
S
0.571
0.009
S


452
19187
CGPG3310
SENSE
0.264
0.001
S
0.51
0.08
T


453
19648
CGPG3134
SENSE
0.218
0.013
S
0.27
0.106
T


454
70354
CGPG3995
SENSE
0.152
0.046
S
0.406
0.029
S


455
70421
CGPG2942
SENSE
0.179
0.225
/
0.581
0.013
S


456
70459
CGPG3758
SENSE
0.187
0.036
S
−0.034
0.884
/


457
70465
CGPG3775
SENSE
−0.009
0.899
/
0.188
0.196
T


458
70683
CGPG4587
SENSE
0.133
0.088
T
0.402
0.014
S


459
70725
CGPG2097
ANTI-SENSE
0.326
0.001
S
0.116
0.548
/


460
70852
CGPG1465
SENSE
0.237
0
S
0.297
0.127
T


461
71112
CGPG934
SENSE
0.199
0.013
S
0.316
0.034
S


462
71127
CGPG945
SENSE
0.097
0.02
S
0.4
0.054
T


463
71132
CGPG1561
SENSE
0.195
0.02
S
0.08
0.524
/


464
71217
CGPG95
SENSE
0.234
0
S
0.566
0.036
S


465
71645
CGPG4688
SENSE
0.475
0.003
S
0.361
0.133
T


466
71726
CGPG3894
SENSE
0.223
0.056
T
0.458
0.033
S


467
72432
CGPG4562
SENSE
0.209
0
S
0.581
0.041
S


468
72450
CGPG4732
SENSE
0.335
0
S
0.79
0
S


469
72455
CGPG4742
SENSE
0.278
0.019
S
0.482
0.051
T


470
72727
CGPG5522
SENSE
0.123
0.002
S
0.315
0.004
S


471
72817
CGPG4987
SENSE
0.254
0.023
S
0.485
0
S


472
72992
CGPG5777
SENSE
0.219
0.023
S
0.664
0.015
S


473
73007
CGPG5760
SENSE
0.139
0.093
T
0.462
0.008
S


474
73073
CGPG5688
SENSE
0.164
0.022
S
0.285
0.247
/


475
73506
CGPG6496
SENSE
0.512
0
S
0.986
0
S


476
74107
CGPG6590
SENSE
0.282
0.002
S
0.538
0.057
T


477
74117
CGPG6575
SENSE
0.211
0.002
S
0.449
0.005
S


478
74131
CGPG6592
SENSE
0.142
0.047
S
0.586
0.003
S


479
74344
CGPG5929
SENSE
0.27
0.01
S
0.474
0.105
T


323
13502
CGPG1354
SENSE
0.57
0.002
S
0.625
0
S


330
18259
CGPG3368
SENSE
0.226
0.026
S
0.551
0.021
S


361
19833
CGPG4074
SENSE
0.259
0
S
0.472
0.016
S


334
70417
CGPG3427
SENSE
0.187
0.056
T
0.113
0.65
/


273
70423
CGPG3165
SENSE
0.03
0.747
/
0.185
0.007
S


515
70435
CGPG3701
SENSE
0.131
0.051
T
0.394
0.014
S


493
70601
CGPG2917
SENSE
0.177
0
S
0.365
0.063
T


291
70696
CGPG4590
SENSE
0.079
0.205
/
0.331
0.024
S


365
70738
CGPG3195
SENSE
0.012
0.864
/
0.472
0.003
S


293
70740
CGPG3700
SENSE
0.103
0.082
T
0.387
0.021
S


419
71134
CGPG817
SENSE
0.063
0.548
/
0.279
0.066
T


422
72086
CGPG5236
SENSE
0.083
0.234
/
0.398
0.05
S


505
72463
CGPG4760
SENSE
−0.131
0.416
/
0.422
0.003
S


285
73014
CGPG5692
SENSE
0.11
0.391
/
0.373
0.096
T


521
73488
CGPG6394
SENSE
0.018
0.793
/
0.398
0.1
T


299
73504
CGPG6480
SENSE
−0.152
0.479
/
0.538
0.012
S


300
73507
CGPG6504
SENSE
0.15
0.002
S
0.053
0.854
/


301
73573
CGPG6462
SENSE
0.112
0.07
T
0.375
0.006
S


302
73586
CGPG6471
SENSE
0.309
0
S
0.611
0
S


303
73770
CGPG5435
SENSE
0.21
0.059
T
0.281
0.484
/


509
74103
CGPG6558
SENSE
0.374
0
S
0.561
0.024
S


428
74140
CGPG6569
SENSE
0.16
0.017
S
0.376
0.066
T


527
74262
CGPG5353
SENSE
0.203
0.012
S
0.375
0.045
S


430
74265
CGPG5356
SENSE
0.107
0.259
T
0.432
0.067
T


529
74302
CGPG5384
SENSE
0.115
0.101
T
0.269
0.056
T


431
74369
CGPG6076
SENSE
0.138
0.03
S
0.195
0.21
/


534
74465
CGPG6692
SENSE
0.2
0.02
S
0.688
0.042
S


507
74572
CGPG6640
SENSE
0.162
0.023
S
0.617
0
S


314
74583
CGPG6649
SENSE
0.144
0.023
S
0.403
0.008
S





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement as compared to the reference with p < 0.2


/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset






J. Late Plant Growth And Development Screen

This example sets forth a soil based phenotypic platform to identify genes that confer advantages in the processes of leaf development, flowering production and seed maturity to plants.
Arabidopsis plants were grown on a commercial potting mixture (Metro Mix 360, Scotts Co., Marysville, Ohio) consisting of 30-40% medium grade horticultural vermiculite, 35-55% sphagnum peat moss, 10-20% processed bark ash, I-15% pine bark and a starter nutrient charge. Soil was supplemented with Osmocote time-release fertilizer at a rate of 30 mg/ft3. T2 seeds were imbibed in 1% agarose solution for 3 days at 4° C. and then sown at a density of 5 per 21⁄2′′ pot. Thirty-two pots were ordered in a 4 by 8 grid in standard greenhouse flat. Plants were grown in environmentally controlled rooms under a 16 h day length with an average light intensity of  ̃200 μmoles/m2/s. Day and night temperature set points were 22° C. and 20° C., respectively. Humidity was maintained at 65%. Plants were watered by sub-irrigation every two days on average until mid-flowering, at which point the plants were watered daily until flowering was complete.
Application of the herbicide glufosinate was performed to select T2 individuals containing the target transgene. A single application of glufosinate was applied when the first true leaves were visible. Each pot was thinned to leave a single glufosinate-resistant seedling  ̃3 days after the selection was applied.
The rosette radius was measured at day 25. The silique length was measured at day 40. The plant parts were harvested at day 49 for dry weight measurements if flowering production was stopped. Otherwise, the dry weights of rosette and silique were carried out at day 53. The seeds were harvested at day 58. All measurements were analyzed as quantitative responses according to example 1M.
Table 13 provides a list of recombinant DNA constructs that improve late plant growth and development.













TABLE 13









Rosette Dry


Silique Dry



Pep

Weight
Rosette Radius
Seed Dry Weight
Weight
Slilique Length
























SEQ



p-


p-


p-


p-


p-



ID
Construct_id
Orientation
delta
value
c
delta
value
c
delta
value
c
delta
value
c
delta
value
c



























480
14320
SENSE
−0.145
0.94
/
0.137
0.038
S
−0.702
1
/
0.477
0.002
S
0.016
0.276
/


481
16756
SENSE
0.485
0.016
S
0.223
0.025
S
0.148
0.042
S
0.481
0.002
S
0.147
0.013
S


482
17448
SENSE
−0.288
0.991
/
−0.054
0.829
/
0.376
0.008
S
0.185
0.034
S
−0.064
0.981
/


483
17633
SENSE
0.018
0.359
/
0.13
0.106
T
0.416
0.055
T
0.399
0.044
S
0.116
0.08
T


484
18876
SENSE
0.257
0.016
S
0.026
0.448
/
−0.213
0.786
/
0.388
0
S
−0.015
0.705
/


485
19120
ANTI-
−0.252
0.936
/
0.022
0.006
S
−1.042
0.95
/
0.165
0.076
T
0.046
0.021
S




SENSE


486
19221
SENSE
−0.316
0.986
/
0.153
0.097
T
−0.35
0.903
/
0.394
0.068
T
0.183
0.028
S


487
70206
SENSE
0.125
0
S
0.074
0.18
T
0.712
0.022
S
0.12
0.026
S
0.019
0.403
/


488
70223
SENSE
0.197
0.026
S
0.23
0.018
S
−0.781
0.998
/
0.134
0.039
S
−0.205
0.915
/


489
70347
SENSE
0.156
0.038
S
−0.082
0.868
/
−0.153
0.752
/
0.405
0.006
S
0.056
0.068
T


490
70406
SENSE
−0.275
0.948
/
−0.245
0.992
/
0.759
0.025
S
−0.282
0.949
/
−0.121
0.939
/


491
70469
SENSE
0.032
0.392
/
0.348
0.004
S
−0.733
0.996
/
0.325
0.059
T
−0.141
0.922
/


492
70564
SENSE
0.17
0.037
S
0.051
0.234
/
0.772
0
S
−0.381
0.977
/
−0.015
0.655
/


493
70601
SENSE
0.231
0.086
T
0.247
0.004
S
−0.257
0.959
/
0.323
0.024
S
0.082
0.03
S


494
70612
SENSE
0.053
0.112
T
0.082
0.096
T
1.049
0.011
S
−0.212
0.992
/
0.07
0.004
S


495
70720
ANTI-
−0.16
0.898
/
0.028
0.384
/
1.312
0.009
S
0.219
0.121
T
0.128
0.018
S




SENSE


496
70735
SENSE
−0.058
0.672
/
0.156
0.087
T
0.421
0.036
S
0.532
0.003
S
0.033
0.038
S


497
70846
SENSE
0.086
0.255
/
0.134
0.063
T
−0.33
0.918
/
0.142
0.151
T
0.011
0.439
/


498
70923
SENSE
0.484
0.011
S
0.108
0.081
T
−0.4
0.844
/
0.091
0.198
T
0.099
0.001
S


499
71149
SENSE
−1.085
0.993
/
−0.043
0.681
/
0.346
0.017
S
0.136
0.133
T
0.066
0.077
T


500
71608
SENSE
−0.849
0.907
/
−0.132
0.976
/
0.816
0.006
S
0.279
0.004
S
0.038
0.238
/


501
71739
SENSE
−0.275
0.937
/
−0.107
0.955
/
0.334
0
S
−0.075
0.815
/
−0.119
0.874
/


502
72014
SENSE
−0.038
0.94
/
0.07
0.278
/
0.732
0.06
T
−0.026
0.584
/
−0.013
0.596
/


503
72051
SENSE
0.026
0.28
/
0.311
0.003
S
0.222
0.236
/
0.453
0.009
S
0.052
0.168
T


432
70217
SENSE
−0.202
0.893
/
0.203
0.024
S
−0.079
0.743
/
0.27
0.07
T
−0.063
0.856
/


454
70354
SENSE
0.134
0.147
T
−0.119
0.684
/
0.48
0.02
S
−0.119
0.736
/
0.014
0.3
/


334
70417
SENSE
−0.35
0.988
/
−0.041
0.637
/
0.69
0.012
S
−0.136
0.978
/
0.004
0.411
/


515
70435
SENSE
0.664
0.014
S
0.146
0.036
S
0.138
0.097
T
0.33
0.038
S
0.039
0.16
T


457
70465
SENSE
−0.106
0.79
/
/
/
/
0.883
0.001
S
−0.27
0.945
/
−0.097
0.818
/


460
70852
SENSE
0.178
0.031
S
0.145
0.034
S
−0.525
0.904
/
−0.315
0.861
/
−0.059
0.774
/


405
71508
SENSE
0.195
0.162
T
0.251
0.017
S
−0.515
0.929
/
−0.322
0.997
/
−0.196
0.982
/


295
71835
SENSE
0.139
0.055
T
0.163
0.049
S
0.538
0.019
S
0.27
0.068
T
0
0.496
/


467
72432
SENSE
0.146
0.021
S
0.139
0.02
S
0.325
0.012
S
0.074
0.149
T
−0.106
0.879
/


443
72453
SENSE
0.204
0.037
S
0.116
0.046
S
−2.198
0.995
/
0.016
0.448
/
−0.013
0.534
/


433
72711
SENSE
0.292
0.058
T
0.143
0.024
S
−0.114
0.76
/
−0.04
0.667
/
−0.093
0.946
/


449
72969
SENSE
0.046
0.072
T
−0.158
0.887
/
0.39
0.031
S
0.477
0.001
S
0.095
0.054
T


426
72987
SENSE
0.385
0.006
S
/
/
/
0.098
0.104
T
0.153
0.016
S
0.057
0.061
T


525
74022
SENSE
0.11
0.226
/
−0.069
0.844
/
0.71
0.009
S
−0.05
0.613
/
−0.004
0.544
/


287
74251
SENSE
−0.56
0.961
/
−0.184
0.916
/
0.611
0.001
S
0.174
0.229
/
−0.137
0.923
/


537
74507
SENSE
0.255
0.017
S
0.178
0.03
S
0.318
0.032
S
0.188
0.011
S
0.013
0.078
T


313
74512
SENSE
−0.247
0.953
/
0.107
0.015
S
−0.073
0.86
/
0.113
0.034
S
0.05
0.175
T





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents data points not determined or the transgenic plants showed a trend of trait improvement compared to the reference with p < 0.2


/: represents the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset






K. Limited Nitrogen Tolerance Screen

Under low nitrogen conditions, Arabidopsis seedlings become chlorotic and have less biomass. This example sets forth the limited nitrogen tolerance screen to identify Arabidopsis plants transformed with the gene of interest that are altered in their ability to accumulate biomass and/or retain chlorophyll under low nitrogen condition.
T2 seeds were plated on glufosinate selection plates containing 0.5×N-Free Hoagland's T 0.1 mM NH4NO3 T 0.1% sucrose T 1% phytagel media and grown under standard light and temperature conditions. At 12 days of growth, plants were scored for seedling status (i.e., viable or non-viable) and root length. After 21 days of growth, plants were scored for visual color, seedling weight, number of green leaves, number of rosette leaves, root length and formation of flowering buds. A photograph of each plant was also taken at this time point.
The seedling weight and root length were analyzed as quantitative responses according to example 1M. The number green leaves, the number of rosette leaves and the flowerbud formation were analyzed as qualitative responses according to example 1L.
Table 14 provides a list of recombinant DNA constructs that improve low nitrogen availability tolerance in plants.













TABLE 14









Flowerbud
Number of green

Number of




formation
leaves
Root Length
rosette leaves
Seedling Weight
























Pep SEQ


RS
p-

RS
p-


p-

RS
p-


p-



ID
Construct_id
Orientation
mean
value
c
mean
value
c
delta
value
c
mean
value
c
delta
value
c



























375
72963
SENSE
1.1
0.004
S
0.293
0.021
S
−0.446
0.002
S
−0.246
0.786
/
0.137
0.001
S


389
70437
SENSE
−0.28
0.982
/
−0.08
0.769
/
0.259
0.006
S
0.5
0.005
S
0.133
0.003
S


390
71633
SENSE
0.26
0.26
/
0.254
0.114
T
−0.1
0.539
/
0.647
0.06
T
0.106
0.023
S


391
72948
SENSE
0.587
0.06
T
0.539
0.029
S
−0.237
0.003
S
0.479
0.1
T
0.078
0.121
T


392
72519
SENSE
0.749
0.033
S
0.209
0.104
T
−0.09
0.274
/
0.276
0.264
/
0.116
0.006
S


393
10475
SENSE
1.256
0.026
S
0.588
0.005
S
−0.378
0.002
S
0.081
0.319
/
0.018
0.75
/


394
11120
ANTI-
0.795
0.033
S
0.608
0.015
S
−0.45
0.001
S
0.287
0.106
T
−0.041
0.387
/




SENSE


395
19736
SENSE
−0.24
0.907
/
0.355
0.033
S
0.014
0.864
/
0.64
0.006
S
−0.075
0.005
/


396
71606
SENSE
0.605
0.088
T
0.176
0.11
T
−0.033
0.708
/
1.239
0.005
S
0.133
0.002
S


397
71840
SENSE
0.408
0.235
/
0.879
0.006
S
−0.137
0.248
/
0.524
0.032
S
0.066
0.198
T


398
74240
SENSE
−0.06
0.602
/
0.13
0.076
T
−0.107
0.306
/
0.714
0.034
S
0.108
0.002
S


399
74331
SENSE
−0.44
1
/
0.054
0.203
/
0.132
0.055
T
1.045
0.021
S
0.134
0.003
S


400
74610
SENSE
−0.59
1
/
−0.08
0.922
/
0.289
0
S
1.241
0.017
S
0.137
0.001
S


401
75527
SENSE
0.242
0.228
/
0.376
0.028
S
−0.183
0.045
/
0.352
0.083
T
−0.005
0.8
/


316
71811
SENSE
−0.45
0.91
/
/
/
/
−0.112
0.202
/
0.438
0.014
S
−0.054
0.161
/


505
72463
SENSE
−0.16
0.976
/
/
/
/
0.112
0.109
T
0.366
0.024
S
0.13
0.006
S


351
75701
SENSE
0.736
0.048
S
0.07
0.861
/
−0.4
0.018
S
/
/
/
−0.109
0.193
/





S: represents the transgenic plants showed statistically significant trait improvement as compared to the reference (p < 0.05)


T: represents the transgenic plants showed a trend of trait improvement compared than the reference with p < 0.2


/: represents data points not determined or the transgenic plants didn't show any alteration or had unfavorable change in traits examined as compared to the reference in the current dataset






L. Statistic analysis for qualitative responses

Table 15 provides a list of responses that were analyzed as qualitative responses










TABLE 15





response
Screen
categories (success vs. failure)







wilting response Risk
Soil drought tolerance screen
non-wilted vs. wilted


Score


growth stage at day 14
heat stress tolerance screen
50% of plants reach stage1.03 vs. not


growth stage at day 14
salt stress tolerance screen
50% of plants reach stage1.03 vs. not


growth stage at day 14
PEG induced osmotic stress tolerance
50% of plants reach stage1.03 vs. not



screen


growth stage at day 7
cold germination tolerance screen
50% of plants reach stage 0.5 vs. not


number of rosette leaves
Shade tolerance screen
5 leaves appeared vs. not


at day 23


flower bud formation at
Shade tolerance screen
flower buds appear vs. not


day 23


leaf angle at day 23
Shade tolerance screen
>60 degree vs. <60 degree


number of green leaves at
limited nitrogen tolerance screen
6 or 7 leaves appeared vs. not


day 21


number of rosette leaves
limited nitrogen tolerance screen
6 or 7 leaves appeared vs. not


at day 21


Flower bud formation at
limited nitrogen tolerance screen
flower buds appear vs. not


day 21









Plants were grouped into transgenic and reference groups and were scored as success or failure according to criteria in Table 15. First, the risk (R) was calculated, which is the proportion of plants that were scored as of failure plants within the group. Then the relative risk (RR) was calculated as the ratio of R (transgenic) to R (reference). Risk score (RS) was calculated as −log2RR. Subsequently the risk scores from multiple events for each transgene of interest were evaluated for statistical significance by t-test using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). RS with a value greater than 0 indicates that the transgenic plants perform better than the reference. RS with a value less than 0 indicates that the transgenic plants perform worse than the reference. The RS with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.
M. Statistic Analysis for Quantitative Responses
Table 16 provides a list of responses that were analyzed as quantitative responses.








TABLE 16





response
screen







seed yield
Soil drought stress tolerance screen


seedling weight at day 14
heat stress tolerance screen


root length at day 14
heat stress tolerance screen


seedling weight at day 14
salt stress tolerance screen


root length at day 14
salt stress tolerance screen


root length at day 11
salt stress tolerance screen


seedling weight at day 14
PEG induced osmotic stress tolerance screen


root length at day 11
PEG induced osmotic stress tolerance screen


root length at day 14
PEG induced osmotic stress tolerance screen


rosette area at day 8
cold shock tolerance screen


rosette area at day28
cold shock tolerance screen


difference in rosette area
cold shock tolerance screen


from day 8 to day 28


root length at day 28
cold germination tolerance screen


seedling weight at day 23
Shade tolerance screen


petiole length at day 23
Shade tolerance screen


root length at day 14
Early plant growth and development screen


Seedling weight at day 14
Early plant growth and development screen


Rosette dry weight at
Late plant growth and development screen


day 53


rosette radius at day 25
Late plant growth and development screen


seed dry weight at day 58
Late plant growth and development screen


silique dry weight at day 53
Late plant growth and development screen


silique length at day 40
Late plant growth and development screen


Seedling weight at day 21
Limited nitrogen tolerance screen


Root length at day 21
Limited nitrogen tolerance screen









The measurements (M) of each plant were transformed by log2 calculation. The Delta was calculated as log2M(transgenic)−log2M(reference). Subsequently the mean delta from multiple events of the transgene of interest was evaluated for statistical significance by t-test using S-PLUS statistical software (S-PLUS 6, Guide to statistics, Insightful, Seattle, Wash., USA). The Delta with a value greater than 0 indicates that the transgenic plants perform better than the reference. The Delta with a value less than 0 indicates that the transgenic plants perform worse than the reference. The Delta with a value equal to 0 indicates that the performance of the transgenic plants and the reference don't show any difference.
Example 2
Identification of Homologs
A BLAST searchable “All Protein Database” was constructed of known protein sequences using a proprietary sequence database and the National Center for Biotechnology Information (NCBI) non-redundant amino acid database (nr.aa). For each organism from which a DNA sequence provided herein was obtained, an “Organism Protein Database” was constructed of known protein sequences of the organism; the Organism Protein Database is a subset of the All Protein Database based on the NCBI taxonomy ID for the organism.
The All Protein Database was queried using amino acid sequence of cognate protein for gene DNA used in trait-improving recombinant DNA, i.e., sequences of SEQ ID NO: 240 through SEQ ID NO: 478 using “blastp” with E-value cutoff of 1e-8. Up to 1000 top hits were kept, and separated by organism names. For each organism other than that of the query sequence, a list was kept for hits from the query organism itself with a more significant E-value than the best hit of the organism. The list contains likely duplicated genes, and is referred to as the Core List. Another list was kept for all the hits from each organism, sorted by E-value, and referred to as the Hit List.
The Organism Protein Database was queried using amino acid sequences of SEQ ID NO: 270 through SEQ ID NO: 538 using “blastp” with E-value cutoff of 1e-4. Up to 1000 top hits were kept. A BLAST searchable database was constructed based on these hits, and is referred to as “SubDB”. SubDB was queried with each sequence in the Hit List using “blastp” with E-value cutoff of 1e-8. The hit with the best E-value was compared with the Core List from the corresponding organism. The hit is deemed a likely ortholog if it belongs to the Core List, otherwise it is deemed not a likely ortholog and there is no further search of sequences in the Hit List for the same organism. Likely orthologs from a large number of distinct organisms were identified and are reported by amino acid sequences of SEQ ID NO: 539 to SEQ ID NO: 22568. The relationship of the homologs to the identified trait-improving genes on an amino acid sequence basis is found in Table 17 where the amino acid sequence of a protein encoded by a trait-improving DNA, e.g., SEQ ID NO:270, is followed by the amino acid sequences of protein encoded by homologous genes, e.g., SEQ ID NO:19844, 4248, 2761, 15944, etc. The source organism of each homolog is reported in the Sequence Listing.







TABLE 17







Sequence IDs for homolog proteins








Seq ID NO:
homolog Seq ID NOs























270:
19844
4248
2761
15944
11776
16144
10470
9742
6776
1010
2285
16333




9154
20620
16454
20025
8388
10646
1208
6001
1706
2448
14768
10226



12626
19846
9302
17295
17794
6354
5098
1789
6430
17749
821
10109



7542
17855
15562
17462


 


271:
1715
5418
7208
19338
7440
711
8113
17151
17592
18879
4807
8671



9936
11315
10681
3177
10519
6830
13563
12162
15155
1860
18072
20945



6715
15032
5192
10928


 


272:
0


 


273:
13771
8553
4219
10043
10800
8345
17501
13569
954
17197
6188
3760



13267
16169
8132
2667
11216
15637
4652
2270
10309
5708
18374
9446



12844
7790
7569
4786
9725
14187
12859
16948
18626
13741
5525
7877



4550
15544
9706
7616
14358
15163
13182
14560
16722
1129
1472
4261



10693
20144
6437
21413
17893
17984
17116
9925
19953
20648
983
2837



5663
2943
10465
1841
12497
6435
14763
13495
12676
7513
8363
16389



8162
7945
14956
15029
12433
22241
16071
13003
16940
18847
12354
7732



14013
5735
11505
8833
17658
16048
17609
575
6641
6331
3738
10842



18927
21518
20097
14117
5309
13744
15880
19484
9648
22509
1221
15515



6785
852
6466
17423
14164
658
8704
16710
19375
3306
14050
16883



16322
1722
15481
13636
10680
6347
3552
8885
21794
17703
22557
14777



21189
13711
3601
3968
3692
4003
20044
12943
19749
1865
6355
14902



6137
22370
19468
10410
21460
10451
17175
20965
12916
1206
6796
11329



9139
11008
10569
9058
7988
19743
20088
14111
8231
4522
18497
11952



2866
15466
3609
2403
16796
13539
14806
5364
12620
20699
12940
15426



4409
12452
5296
9156
629
12665
20947
3649
7530


 


274:
16167
5178
2412
10455
20036
11246
19666
6400
5573
22539
8547
21845



2413
20290
4036
19351
5886
6071
17184
9738


 


275:
14665
16694
12678
14928
21489
7918
1571
3959
2490
2517
14615
3788



10022
16096
21248
13293
8541
13446
6120
4360
3812
15574
18938
19203



2284
2215
10054
14052
9653
10183
17752
20776
4240
22343
8270
9192



17217
7374
12141
20657
7674
6445
2522


 


276:
12496
3460
13599
7043
9150
1664


 


277:
16949
9640
8150
2014
12188
5779
17876
14612
18293
11053
15958
15263



18370
20984
13094
18734
7380
10318
21641
12737
13028
20561
7087
10686



9894
7528
12573
16043
14846
20513
2802
8897
14716
10257
16407
2727



6151
1484
6831
16916
10146
17756
13193
7670
15946
7750
9397
20046



12547
5399
18644
11883
12531
12530
17188
2130
3805
17493
16821
10181



3639
3934
1419
780


 


278:
16531
9228
5799
19821
10980
17656
3449
19982
13335
20959
11238
13084



10281
17610
19623
17614
9736
21375
19978
5859
4943
12390
18806
1349



8759
21741
20400
13707
9170
15899
11361
4333
10631
12909
2136
12776



20323
7676
5847
9065
19902
4545
6768
546
9246
14134
4442
20731



9931
6700
8677
19305
4828
3655
17550
7579
21629
9044
19475
637



4209
3519
3873
15884
12247
10177
1407
10312
15957
16955
17469
20241



7267
21862
16864
22192
19599
6365
12324
10985
6424
6449
14564
3115



14885
10603
911
18609
3359
7059
2851
5801
9613
8391
18695
7987



14964
1081
15171
1312
14747
3060
13390
22115
5060
16536
19729
13468



11109
21989
2230
9462
9096
18775
10721
11999
8340
16607
22199
8687



3652
8147
22073
4090
3491
20506
7835
10890
6781
7839
14478
16371



579
6442
9721
5423
2566
7876
17580
2023
13164
9424
14096
8115



15304
14818
7282
6422
4841
10106
1075
12995
9297
21280
2309
895



12373
5366
22159
4499
737
11369
4308
13974
2794
12771
16584
9250



6930
8792
10185
17718
13148
5054
22383
9674
9868
21546
15881
20000



19029
4938
14024
15026
15535
16386
15402
21036
12653
9490
4170
11551



3711
4179
18619
16370
14161
11294
9094
2689
21881


 


279:
13577
10344
17371
5840
13904
12689
11795
21912
17226
14519
8854
14229



7998
8326
1400
16163
11785
10117
21511
21043
18205
2933
2043
22181



21628
20090
18018
20578
19081
11011
4768
2834
4635
13475
6306
13119



1671
21348
16926
20801
6361
4269
4366
15615
941
7046
3166
16526



6250
4790
825
16541
14270
3574
8127
18015
13147
17950
15243
6671



20662
3487
11787
6762
5488
7117
9417
15702
4859
20527
19240
17200



7115
19523
12823
9115
11770
20530
4791
20334
11019
20645
14383
1914



8945
15358
9347
2496
3098
11604
16441
19352
18451
12451
8877
15655



791
12609
12588
19785
18328
8143
22185
1488
10212
14002
16826
11779



1890
3879
20130
21633
20930
17222
2691
18852
7120
7853
644
21495



14630
21041
5676
8447
16750
10436
4523
2686
8237
2948
13559
19008



3686
18465
3895
7416
10170
18867
14574
1159


 


280:
16310
3796
10205
19486
13581
11022
21960
15598
12604
9662
20210
21764



8626
17323
11666
21316
6121
7438
982
2135
17499
12340
20711
6553



10296
17874
914
17716
3723


 


281:
20551
21096
13717
4006
19762
10017
14425
17785
6291
3855
15232
11917



21856
18564
19010
3910
13621
19087
670
10539
851
5698
9112
12694



14218
11947
18104
21227
22348
14178
1862
9829
9325
14533
13149
8679



17127
6507
10375
2782
3357
594
7548
9316
5728
7981
8022
13688



1294
13817
21893
10781
5263
11292
5492
9542
8000
16102
6328
11687



13695
15807
4068
3478
6486
13660
22165
17881
19166
3613
7013
6393



15983
17688
5124
4243
19684
17008
12366
7161
20062
12194
15870
14385



9124
9865
6211
628
6448
700
8869
17941
10697
21134
5586
4469



21167
855
20538
9251
1036
10678
1977
17337
4575
19974
3520
10195



14572
3870
21293
19011
12921
20120
11647
15054
13976
21163
20362
12988



15636
2345
4740
3205
17504
1953
15208
16834
7654
15907
8961


 


282:
19261
3860
7076
12616
1790
4886
9735
12611
20478
4501
18874
7032



18024
7225
4544
11443
2127
19283
7367
1338
4482
15213
20554
3826



14978
21769
11755
4250
15506
20020
6593
1286
20750
18985
16069
4571



22536
3773
11152
9745
13196
2190
8120
7914
16863
11987
16172
15399



14422
12490
8076
17180
19067
14493
13105
16459
18285
15863
14085
18130



11566
17352
20003
2995
5386
8757
19103
15685
20563
18739
20815
19454



7820
20771
7972


 


283:
0


 


284:
10750
5276
3894
3486
12240
18158
12170
15393
9765
11266
5031
2792



9334
20684
1144
13799
10858
16622
20849
22001
6897
17710
15401
18589



9550
1757
10249
21993
2001
19689
15058
4297
19990
643
11414
18208



19995


 


285:
17916
15231
15741
15829
4645
21977
10291
1806
21573
474
6018
2663



8036
9618
16693
3960
15864
14578
17125
15924
21826
13440
17249
8650



20159
1986
15742
19706
22092
8766
6813
17830
10853
21281
13394
5285



8139
21004
14220
17563
2086
2488
1597
4698
13233
4654
1250
15737



2907
1469
9957
13288
6516
22526
16496
14873
10471
18290
3086
11953



18592
3185
9418
17135
8081
9593
19180
4673
7979
16544
13933
1300



16782
15551
8460
15960
3405
13997
1566
21046
8636
17134
6512
6596



13346
15639
14396
9252
12093
21591
15042
6953
18637
16784
22523
6262



16933
22448
4612
19863
6076
4133
19601
3344
12192
16828
17089
19303



6118
15088
14986
21070
771
3291
2153
21234
18173
11970
21215
10644



20638
4377
21183
9519
13810
10948
17764
3781
21029
16613
18091
6526



5846
22213
22003
20765
3801
21866
21771
14860
861
6743
5007
5529



14267
14880
21391
10210
5693
5970
3793
15855
1007
13001
6878
9875



16912
19329
13614
10333
13714
6903
21112
8204
1133
21262
16852
15703



21338
6248
21547
15242
13567
16788
11020
18655
10528
19496
17440
22414



17480
8142
7760
20388
2829
16249
12914
2569
14595
7096
6689
12534



6105
16041
9242
9145
1552
10313
1379
9596
11771
5820
593
15445



3268
14744
18410
6984
10872
10053
9713
8837
1383
4305
10421
2944



20363
19120
7463
16753
20969
18430
12905
10227
11066
6057
13677
18640



4083
1527
19285
5385
17557
20851
15693
17304
1683
14391
15965
19854



8425
14916


 


286:
16099
8075
8242
429
13036
10217
18174
507
11062
15313
16399
14594



3820
5216
5717
10394
20464
15374
15526
18461
17556
14810
4502
12391



11462
14924
1652
18690
9504
13188
8137
3327
12056
2537
4030
1064



15390
6367
19001
8581
6859
7602
14778
7681
18172
18107
12112
7417



13896
2465
12130
19906
4949
17359
16220
13607
526
20229
16469
10705



15889
16978
8246
16554
18467
9002
9780
17882
21655
20242
21051
3977



19627
9063
10810
15051
5705
9204
11991
9915
15840
16148
14589
6171



6614
20389
6007
6226
5373
19830
9030
14898
11912
16971
18279
19602



13078
18533
1840
4491
7812
8977
13027
6371
15170
21310
6443
9755



12535
2819
14876
3052
12267
8377
3804
7983
22450
22167
22369
21674



891
13549
7113
14532
11064
9354
7858
8394
2489
17243
2044
9345



6836
2341
19148
17763
13516
14240
9032
15489
8304
7822
17083
9993



8188
5481
11010
1093
7789
21699
21306
11113
8956
18128
7880
3416



2857
17664
21589
11961
19154
11542
4812
7486
21609
5307
21506
3533



6663
4677
18511
4730
696
9569
17797
18687
7097
20259
15252
13221



3425
3261
450
6778
14692
22502
14915
21157
20371
3563
12591
16308



7626
4212
19798
16130
21296
2832
830
14553
14135
6434
18295
17956



20230
476
10392
12043
3289
20779
1281
17745
21065
5487
3754
8587



2882
12578
11548
12705
15830
16586
7955
15449
988
20859
1768
11129



6547
22518
5552
9854
11376
6669
5359
9132
10964
22087
1879
17298



13137
3333
20301
13752
19370
2425
6946
19538
14273
8003
15340
6427



17165
15603
12419
10809
13658
14064
4221
7387
3386
13284
20981
2316



3116
8418
3578
6508
18217
992
22249
14855
13442
3471
19949
5277



19885
2816
10917
21954
9436
18538
18659
12816
12308
20364
15943
2649



2515
14701
9329
8193
17579
22076
8890
21073
8376
15177
10882
859



5990
9814
16053
12078
3144
5209
13848
22428
19076
9604
5454
3141



17056
14918
953
4088
6615
15729
11270
5837
14568
12544
17630
2371



4694
12396
15369
16597
3697
10741
13396
21621
16662
8841
11120
14081



13456
21472
7487
12898
12787
7866
11748
8692
9484
2336
15204
6565



14320
2978
9294
7482
9493
1277
4576
15513
21001
3242
4952
7813



7306
12296
12021
6645
21060
9286
22489
6264
13745
16653
19069
7780



15219
16969
9271
18873
14762
10802
10479
16663
3678
16713
7751
13703



3630
4691
9472
10709
8542
7060
6112
22457
21974
20476
7333
6482



14526
7151
2644
835
10655
12264
17372
788
15452
14361
11678
2754



15289
14415
9468
1673
2117
17672
3643
6809
9280
22095
18903
3706



15256
18593
5764
11115
12583
11568
622
13613
2331
5136
8073
15998



5630
10889
9673
9014
7950
6748
9771
11716
18929
14842
6789
1773



981
18575
19878
21485
14269
9584
12794
21399
20485
9218
18691
3350



18263
4846
544
19667
9933
4140
1318
20418
11128
20105
16734
2376



15699
7061
4232
15357
14036
5339
7107
19030
7165
21370
12103
4848



13211
22530
15360
12863
9975
6398
14067
16683
21170
1924
890
8155



15866
10131
7187
4332
1235
20330
12927
7088
5099
2302
12424
8303



17466
14322
11383
2282
14043
15956
1818
3414
12982
18548
15665
10961



21084
10824
18440
16819
3730
13940
18821
14864
19607
7969
6546
16771



5441
8459
18266
5000
15749
13014
14274
8444
4707
13097
15930
6281



18662
7992
9661
19875
11156
3619
14086
20948
16946
3456
6143
6097



786
6709
13766
5954
9650
17926
9083
20839
17473
5462
22351
12788



5453
13277
11817
9672
8255
6463
15113
9053
15528
1319
926
17317



6368
7704
18694
3644
12154
3794
21257
21638
18386
18111
21498
10731



13776
5539
14530
20282
7762
9529
17675
15191
12380
14865
15825
2818



16442
10262
10965
15558
5973
22476
19408
5773
5016
20819
18982
19997



4572
640
19168
17905
2970
2132
20312
4655
16568
21093
13882
5730



7976
7079
13035
3505
2800
12522
8939
11219
2155
15905
3206
2017



15587
6277
1297
1423
9549
6119
18728
12545
2037
11704
9571
21379



13089
11197
13107
22367
2420
17943
7599
5997
1801
21374
16528
8722



16672


 


287:
8532
19537
5344
9149
21995
6924
15909
4097
11901
8844
7649
2133



2202
5183
18294
19005
11338
19082
14317


 


288:
19787
18784
5642
15617
18773
2850
21740
18774
8659
17768
4774
15786



13450
5349
3435
11594
3337
19500
18011
5026
20455
17036
22287
17395



9621
6514
1386
11394
7035
21823
15347
11387
15362
4210
5017
10279



19441
22361
7943
7642
8141
16188
15181
3603
7428
2749
21346
17018



10086


 


289:
652
22063
8339
21393
2555
8373
21778
11080
15895
10755
10659
16023



19651
17328
17996
10829


 


290:
7684
9445
17343
1187
6793
10461
6245
10467
20367
12284


 


291:
19558
4465
12410
21267
12416


 


292:
13619
7334
20532
21542
12253
16189
8764
2507
21921
6586
19095
9485



5381
5599
12372
14529
13331
15295
19004
1397
21520
14556
20510
22236



2986
7080
8521
6732
13272
16558
4313
20499
22483
14588
9523
7470



18752
12181
20429
12348
695
10907
14321
14033
18144
3919
8802
20993



8165
3312
17714
14662
16218
6883
15696
5826
8318
13408
11637
4264



1882
4226
19194
12589
20866
1376
21972
8106
9328
11225
6391
8315



20980
16513
6488
21387
10825
871
22553
7860
20986
5123
7178
8148



19174
5633
3500
7737
17944
19517
19101
2424
1678
1758
16439
22408



11623
7905
18353
18641
17321
18471
6775
8307
11843
9722
15205
20080



16600
6416
10161
8449
8858
14419
19903
14156
15652
13343
15817
3739



12654
14241
7430
8851
6919
22017
6531
1962
3371
18557
2125
6815



15392
19947
6480
15023
1479
1138
21122
20521
21201
1677
17815
9610



8319
5949
7617
10353
9342
4915
6495
1682
14875
17940
1204
13538



16452
19603
8029
2886
14466
19507
12125
6822
10351
4123
9222
9605



18244
18276
17153
17212
12619
14496
11337
8683
15935
10084
14862
5228



2090
99138
702
17034
5542
16281
16905
2247
17007
15480
13602
16745



3223
6163
1245
10228
21595
18522
6698
5750
8543
21981
15735
3961



16417


 


293:
10002
18611
6406
14073
5127
8351
1241
12550
10077
11772
10606
3231



10112
15857
13002
6686
8210
8327
4270
16125
8696
5110
12311
7006



19199
6994
12597
5342
12722
21575
5723
2670
6046
12012


 


294:
9532
10331
15934
2046
6293
19503
22316
15147
14129
5699
7047
13130



2151
1110
20162
9143
9176
4233
8431
6088
7434
18942
5356
18099



10361
3212
21551
20873
2419
8077
3140
13009
21068
6829
20236
14326



6208
847
2330
15785
1052
14929
4869
4567
16757
19546
10070
1727



6317
8558
3880
18261
6409
6931
17666
21501
12516
16760
16947
3896



3358
6554
14909
1974
4835
20560
11275
2659
12987
18209
16896
15952



20752
15130
15569
7406
13923
1051
11602
3912
10042
4592
22204
2430



1382
18088
15862
20314
9057
10845
5041
7582
21742
12835
17242
15658



8522
22493
7831
1594
13654
7414
12151
12808
14165
15368
17979
10215



8018
22247
22220
7254
654
20380
2845
5061
7274
5018
9568
9818



1494
3725
11235
6804
8938
12700
17001
19302
11190
4028
3173


295:
21192


 


296:
10114
6052
18680
17170
11936
20081
9148
3938
18585
12840
14672
14951



1544
20775
4870
8723
3360
19138
1416
1901
18744
2625
951
6936



16543
3550
9203
11070
9360
21434
3920
1693
10869
11881
12895
1003



5405
14684
21089
14531
21626
20145
17875
7549
2428
2613
18036
1421



18003
6973
20577
12790
22102
3375
13691
15302
6589
15430
18154
16596



19645
21006
17187
14709
19975
1854
17428
11844
10033
1792
934
845



5533
21419
6582
2314
19307
8902
8278
20861
7030
10654
9636
16002



2969
15306
5928
1894
592
13534
4985
5623
5150
2342
22174
1686



2688
10718
20383
19440
19860
618
22308
10355
12565
8585
15993
12725



22563
5578
14369
13712
9428
20205
10767
1546
5387
8364
18905
18948



2545
17156
20131
22338
2223
7037
15860
5111
16844
14702
1634
13698



16173
17860
16802
21238
20479
18313
18815
16556
12453
18481
19778
12153



19470
20701
1477
19657
2220
5033
6117
12286
12326
6566
16110
13469



17077
20231
4058
9834
3780
19094
6308
9548
1549
21687
13080
7340



18746
2266
11139
2005
14735
5543
4941
2921
8468
19501
3139
16192



10723
6333
11355
3845
11452
19385
14092
1395
5545
2216
10682
21311



15661
21100
2597 14697
9008
17498
17514
9946
20269
5669
5062
15447



2775
6975
12793
12639
8185
19766
8932
4278
14889
4981
22471
5952



6818
16336
21140
14098
21953
4979
13968
16410
18878
9696
4978
16146



2609
22097
16986
20921
22527
16920
18126
16034
5362
933
15779
16770



1772
9517
6866
16982
19232
19937


 


297:
9912
18441
5938
12696
19188
22233
2840
10426
16052
9508
12778
15536



19286
9563
19034
20209
15518
6498
21608
11026
14044
10165
12621
12028



16923
21297
5646
16170
12477
16660
1378
2624
3809
12760
16057
7256



15500
18959
10317
10326
5977
4830
1143
1499
10983
2571
15543
13839



12370
9041
1331
8508
16831
13832
4303
18599
21584
9787
1548
8292



1560
18466
11683
17762
17468
9815
4374
18826
20637
18709
16467
7307



16512
904
18899
17113
10700
5006
22084
5463
20856
18792
1535
13608



11312
8634
19381
11435
870
7232
5079
6299
13314
10487
15382
14552



14878
20590
14820
7911
8924
12070
7581
2122
18063
8338
12227
14479



3229
17500
9776
16213
12767
17455
5438
1839 9982
4649


 


298:
18555
7838
6652
1982
15254
2218
6543
13709
5154
14481
4355
8406



21382
21663
19102


 


299:
19421
10037
3803
5108
3343
20336
11002
8946
12661
9168
15378
18020



16075
5607
19118
22336
19724
8555
2295
21579
1259
8494
18083
960



13560
20787
22477
19264
6401
8583
9620
5248
701
10759
22290
2593



21024
17543
1417
15845
17793
18019
6222
10575
1775
8903
15288
2885



7993
20494
10001
13641
11853
614
12502
13330
17952
13069
9420
1771



17636
13434
5588
2165
19494
4998
1751
16904
10306
3123
12413
16515



21706
9144
19364
2394
2677
15207
13710
9045
11258
1042
11230
13140



18738
15056
11058
930
22417
8107
16349
7322
16450
15822
16451
6646



18926
2197
302
8630
17924
14996
19753
4515
10245
7300
6710
21723



12990
8111
14791
13977
19581
12911
22090
603
15053
14851
21092
14284



20327
13846
9587
21947
7441
12133
14923
3660
13939
6002
5590
11909



22216
4929
14884
9941
9040
12896
7903
21683
11508
22006
11550
5658



2777
9977
15523
5791
21963
16141
18240
5322
2499
19702
9292
20325



5274
9028
10531
1688
20146
5153
7485
18445
4165
5614
10277
16781



4563
3161
6533
5957
7089
2687
7105
21351
21515
22453
19592
7173



1968
19358
11469
8780
17266
7586
12142
3066
7281
14577
9430
3270



13924
12341
15761
12637
10962
2998
19035
4877
659
9821
20906
12168



1736
20717
2543
16421
1821
18042
14352
15050
8399
19775
21325
7525



17680
18001
19979
18487
6598
5555
10444
21216
21128
8987
1493
17533



16118
1639
12595
6855
22098
18912
3290
5745
6378
9285
11287
14670



11840
9243
9669
14995
13742
20118
9371
11429
12747
19245
1590
13573



3695
20005
2712
20008
9988
1732
20129
19987


 


300:
16422
16652
2702
1006
429
10217
533
507
306
535
478
10780



18650
18461
13998
11343
7459
5240
10390
15192
6367
19001
17659
17306



18166
3899
19411
17674
6453
14183
9624
20799
11585
14626
21314
12049



7812
18212
3745
13797
8320
4940
22404
21496
12527
18023
6674
2559



15926
17879
9075
14547
21758
1689
16481
15621
18121
13519
12731
5774



10662
11761
10902
17148
12381
2857
17664
21589
11961
19154
6201
21437



11195
20933
21506
3533
4677
18511
4730
696
9569
17797
18687
7097



20259
15252
13221
450
11335
20777
12713
22502
14915
21157
20371
3563



12591
16308
7626
4212
19798
19458
5691
18292
16130
21296
2832
830



14553
6434
18295
17956
829
12043
20779
1281
17745
1768
6547
16586



7955
12705
11376
15449
988
20859
11129
22518
5552
9854
6669
5359



9132
10964
1879
22087
13137
17298
3333
13752
20301
19370
2425
6946



19538
14273
17165
15340
6427
15603
12419
10809
8003
13658
14064
4221



7387
13284
20981
2316
3116
8418
3578
3386
6508
18217
22249
992



13442
3471
14855
19949
5277
19885
2816
10917
9436
18538
18659
12816



12308
15943
2515
20364
14701
8193
17579
22076
8890
2649
9329
21073



8376
15177
10882
859
5990
9814
16053
5209
3144
13848
22428
19076



5454
17056
14918
4088
11270
5837
9604
22455
19840
801
12234
21301



1149
953
9960
1656
3755
12278
22119
21098
1005
10264
13037
11702



20954
5581
5063
1118
6454
20632
1696
19474
14911
9381
4111
13374



2932
3071
18551
22362
1431
12923
6509
19229
14012
5372
12362
17380



20272
16391
13395
5132
901
9540
19228
14568
17630
2371
4694
12396



15369
16597
3697
10741
16309
4927
13396
21621
8841
16662
11120
14081



8692
9484
15204
6565
14320
2978
9294
7482
9493
1277
4952
7813



7306
12296
12021
6645
9286
22489
13745
16653
19069
7780
15219
16969



14762
18330
10802
10479
16663
3678
16713
7751
13703
3630
4691
9472



10709
8542
7060
6112
22457
21974
20476
7333
6482
14526
7151
2644



835
10655
12264
9315
2786
16253
9488
5634
17372
788
9280
22095



18903
3706
15256
18593
5764
11115
12583
11568
13613
2331
5136
8073



15998
5630
11304
19137
5817
5580
18341
8588
12540
2454
4970
17445



2401
11869
6193
21516
10889
5190
13207
16465
9673
9771
11716
18575



9584
12794
21399
20485
9218
18691
3350
18263
4846
544
19667
9933



4140
1318
20418
11128
20105
16734
2376
15699
7061
4232
15357
14036



5339
7107
19030
7165
21370
12103
4848
13211
22530
15360
12863
9975



6398
14067
16683
21170
1924
890
8155
15866
10131
7187
4332
1235



20330
12927
7088
5099
2302
12424
8303
17466
14322
11383
2282
15956



3414
12982
18548
15665
10961
21084
10824
18440
16819
3730
13940
18821



14864
1818
19607
7969
6546
16771
5441
8459
18266
5000
15749
13014



14274
8444
4707
13097
15930
11872
2621
7158
6942
18502
5408
10837



21928
13800
5188
19614
16117
4719
19714
1833
13499
3107
21492
12503



21929
7921
1429
14398
22189
6439
12993
12307
6650
18994
747
5932



18630
6683
1921
15651
5594
6958
13597
19763
10097
14124
14687
1094



5780
7770
7688
15110
5797
7907
21169
3329
12627
4065
19882
22067



19211
2061
7038
8909
16914
13129
19881
13235
16317
1551
14888
1201



20264
7800
7526
2209
5887
10798
15317
10223
11351
10105
10661
15014



2908
16412
22277
19851
12003
19616
11003
7768
6166
4620
13850
16231



7016
20541
3458
1240
15787
20099
9282
11480
4994
6281
18662
7992



9661
19875
11156
3619
14086
20948
16946
3456
6143
786
6709
5954



17926
20839
22351
13277
15113
6368
7704
18694
3644
12154
3794
21257



21638
18386
18111
21498
10731
13776
5539
14530
20282
7762
9529
17675



15191
12380
14865
15825
2818
16442
5901
8220
18578
13297
2495
21913



4526
16085
10965
15558
18982
2984
11245
17095
7536
5117
2132
20312



4655
16568
21093
13882
5730
7976
7079
13035
6801
2800
5619
10126



12545
2037
11704
9571
21379
3712
7685
4483
18456
22341
11197
13107



16006
21848
20337


 


301:
19000
5175
17102
15893
7025
2120
12938
12299
15442
11496
10074
5733



14018
4497
12860
17771
17375
8161
11868
22009
7849
15996
13313
6451



9249
12173
13520
22245
9579
19128
12786
1276
5812
2509
15030
5404



7335
18498
6363
16355
19266
14084
8908
19966
8863
19169
539
3459



13467
15720
2898
9093
9837
17258
1976
20125
1460
8614
10175
5871



15351
6426
7149
12032
11271
8224
11194
11907
4519
3493
18304
22188



18791
10516
18657
11078
6908
20626
14734
4564
18762
9992
1434
6230



9846
4840
12934
18658
3437
11327
16354
4241
1568
14609
10156
19085



969
13387
20299
14003
18420
8635
5706
21190
1432
6124
1526
18029



16093
11179
1324
22474
8016
22059
5181
15454
1309
4573
20643
18651



6220
9683
15336
12171
3835
20676
15459
11187
6374
19257
8887
12249



15479
8309
2813
3769
17901
16142
2063
6092
19535
21118
1210
8401



7375
9531
10590
7738
17854
1657
9700
15494
7894
15287
1226
6551



13070
19534
13610
1802
22458
17237
8915
14991
3591
9885
9545
21263



14344
19754
11799
10504
11873
18078
16699
19633
15816
12846
4878
20466



21081
22381
17475
18403
21905
22085
18453
5736
17957
4021
7834
14499



2154
13058
11572
3583
10692
7183
16133
15670
12332
1756
16507
18601



11306
18332
20634
7994
12919
9064
5045
14968
9551
2414
1646
4753



1805
1502
18175
18991
19442
17821
1888
18587
10549
18855
4858
7028



14908
22205
5964
4933
4340
5271
927
8374
3316
9426
12837
20014



16546
20576
5008
9914
10822
13163
7197
6338
17347
21212
3445
14444



561
1401
13989
15266
20426
12281
7477
7453
766
14757
19524
4093



17235
17808
16762
10080
9217
14800
9794
18834
2718
6390
20406
7926



924
20355
13005
19727
8981
4683
7215
4131
6381
3407
11741
12599



16185
18844
16344
19195
11057
8355
8958
11977
19693
20196
6610
14503



22133
4744


 


302:
8946
9168
14628
299
6214
4182
16075
11899
18083
13560
20787
22477



16789
19264
6401
9620
5248
701
22290
21024
4074
1417
17543
11432



17793
6222
18019
21867
3019
20723
20357
10575
3819
6741
17742
1775



8903
15288
14774
2885
20494
20292
10001
11853
13330
17952
16001
17214



1751
16904
10306
20788
12413
1843
16515
21706
2394
15207
18738
15056



9630
930
15269
6646
18926
4515
7300
14791
6976
603
21092
2483



21947
7441
14923
3660
12133
22216
4929
5590
11909
14884
9941
6002



9040
13939
12896
7903
14465
21683
11508
8978
22006
5322
21963
2499



2777
18240
5791
16141
9977
5658
15523
9292
11550
19702
1688
20325



5274
10531
9028
20146
5614
7485
10844
2674
18445
4072
7836
5153



4165
15169
10277
16781
7814
3161
5957
6533
4563
7089
2687
21351



21515
22453
19592
7105
7173
1968
19358
10994
20056
7763
9851
13457



8780
11469
7586
17266
3066
12142
9430
14577
13924
7281
3270
19033



15761
12341
2998
12637
2543
10962
19035
20906
4877
659
9821
12168



20717
14352
1821
18042
16421
1736
15050
8399
19775
17680
18001
21325



6598
18487
15537
14525
21216
7525
19979
5555
10444
11512
21128
1493



8987
17533
22098
6855
12595
18912
15079
5745
6378
9285
11287
14670



11840
14995
13573
3695
19886
12402
20005
2712
9988
1732
1437


 


303:
11613
15600
900
21525
7534
8911
20350
17575
7154
10397
5219
15446



9261
1171
16015


 


304:
14477
14507
15421
17494
18062
2648
7991
15716
18037
11158
2783
2025



16965
22007
22121
19530
11719
9318
11530
17738
1299
21214
12334
21809



4864
10621
8796
1194
4280
19732
18120
15953
2967
3266
19426
6571



6888
17781
1697
10045
1251
7276
7126
12814
4960
7248
3598
17583



17847
7140
8316
18820
21056
7036
15143
13926
18581
5501
1142
3253



7339
4338
10287
9635
11127
20511
19012
13060
4347
12691
12055
20545



21425
3704
15743
19675
16542
16559
21229
11524
9274
11075
8804
17224



3133
3607
10123
13418
2973
11620
15872
11732
19109
740
1359
17419



6843
1680
16067
3406
4022
6395
9511
2595
20688
4623
22346
2369



17333
5395
11866
17305
10611
7631
15733
18432
1959
6814
6323
13183



9078
3032
21942
3286
13158
20759
22152
18157
1337
3062
16447
2772



10847
14538
4797
21087
7452
19143
16659
16530
16620
11651
17070
1585



22147
10166
6601
22431
15463
10652
13842
11862
1897
19136
9220
17946



12697
22065
3138
16162
17146
7667
19062
9071
11793
11610
7968
7594



7221
10376
5672
11711
3151
4084
14756
8726
18769
3480
10028
2838



20595
20275
1292
17594
12254
4853
20642
8186
16379
11759
11502
8591



4288
11992
15333
9201
14724
3846
15765
5294
9934
3608
4038
2297



1916
7144
15894
19202
4614
3319
11943
21007
5703
7304
9147
4298



11434
11341
10440
4734
13768
7039
7695
14607
13737
10493
13662
16005



5151
2008
20772
4271
19954
15488
16991
11303
17894
5027
7608
5609



16228
18306
2556
13676
8198
5829
17178
6626
16081
16487
20616
939



20854
11693
16083
22378
12968
16437
7624
6039
10274
13230
16495
10978



13806
5631
6912
22114
2955
21137
8847
12918
16985
22400
13954
16430



1905
19874
9524
6234
15225
4942
7297
20802
5035
10180
21875
20321



12454
9019
4029
11248
16017
3758
13953
2462
4887
8042
7962
11126



15081
22027
16352
2791
1889
15457
8559
17058
7622
4393
1128
8437



21033
5138
12634
1952
5842
21247
15955
3808
10420
14719
17939
4794



21353
19950
18656
13160
3900
11932
16466
13358
19797
5206
2289
9733



10875
12426
15784
10942
12064
7723
14341
5120
18048
8171
11041
17196



16314
20434
21075
20432
4222
8716
4904
18464
630
8403
6184
18819



17228
16302
10762
9202
16022
17350
15237
12703
11159
17792
4597
7291



15512
1072
6812
15612
4183
5466
17341
9980
4892
1108
1406
22046



1630
11923
17859
5617
18198
5043
6967
20318
22123
1264
12469
12218



10452
13091
8047
6109
14712
428
22157
7745
16695
22271
6934
7635



18516
6231
1574


 


305:
8099
12755
17111
978
1006
1542
15083
18937
12887
2035
10217
535



533
507
306
478
8894
18461
12108
1501
12486
21909
7034
7130



11072
14307
10425
15925
19332
1367
13146
7730
1937
9628
18825
609



18335
21615
4370
7330
8575
15629
12213
5331
3191
19367
4875
5240



11492
21592
7018
6367
19001
20564
13807
16754
21328
19708
4343
9940



1066
2399
12207
1629
18478
7812
13667
3746
5720
16976
18312
3470



5618
19921
17739
4546
14856
18520
3007
531
7912
8917
11529
6283



8622
8288
17072
4299
7840
5830
6060
19606
3409
7000
8429
19346



2857
17664
21589
11961
19154
18239
18127
11549
16493
21437
11195
20933



21506
1079
9281
838
3533
4677
18511
4730
696
9569
17797
18687



7097
20259
15252
13221
450
11335
20777
12713
22502
14915
21157
20371



12591
3563
16308
7626
4212
19798
19458
5691
18292
16130
2832
21296



830
14553
6434
18295
17956
829
476
12043
20779
1281
17745
7955



9854
988
20859
6669
5359
9132
16586
12705
15449
1768
11129
6547



22518
5552
11376
10964
1879
22087
13137
17298
19370
3333
13752
20301



2425
6946
19538
14273
15340
6427
17165
15603
12419
8003
10809
13658



14064
4221
7387
13284
20981
2316
3116
8418
3386
6508
19949
18217



3578
22249
992
18538
13442
3471
14855
5277
19885
2816
10917
9436



18659
12816
12308
15943
2515
20364
14701
8193
17579
22076
8890
2649



9329
21073
8376
15177
10882
859
5990
9814
16053
5454
5209
3144



9604
13848
22428
19076
17056
14918
4088
9960
11270
5837
22455
19840



12234
21301
1149
801
953
1656
20632
3755
12278
22119
21098
1005



10264
13037
11702
20954
5581
5063
1118
14568
17630
2371
4694
12396



15369
16597
3697
10741
16309
4927
13396
21621
8841
16662
11120
14081



8692
9484
15514
10745
15204
6565
14320
2978
9294
7482
9493
1277



4952
7813
7306
12296
12021
6645
9286
22489
13745
16653
19069
7780



15219
16969
14762
18330
10280
10802
10479
16663
3678
7751
16713
13703



3630
4691
9472
10709
8542
7060
6112
22457
21974
5278
4682
10694



19198
20476
7333
6482
14526
7151
2644
10655
12264
835
9315
2786



16253
9488
5634
17372
788
15256
22095
18903
3706
9280
18593
5764



12583
11568
13613
2331
5136
8073
5630
11115
11304
15998
19137
5817



5580
18341
2454
8588
12540
4970
17445
2401
11869
7142
4488
19396



10889
5190
13207
16465
9673
9771
11716
16290
14313
9218
15665
15699



5441
20485
18548
18575
18821
3350
18263
20418
11128
16734
19030
21370



13211
22530
15360
6398
14067
21170
15866
10131
4332
1235
12927
7088



5099
2302
12424
9584
8303
11383
2282
8459
3414
15749
18691
12982



12794
10961
10824
18440
16819
9975
21399
8444
4707
13097
15930
11872



2621
7158
14864
6942
19607
7969
6546
5408
18266
10837
21928
13800



21492
5339
20330
1833
4846
544
19667
9933
4140
1318
20105
2376



7061
4232
15357
14036
7107
7165
12103
4848
12863
16683
1924
3730



890
8155
7187
17466
14322
14398
22189
15956
21084
16117
13940
1818



19714
16771
6683
1921
5000
13014
14274
18502
6650
13499
19763
10097



5188
19614
4719
3107
5780
12503
21929
7921
1429
5797
6439
12993



12307
15651
18994
747
5932
18630
7907
5594
6958
3329
4065
19882



13597
14124
14687
7770
1094
7688
15110
21169
12627
22067
19211
16914



2061
1201
19616
7038
8909
13129
19881
16317
1551
13235
14888
20264



7800
7526
2209
11351
5887
10798
10105
15317
10223
10661
15014
2908



16412
15519
22277
19851
12003
6166
7768
11003
20541
3458
1240
15787



20099
9282
11480
4994
6281
18662
3619
7992
9661
19875
14086
11156



20948
16946
3456
6143
786
6709
5954
17926
20839
22351
13277
13192



15113
6368
18694
3644
12154
3794
21257
21638
7704
21498
14865
18386



18111
10731
13776
5539
14530
20282
7762
9529
17675
15191
12380
16442



15825
2818
8220
5901
18578
13297
2495
21913
4526
16085
10965
15558



21784
775
7504
10887
3135
11204
15835
17679
3002
18189
7516
6483



9518
14034
11913
9964
2126
10118
3160
10150
10347
14405
15486
7545



12799
13960
1340
3431
18982
2984
15366
2275
6738
11245
5295
2132



20312
4655
16568
21093
13882
5730
7976
7079
13035
18086
2800
4062



8280
13990
6955
16633
12655
12545
2037
11704
9571
21379
3712
7685



4483
22341
18456
11197
13107
16006
9320
19090


 


306:
14420
1006
429
10217
10190
2439
478
314
18461
5240
6367
19001



3122
4403
526
11601
7812
6192
17664
2857
21589
11961
19154
21437



11195
20933
21506
3533
4677
18511
4730
696
9569
17797
18687
7097



20259
15252
13221
450
11335
20777
12713
22502
14915
21157
20371
3563



12591
16308
7626
4212
19798
19458
5691
18292
5019
17572
15704
16130



21296
2832
830
14553
6434
18295
17956
829
476
12043
20779
1281



17745
16586
7955
12705
15449
988
20859
1768
11129
6547
22518
5552



9854
11376
6669
5359
9132
10964
1879
14844
22087
13137
17298
3333



13752
20301
19370
2425
6946
19538
14273
15340
6427
17165
15603
12419



18044
10809
8003
13658
14064
4221
7387
13284
20981
19949
2316
3116



8418
3386
6508
18217
3578
22249
992
14855
13442
3471
5277
19885



8739
2816
10917
15943
9436
18538
1973
18659
12816
12308
2515
20364



14701
8193
17579
22076
8890
2649
9329
21073
8376
15177
10882
859



5990
9814
16053
5209
3144
13848
22428
19076
14918
5454
17056
4088



11270
5837
9604
953
22455
19840
12234
1149
801
21301
9960
1656



3755
12278
22119
21098
1005
10264
13037
11702
20954
5581
5063
1118



6454
20632
1696
19474
14911
9381
22362
4111
13374
2932
3071
18551



1431
12923
6509
19229
14012
5372
12362
17380
20272
16391
901
13395



5132
9540
19228
6169
11589
13306
3179
14568
17630
2371
4694
12396



15369
11784
16597
3697
10741
16309
4927
13396
21621
16662
8841
11120



14081
8692
9484
15204
6565
14320
2978
9294
7482
9493
1277
4952



7813
7306
12296
12021
6645
9286
22489
13745
16653
19069
7780
15219



16969
14762
18330
10280
10802
10479
16663
3678
16713
7751
13703
3630



4691
9472
10709
8542
7060
6112
22457
21974
20476
7333
6482
14526



7151
2644
835
10655
12264
9315
2786
16253
9488
5634
17372
788



18247
9280
22095
18903
3706
15256
18593
5764
11115
12583
11568
13613



2331
5136
8073
15998
5630
11304
19137
5817
5580
18341
8588
12540



2454
3016
4970
17445
2401
11869
6193
21516
2447
10889
5190
13207



16465
9673
9771
11716
13123
18575
9584
12794
21399
20485
9218
18691



3350
18263
4846
544
19667
9933
4140
1318
20418
11128
20105
16734



2376
15699
7061
4232
15357
14036
5339
7107
19030
7165
21370
12103



4848
13211
22530
15360
12863
9975
6398
14067
16683
21170
1924
890



8155
15866
10131
7187
4332
1235
20330
12927
7088
5099
2302
12424



8303
17466
14322
11383
2282
14274
15956
3414
12982
18548
15665
10961



21084
10824
18440
16819
3730
13940
18821
14864
1818
19607
7969
6546



16771
5441
8459
18266
5000
15749
13014
8444
4707
13097
15930
11872



2621
7158
6942
18502
5408
10837
21928
13800
5188
19614
16117
4719



1833
13499
3107
21492
12503
21929
7921
1429
14398
22189
6439
12993



12307
19714
6650
18994
747
5932
18630
6683
1921
15651
19882
5594



6958
13597
19763
10097
14124
14687
1094
5780
7770
7688
15110
5797



7907
21169
3329
4065
12627
22067
19211
2061
13235
7038
8909
16914



13129
16317
19881
1551
14888
1201
20264
7800
7526
2209
5887
10798



15317
10223
11351
10105
10661
15014
2908
16412
12003
22277
19851
19616



11003
7768
6166
13850
4620
16231
7016
2717
20886
14522
5254
15519



20541
3458
1240
15787
20099
9282
11480
4994
6281
18662
7992
9661



19875
11156
3619
14086
20948
16946
3456
6143
786
6709
5954
17926



20839
22351
4726
13277
15113
6368
7704
18694
3644
12154
3794
21257



21638
18386
18111
21498
10731
13776
5539
14530
20282
7762
9529
17675



15191
12380
14865
15825
2818
16442
5901
8220
18578
13297
2495
21913



4526
16085
10965
15558
18982
2984
11245
11137
12977
2132
20312
4655



16568
21093
13882
5730
7976
7079
13035
2800
7101
509
307
305



12545
2037
11704
9571
21379
3712
7685
4483
18456
22341
11197
13107



14927
16006
532
428
372


 


307:
3349
11861
16061
5336
12949
18887
6597
3983
22099
2324
447
4512



11806
9383
17441
7957
16578
12877
12285
6745
8158
22143
14055
16201



1754
1561
18005
11717
9710
6672
1451
10276
2243
21572
5576
13128



10885
6027
17037
15496
10454
6068
15308
2899
11018
14627
20697
3974



7859
15270
15938
5811
2698
16879
8859
21272
14729
18033
840
5497



13669
21714
19513
5502
16413
15747
1844
4651
13831
22094
8195
21288



3065
16776
4822
18118
19091
5553
14834
21349
12572
16815
8955
13075



12733
4648
22049
5403
1029
1877
22486
14277
19152
1410
21143
7372



21858
17690
6467
9703
12831
4606
4506
19173
2942
1265
17141
18407



8705
18889
4746
3811
9106
17506
7669
11911
20449
10409
22212
9181



21792
22305
9003
8407
9234
9976
16656
2745
18946
4081
1944
12668



7458
13728
15650
11471
11697
10966
5324
9173
19342
21172
9379
17192



21538
8949
22434
3555
19594
13734
9786
21821
21772
21456
21196
9187



10524
11998
20944
6938
7779
8979
13537
5360
18851
21252
15297
11734



1781
17886
3931
16508
9825
5806
12904
4987
1438
8935
16959
16382



1164
22141
2007
17210
14488
6399
14781
6373
22109
599
9398
9473



5195
4504
5963
14256
6939
6345
1162
18406
17784
1442
4331
6935



5879
18326
2240
20428
19414
11272
14731
3444
1809
21833
14869
12121



5793
8845
20490
14861
12563
4528
17811
1567
9570
733
11892
13985



11612
1512
1070
2951
19948
9341
9353
1718
16198
8546
15048
9919



20674
3909
10843
16432
10218
15654
4043
14847
6714
8707
9886
20997



22088
13038
8037
9178
5352
3851
6484
16180
20444
13259
20733
21480



17651
22149
13792
13689
6933
21822
7825
13935
22010
15175
7093
21104



21571
14166
13059
1320
19388
12387
18647
10615
2572
16738
17277
10671



13485
4638
2352
7397
5048
13275
7941
11316
9127
6631
17029
14783



5081
2879
3446
12504
11878
2839
3228
19752
1088
1402
6179
16798



22525
16859
10813
2895
20050
10689
9920
13359
6382
16804
22048
15372



17024
19135
2131
20708
19186
7679
13583
7611
8668
2471
18443
8080



14254
18267
2356
17724
10645
21850
7740
18043
7128
21146
15408
8078



2828
15730
8513
18968
650
14046
3085
13243
11222
15011
1105
3976



1507
15891
10269
17937
6229
13505
13897
17262
1497
9774
5505
5939



7480
3041
12199
18054
15132
17133
9987
21835
578
22529
20727
13110



17553
15284
5186
15066
12304
21091
9863
4969
14128
13927
2253
22173



20557
7177
21588
7134
20606
17839
15095
21594
19310
4399
21404
1167



2570
8102
16155
14831
9194
9119
2558
21715
11856
11165
7573
10855



15158
22322
8996
12383
18252
20458
20237
9891
4099
16104
8881
12231



2561
647
22387
19765
4615
21305
15139
4964
20281
11812
22153
16216



18833
21797
7671
15097
11028
2172
17562
12574
2664
9816
12353
2954



21428
12735
2392
5226
8680
17177
18591
20343
15667
21341
11930
10157



19825
5210
7091
22444
3844
717
18902
14808
11659
8670
1785
8233



21693
8835
7167
12659
3869
22226
15589
13405
5280
13156
10456
10253



16881
14660
15728
6461
7132
8140
20493
14049
12322
15683
846
12989



4867
5866
20584
15020
1964
20885
21697
12785
2196
12335
15689
9927



763
1531
20138
11358
1132
19079
14162
5361
7543
18763
17112
21617



7915
20412
1643
20533
8020
9158
2930
19107
21064
3201
11077
14726



20135
9411
15348
9402
12669
15995
8168
7755
3112
12219
551
21933



8916
13876
14513
6877
8597
18116
8268
9085
9769
11890
21961
11709



10758
12800
8640
1212
10509
21023
11267
21751
1557
18735
18051
21254



15200
5771
17912
3381
18702
19564
12819
17593
8712
8128
1909
6496



14338
16091
948
17565
16614
18595
11540
18075
9306
5464
18421
10608



12167
17453
9268
11122
12880
13525
21522
5843
7885
10162
18002
13496



9474
11489
14139
5078
13260
10989
5480
3428
22424
15138
4252
2826



5882
20175
15811
16116
7879
12274
19326
5318
8325
1069
22306
6156



14294
21302
22394
4521
10893
5702
4766
568
15271
1077
17778
9726



14452
21497
5648
10532
15330
3647
8350
19227
17520
19834
16427
12852



9016
11208
9994
5906
20197
1115
15244
22452
15164
4258
6471
14545



6910
6417
21796
17471
17991
14998
15383
3215
10382
2580
8289
8452



12605
11475
6041
3868
2991
13102
15010
21365
21059
6894
21208
11757


 


308:
13659
8503
5417
2636
18255
14258
18186
14480
2997
12520
20393
16958



14070
11296
3421
20754
17033
17928


 


309:
22368
4230
16648
2237
2516
16152
10415
5473
14703
19022
18480
3997



17101
4156
10514
13955
12976
4059


 


310:
4051
3527
13345
2678
8550
12739
5968
11252
1249
13757
11908
13883



642
18741
863
15128
20888
14323
4672
8571
16644
10920
80042
2023



10377
10056
4837
19713
7234
1632
16113
13478
12526
19784
19698
2701



14658
15220
8365
18917
19359
21121
8529
17053
2009
2989
6906
5789



4862
8968
21524
13803
21337
5536
3377
936
20910
2445
21144
20276



15766
10716
15947
17616
7904
2437
12791
8028
5119
21686
20353
6765



13032
12566
2929
19589
6025
12404
5688
7908
7436
18460
14549
1975



9403
18910
15871
5414
21648
22373
15001
8960
8512
14839
13626
2150



18521
4687
4104
15162
19653


 


311:
7246
7633
13566
4922
7189
6648
3075
2678
8937
18998
20791
2442



4593
2102
13757
8484
11908
21901
9697
3719
21432
10971
8004
22023



10056
4837
6659
9021
8001
9591
4169
18319
12526
20405
19739
12836



12342
14209
15108
19698
21200
14093
4820
21147
16711
686
18917
21915



21121
8529
17053
2009
2989
6906
12853
7577
6127
14095
21524
6916



13803
7336
19598
11276
21713
14811
21337
20910
936
2445
11616
15947



17616
8331
11034
19471
5916
17862
12115
14492
18721
8414
19179
5371



9277
5119
17025
20374
3996
5057
1399
16384
9470
18719
18399
3401



8557
2158
3928
19925
15780
1172
19003
4754
6752
7557
21304
14324



15694
2929
15268
11094
19589
8713
6025
14222
14987
11178
3467
7373



1975
9403
2856
8286
16534
8056
9369
2719
15871
5414
22373
21648



1955
5457
18895
17041
22161
13812
8512
14839
13626
18521
21678
2002



4687
18560
4104
7854
18098
13647
15831
11038
19653
8205


 


312:
12932
12556
10612
8324
20661
21261
10886
21109
3162
13645
12519
14154



4627
9952
7890
12288
13254
22516
21541
11332
12510
6556
17293
7951



6797
12554
2010


 


313:
15796
4326
10345
13029
13317
20973
19570
14613
13103
3759
11764
3247



11747
9513
3029
20696
20193
9454
5495
18809
16012
8066
7668
15744



1836
6499
12779
2481
14822


 


314:
7479
533
507
535
306
12575
15642
18461
5073
9804
18997
8666



3659
6320
6367
19001
6157
11916
2258
21356
9857
2163
2584
3331



20086
6423
4662
3838
14933
8860
19820
15915
7403
4850
4714
9730



12137
14919
12016
19643
3913
17315
7812
21939
13951
1478
7199
10536



9491
3917
15376
4608
20333
7216
21640
8527
16880
19870
10959
4725



15069
5944
17598
6786
14232
4110
10546
16887
4811
15616
10586
10067



11756
20433
13391
16270
10468
7655
21719
5755
2857
17664
21589
11961



18800
19154
879
12644
16878
2030
4833
5428
3533
8904
4677
18511



4730
696
9569
17797
18687
7097
20259
15252
13221
450
22502
14915



21157
20371
3563
12591
16308
7626
4212
19798
19458
5691
18292
16130



21296
2832
830
14553
6434
18295
476
12043
20779
1281
17745
16586



7955
12705
15449
988
20859
1768
11129
6547
22518
5552
9854
11376



6669
5359
9132
10964
1879
22087
13137
17298
3333
13752
20301
19370



2425
6946
19538
14273
15340
6427
17165
15603
12419
10809
8003
13658



14064
4221
7387
13284
20981
2316
3116
8418
3386
6508
18217
3578



22249
992
13442
3471
14855
19949
5277
19885
2816
10917
9436
18538



18659
12816
12308
15943
2515
20364
14701
8193
17579
22076
8890
2649



9329
21073
8376
15177
10882
859
5990
9814
16053
5209
3144
13848



22428
19076
14568
4694
12396
15369
16597
3697
10741
13396
21621
8841



16662
11120
14081
15204
6565
14320
2978
9294
7482
9493
1277
7813



7306
12296
12021
6645
9286
22489
13745
16653
19069
7780
15219
16969



14762
10802
10479
16663
3678
16713
7751
13703
3630
4691
9472
10709



8542
7060
6112
22457
21974
20476
7333
6482
14526
7151
2644
835



10655
12264
788
9673
9771
11716
18575
9584
12794
21399
6281
18662



7992
9661
19875
11156
3619
14086
786
20948
16946
3456
6143
6709



5954
17926
20839
22351
13277
10965
15558
8891
10152
15189
13161
3256



13226
1748
21271
22258
10136
18982
20726
10154
17115
12652
1426
12623



4475
16706
17850
21904
545
2132
20312
4655
16568
21093
13882
5730



7976
7079
13035
7273
14619
2800
12728
15228
19263
10088
305
16894



6203
22106
19049
10427
11733
8133
372
12866
13782


 


315:
3342
12667
2100
12617
1136
6158
17567
4891
9212
13652
2310
14039



8257
4890
20474
16825
8834
4127
1369
14688
17548


 


316:
10331
15934
2046
6293
19503
17105
13130
7604
20972
611
2151
19670



1110
20162
9143
9176
19023
19084
18099
5356
15439
3212
10361
21551



20873
18300
6915
11130
19131
17963
7910
4920
4372
18216
8334
5551



11009
21068
13009
6829
20236
2330
15785
1052
14929
4869
4064
14251



6409
4771
21722
3477
22283
4071
13419
2771
12516
2855
6554
5053



15301
13629
22091
9959
8014
18394
7184
5437
10014
15912
7764
9611



13654
12151
12808
1755
10215
21979
12492
8018
7254
20380
2845
654



5061
7274
9568
5018
11235
6804
8938
12700
17001


 


317:
7147
17102
22335
21107
13529
17232
12299
15442
10074
12860
8161
6451



9249
12173
13520
2381
7179
2397
4681
8032
10026
17837
1679
750



13467
22304
9093
12920
8746
12849
8975
8614
11049
15351
6414
11194



18365
22000
10060
950
10516
16860
18657
11078
20626
15349
9992
11321



16068
13071
20575
10629
4241
12058
21534
18040
574
21837
6124
1526



12392
2919
22474
21550
1309
19435
13170
2632
14407
19257
15479
17487



7797
19535
13125
7697
9338
11284
15648
2144
9885
3591
21263
9545



14344
6947
19431
21279
4756
13704
4125
11670
6044
22381
21905
15904



14499
10692
12332
1756
4806
18332
7994
9064
18991
17391
20098
22081



5015
11957
13991
17712
22029
21507
3779
6619
4824
1847
11561
2927



7497
1618
16567
1625
1011
14504
1983
2353
13906
16938
11796
2922



13819
606
3676
2458
20599
20531
13948
7520
12498
13705
8646
9042



9750
14969
563
1092
8562
6341
3562
11565
15599
16895
12974
1939



16577
1711
21985
5754
19800
15748
4371
12415
2463
4827
5656
6520



14881
1513
19711
6790
20528
13821
9526
8611
6058
17821
1888
18587



4858
18855
7028
14908
22205
5964
4340
5271
17773
927
8374
3316



2666
20014
21527
9914
10822
7197
21212
13163
13989
17347
15266
1401



6338
14444
3445
561
9349
20565
2095
7453
18711
19524
4093
8735



14800
9217
16762
10080
19195
11057
11977
21372
20196
6610
14503
22133



19181


 


318:
4562
9606
1444
14309
10107
21463
18996
14333
1334
13632
22339
19891



15309
3134
19973
17693
15433
13252
20233
7289
15373
8208
21182
21283



14523
6862
8920
22389
7427
19075
14622
18190
16360
12132
1586
14883



3538
22299
4909
6348
9295
11703
6913
16728
19809
21668
18897
20235



616
10625
12272
10812
4295
2449
11240
20715
10788
12331


 


319:
11506
8025
19504
17096
9236
5967
13265
13150
20345
2575
15093
720



9238
20846
7110
18462
16689
3666
13238
20249
22504
5681
15821
11308



7263
2587
3204
8488
1135
3170
19880
7687
4736
5783
20141
8454



11622
3641
10121
15474
15016
3635
12476
2148
6474
12754
10278
4251



2157
17396
18150
5472
2435
9967
3440
12802
21724
16131
7711
10389



5452
18194
15428
5298
16650
13697
3148
15338
9355
4542
7607
12624



9835
6871
1450
17202
4346
18156
2256
5469
11562
11593
8125
13465



21018
14122
6978
1534
5284
19934
13992
9438
9860
16616
13159
22237



9239
12122
7442
20605
16637
625
10834
2546
3875
5692
13941


 


320:
5584
14500
1413
1303
11425
12432
20553
5678
19728
1702
21840
16132



13978
8053
11638
17005
7048
12602
12897
18092
1532
16921
11347
8929



2022
22014
21806
20100
17247
5940
13718
10507
20917
13044
3334
11629



3850
19276
8082
15982
2091
8247
2827
4647
21352
16763
13875
2729



11068
14988
22366
6081
6500
17279
7348
9062
12873
13199
7794
6832



16726
7795
12202
12467
5788
10596
12756
21948
11263
10951
13316
8432



13127
9984
13493
8019
12038
1826
14472
9125
4955
19548
16684
5905



16785
3169
20313
12232
10211
6536
20084
4322
19294


 


321:
11679
7712
4765
2778
10428
15613
13759
8748
15529
4789
4143
22492



10520
5899
5524
1150
15997
6728
7250
19339
3882
2486
16390
13804



4361
20834
20825
586
2032
2861
14363
17505
15033
6284
9806
19295



8378
20979
6962
22356
6126
18081
16902
16016
11797
14905
12294
1932



4974
560
4872
19916
7886
18272
11004
21567
6122
3929
10292
20964



14636
4316
16294
16670
6202
3283
3979
708
10683
3722
9024
10315



7707
21597
12187
21031
11517
17433
1323
3512
20375
12464
21941
10366



13212
15089
3993
3918
17676
18119
2709
17107
15788


 


322:
10548
4897
5425
7818
3878
10642
3310
16365
9020
12789
17219
2142



13865
19002
946
18211


 


323:
12932
12556
10612
8471
9836
7200
3244
4931
6155
11441
5925
9952



1283
20609
2129
3224
8050
2693
11630
8765
8258
10271
17801
10768



7008
3010
16997
6501
20840
3978
2010


 


324:
10963
8235
5742
21966
17140


 


325:
15756
6758
15758
5287
19343
4588
17303
13336
21407
16436
6718


 


326:
16712
1166
15865
2733
17246
15303
1381
13492
6572
7728
13605
12382



9290
21152
7868
21359
6654
2544


 


327:
12913
16464
16273
20905
3701
8623
4539
11237
7785
12606
12734
1726



4555
7538
13748
14436
18787
11199
15234
14408
15929
7578
1919
8057



11210


 


328:
14125
20217
16758
5776
15086
18339
9344
13845
5197
20795
5897
9137



9973
704
18058
11958
7252
2114
7827
5752
4689
7592
15102
22013



12505
15413
5397
16345
11067
11445
21494
14366
3450
8189
13758
10214



21577
10815
1385
19042


 


329:
3497
2714
8101
4216
8074
1106
1600


 


330:
11223
1913
6535
17340
17586
15949
10628
5279
16989
13504
22441
20437



16847
20368
18727
20627
677
8442
2847
12004
5478


 


331:
13633
5900
8539
14672
12233
964
10478
20775
5875
5805
1804
1598



8760
4906
10137
12276
20894
3902
1838
1416
13999
8927
20239
21067



2846
1536
951
9346
9547
21436
4596
12781
4010
11786
8601
10761



6187
19637
20291
12933
1404
16680
16262
4172
6985
11107
10370
17700



13722
7971
3488
17731
14386
6750
13862
2765
17564
11582
6358
16691



21558
16323
14350
16234
10634
8984
7275
21757
12586
15643
921
10884



20529
8362
4086
15778
8213
645
8641
14759
21903
9293
2872
7350



12282
7277
21911
19806
10064
12113
15339
20238
1645
15286
17911
15808



9799
12685
4914
19213
10728
2852
4839
20536
7995
7379
15240
12971



591
20690
5666
5145
10145
1126
13494
13052
13798
22187
6567
2224



22255
22276
19919
8963
4880
11857
15344
20168
20601
21712
7009
15363



10724
8126
7044
2098
12067
13898
7690
17967
22554


 


332:
4051
7246
14199
4757
2211
19939
7119
18627
18219
10672
19725
20509



7686
2989
10561
7284
936
20910
3464
21563
5884
11537
21063
1972



3103
11411
17085
21614
20041
6920
896
19271
21101
6679
18617
19003



7557
2937
8512


 


333:
11864
18401
7916
16388
781
8033
2364
22015
9748
2475
765
14146



13993
16445
7070
19406
13139
11260
21048
22437
16423
12388
15277
9405



13452
21701
15472
21519
18268
18140
17189
10852
15461
8741
9641
20297



21384
9522
16319
4702
6436
9574
18726
3628
16593
18634
13820
16639



4189
17966
3524
13210
12214
13114
18483
11648
5974
15580
4570
17225



19309
11814
7466
15434
16431
13455
9128
17770
17597
11744
5668
18077



20332
12252
6487
5722
19526
13902
14620
19013
12349
14056
3612
8715



15923
4913
11247
1777
16114
9983
8124
7153
20006
10025
1074
21879



4977
19631
8517
5456
9877
4139
21162
4364
7696
9702
1769
9365



5341
7159
5323
19419
19967
19719
15541
14191
10391
612
17071
22520



2596
16797
826
17040
18360
6069
16301
22129
12523
19596
3074
6712



12666
21205
18284
21965


 


334:
22427
862
20385
6265
12399
22070
17740
7828
4067
1153
11326
2935



9841
19586
16621
4285
4474
5816
1468
12636
2870
12256
15396
20978



13936
20481
9196
15850
17759
14083
17877
11918
14099
22052
4350
14967



3030
4936
6573
20255
16179
17938
6661
14748
18841
8544
17559
16186



13096
10817
20956
15131
16981
20307
6896
9262
16287
14784
4658
18310



6506
1358
8689
18061
6280
1214
4418
13166
8421
2972
19253
6332



1033
16181
10378
16100
12772
1621
974
9705
11370
21857
9111
13663



6502
15769
18274
20182
18579
16062
18202
10290
15049
10207
10284
15057



16286
790
10968
5513
1193
17665
4712
12568
13764
17032
778
11095



19866
6900
21331
931
14040
5675
18493
5449
2779
10498
12780
10602



10664
9704
2890
2696
4265
20376
6349
5782
11580
15846
4001
15619



21400
16911
11079
4939
15709
14567
17601
14494
2988
2987
3279
14867



3013
15708


 


335:
15578
12136
19850
745
14354
11893
10400
16221
5102
12842
19938
8836



6356


 


336:
1655
8875
17969
16217
19832
4954
10256
13907
2280
7705
2896
5571



6016
2423
14230
7769
16575
6956
11570
638
22347
12730
3084
7136



21153
979
18133
14470
4762
13775
13517
18643
14115
20989
4195
14211



17841
21732
2378
6456
2604
16482
9054
9876
6696
20958
9729
13852



22311
12997
5664
17434
511
11309
2192
7514
21788
10558
17846
13447



19287
9827
17314
19480
8685
19289
9913
16783
7798
15355
21362


 


337:
16000
2106
21931
7956
15630
12717
10346
15324
20316
3593
13203
3167



2799
12590
13815
4459
3056
18383
12953
14071
22160
2913
15509
16846



14685
8814
5931
19964
20780
14871
13615
6779
997


 


338:
20669
3012
4857
6538
14648
21791
21544
15892
20656
17483
5020
19379



16363
12470
5494
9395
17737
5910
3081
8480
20365
7510
1412
19099



8801
14807
16161
2393
10298
8123
1239
10701
8769
20670
18966
739



17909
2578
9830
13785
17449
6339
13378
17851
10673
17975
18270
18869



20842
16312
5144
8392
13794
20142
22008
4642
7811
9505
7022
21431



7054
13460
4715
13880
15813
16187
18556
12071
5168
4445
8109
6238



19089
10095
2361
20340
16200
16747
6028
22386
10599
2796
3476
14418



7731
5135
20971
13422
21726


 


339:
2298
2436
19901
14207
19971
5786
15380
16523
17629
1543
2607
3939



13893
13623
19506
1858
2400
20267
18864
3159
17867
6744
4520
5184



1289
5953
16468
8962
5695
6006
21230
5243
4876
2057
22012
13975



18334


 


340:
5094
13921
9689
10104
19369
17978
21082
26901
7645
3853
21213
17435



20373
10839
8089
4282
11406
17050
9439
11816
3709
8068
13476
14133



17945
20398
2090
4164
2229
4799
13949
13578
8589
10573
17122
18689



4604
7491
11399
9657
1351
17934
21685
9266
20395
18832
4616
13018



17935
782
21358
4923
2048
7355
19368
10540
3418
15391
6352
1454



584
6023
7278
13416
7550
12889
8632
13053
6040
1583
16498
7544



2590
9046
601
4785
7174
3594
18195
11021
9022
8275
1685
3624


 


341:
13668
15794
6342
14448
9278
20512
8200
16696
13521
1647
7170
15662



12079
14698
21329
1853
5038
11976
2478
11937
22112
21927
3832
2707



4810
16842
15999
3541
7952
9937
1893
9025
19963
10155
18805
2652



3473
21709
10794
17386
16395
9463
14603
17255
17919
4407
21734
1453



17376
5269
14505
22325
7898
5201
11700
7509
3737
5907
11800
14887



18221
3408
1391
6603
945
2741
11668
22559
13979
16492
19717
12052



13678
12316
20893
10627
18110
9396
17457
12447
17706
4188
12736
10213



5657
1361
17750
2164
20757
10342
5557
13427
1459
5874
11035
14177



18713
8770
13138
12686
12216
4228
12420
12044
4116
20177
7717
20582



6544
20287
13392
5213
21383
20420
12277
610
7242
13209
21266
15605



17695
1713
6386
7399
3771
15478
14510
14171
22266
6037
14656
20179



7964
7223
20189
1994
21418
7850
8756
18014
14079
726
12260
21444



10406
2633
8511
11121
10904
3092
2853
17906
16524
8187
17998
20778



2286
20199
3729
6145
21008
2565
12962
1257
15354
10610
7157
1796



6315
17300
22423
14435
16471
16628
6594
21171
9956
9842
18573
17327



8572
9431
16077
13255
10967
18428
7421
19940
17552
22215
6311
22128



15501
21897
19865
6983
7587
17139
17241
18872
10791
18810
8812
5978



20019
19994
6664
13673
2650
8721
16038
14722
18896
21111
2762
17606



10806
18027
9130
18559
12287
11513
6131
3172
1990
4027
5292
3287



13481
11191
19301
11297
10021
9171
14682
17190
14540
1140
8118


 


342:
2631
10657
18699
2536
6440
2600
16629
20719
4729
7634
14681
5022



8936
10883
18494
6327
9575
8157
8096
14112
2076
9896
11151
16731



2204
9035


 


343:
2755
21932
4777
6313
21861
12723
19508
1200
15989
16994
5687
6685



14644
15427
18561
20955
5319
10512
18861
4752
4558
9237
14495
2731



18748
20853
21624
4158
6682
15121
13328
11123
17204
3588
8417
12524



14426
12139
5914
22397
13082
3579
4217
4229
10653
20451
3376
17804



6004
10303
2139
15227
11831
18631
19845
12159
11935
18000
18066
13477



5998
16773
6555
6113
18981
769
10905
22461
18369
7501
12698
17209



22284
15974
3000
1328
15319
13157
21846
21899
1244
17408
7057
674



11714
14760
3856
22403
13952
5958
18866
16803
5945
7999
10446
17518



21309
12969
5105
10656
10777
18972
14634
7566
9182
10453
6623
6029



3857
1041
17997
10387
10643
1035
3246
9229
18431
9219
13687
4600



14823
1650
1911
1506
20935
12204
6588
14467
12894
12964
21745
4773



4206
8608
11403
21291
15104
9577
22470
10395
7106
18085
1045
18438



20164
5627
6989
3125
20664
9899
17477
20384
8914
13339
21568
14346



15753
18139
2610
22040
12680
1930
10147
11672
21762
14042
4281
8445



18322
7765
11544
8117
10562
17367
2534
14153
16918
7771
16968
14118



14857
20095
4419
15545
5134
11331
9802
4911
9938
3150
22321
8071



13544
4128
7168
8967
22547
3271
5965
9073
16529
16953
7176
17918



1026
16418
11579
13510
11163
21202
11509
15037
10348
12555
7467
9767



15877
20472
13410
15361
12759
10511
8273
5946
3774
8121


 


344:
0


 


345:
11342
13022
13189
13375
19355
4603
9001
693
10423
19460
11989
3379



20654
19018
22196


 


346:
4152
1714
20305
10236
21335
10203
4070
20587
13194
15640
10242
11539



16862
1124
10029
12355
19807
3466
21983
17696
21768
12283
20617
17748



17513
7821
7744
10704
11320
3178
16909
21864
9099
11919


 


347:
15403
7222
17930
22425
8943
8767
5431
9412
9840
7098
10706
20039



19399
18679
3063
13922
12891
19177
14765
8888
3766
15264
15112
8346



19249
9496
8467
12097
7066
13354
10438
19683
1867
13713
11392
20463



8419
9530
13590
19828
17789
3181
19673
20308
20048


 


348:
22289
699
7239
11768
21811
2946
15818
18586
10674
14308
3720
10934



14107
19528
13730
3925
20977
22047
4398
22391
16849
20631


 


349:
6607
11545
4182
16789
10863
11170
7754
13889
17668
6741
14774
8116



16721
683
13808
10075
2147
13320
7970
20540
8978
11576
16025
12292



10319
5002
13246
7075
22235
11091
9999
20403
14955
7021
7400
15424



1403
1576
15201
1547
21389
2191
20891
18977
17393
11512
10958
14007



6534
12539
20887
4792


 


350:
21637
9766
13473
10430
2373
10229
22472
2533
8573
9183


 


351:
21639
6558
15717
6583
1489
21115
14590
22491
22170
13729
14708
11897



9169
16003
16657
18303
2503
7619
9667
19462


 


352:
14832
4680
730
8944
19323
9756
8807
16549
6800
8830
14997
16942



1293
1086
19167
624
11497
15185
11336
10574
6438
21022
10877
3498



1533
10113
8352
18565
10340
12363
3314
18700
8755
21000
5888
14051



11660
10255
20975
16898
1028
4899
9807
15549
1039
16373
21500
3757



4390
13334
5158
7861
19157
5010
21692
13142
13526
12155
3025
15524



4634
18132
20879
11836
6517
5390
6944
17642
10919
12777
22312
12374



12089
2107
8665
10063
15827
10534
19377
14693
4078
16983
7351
21180


 


353:
9803
5872
20880
7736
9394
19214
20066
4813
3867
8129
6810
8821



14360
9758
10208
22542
16794
8724
2461
5260
11314
17374
20064
22105



20232
17121
17288
16874
14516
1119
2781
15198
20079
14792
3439
22298



12105
21450


 


354:
6321
5225
13406
4040
4448
20067
13787
8883
20377
876
9126
21945



4701
11644


 


355:
8510
12886
3986
1767
22183
9676
17318
13827
6552
22440
15136
22229



15255
8556


 


356:
20329
7584
18151
12782
19780
869
1234
4543
4443
6003
10879
6195



14766
2261
5303
18455
14900
13423
4770
7064
4996


 


357:
1090
22239
4778
3892
10096
3089
20675
8225
3300
3785
10016
16285


 


358:
7246
7633
13566
4922
7189
8550
21432
6659
8001
9021
4169
9591



18319
13478
12836
12342
14209
15108
19698
21556
16875
20650
5851
17486



6249
13780
15776
11726
21200
14093
18917
21915
21121
8529
17053
2009



2989
6906
7928
14192
7577
6127
6916
13803
19598
11276
7336
21713



21337
2445
11616
17616
21069
3827
12208
819
16095
11103
17030
6075



5459
17025
20374
5057
1399
3996
16384
3401
8557
9470
18719
18399



2158
3928
19925
15780
1172
19003
4754
6752
7557
15694
2929
15268



8286
16534
8056
2719
1955
18895
5457
17041
22161
8512
21678
7854



13647
18098
15831
11038
15162
8205


 


359:
11518
14223
17501
6542
13569
4821
16169
8132
15637
2270
10309
5708



18374
9446
7790
12784
7569
14187
5525
7188
21691
15258
4550
7616



5261
15163
14560
20144
10693
12244
21413
6135
7464
11142
13491
17655



15676
14174
18333
13472
20648
6435
14763
13495
12676
18906
12036
16066



21593
21667
14370
16048
17609
19658
5604
575
21518
5309
13744
15880



19484
6704
658
8704
16710
12907
19375
3306
19468
11042
39841
7175



20965
1206
9155
20970
6796
17057
11008
18605
3353
4650
9058
7988



19743
8369
3654
18497
11952
2866
16796
8504
20034
19086
4409
12452



12908
7530


 


360:
13327
3981
5070
16490
20204
5909
15321
10310
12746
5760
6045
1730



1018
15233
13486
17387
20991
5406
13208
21716
9902
21763
22315
6472



824
1364
18250
18767
12499
21700
3665
8788
19915
12114
17927
19363


 


361:
13771
10043
10800
17501
13569
6854
4637
16169
8132
2667
15637
2270



10309
18374
5708
9446
9291
12844
7790
12784
10720
9725
13741
5525



15920
7877
7188
21691
15258
6705
4550
14008
15544
9706
7616
14358



13182
14560
16722
1472
4000
1388
4261
10984
9823
21413
4976
9514



16806
9972
9224
13061
15247
17984
9925
19953
10247
2676
11680
9751



7643
12752
14983
15727
20648
3200
6435
14763
13495
13003
16940
7214



15976
5735
6270
22352
12931
14053
16048
17609
575
18286
21834
10842



18927
21518
20097
5309
13744
15880
19484
658
8704
16710
19375
3306



6347
19468
10410
10451
11618
20965
12916
1206
20970
11008
1237
7988



19743
20088
8231
4522
18497
11952
2866
15466
3609
2403
16796
13539



14806
5364
12620
20699
15426
4409
12452
5296
3102
20918
9156
12665



629
20947
3649
7530


 


362:
1670
8427
4513
13628
17835
1981
20663
1254
19692
11385
11148
18372



18636
1663
18409
21940
17744
17838
14190
16086
18411
682


 


363:
2344
7205
10404
18090
3530
22282
2138
1522
5878
7220
18971
21529



18900
21077
6911
22318
1047
9881
13404
7614
13515
13092
13300
20784



3818
8910
19715
892
11279
3262


 


364:
7108
14914
4339
11633
1095
18100
17413
19557
4087
5710
14536
8563


 


365:
16767
7229
7241
19193
14780
8455
1669
2113
16394
13341
4965
20987



21669
16094
13151
2242
9452
6246
4432
1828
6419
11839
6713
3448



5129
7881
21433
18954
18710
15718
12146
16149
12186
2849
17256
2464



11803
21181
15484
8463


 


366:
6504
7524
14750
15585
13755
6170
12273
16565
1049
12336
18307
21260



18677
18863
18346
15597
19755
3751
19920
2278
12086
1347
858
13286



14325
1020
15133
10620
2444
3050
16139
16876
1573


 


367:
16254
15810
10496


 


368:
4014
5426
1644
4368
10252
8347
14623
6336
3143
17492
2883
8191



13701
14972
3068
11055
13245
907
1811
17743
3222
22379
5180
8300



10258
19899
11656
9989


 


369:
12917
22561
8545
4583
16643
6177
17097
3916
4541
14252
13870
6493



5606
20188
10820
20279
8970
21193


 


370:
4823
10748
15527
15435
796
14678
9764
10076
11244
9175
7609
5115



21377
11924
10595
20985
18928
3164
9788
11682
14840
9374
7368
12999



6258
1876
19771
13726
657
7929
12344
4713
4417
19488
11641
3883



6787
11345
16897
10763
19559
1322


 


371:
7873
16841
5024
667
14126
19489
20718
20114
6307
13749
4296
12328



20713
14817
9449
17512
12753
3363
11373
19490
20600
14723
1330
10565



10981
22124
10789
16941
4863
19070
12635
18547
1307
13095
22242
5955



10082
21555
19391
1056
17810
15497
12220
6532
15511
7786
2790
6035



18667
7196
17474
2121
6729
19563
5810
3048
746
3566
16497
12050



8774
22138
1519
13512
22303
2143
10708
12991
12651
3454
16182
10091



18474
18979
19291
18720
9390
8536
17155
6630
10710
15964
7870
4033



10458
1637
16790
15145
11065
1445
18055
8688
19251
19006
13222
19365



2339
7595
4454
13908
19220
15364
18914
10062
2726
22250
16571
5841



19972
14541
2092
17011
19981
20191
13421
15044
15913
3884
17551
14501



21005
6049
4670
18512
20116
22469
12494
12091
21526
21355
8895
11631



16944
11810
10807
21779
906
756
15963
10502
12400
1579
15470
8436



8930
2675
20539
7266
19290
13829
1855
2679
8784
9380
16462
6530



22546
22416
17960
15631
1720
21887
5290
18327
18553
4590
7391
7917



14645
15614
13247
14642
12300
11299
19418
1740
14311
18764
7547
14528



635
9762
6404
17436
12422
16799
6528
15127
16603
13586
8483
3332



2647
22390
2094
17405
13174
3330
5025
17812
3810
19126
12711
9225



18885
11043
21479
7774
21789
17915
1800
13040
16606
13023
2103
2956



11730
6062
11525
15548
21598
14233
4710
17481
6481
20502
21631
14591



703
7865
21847
18542
17238
15120
19632
2874
12903
20473
16809
19278



15968
14351
21898
19284
8790
7247
6870
19794
764
16079
17554
10015



14157
5749
18287
4764
3645
13045
14959
2521
18913
21473
9257
12508



4304
1449
12138
6240
11499
7386
10591
20874
4447
5379
5272
10120



8267
6643
6108
22137
17643
4910
15972
4782
15186
2068
5528
12337



11658
13756
3142
15763
12030
13236
17079
5380
18622
5896
4307
3158



17067
12487
14339
613
6726
15106
15675
11379
7371
5597
12017
17274



14824
18417
20721
11498
16295
15954
12677
9580
12641
17409
19499
9907



10576
21600
1559
1247
18152
17273
13554
16429
16780
20611
10103
3507



20153
6861
13556
3496
16183
11289
3492
3006
5273
3374
9357
22451



22004
19394
18283
11166
15318
8768
18770
9309
16332
16630
17527
9479



8561
16576
10695
18749
17951
10930
9616
3752
10469
2422
4538
18515



20865
7082
1739
12250
6190
16535
9858
7630
15712
2642
22497
1808



8526
18181
7296
21659
16761
14816
21998
13413
14852
10402
11131
6668



16521
11910
2019
5412
11257
11346
4369
17646
5667
12585
1224
11092



5532
10200
16673
17220
17006
6359
17169
14019
20289
17599
5659
9944



9538
3683
3521
19933
16463
13655
5686
21698
20758
784
11603
2553



8439
21559
7563
14314
13522
15633
1741
1518
15412
5891
1375
2785



14686
15611
21250
11653
21545
17884
18298
2764
3864
12813
11218
5410



16378
1570
7383
9592
15460
16787
1050
10032
6964
19685
13788
11507



3504
17418
3664
20387
12935
8777
20035
14635
11626
3618
6519
743



1923
12738
13872
12436
5348
10557
14330
1015
17903
15405
4174
2069



16438
1021
19206
4166
13172
10851
2780
1326
13679
7285
11220
4129



8228
10294
19721
22543
1447
5468
4193
6618
3849
14474
3736
3595



18582


 


372:
1006
429
14179
7479
10217
535
533
507
306
478
12575
314



2254
5697
5950
15642
18461
5073
9804
18997
8666
5240
3659
6320



6367
19001
6157
11916
21356
2258
11071
9857
2163
2584
3331
526



15153
20086
6423
4662
3838
14933
8860
19820
15915
7403
13646
4850



4714
9730
12137
14919
12016
19643
3913
17315
7812
21939
13951
1478



7199
10536
9491
3917
18676
15376
4608
16936
20333
1999
7216
1589



17342
21640
8527
5069
16880
19870
10959
4725
15069
13329
20496
5944



17598
6786
7612
14232
18318
534
531
286
4110
10546
16887
4811



15616
10586
10067
4095
692
11756
20433
13391
9954
10468
16270
7655



21719
5755
4146
2857
17664
21589
11961
18800
19154
879
12644
16878



2030
4833
21437
5428
11195
20933
21506
3533
8904
13072
4677
18511



4730
696
9569
17797
18687
7097
20259
15252
13221
450
11335
20777



12713
22502
14915
21157
20371
3563
12591
16308
7626
4212
19798
19458



5691
18292
16130
21296
2832
830
14553
6434
18295
17956
829
476



12043
20779
1281
17745
16586
7955
12705
15449
988
20859
1768
11129



6547
22518
5552
9854
11376
6669
5359
9132
10964
1879
22087
13137



17298
3333
13752
20301
19370
2425
6946
19538
14273
15340
6427
17165



15603
12419
10809
8003
13658
4221
14064
22249
7387
13284
20981
2316



3116
8418
3386
6508
18217
3578
992
13442
3471
14855
19949
5277



19885
2816
10917
9436
18538
18659
12816
12308
15943
2515
20364
14701



8193
17579
22076
8890
2649
9329
21073
8376
15177
10882
859
16053



5990
9814
14918
5209
3144
13848
22428
19076
5454
17056
4088
11270



5837
9604
22455
19840
1656
12234
1149
801
953
9960
21301
5581



3755
12278
22119
21098
1005
10264
13037
11702
20954
5063
1118
6454



20632
1696
19474
14911
9381
4111
13374
2932
3071
18551
22362
12923



1431
6509
19229
14012
5372
12362
17380
20272
16391
9540
13395
5132



901
19228
14568
17630
2371
4694
12396
15369
16597
3697
10741
16309



4927
13396
21621
8841
16662
11120
14081
8692
9484
15204
6565
14320



2978
9294
7482
9493
1277
4952
7813
7306
12296
12021
6645
9286



22489
13745
16653
19069
7780
15219
16969
14762
18330
10802
10479
16663



3678
16713
7751
13703
3630
4691
9472
10709
8542
7060
6112
22457



21974
20476
7333
6482
14526
7151
2644
835
10655
12264
9315
2786



16253
9488
5634
17372
788
9280
22095
18903
3706
15256
18593
5764



11115
12583
11568
13613
2331
5136
8073
15998
5630
11304
19137
5817



5580
18341
8588
12540
2454
4970
17445
2401
11869
6193
21516
10889



5190
13207
16465
9673
9771
11716
18575
9584
12794
21399
20485
9218



18691
14036
3350
18263
4846
544
19667
9933
4140
1318
20418
11128



20105
16734
2376
15699
7061
4232
15357
5339
7107
19030
7165
21370



12103
4848
13211
22530
15360
12863
9975
6398
14067
16683
21170
1924



890
8155
15866
10131
7187
4332
1235
20330
12927
7088
5099
2302



12424
8303
17466
14322
11383
2282
15956
3414
12982
18548
15665
10961



21084
10824
18440
16819
3730
13940
18821
14864
1818
19607
7969
6546



16771
5441
8459
18266
5000
15749
13014
14274
8444
4707
13097
15930



11872
2621
7158
6942
18502
5408
10837
21928
13800
5188
19614
16117



4719
1833
13499
3107
21492
12503
21929
7921
1429
14398
22189
6439



12993
12307
19714
6650
18994
747
5932
18630
6683
1921
15651
5594



6958
13597
19763
10097
19882
14124
14687
1094
5780
7770
7688
15110



5797
7907
21169
3329
12627
4065
22067
19211
2061
7038
8909
16914



13129
19881
16317
1551
13235
14888
1201
20264
7800
7526
2209
5887



10798
15317
10223
11351
10105
10661
15014
2908
16412
22277
19851
12003



19616
11003
7768
6166
4620
13850
16231
7016
20541
3458
1240
15787



20099
9282
11480
4994
6281
18662
7992
9661
19875
11156
3619
14086



20948
16946
3456
6143
786
6709
5954
17926
20839
22351
13277
15113



6368
7704
18694
3644
12154
3794
21257
21638
18386
18111
21498
10731



13776
5539
14530
20282
7762
9529
17675
15191
12380
14865
15825
2818



16442
5901
8220
18578
13297
2495
21913
4526
16085
10965
15558
8891



13161
15189
10152
3256
22258
1748
21271
13226
10136
22036
22190
18982



10154
20726
17115
12652
2984
12623
1426
4475
16706
17850
21904
11245



427
304
477
545
2132
20312
4655
16568
21093
13882
5730
7976



7079
13035
14619
7273
2800
12728
15228
8972
19263
509
305
16894



12545
2037
11704
9571
21379
3712
7685
4483
18456
22341
11197
13107



22106
6203
19049
10427
16006
11733
8133
2264
12866
13782


 


373:
21829
10688
1896
2695
15238
6923
13321
678
19909
6620
21886
4554



4937
9312
12000
1954
4679
2233
18331
21411


 


374:
6020
13479
5421
1087
1989
14406
16303
744
882
12075
4026
22268



4422
9416
14897
9425
12945
10738
8798
854
13950
9451
21908
8474



22358
10772
3605
17605
21264
20769
15607
2985
11015
4905
7656
19436



14257
8375
8062
4389
12310
14819
10371
1341
10899
5014
18145
16619



22100
13463
2860
1177
15365
14952
17870
14006
9642
5832
1504
11884



15546
4286
20157
10385
20872
9791
8771
2815
22262
21484
5747
8299



21256
22118
17091
16759
13043
22140
18776
20913
940
1270
18159
1798



2743
20782
13248
7678
22155
12200
20855
14226
12099
17769
164191
2580



3054
20908
14508
1936
4781
6302
8832
7652
17179
5548
11581
15473



20439
4751
15772
5864
2710
20430
12577
14048
13591
22217
16257
11511



9747
18047
5923
8265
7628
7942
7892
18958
3784
7603
8156
22139



12033
735


 


375:
17388
7507
6853
5756
19170
605
19514
774
18117
6385
7365
5325



15832
13388
6982
805
9639
15679
21883


 


376:
6489
1778
6165
13947
15105
6175
12001
16671
13761
6928
4769
20271



21491
10938
7900
12795
10505
14143
12592
21354
13777
3510
5052
16591



12548
849
3088
6774
12148
9161
12039
2189
17027
1969
2627
11841



9768
10979
13916
11981
6032
1926
3963
5427
18199
9453
4199
2366



9495
7328


 


377:
15533
9191
10043
2753
19146
7790
4261
10984
17984
14174
13472
11804



22074
3317
10460
14013
11181
20629
17263
5966
14272
21460
17175
8369



11952
21759
11479
16405
2498
10527


 


378:
22075
3099
19424
9211
2977
18514
18017
15584
2234
15782
4438
4584



3716
9235
16307
16304
1846
2682
20821
956
5704
7923
18167
11822



6140
3047
10275
21133
18224
5583
15900
13725
19275
17302
20181
18021



3015
5962
12164
9319
20992
4622
8799
22151
20338
14742
22201
10555



17777
10102
7163
15444
9363
21307
8269
10110
4429
6632
20691
9638



14736
14020
10138
20244
11555
5116
14225
2312
21414
10821
2249
22550



13699
938
20457
5430
12992
13398


 


379:
2406
15415
587
9691
5895
21357
17711
19549
17251
13715


 


380:
10085
21760
4289
13995
7446
9558
14863
11606
20045
17971
9039
18616



9373
14535
4825
12951
3787
17677
5214
18052
6875
18135
21630
5232



20890
1770
10991
8146
11813
4697
17003
7055


 


381:
21131
21645
7243
20820
5674
11731
6667
10151
4817
4113
1832
17312



4832
4163
2924
20194
9682
19740
4054
16537
8531
19852
9450
8277



12513
18904
5869
17443
18723
22349
1228
589
14210
7986
10598
6036



822
5873
20860
8502
1122
16499
3922
18794
17929
19216
19270
21895



2758
12314
2468
11214
6717
21474
11827
3203
6754
13557
17019
1712



19477
13019
5645
10923
3817
6215
7503
816
4760
3589
12238
12773



10459
10744
9856
10749
18103
19048
3457
21457
19189
16910
16867
19415



16265
3682
16610
16470
16356
21013
5777
8013
18006


 


382:
19957
3226
10525
8530
7982
3708
16506
20265
19422
17319
17973
16769



6887
12979
5654
18163


 


383:
8694
8251
3871
9924
7519
16224
22555
14009


 


384:
12702
17415
4664
4449
4325
19760
21746
3267
10368
3093
11350
3531



16380
21838
19608
11587
8367
20351
2902
19733
18161
18210
22500
20226



13535
5126
18613
16364
11894
11183
4057
11226
17119
9098
3211
2185



20288
20489
21647
4103
11973
11885
10260
12393
7832
4511
20170
1928



13833
21900
14890
1027
7922
6811
5790
14015
17065
6010
13795
9245



9566
11876
8337
15174
2263
19217
5530
9598
9388
1659
10101
17925



9717
14228
12740
14255
18590
12269
14674
17257
22064
8551
1059
11112



5338
15072
18204
15279
6969
14944
11583
18391
14690
6503
2317
785



6692
22080
20243
22421
4743
11968
5398
1750
12965
11391
12878
4721



9384
20075
5074
2118
14173
6821
5012
9798
11794
9811
10818
7495



4569
11830
12957
2497
1617
4507
10667
12465
5796
18993
12481
3573



13319
20523
21343
19968
8041
15867
18712
11232
18921
4109
16995
2484



14332
5833
21284
1405
18264
14069
17528
20295
17890
20822
18580
1268



2953
21324
21132
13085
21661
13618
87813
234
5382
11592
6749
16229



5848
4012
12378
1929
19687
9013
10182
7228
2028
14882
10560
6987



11978
10065
16975
15791
15013
5286
17958
17087
8422
21303
2200
3761



1089
18218
15951
21747
17124
2300
8857
8486
8383
19233
3427
1197



12998
7171
8654
10108
13462
17054
18871
15851
8291
18960
4494
15966



22488
18543
7773
12235
12178
22041
19129
20411
2027
19172
15806
6469



6370
22096
4105
13426
4809
19640
4659
12156
13332
9311
588
10476



22560
15961
14413
21548
7803
12072
17825
16719
17270
21888
3990
11256



4092
19664
21590
20602
20735
3685
16999
11453
18129
8997
1186
3952



7692
11037
17899
10950
13564
21682
15755
6178
12242
16297
14421
11436



3560
21531
1486
13983
14970
9231
6305
2459
19412
13256
16829
9649



11045
1317
16681
21053
19661
21038
7395
6841
1114
9000
14705
10494



9108
17796
20404
8446
10945
13307
15299
1073
16289
6701
11667
17949



18645
17044
20488
19663
20206
14400
4114
15577
20198
18423
6253
6078



16870
15568
10782
8621
17031
19360
1692
11227
9123
16836


 


385:
2321
2906
21872
10635
21078
7065
15156
1425
16772
13825
20591
13813



12899
1192
19892
18999
8889
11465
20504
3946
12048
20666
9727
18484



4435
1280
12630
17995
1807
1366
12411
2542
17186
15294
18206
15878



13956
14389
4053
13057
11138
9564
13365
14654
2060
6116
1599
22293



3117
16686
18549
738
1017
18097
20366
10906
7412
972
3303
9331



22033
22401
5340
18297
5439
11324
19746
22005
10666
2640
21017
10814



15503
15916
4700
10384
8762
17479
12301
18760
11416
17765


 


386:
7205
16239
22108
14974
15094
21071
5878
17987
14219
8882
17848
8491



10730
10766
11279
3262
17261


 


387:
11850
8199
14384
16243
558
13030
19215
3479
17782
5827
19788
4416



9270
4145
3077
7366
3904
3442
6662
14375
15690
11650
11821
1949



1649
4587
11211
6236
21165
7837
18707
13824
13754
3121
9516
1966



16645
15795
15859
18232
16579
16020
15502
22210
11865
21270
5088
2239



6314
11933
1605
15281
22051
13665
9116
6153
21677
5334
21914
20518



6808
1927
14427
10241
5400
1667
20796
14866
19395
5422
7965
11136



5440
20442
14551
4855
21855
2066
15805
15888
3582
2334
3800
4016



7072
16877
16230
17326
7806
2071
7990
18865
641
16590
21955
17280



17308
2396
13681
3877
6148
11519


 


388:
3596
7515
17959
3549
15024
13576
15991
5407
13215
8370
14374
9790



11193
17162
12026
4861
2328
9153
3383
2598
20929
5662
2982
14262



15841
5775
18375


 


389:
1348
15757
11966
16108
15222
19104
21321
4489
18184
6470
8478
20190



12628
857
9167
18660
16328
21136
12320
10268
9965
9361
754
20342



17110
8283
11192
16702
18986
16654
9350
12474
2031
22089
14245
18814



2961
13231
21209
11352
13736
3385
5559
5208
19587
21576
13363
21166



21877
19184
16154
13751
7235
18147
10895
7829
1343
5632
11790
12936



5995
4717
22228
14767
9074
21907
10830
11760
8348
21924
13987
8023



20096
10747
11879
2097
22475
2738
9421
8592
11715
20682
10679
9928


 


390:
11664
19373
14010
12174
19349
11769
4498
2274
21231
2630
10572
10953



8931
12005
7156
11098
17092
4856
20031
3585
18340
15441
19792
8259



5870
21654
18857
1161
9343
17931
22246
9364


 


391:
10049
13487
22261
19856
31197
34221
599
5351
21514
16751
9423
5727



5042
8667
12431
7989
2965
4617
5682
19205
18518
12013
6053
9576



5713
11119
9862
16190


 


392:
18732
10751
14514
17429
18757
4995
12463
17063
16460
10638
21916
17344



6287
10622
9084
7641
13773
14486
3055
1922
8058
12612
3059
4605



19053
5881
16145
10408
6114
13634
14741
604
18842
18868
21777
10350



10325
1510
2222
7494
12825
2470
17446
15874
22549
13588
17843
20862


 


393:
14137
6491
1356
15653
9544
17800
4657
6563
18761
19273
9502
12445



4359
12437
16199
18995
2176
8009
10587
4063
4005
20824
17620
19815



19234
17093
19511
10545
5626
9305
16800
12265
9897
6943
20358
12184



3707
16211
21688
10582
3368
2742
2365
10339
3898
8612
3028
11721



15331
5828
13918
3634
19884
16538
1111
1554
14399
14371
5104
1829



8104
17482
13063
16292
21720
7729
11408
5629
16271
21292
11860
3424



9033
21268
21695
15296
18354
4934
13381
16156
13739
13034
4472
20876



17313
16924
12087
18839
13963
22413
3248
944
6344
11007
8619
11418



9828
5268
14031
21703
8516
10388
866
2809
7449
17438
17555
16805



1636
15432
1152
10419
17275
12821
6115
15623
5603
15002
12960


 


394:
3202
16855
14393
16047
12109
20443
2004
17625
4349
19221
15436
5819



17066
19649
12670
1965
14910
21969
9893
3600
12536
3822
12850
4180



6132
13648
21798
18884
15981
21322
10336
20424
7005
13279
15017
2477



13011
10124
4373
14416
7377
13006
8088
4485
10568
6181
12408
4013



2391
10416
1220
14803
9678
2564
13861
16954
3199
4561
21312
6627



14292
4410
5823
16748
12403
14414
8669
12884
734
17278
562
6518



1881
4518
21380
6494
15852
17368
13840
12084
19239
3091
7517
5984



14268
4629
5191
19861
16641
17406
4144
18973
21415
21258
6950
14555



7041
13342
18344
19371
17541
1834
8664
16235
11478
14116
6540
10401



15100
22060
16196
11176
13397
19092
19747
10990
13435
9728
7131
8313



8349
9269
11964
1483
16810
1500
2320
14402
11184
3721
1256
10000



920
14238
8919
5685
20117
4235
9693
6412
5084
10715
7364
9817



5924
6015
15928
18253
21113
17516
623
7726
7224
14801
7682
8966



18692
11054
14527


 


395:
15084
19366
8811
1190
6727
4321
12649
19433
12507
3995
12015
20152



21429
17657
22463
5534
8079
16111
6899
3734
13762
19928
19272
17507



6452
12037
16857
11523
12569
6792
18238
5763
14939
12699
21275
3930



16987
2452
2193
21219
4796
14984
6885
7888
6130
13135
14954
1935



2387
6161
8499
19730
6609
2287
21882
2634
15588
11487
4376
17509



21610
2093
18802
7506
11993
4406
20544
8974
1016
16510
12662
20736



14836
15691
17920
7734
11742
21957
21727
7743
12157


 


396:
14699
6724
12500
6101


 


397:
4537
6708
17900
6960
21012
6873
21604
2141
7919
10956
4205
10702



6611
13911
7162
10797
21569
16752
6012
6869


 


398:
16076
2321
4451
19562
5982
1411
2156
12558
8727
16772
14543
9800



22191
22531
13050
12479
8829
9890
18225
2623
2538
1627
16157
3656



21344
15134
15798
9715
5142
2700
1902
22110
11093
7017
1409
20559



10443
9498
11500
15719


 


399:
5094
13921
11673
21082
2690
17779
21361
3853
21213
10839
20373
8089



10464
18576
8718
15705
2229
4135
17410
12062
1263
10684
4844
14376



13578
8589
10573
17122
19674
11088
5570
7753
17311
3616
11735
4466



20847
13877
22288
22274
8170
8179
8272
5234
21652
20395
3764
17831



14489
8729
13018
2082
3516
719
14264
7662
13451
8424
15025
6352



2590
8632
13053
6040
1583
16498
5182
601
17476
6142
7174
3594



4785
18195
11021
9022
3624
8275
1685


 


400:
20186
17002
688
20774
5087
2884
14965
22528
18781
1690
4912
5577



11728
16448
16807
10976
1638
15429
8957
580
4493
5637
5091
16774



7160
16922
15404
15627
18041
10475
11189
17861
6458
12796
2292
15539



10144
3302
20470
15550
17536
14443
18302
11535
18222
3339
19330
20436



12330
4213
11378
13719
1446
18705
18359
16604
7847
4055
1915
4452



7369


 


401:
8043
17358
11675
2116
16697
17985
22056
14520
11577
10169
8676
13033



5205
2318
6687
11694
2175
13809
11141
20423
6267
21105
10537
11944



20585
17725
18065
11722
4293
11413
1336
17250
5565
20625
13141
8797



11621
2433
14925
19876
20907
16241
14430
8061
5447
21832
6570
13938



7924
2863
13858
15223
21239
17416
17732


 


402:
12881
6207
13353
11751
15876
1666
22177
13826
8030
20136
19068
10849



6185
15941
3633
6034
14463
19984
9404
13322


 


403:
6523
1729
4595
16330
20515
15902
19588
3043
18919
11708
20497
2327



1441
669
18540
19877
6224
20250
22038
16853
15290
17729
14201
6522



6639
958
6067
9352
10585
13076
22253
20296
15055
16741
856
13376



4656
741
7600
3292
2041
12712
9536
7469
18070
17534
11560
1612



12461
21026
10734
16245
11563
6734
14728
2065
12210
7856
6660
21451



19555
3908
11196
7863
3537
4208
19879
1538
19512
18260
12688
20261



4516
10861
13969
17758
753
6479
4375
12255
14160
16212
16720
16343



8787
1275
20369
8862
14259
6209
11213
8389
15695
8160
17885
1848



22162
17147
13696
14312
4009
8381
15581
21090
8703
15075
12930
5299



2236
11851
22418
6073
6225
7353
12257
3365
8254
20943
7181
15879



9322
10783
967
9671
5003
19304
21876


 


404:
20121
4845
16755
15934
12656
20703
22316
10903
1987
15141
15570
565



19322
15992
21094
9068
15986
19707
21551
8077
3154
5118
3193
22498



2768
10551
21158
3298
8643
5107
10916
16268
21127
14373
13081
4348



2952
5053
3842
20761
1985
10588
14743
18380
9448
6324
8451
11229



12521
5321
13117
13654
3189
9466
973
16177
20685
18436
6742
8800



8329
17207
10301
16839
21523
20549
8368
16906
21295
16634
14165
16440



18858
11979
19688
11752
20704
8049
5101
12140
6150
18457
17776
10823



1055
10714
2685
17863
16832
18771
9387
18448
11154
16274
12172
16594



19936
10508
11531
11286
3525
16791
20273
17444
20714
13542
11356
22016



9568
15624
2045
8938
12700
17001


 


405:
15903
14278
19149
8514
3419
8145
3547
17612
11032
20571
12663


 


406:
17617
22319
7205
6865
16239
9494
6282
3807
1614
20370
12460
4224



10012
14713
14403
11097
4784
11736
5165
8086
18191
18915
19519
8491



10730
11279
3262


 


407:
2891
3958
11588
15385
3069
2735
20029
11448
11546


 


408:
9532
10331
19503
14129
5699
15147
7047
5185
7604
2151
19670
9143



1141
10189
18230
4233
9176
19023
18942
18099
5356
3212
10361
20873



12438
9248
1233
17267
19131
21068
13009
6829
20236
14326
6208
21773



15785
1052
14929
4869
4397
13900
6409
3254
21501
12516
1974
20560



11275
2659
5053
9051
18988
22150
21521
17242
12835
5328
12151
12808



14165
1755
10215
22220
22247
7254
20380
5061
2845
654
5018
7274



9568
11235
6804
8938
3173
12045
19302
4028
11190
14318


 


409:
17878
16387
12478
9552
20335
20588
14328
20566
9985
11470
9847
3078



10774
13004
16036
17980
12297
19736
12682
20732
15994
18840
14761
7615



3382
13301
14704
1253
8175
1203
14879
16511
18949
17910
4951
12305



12551
984
5460
6545
17611
15062
3611
21636
4337
22142
17887
5729



13561
7326
7817
4775
12683
18165
11442


 


410:
19444
20278
7007
15467
10031
19083
20001
8653
7474
9943
2052
11515



1185
15759
16848
7867
18893
18562
4415
7309
16700
646
22398
21783



11681
20677
13180
18105
19115
9525
21934
4239
9535
3136
7961
4042



17090
13917
13550
6807
15815
5083
14397
15406
5715
18413
1545
1181



18323
18109
4215
21611
15464
20382
18532
10295
3243
557
7871
21405



21910
742
17206
11102
8305
8131
8342
13021
21027
6288
21249
8657



3604
2514
17634
15990
20228
10314
8380
7680
4394
11472
9447
8886



14484
732
21164
5885
1582
18950
4256
13943
10878
4921
18817
9457



13643
5491
10600
6106
7932
12303
8523
7259
15802
15381
10690
5244



2547
9595
13530
3278
7558
787
14423
1602
7317
8244
875
22340



2864
14957
7393
18265
8733
9103
19853
11368
18367
16756
15487
19985



15688
15114
9775
5561
11959
3943
9409
7846
14829
11027
10403
18628



18693
10737
1258
16202
22195
7356
22522
660
14611
1131
8690
1624



19314
19584
2161
13946
12833
2228
22353
20610
5071
22372
14247
6017



7666
12648
14029
21735
14114
20694
13015
18234
681
10081
11147
18808



19321
6914
2616
12379
10311
15491
18766
3328
17588
18807
17753
12618



11319
20579
10231
2975
15216
2793
16097
1795
18974
14473
20268
21825



19495
18545
14979
5483
4788
9820
13325
2386
8747
14646
4173
8861



11690
17653
8169
5892
19300
15684
9216
665
16990
4535
11520
8301



5772
10373
13686
13843
9665
19610
10235
17272
17334
22154
18621
865



3789
1076
18122
12966
12081
14558
1943
10356
2299
11243
18708
10234



14621
11538
20401
7508
14752
11476
8779
13046
15566
2029
5433
2096



1102
12085
13461
16044
8180


 


411:
9901
16263


 


412:
2162
18233
20049
12022
11201
15202
8052
7238
12584
4327
7460
9562



19525
21286
14793
5936
10320
1414
6411
4718
9260
8095
16930
16698



18606
2323
7175
13143
20113
14775
19888
9560
2539
9038
9327
18125



11362
1014
9813
17983
8842
7807
2893
11880
14342
9378
3540
3096



19574
14434
16029
13644
4168
6269
3249
12029
19054
8470
14290
17635



13553
18138
15398
9069
18935
2699
5624
1368
7752
20623
8011
16598



2959
7392
16359
2033
13502
14870
6425
15173
5707
18089
13368
20148



5202
12293
7625
10367
22375
9160
16642
1146
8644
18822
6890
4761



16943
2968
15397
10530
4187
21770
6917
9741
21455
11557
12319
13100



16735
11024
6580
16704
17268
12608
10727
17252
2824
12882
11405
20525



6174
16284
14249
11514
4780
19770
4323
7269
18976
6189
4008
10736



14087
19567
22011
14971
14023
17424
2305
22078
3592
16956
7560
1738



12942
21285
20899
11460
19580
21557
17786
17161
3227
5470
5918
1584



6167
21290
18980
5133
22328
17322
3423
15190
20764
18753
17010
16011



3336
4391
7094
15896
7833
16618
16299
7848
21863
6217
20647
1230



22513
18373
7471
12642
20594
15221
18254
7661
18439
4382
21156
10463



3841
9623
18957
1044
10437
20165
17833
1374
15273
7844
19571
16820



11643
1575
1920
1695
11209
7958
10668
13971
5416
7100
13690
4695



12664
13185
8604
18811
916
3631
20925
6998
12127
1325
18046
17290



2206
18785
10243
13121
8060
8498
7423
7638
19073
2589
3866
17454



20816
16178
20033
1467
16026
8181
1941
8720
17669
4852
1609
20526



4534
20245
18223
21627
17568
18939
13054
14168
5856
18898
11014
19510



9810
12845
4047
20300
8236
15824
1098
16088
13424
14404
7164
14512



21944
14356
18936
12143
10894
5086
16950
18427
2265
12701
10676
14700



636
7556
1255
21562
20004
4018
14365
7792
21836
22515
4626
7954



2981
3277
20156
14224
17076
11402
8579
1038
19802
8390
4147
20742



19929
17647
3352
12885
17628
22490
3315
8629
6094
6966
14651
5256



8279
11688
6703
11108
1457
14601
9351
8986
2075
8490
2577
15644



14025
2586
2646
3999
9581
14960
19190
15504
18393
7405
2505
7583



10066
9356
8520
21298
7640
20817
21774
15316
7810
6357
19520
17972



21917
15777
13859
21642
3703
5853
1354
21366
12726
4471
1978
4480



2810
5335
21970
14779
22384
3393
5141
2928
5815
5890
8839
5761



19941
2036
7211
760
9097
1184
5514
15671
4895
2808
12236
11124



11100
16580
8441
6767
10567
19813
7882
20630
8524
3825
22209
13732



18168
5582
4896
6403
15710
3400
12466
7217
21299
649
918
4342



3021
10027
21150
13145
6798
20339
2003
8395
18183
5409
11431
19105



15632
1529
11293
12867
2841
12448
8092
20698
17823
17746
9681
10359



16092
22422


 


413:
22034
8322
12008
13273
812
11765
21273
20302
7255
570
20174
13723



6666
22086
18541
9801
12646
17511
3238
4002
10019
21775
19200
1284



18777
16126
10941
18730
16545
2655
10677
1823
3378
22507
22487
2105



21401
16277
16134
17411
11463
10282
1387
17316
814
12211
10675
5919



2531
8234
15950
13303
20240
15142
6343
17332
698
8256
18799
16961



7465
15483
15126
11265
1248
13144
4991
910
12748
14534
20200
1113



9537
549
10854
14460
6577
14432
1556
6735
19416
16300
21874
14903



5716
1511
13958
10119
16522
6561
5477
12441
6266
14367
2568
7559



19472
19810
11611
12517
13165
12364
21402
12768
9589
5941
15456
1565



5435
14242
21793
4776
7231
1961
19032
1380
19110
4711
18525
20467



16120
1422
22131
18435
17158
21488
4267
21287
8136
21470
13657
7855



7462
20952
6100
21395
1091
7935
12221
4220
4275
22272
11605
1109



6087
13735
21333
11724
18084
1628
1346
8072
15856
19515
2451
14016



9102
7499
20103
6129
8681
14265
22363
10518
18395
20658
15572
20900



17981
16009
11950
11074
707
8846
9190
18652
12428
10283
19835
9740



19502


 


414:
6834
6684
1593
14718
639
18288
5930
12385
11749
15764
10482
14872



20500
17253
17922
10034
9808
1699
3559
8697
16121
8791
12687
718



18476
13223
1062
17385
11440


 


415:
13988
14945
6635
15700
20023
11536
17004
12834
5317
19374
10116
7960



5032
17692
21336
1492
19786
6860
14502
20681
5259
9724
20051
567



3863
6677
19709
7411
16573


 


416:
5198
4396
18094
11888
17143
5694
21019
8021
21392
3175
21815
18035



12729
6083
4640
17336
18325
1938
5227
15019
16822
2110
2441
1780



11073
2067


 


417:
4094
4249
6447
4266
21799
11386
7114
19998
8507
4118
4930
21755



7422
470
10048
18342
16272


 


418:
12195
18355
21055
18016
20402
12963
12102
17702
3106
18678
18424
22227



19413
1979
17990
22510
17357
1360
17490
9376
2639
8398
14303
1563



9512
20310
20534
2405
3798
12350
16708
19648
22564
959
8091
13262



18778


 


419:
8007
9185
12493
17152
21173
10594
13523
16563
10079
2431
10753
19487



20147
9647
14147
9929
1830
1654
17021
19317
3147
1112
10779
8538



3237
6408
13120
3049
22221
899
15039
8907
16324
4879
3957
783



19624
12762
8261
15668
12832
19114
10457
3297
5741
17704
16175
15159



6999
11766
8413
11239
8954
8566
11458
11367
8923
11782
3384
18544



8298
18012
21849
14689
1883
11942
728
1701
15090
13910
20183
12952



1872
3776
5981
21830
14524
4742
9043
15217
19665
12092
13500
7051



5615
9310
10058
672
11082
16588
21831


 


420:
11518
6542
21976
16169
2667
11216
15637
2270
12844
12784
7569
16948



13741
15920
7877
4550
7616
14358
13182
14560
4000
4261
10693
20144



8084
21413
5610
16074
7572
9778
1745
15641
21565
16376
10773
18848



1071
17984
9925
22043
15210
10247
20648
3200
983
20360
6435
14763



13495
12676
12528
8206
20705
3157
4565
9878
16048
17609
21518
13885



7244
5309
13744
15880
19484
658
8704
16710
12907
7984
6137
22370



19468
10410
9826
17175
20965
6796
9139
11329
17057
3353
18605
9058



8231
2866
8504
16796
20034
12940
6134
15426
4409
917
9156
12665



629
12908
7530


 


421:
15638
19372
18500
2446
1148
7739
10757
13616
14797
4533
4130
21278



9633
2203
20379
16812
11738
4453
78751
79891
1775
6848
21289
15564



17637
18961
2109
17883
5751
4470
13318
19225
8496
999
19409
7694



4874
3523
21721
18661
5643
6235
15475
4993
8534
2734
16838
841



8087
7343
11926
5112
4748
2697
17805
22050
13444
20480
12313
19959



1236
14235
21517
18071
19134
12869
2268
16339
6198
11473
8372
13642



19849
19983
19150
7473
17895
14624
12838
18434
8867
10445
17390
4706



4050
4847
16631
13356
6505
18534
1687
1794


 


422:
5236
21619
6397
2990
20047
22291
6084
2760
5267
5082
12765
16136



9918
13383
21449
6268
20961
21232
5855
7138
1995
14576
1013
12134



10069
15073
6212
15451
8899
18412
4849
4386
4988
16988
4076
12237



5275
16208
20027
947
6757
10538
21003
4705
12576
20998
19553
17590



14359
11013
19927
6707
17088
18975
18429
14985
13721
17584
21718
20322



1390
19701
8055
6576
18610
20621
22331
12128
6286
7906
19843
18623



20065
11146
18095
21935
18101
573
11564
9180
14490
7031
11030
15407



20068
22044
7315
18849
17381
20667
5814
8717
2914
14041
6827
11083



22344
21424
1648
14123
11982
18740
20583
4741
19612
1957
6206
19071



15555
14347
15150
22534
1958
16979
5465
17615
13167
9714
3221
17417



1787
20324
4795
20150
11040
19333
5877
2311
2374
14089
20555
1581



853
11842
5451
3675
12229
8405


 


423:
7073
8473
19204
13490
15419
10302
3067
4727
8682
22082
19922
8178



18347
11384
16646
9838


 


424:
21999
10083
17047
19988
17430
10414
4089
1514
16232
9556
12818
19256



18022
1716
9583
12552
17581
13890
9464
2246
15706
18577
15783
9368


 


425:
8789
9460
12638
14787
661
13830
20570
19781
2510
17126
17660
1608



2111
19080
6968
16599
5238
20160
4150
9643
16795
2152
20203
17640



20673
20994
13131
12812
8991
987
22355
19043
18108
15045
14625
18638



2854
600
13108
14921
11424
11994
19459
3294
9066
11477
13026
15931



6981
9953
20837
1046
8701
4413
3741
4408


 


426:
6366
17652
5554
20635
17360
4793
14236
21110
8866
5732
11691
19410


 


427:
1006
14786
20756
8495
10217
5541
16973
535
3260
11264
12854
314



18315
18461
5544
18823
5240
2104
12358
16966
17291
6367
19001
15260



3597
5549
20568
10778
12440
1988
13743
11485
1783
18629
526
17425



9565
20151
9300
11185
5246
15752
2084
18185
2918
8400
3763
3837



5746
20378
7812
13638
17369
4080
10663
17802
2070
4989
7198
21781



14194
4437
8103
12106
16298
13285
19237
5066
19712
16970
17382
14671



9686
5137
13854
11867
2123
3011
15148
11900
22193
17908
9773
16561



14739
22164
2058
837
20811
16636
19327
18664
13237
18684
19318
3797



6139
2857
17664
21589
11961
19154
21437
16338
11195
20933
21506
3533



9255
4677
18511
4730
696
9569
17797
18687
7097
20259
4425
3003



22502
14915
21157
1139
2541
4460
16130
21296
2832
830
14553
6434



18295
17956
829
476
12043
3677
7659
5486
9761
5540
8443
15590



15367
14935
4602
18472
9627
7490
20779
1281
14568
17630
2371
4694



12396
15369
16597
3697
10741
16309
4927
13396
2277
21198
5308
21621



8841
16662
11120
14081
7321
10424
15204
6565
14320
2978
9294
7482



9493
1277
4952
7813
7306
12296
12021
6645
9286
22489
15165
9809



16089
15681
13745
16653
19069
7780
12587
19236
8110
4594
14762
10802



10479
16663
3678
16713
7751
13703
3630
4691
9472
10709
8542
7060



6112
22457
21974
20476
7333
6482
14526
7151
2644
835
10655
12264



9315
2786
16253
9488
5634
17372
10399
10547
16039
14345
5365
10379



14566
788
5943
10889
5190
13207
16465
10488
17136
6257
8306
2432



11948
16340
15326
5415
9673
15586
20108
20541
3458
1240
15787
20099



9282
11480
4994
9189
12928
10743
7111
16707
19389
2889
19161
9422



21510
10965
22018
15558
4315
21926
12010
3187
21634
19556
2333
13869



18982
20495
2984
2763
5448
20219
12749
2389
7476
15067
3515
5375



1465
12770
18281
1243
13198
9265
1763
1615
5056
11143
12570
11245



2293
2132
20312
4655
16568
21093
13882
5730
7976
7079
13035
16007



2800
3186
7053
15790
7591
22480
20744
22093
6055
15875
305
12545



2037
11704
9571
21379
3712
7685
4483
18456
22341
10909
11197
13107



16635
16006
372
18722
18324


 


428:
14507
15421
14477
17494
566
7991
15716
19769
6346
11158
2783
18037



18920
14200
1991
21228
2025
16965
22007
22121
19530
17216
16560
11719



9318
17738
1299
21214
12334
12769
13914
10771
21809
10996
18789
3482



8428
15625
18025
21461
4486
4864
18259
1869
18894
5076
10621
8796



15953
1037
18120
21047
8656
2967
13154
6569
12338
19732
3033
10633



15359
15028
16103
12423
19646
3561
2797
6835
16222
3266
19426
6571



21319
4126
22537
6888
17781
1697
10045
1251
7003
7276
19243
14694



15687
11467
11444
12814
7126
7580
4960
7248
3598
14355
7140
17847



8316
17583
16488
4421
19550
8999
1180
18026
20573
8479
18820
21056



7036
15143
13926
18737
18581
16569
13403
1142
3253
7394
4463
11832



14382
7339
16547
15305
10604
4338
16236
16768
4138
18754
442
18246



10039
12847
6541
21176
20831
13811
12065
10287
11127
9635
20511
19012



13060
12724
898
4347
8702
12055
21425
20545
19336
8006
14571
5579



15743
3704
19675
16542
16559
21229
11524
9274
11075
8804
4132
17224



3133
3607
12684
13418
10123
13007
14891
18308
10517
2973
11620
15395



10374
3168
3051
15872
11732
9301
740
19109
1359
17419
10051
6843



1680
5509
10865
11707
16067
22296
1000
3406
18908
8719
9844
20469



12031
11698
9632
4041
4022
2595
20688
6395
9511
16615
4623
22346



2369
17333
14975
19292
12095
14281
11866
5395
16733
3840
17783
15322



17305
10611
1428
7631
15733
5563
18432
3777
1959
18192
1274
9859



16608
6676
5894
4159
21902
6323
6814
7759
17670
10954
17330
3090



8154
15122
14026
9078
475
20224
15939
13183
2171
20320
12959
18620



6196
16678
13224
21618
9870
17904
435
11481
4738
13205
11111
6886



4385
10224
21396
16032
22294
3286
13158
7249
8239
1725
17508
13407



19123
1165
2912
20759
22152
18157
16447
2772
12222
10847
14538
17604



14596
20901
4797
3679
21087
19143
7452
9348
17993
21440
14394
16530



16659
10434
15793
9088
17239
4831
6340
2684
689
15731
9528
16620



17353
14180
11651
17070
2073
1585
8281
12298
22147
10166
22431
15463



6601
8700
16540
2993
4260
9664
10652
15975
13842
14457
15948
11412



5572
7389
11862
1897
19136
11526
10222
1819
9220
12697
22065
20558



17946
2983
18633
13258
13996
5429
20450
10529
15553
3208
12002
14696



12270
16162
9071
7594
17146
7667
11793
2416
11610
19062
19931
6868



7221
5047
7968
10376
5672
2355
11711
4084
8726
14756
9731
18769



5680
10960
10209
17713
4926
14217
6624
9314
3151
6850
4560
18134



17898
12386
5636
3480
10028
2838
20595
1292
20275
12254
17594
4853



20642
8186
16379
10383
4591
11759
11502
8591
4288
11992
17721
15333



9201
12553
14724
3846
22481
1723
15765
17537
17720
5294
9934
3608



16724
3395
2370
22184
6889
3455
7902
13093
4038
2297
7376
1916



7144
16736
15894
19202
4614
10304
12063
3319
11943
21007
5703
6099



3369
4162
18750
20614
3762
6621
7015
7304
4298
9147
11434
11341



10440
4734
13768
14607
7695
13737
10493
7039
16005
5151
2008
20772



4271
19954
7608
5609
5027
11303
8198
15488
16991
16228
17894
18306



2556
6626
20616
16083
12968
22378
16437
7624
19737
2476
20184
8948



13230
16495
6039
10274
10978
13806
5631
6912
22114
2955
21137
13954



22400
1905
19874
16430
12918
16985
8847
4942
18505
6234
9524
15225



3274
6369
1408
7297
20802
5035
20321
12454
13377
9019
4029
11248



3758
16017
6780
2467
8343
727
18768
2737
1889
15457
2791
8559



7622
17058
4393
1128
13953
2462
12634
4887
5138
7962
11126
15081



22027
8042
16352
21033
8437
1952
10420
21247
20139
11118
19561
15955



3808
17939
13239
4794
21353
13160
11932
16466
19950
18656
9789
3900



11330
1626
21761
16275
839
16058
1199
5355
10804
10251
19141
19986



19797
13358
5206
9701
1661
11677
2289
10875
9733
12426
10942
7723



15784
12064
14341
5120
6223
5983
18048
8171
11041
10670
17196
16314



20434
21075
20432
1709
8716
4904
18464
630
8403
6184
18819
13412



17228
16302
10762
4918
9392
16256
4056
5523
9202
16022
17350
304



477
437
537
12703
15237
17792
11159
4597
7291
15512
1072
6812



15476
15612
4183
5466
17341
12261
10729
21044
4892
1108
1406
1630



5617
11923
17859
18198
6967
1264
20318
12469
22123
10452
5701
13091



15516
2225
1997
712
14602
8047
16361
6109
14712
22157
7745
16695



22271
11050
6934
6231
7635
1574
21574
18516
4245


 


429:
4085
2527
1065
15525
14455
18955
8686
14616
12315
16572
15135
18501



11081
5251
21737
20520
691
6761
19795
13031
3691
6753
3599
17478



19773
21114
6014
3843
12406
18706
4803
19871
5593
19334
19269
16267



7830
3347
21925
18962
16676
22334
12165
16013
9753
7457
5446
17495



20783
3532
19992
4641
17715
4676
10300
11655
22264
21804
2482
3288



11375
10949
13571
5392
22506
4117
12147
9177
12485
12082
14102
16550



8227
7887
9151
10009
2194
8035
3263
2456
10489
4324
17182
18539



10448
18491
18289
3508
9164
17132
9114
3276
7050
5077
679
14103



7303
20808
18812
5767
9247
20679
11242
19578
10187
6168
13518
22465



10647
6086
8411
19641
21890
15074
12875
17324
6537
5971
5621
4007



3671
5808
7390
2901
10617
17896
3947
21371
18983
19065
5064
8637



18056
8308
7938
11745
19805
2000
1635
14446
18226
8264
6511
6429



20857
18717
10669
18388
21752
2576
14131
18309
6243
2219
5023
15274



22359
8379
1196
17230
21410
4530
10035
18136
20678
8008
3975
22206



7027
14101
9482
8164
18390
11397
4582
20372
17760
16433
21946
13871



7967
9095
17117
9950
6587
14695
5445
3749
17681
19020
11758
8793



9612
10927
2881
20172
17530
3965
10328
18563
5640
17022
14773
3998



19518
6413
13513
21860
10059
4735
8090
17595
22329
18064
11254
4553



5196
7698
6952
13389
11495
4755
6529
14647
755
3688
17603
11692



15799
7721
11607
1907
5972
12763
20832
5479
6622
14733
843
8870



17397
3483
11493
19618
5858
18527
4566
20709
6716
19382
5368
13548



7884
20519
2015
18102
11949
19869
7193
21204
8094
22071
1179
8989



687
3082
1061
9091
4149
3083
19222
3949
20094
5822
13984
6059



18698
5004
9193
7842
2013
15901
10055
17023
9839
17902
6839
14943



21717
9782
8458
1749
6940
4589
14640
12339
16830
2964
18200
9685



7385
3184
18909
12110
11069
17845
2560
4950
3891
6893
12525
5770



4468
20897
13934
19823
2551
11904
3681
22519
2294
4619
581
12706



20766
3718
18030
8652
13568
7092
5748
13595
21125
11323
6294
7245



16341
18013
21906
7756
20468
12807
8952
4854
12892
20932
17233
15059



14597
10337
3390
2083
16457
2550
6863
18213
15149
18389
13296
2910



16845
800
9561
7305
5114
20251
14853
21958
20953
13079
4928
12827



18923
12922
12166
4134
8385
16411
6965
15063
11981
6204
2303
10341



9006
17038
3101
4223
12243
5951
19417
14082
8354
18716
5876
1912



10613
2494
17074
6579
21936
20024
9076
15771
8144
18836
21381
752



15962
2074
7618
7564
6180
14316
20422
20988
5743
1333
16210
5635



6971
21853
13791
11654
848
13920
16054
21997
16890
8874
3252
14854



18045
15844
1948
2167
19790
7872
15068
14737
9874
19597
19839
10412



18454
3700
11597
3064
19734
6549
14176
16329
18535
22267
1097
4574



8296
12640
20748
9752
18291
15724
1817
20274
2199
11298
12660
14557



21233
19952
16014
18187
20646
4479
15036
19259
10006
11951
14208
807



17472
22405
14906
11172
3156
4287
8159
7673
17535
1537
14931
1539



22567
5737
13603
19699
9407
1651
6379
10581
19260
3165
14667
7864



9195
1596
13152
16816
7742
6806
1616
17544
17707
20454
6937
3411



18482
20603
12839
21210
2018
11104
7947
15160
8893
5218
15123
19207



2443
10521
17888
19288
11689
13671
1060
7139
4723
16589
12750
20823



21620
13574
8321
3982
8782
19710
7192
18007
8616
10833
19376
1910



15804
21373
14004
10937
1232
14661
15988
3876
11632
4668
17814
12594



19491
10832
9955
715
15438
10841
3640
9007
971
6389
21095
16446



17276
14461
2963
15035
11615
3420
8456
9895
20407
21560
16473
17496



7402
5937
16861
18408
18177
14755
15184
9227
12946
18987
11729
4477



14072
12541
20612
4203
11063
18943
22467
12279
17726
13181
1130
19124



12801
22244
16404
4962
17046
2469
2626
10801
569
7725
2704
8430



18169
17621
14239
22342
20154
12018
6110
3490
20465
12047
553
5605



2628
16250
2059
9087
3888
731
1827
21528
6858
5758
9971
9055



19121
12596
9582
4484
11624
14798
6138
15283
7826
6756
11874
17055



9759
17852
4798
22395
20864
12972
13464
710
16153
11807
13928
12179



4667
18624
20452
2395
16837
8335
12798
16367
2473
10503
15101
11372



17828
16195
20015
2994
16716
1892
19350
3636
17068
17570
11645
1327



7475
20149
10068
20414
14198
6292
22295
12872
21980
3940
12542
18299



9655
2119
18038
8871
8658
17301
11886
2039
3626
2453
8245
11903



20419
2748
18668
6038
20076
11931
18793
3924
4613
6432
14061
11825



10785
4319
20931
20293
12868
20904
13240
9080
5315
18550
5568
3790



19960
18123
4607
21807
11927
7809
10838
3410
7219
3828
9117
4802



17766
7596
18685
14850
2868
2410
17525
18227
5587
748
9922
6817



13414
4122
14886
6795
20797
1302
13981
13919
12280
20227
13694
3432



3581
17689
19868
22144
2368
4292
15152
803
7632
8409
12829
22148



18235
17947
2085
4436
22035
19521
12094
1279
20990
7209
22178
9906



5312
962
12098
7610
20672
14380
1096
4197
11149
11974
5660
10939



21240
21535
6515
17591
13604
20996
6136
21870
19319
4234
6030
1908



3858
15291
7352
15060
2897
10563
1195
7862
18600
13786
13041
4898



2026
19040
19171
16313
14963
5034
1080
22252
3765
15468
11773
5638



12937
5393
13796
9419
21859
15071
8173
759
8785
10233
12251
10936



13190
19896
6977
14271
14981
14593
7058
1904
16109
1104
9497
9142



17439
2429
3251
1743
15595
776
11599
957
18835
15453
11273
11954



11554
9554
14263
13171
11110
17426
11221
20644
11573
20653
13888
8606



21124
21083
881
7084
6656
7589
1372
21363
12603
1875
14364
12080



923
17921
6852
4045
16278
13047
5535
17042
3118
9872
3494
11980



20390
19044
13195
11777
10230
14014
9456
5040
5358
12359
10174
7896



15061
19132
17294
9949
7651
19331
16609
5096
11833
5926
12692
793



7709
17994
10740
21923
4044
18803
7948
12708
4621
7049
15789
3183



19757
20773
18237
13118
11870
7824
12271
20914
19595
2904
7799
5575



11905
17160
7767
15836
1947
9981
9284
16661
19158
10592
7940
12764



14604
1540
8753
2822
16765
15532
1631
9675
17723
5039
9590
12994



21045


 


430:
10036
20702
8519
2198
16727
19676
5461
9855
22269
16042
7071
17892



17842
9521
21420
10703
799
9651
7127
11809
20361
1641
6056
19650



2134
9375
17502
21016
902
22224
8402
19626
7011
22503
19144
15490



2388
16766
6922
16601
9905


 


431:
10556
12984
17708
9241
19697
1495
14030
9734
14027
5836
7290
3964



13438
20803
17948
4643
8743
19246
8822
22019
15937
17296
18828
14438



21756
1588
19060
15098
4646
10462


 


432:
9532
10331
15934
2046
6293
19503
15147
5699
7047
14129
17105
9079



5185
13130
2151
9143
1141
10189
18230
19023
8431
18942
18099
5356



3212
10361
22388
21551
20873
5482
17963
7910
21068
13009
6829
14326



20236
6208
21773
15785
14929
1052
4869
16502
3926
4567
14892
13401



6433
9709
18261
6409
22223
21368
11301
6931
15940
17666
21501
12516



3358
6554
1974
4835
20560
11275
2659
5053
16358
7498
14297
15569



19582
20331
6857
8522
22493
2638
19738
13482
13654
7414
12151
12808



14165
1755
10215
8018
22220
22247
7254
20380
2845
654
7274
5061



5018
9568
9818
1494
3725
11235
10859
8938
12700
17001
19302
11190



4028
12045
3173
14318


 


433:
540
20745
4392
9539
7190
4968
12434
2894
9916
18824
15969
2549



13484
14142
5455
8922
14475
9503
12306
9461
18965
943
1591
20055



3802
8501
6629
7934
6828
21142


 


434:
18584
20810
16027
15628
21406
7354
15327
14802
20743
2149
9585
6022



1273
9506
9718
22409
3023


 


435:
20813
11290
7991
15677
22007
11719
11530
9318
1246
18259
1869
18894



5076
8796
1194
21241
4280
6152
3266
19426
6888
17781
10045
1251



15687
11467
7270
18820
4999
20923
20417
3253
11348
11482
14368
12055



19675
16542
16559
11524
9274
4132
12684
10517
11459
11732
9301
1359



17419
6843
11707
16067
9844
20469
16615
1849
2369
12095
14281
7631



725
21902
14026
3032
7802
1337
3062
21087
17626
17682
15601
15731



689
16620
17353
12317
12298
15292
10166
14717
13048
9220
15209
1054



10376
2355
11711
10028
2838
4853
11759
11502
9201
22481
15765
5294



9934
19837
18485
1903
16398
7376
3319
11943
7304
11341
13768
5151



2008
20772
16083
22378
12968
19737
8948
3528
5985
21137
7297
17939



4794
21353
18048
8171
10670
8716
12196
630
6184
9202
16022
537



15237
12703
1072
5466
12261
21044
4892
1108
11923
17859
5617
712



6109
7745
16695
11050


 


436:
16500
8982
2601
9214
5987
9273
2383
3132
16871
4889
1471


 


437:
14507
14477
15421
18062
17494
2654
566
7991
19769
6346
18037
2783



11158
1991
2025
16965
22007
22121
19530
17738
1299
10771
4864
1869



18259
5076
18894
10621
8796
1194
21241
4280
16103
3266
19426
6888



1697
10045
1251
7276
7003
19243
7580
12814
7126
16291
7248
3598



18820
18737
12671
18581
16569
13403
4999
20923
1142
14188
5501
20417



21625
3253
10604
7339
7394
4463
10287
9635
12724
898
4347
12691



8702
12055
20545
15743
3704
10510
16542
16559
21229
11524
9274
11075



8804
17224
3133
3607
10123
13418
15872
11732
740
19109
1359
17419



10051
6843
5509
1680
11707
10865
22296
16067
1000
18908
3406
9844



20469
8719
4041
6395
20688
22346
4623
17333
14975
11866
3840
16733



5395
17783
17305
10611
7631
1274
3777
16608
21902
3032
10224
13407



20759
22152
18157
1337
16447
12222
2772
10847
17604
4797
20901
21087



19143
7452
8263
17993
16659
22147
10166
2993
14457
11862
9220
12697



16162
17146
7667
9071
7594
19062
11610
5047
11793
10376
5672
2355



3480
10028
20595
20275
1292
17594
12254
20642
4853
8186
16379
11759



11502
8591
4288
15333
9201
12553
3846
15765
17537
5294
9934
16724



3395
2370
4038
2297
1916
7144
12063
3319
11943
4162
7304
4298



9147
11434
11341
10440
4734
13768
5151
2008
20772
4271
19954
22378



12968
22114
2955
21137
20802
5035
10180
17939
4794
16466
19950
18656



13358
19797
9733
18048
8171
11041
10670
17196
16314
21075
20434
20432



4222
8716
4904
18464
630
8403
6184
18819
5523
6647
14751
16022



17350
12703
4597
17792
11159
7291
15512
1072
15476
6812
15612
4183



17341
12261
10729
21044
9980
1108
4892
1406
22046
17859
11923
5617



18198
6967
5043
1264
22123
20318
12469
10452
12218
13091
15516
5701



8047
16361
6109
14712
21918
22157
7745
16695
22271
11050
6934
7635



6231


 


438:
3768
9465
20939
10052
11543
2583
2455
10554
14401
2398
18197
15151



12975
22406
10665
9509
15663
5927
13360
6164
16012
7099
21660
8046



5207
4255
5194
15420
1613
12275
14710
16908
3629
20166
7194
4457



5992
7677
13929
12101
15458
11274
20283
7521
14721


 


439:
14966
4405
7118
6146
8600
7777
22037
13873
8489
21468
8655
19547



10433
17970
9362
20687
16031
887
7010
20077
4253
21851
14561
6562



15040
11590
1611
13370
21623
10658
13700
6147
16428
6376
14491
7527



12266
22380
14286
8408
4247
10369
7295
19540
12488
11421
18418
20410



4446
3422
5527
5306
1906
20728
7396
20941
2529
6606
4873
7218



19654
13098
12189
18969
15325
4309
13598
20706
9719
8328
20122
16518



4685
8609
21086
1946
9979
2746
15560
7370
17671
15455
6005
3018



4686
4101
22066
4236
21839
3651
4990
11914
15738
6771
3325
20546



14826
19572
9615
13650
13899
13584
8742
3543
17869
19591
17545
22430



2960
4200
2077
8177
7719
2622
6263
9849
1470
5989
1311
21426



17824
7804
768
15927
14310
9131
19750
19247
21469
14334
14266
14771



6820
17114
15394
15812
13253
2599
12929
5170
19859
15229
15346
19250



1984
14570
11409
5996
20660
21736
20018
22326
19741
21896
19744
14409



13692
3399
3194
17618
14100
18368
2518
690
20346
14843
6521
4745



19127
22103
15018
18736
20115
1684
21656
3590
20651
2319
16368
7672



3740
21690
1173
6846
5212
19894
8599
13173
8940
12978
16135
15091



18853
20615
1491
18437
15682
13879
16777
19476
17619
1577
7748
13631



13915
10365
12457
15298
8901
8625
5241
7358
14305
18568
18524
14410



11871
21891
15823
20498
6200
9264
16240
19297
18458
2821
14785
3126



14169
5798
9737
21776
19585
3198
14858
22300
7338
8554
11016
18990



9617
18528
8024
16993
22122
4137
8570
22146
12226
9200
11674
21612



14895
9476
16664
3321
16193
18447
16049
5313
18953
8533
11938
13683



5508
2740
18932
19696
7262
8603
9275
18376
10691
20818
7714
1458



12356
7720
7819
17020
17844
13115
5156
10998
6550
16814
12176
777



14663
18952
3941
6256
10163
1231
14652
12351
20934
5346
3674
3417



3413
20689
13348
3105
5424
8660
8772
19183
22278
21459
16873
20072



17128
16743
19690
4218
3991
11096
10955
5602
886
19026
21015
19125



13706
10726
19356
12958
17574
18510
16516
22357
8610
3240
7095
3522



21988
5616
7937
3174
1747
3775
10024
6702
4908
6372
2579
18940



21345
5519
15356
13540
14244
5795
14821
14518
20309
12120
2485
17587



14150
15819
9978
12910
11595
16932
1960
694
14412
11488
18681
8778



12679
17521
8810
5092
21175
2974
13020
10931
21585
2789
4153
18786



18329
1083
19178
2493
21206
9256
15592
13324
16856
12883
15416
22022



685
13962
7186
2183
1824
7815
9340
13838
4490
22377
18686
9359



16226
1355
9864
4384
15337
14450
4671
8942
10589
3962
9323
19836



12509
15215
5193
8404
6144
21817
7541
10871
1525
21274
13622
9110



8628
2177
7874
14877
8824
18554
3163
18907
15713
2835
16426
3153



20949
2770
7004
14833
12848
4690
18093
18783
15352
8130
7593
18583



3501
4178
14120
14899
3694
21340
1642
6921
16503
11048
5058
2817



9969
13347
19955
11925
20475
21679
16040
21148
2936
11490
9879
3668



15510
9259
5266
11317
15129
15828
17404
18074
18271
12632
22568
6154



19613
4529
14306
9441
17517
20968
2859
19635
2252
15293
3989
7716



16280
13326
11344
11586
14234
9625
5215
13361
1156
16246
444
20628



5316
13449
3080
16715
10685
19801
5171
1815
3509
16084
17427
995



9018
10526
18574
10099
13402
17849
6159
17431
3824
555
9659
14148



9887
22551
16739
20950
2267
11455
5169
15137
17589
4048
18400
8026



1816
14675
4107
22512
7319
16725
20176
1188
3250
10266
3394
15649



6275
16233
11157
7931
10836
20212
6842
21580
18201
18602
7675
17976



14515
12856
12375
20806
1835
10204
928
877
18843
14059
6197
22517



12239
14250
21061
548
2492
19311
18449
5757
16595
21416
19162
10349



18182
5204
19779
22446
2313
11619
20729
18442
6595
12083
6350


 


440:
13290
13287
12491
9089
17221
9777
9141
4749
6525
10696
2418


 


441:
5159
5787
3587
20298
1786
13310
1389
10380
15437
20220
15554
14498



1082
9121
21871
18718
1610
17142
16098
20306
17432
20695
8310
15692



2042
20770
15746
11972
20448
7362
5959
17865
8865
4630
3037
6655



4037
16478
11125
6160
10003
14982
9652
7150
5560
21884
7298
3602



9622
20738
13828
4091
21632
1885
10098
5784
19579
11915
1619
4973



4953
15722
18588
9009
3874
3245
15203
22499
1147
19826
15124
8880



21552
2999
14657
21323
6418
1393
5831
1681
17953
9370
9708
13283



7660
9230
20542
6634
21649
6803
7555
12376
14548
16219
9559
21885



10474
5522
13930
9391
12268
15620
22031
14151
12757
13437
1509
3933



21317
8728
11995
13090
16778
17402
11699
16105
1278
2276
10571
834



14094
1564
4767
5915
1665
19312
9166
2756
14136
20976
11340
16028



13738
1229


 


442:
12225
16626
7991
19769
20707
21228
18920
15677
22007
1246
1299
4864



1869
18259
5076
18894
8796
1194
21241
3266
19426
6888
17781
10045



1251
11444
7126
7580
7248
17460
18820
16569
4999
20923
5501
20417



12724
12691
8702
12055
8006
14571
19675
16542
11524
14891
11459
15395



15872
9301
740
19109
15861
1359
17419
10051
6843
5509
16067
22296



18908
12031
4041
3840
17305
10611
1428
10769
7631
21902
14896
20759



22152
18157
1337
10847
14596
3679
21087
17626
10092
3743
21220
22333



4831
12317
17353
2073
22147
9664
11526
9220
2983
5429
13258
10529



12270
3138
6868
10376
2355
11711
9314
6624
17713
17898
12386
10028



20595
10383
4591
11502
11759
11992
15333
9201
12553
15765
5294
4038



7144
1916
10304
3319
11943
7304
9147
4298
11434
11341
9677
12289



5151
20772
19954
22175
19748
3020
16083
19737
21137
3274
6780
14060



10848
7124
17939
21761
1661
9701
6223
5983
18048
10670
1709
8716



12196
630
8582
9202
16022
17350
7291
21223
15476
12261
21044
1108



4892
1406
22046
11923
18198
6967
22123
20318
12469
10452
8047
6109



14712
7745
11374


 


443:
620
12442
20304
1418
14021
9656
19898
1753
11261
6048
9339
20785



14195
2548
20087
4601
12996
2180
21054
8933
8763
20471
14140
10491



10186
6478
12177
17437
1521
8613
20482
15739
2169
12190
15573
15908



1717
6770
16901
15379
7349
10141
17028
12150
9400
11457
15837
15021



17531
12650
16974
10219
14993


 


444:
7118
14966
4405
6146
8600
7777
13873
22037
8489
21468
8655
10433



19547
9362
20687
16031
20077
4253
21851
14561
6562
15040
11590
1611



13370
21623
10658
6147
16428
19540
10369
13700
14491
7527
4247
14286



12266
12488
6376
22380
18418
8408
11421
7295
20410
4446
3422
5527



5306
1906
20728
7396
2529
20941
4873
7218
19654
13098
12189
18969



15325
4309
13598
20706
8328
9719
20122
2746
15560
1946
9979
21086



17671
6005
7370
15455
3018
4101
4686
22066
4236
21839
3651
4990



11914
15738
3325
6771
20546
14826
19572
13650
9615
13899
13584
3543



17869
19591
17545
22430
2960
2077
4200
8177
7719
2622
6263
9849



1470
5989
1311
17824
21426
7804
768
15927
14310
9131
19750
19247



21469
14771
14334
14266
15812
15394
17114
6820
2599
19859
12929
5170



15229
15346
19250
1984
14570
11409
5996
21736
20018
22326
20660
19741



21896
19744
14409
13692
3194
17618
3399
17236
14100
18368
2518
690



20346
14843
15018
1684
18736
20115
21656
3590
20651
1173
5212
6846



8599
19894
13173
12978
8940
16135
15091
18853
20615
1491
15682
13879



16777
19476
17619
1577
7748
13631
13915
10365
15298
12457
8901
8625



14305
18568
18524
14410
11871
21891
6326
13311
6747
13835
5310
2038



11820
1116
18682
439
21539
20042
6396
15465
17351
4069
12600
18504



4527
20683
15823
12851
18338
6200
20498
9264
16240
12538
14814
15115



18458
19297
9737
21776
14169
5798
3126
14785
19585
3198
14858
22300



21664
7338
8554
18990
21738
9617
20013
11016
8024
16993
22122
4137



8570
22146
12226
9200
11674
21612
9476
3321
16049
16193
18447
11938



8533
5508
13683
2740
18932
19696
7262
8603
10691
18376
20818
7714



1970
8639
12180
20962
1458
12356
17844
7720
17020
7819
13115
5156



10998
6550
16814
12176
14663
777
18952
6256
3941
3417
3413
3105



5424
8660
20689
13348
8772
19183
11096
16873
22278
886
21459
3991



19690
4218
5602
3174
1747
6702
4908
10024
2579
6372
18940
5795



3775
14821
5519
15356
13540
14518
21345
14244
20309
12120
2485
9978



17587
11595
12910
15819
14150
694
16932
8778
1960
5092
17521
2770



2183
685
22022
12883
15416
13962
6144
1824
7815
19836
4384
4671



16226
8942
1355
9864
10589
14450
7186
13838
9359
9340
18686
9323



3962
4490
22377
15337
15215
7874
5193
8404
21817
7541
12509
3501



10871
1525
9110
13622
8628
14833
3163
18554
15713
21274
8824
16426



20949
2177
14877
7004
3153
18907
12848
18783
14120
4690
11925
18093



14899
15352
6921
18583
19635
16503
3694
21340
9969
7593
11048
11344



11586
14234
13449
3080
16715
19801
3509
17427
16084
10099
17849
13402



6159
17431
14148
3824
555
9659
22551
9887
2267
16739
20950
11455



5169
15137
16725
1188
20176
3250
10266
3394
15649
6275
16233
11157



7931
10836
20212
6842
17976
21580
18201
7675
18602
14515
12856
12375



20806
1835
928
877
14059
18843
6197
22517
12239
14250
21061
548



2492
19311
18449
5757
16595
10349
18182
5204
19779
22446
2313
11619



20729
18442
6595
12083
6350


 


445:
21014
8217
3568
15315
16602
22197
20633
10719
22273
7927
12557
3214



5391
17035
16717
18176
3239
21949
541
17286
20722
21074
17292
19789



4688
6904
4467
1219
19816
19041
17181
21332
18859
22211
8218
2257



6585
7606
16882
11061
6736
15760
18569
22238
10997
3586
4444
11325



16288
5712
12397
13355
14617
8607
12841
4510
17747
8828
10495
7078



11921
20794
5507
14664
17923
2708
13572
15775
2513
11167
14105
3111



11389
3370
20781
7301
10605
20844
14047
14130
22186
16977
10134
11180



19900
10713
1464
7899
2349
6473
6149
17373
2862
15797
14327
10299



17623
20126
6074
2179
9070
18653
19230
14764
18671
15774
11854
9254



7597
9029
4935
1891
7776
14288
7040
20201
10153
16227
4268
19425



11328
17458
2842
14940
6228
15942
6091
22429
723
13304
7975
9105



16127
19403
11783
823
6252
21376
13980
4302
22514
6008
19617
15008



5641
9634
543
14573
2873
11280
18674
12111
5653
8223
7146
12775



12377
16801
7816
4017
16666
19793
7002


 


446:
10867
18970
4967
22194
5235
18316
19541
18967
16679
12435
17897
14632



9836
7200
3244
4931
6155
724
767
11956
2291
15034
1158
4627



22468
2941
13822
6431
20840
3978
3058
2820
4352
17463
13357
17354



2351
1025
3451
8214
2208


 


447:
3349
11861
16061
20185
18887
5336
6597
12949
13433
7265
3983
19704



21765
4512
9383
17441
11806
19145
7957
1660
16578
8361
12877
12285



6745
14055
16201
22143
8158
1754
1561
9710
18005
11717
18079
2243



21572
10276
6672
1451
5576
13128
10885
6027
6068
15496
10454
833



15308
2899
11018
20697
14627
3974
7859
15270
16400
15938
15725
15423



5811
19619
2698
19513
5502
3862
14005
6242
14353
16413
5739
5131



1844
15747
8386
4651
22360
19139
7085
13831
22094
8195
21288
3065



16776
4822
18118
19091
5553
12572
14834
21349
16815
8955
13075
12733



4648
21653
22049
5403
1029
1877
1485
22486
14277
19152
7372
4606



21858
17690
9703
12831
6467
10808
13527
19173
11175
11657
2942
16325



1265
17141
11133
17532
18407
20507
14912
8705
18889
792
12024
4746



6954
5377
14106
3811
9106
7669
17506
18207
11911
10409
20449
9181



18488
21792
22305
9003
12668
7458
11471
11697
19342
10966
5324
17192



9173
21172
21538
9379
8949
22434
3555
19594
13734
5195
4504
5963



14256
6939
6345
1162
18406
2357
17784
1442
4331
6935
5879
18326



2240
20428
19414
11272
3444
14731
8631
1809
21833
14869
9442
3632



12121
8845
20490
19745
13155
12792
12563
14861
14746
14813
4528
17811



1567
2911
10316
11855
21730
13905
22042
3872
20386
9570
6304
9122



6011
10819
2612
733
11892
20852
15230
16137
13985
13024
11612
8752



1512
3948
22301
14196
1070
2951
8433
842
12944
19948
22053
9341



9353
1718
16198
8546
15048
9919
20674
21812
3909
10843
16432
15654



4043
6714
14847
8707
9886
20997
22088
13038
8037
9178
2878
21388



5352
3851
6484
13259
20733
16180
20444
5834
17651
21480
13792
22149



13049
13689
6933
5650
21822
22010
13935
7093
15175
21104
21571
13059



14166
1320
14676
19388
12387
10615
2572
18647
16738
21225
7210
15278



11859
4902
21097
13308
18378
12809
17277
1707
10671
4638
13485
7397



2352
5048
7941
13275
5252
11316
3053
9127
6631
5081
2879
17029



14783
12504
3446
5574
11878
2839
3228
16798
6179
1088
1402
19752



22525
21445
16859
10813
2895
8708
2051
20050
10689
13359
6382
9920



16804
3557
13937
22048
15372
19186
2356
21850
10645
17724
7740
18043



18443
14254
8080
7679
18267
13583
7611
8668
2471
7128
21146
15408



1163
8078
5556
13897
17262
5961
22314
15730
18968
650
8513
11528



7068
3085
14046
13243
11222
15011
3976
1105
15891
1507
6229
10269



17937
9774
5505
5939
7480
1497
3041
15132
9987
18054
12199
21835



22529
20727
7448
2979
5186
13110
17553
15284
15066
21091
12304
13927



2253
22173
14128
9863
4969
20557
7177
21588
7134
20606
17839
15095



4399
2214
21594
19310
3008
2570
8102
16155
4099
18252
20237
9891



20458
14831
9119
9194
7573
11856
2558
21715
10855
5766
15158
3389



1154
11165
21797
3985
15097
8881
12231
16104
647
19765
22387
4155



2561
4615
21305
15139
4964
16030
20281
8285
15689
12785
2196
1531



12335
20138
9927
1132
4098
6925
14275
10776
22136
15697
16176
15977



2664
9816
17562
11028
11930
10157
8670
12353
12574
2954
21428
12735



2392
5226
8680
17177
8835
7167
15667
18591
21341
2172
3869
15589



20343
19825
5210
7091
22444
3844
717
18902
14808
11659
1785
13405



22226
8233
21693
10456
13156
14660
12659
6461
10253
7132
5280
16881



15728
12322
846
14049
12744
8140
20493
15683
11033
12989
5866
18113



1964
14109
15020
20584
17016
18813
5845
7915
21617
20412
1643
20533



8020
9158
19107
2930
21064
15660
21586
18572
10523
4610
13545
7585



1761
2480
16383
16486
1541
8462
2592
3201
11077
14726
20135
9411



9402
12669
15995
15348
7755
8168
551
8916
3112
13876
21933
12219



14513
6877
8898
8597
18116
8268
9085
9769
11890
21961
11709
10758



12800
8640
1212
10509
21023
21751
11267
1557
18735
18051
21254
15200



5771
17912
3381
18702
19564
12819
15172
17593
8712
8128
1909
6496



14338
16091
948
17565
16614
18595
11540
18075
9306
10608
12167
9268



17453
12880
21522
13525
10162
5843
7885
18002
13496
9474
11489
13260



14139
10989
5078
5480
16165
3428
2826
5882
20175
15811
16116
7879



5318
8325
1069
22306
21302
6156
22394
14294
5702
4766
568
15271



1077
16409
17778
9726
14452
21497
5648
10532
3647
15330
8350
18444



19227
19834
3890
21780
12302
13191
21151
13104
12185
20101
10857
11960



7382
18469
4948
11036
12852
11208
9994
14197
5906
16060
20197
3576



1115
6471
4258
14545
21796
17471
17991
14998
3215
307
10382
6929



2580
6731
8289
9480
9118
11333
12900
8452
12605
11475
19577
6041



2991
13102
15010
21059
21365
6894
11757
21208


 


448:
17617
22319
7205
5436
22282
1508
1614
12460
8290
21670
8595
18790



19872
21481
5282
5245
2363
9283
17848
10766
892
11279
3262


 


449:
13633
5900
8539
14672
20311
964
12233
10478
5805
8760
4906
10137



3902
1838
13999
21067
4596
12781
4010
10761
8601
11786
6187
19637



20291
12933
1404
16680
16262
4172
11107
2757
4381
11802
14386
6750



3881
20169
3124
11582
4779
14837
16691
21558
16323
14350
7275
16234



10634
8984
12657
3036
20253
12586
15643
8362
20529
4086
15778
15634



10975
7311
15107
8618
8213
4431
2716
12985
488
2869
18696
12025



7468
6477
4900
14759
645
8641
3783
17104
16899
21903
9293
2872



7277
12282
7350
15064
2602
14650
13112
15517
7433
889
15267
21911



19806
10064
15339
1645
20835
16525
12947
16705
22317
19397
18277
11661



16082
8992
9337
17911
12718
11845
12581
12948
1001
4151
15609
5506



19445
5163
11501
20741
15240
10944
3217
5255
11017
20435
15575
13834



12427
2375
8287
6080
20581
16884
20690
12613
4380
15312
5145
10145



1126
13494
15530
15431
22255
22276
19919
8963
4880
20168
20601
7009



15363
10724
8126
7044
2098
13898
22554
17967


 


450:
1006
429
10217
535
507
533
306
478
314
12870
18461
5240



6367
19001
17394
8913
526
17484
7812
286
534
531
300
21030



2857
17664
21589
11961
19154
21437
11195
20933
21506
3533
4677
18511



4730
696
9569
17797
18687
7097
20259
15252
13221
22502
14915
21157



22126
18059
20371
3563
12591
16308
7626
4212
20262
19798
19458
5691



18292
11428
18877
16130
21296
2832
830
14553
6434
18295
17956
829



476
12043
9077
20779
1281
17745
16586
7955
12705
15449
988
20859



1768
11129
6547
22518
5552
9854
11376
6669
5359
9132
10964
1879



22087
13137
17298
3333
13752
20301
19370
2425
6946
19538
14273
15340



6427
17165
15603
12419
10809
8003
13658
18296
14064
4221
14568
17630



2371
4694
12396
15369
16597
3697
10741
16309
4927
13396
9785
21621



8841
16662
11120
14081
15204
6565
14320
2978
9294
7482
9493
1277



4952
7813
7306
12296
12021
6645
9286
22489
13745
16653
19069
7780



15219
16969
14762
18330
10802
10479
16663
3678
16713
7751
13703
3630



4691
9472
10709
8542
7060
6112
22457
21974
20476
7333
6482
14526



7151
2644
835
10655
12264
9315
2786
16253
9488
5634
17372
16548



13727
19096
10973
788
10889
5190
13207
16465
13178
9673
5374
9771



11716
10164
20541
3458
1240
15787
20099
9282
11480
4994
4865
1120



6281
18662
7992
9661
19875
11156
3619
14086
20948
16946
3456
6143



786
6709
5954
17926
20839
22351
13277
10965
15558
18982
2984
11245



2132
20312
4655
16568
21093
13882
5730
7976
7079
13035
2800
305



307
509
12545
2037
11704
9571
21379
3712
7685
4483
18456
22341



3094
11197
13107
16006
532


 


451:
3345
751
19630
15890
21334
2502
16406
10364
15556
10330
10995
14378



13458
16021
7590
20173
3502
572
19897
3673
12693
3848
6462
21994



20804
17582
4276
16244
8615
17497
11060
9796
2390
21178
7166
2759



19905
12817
14186
14243
5490
6784
5792
13470
1101
12561
6617
11241



6402
9027
20751
1813
16709
11278
10008
3046
7793
13443
5592
8736



3403
14205
19027
2511
14794
10004
16347
3927
13109
8732
21235
10480



19155
2047
21601
2804
11826
19016
20937
21978
13295
3255
17452
6272



19822
8274
14395
20792
19448
1967
4644
19536
3230
8051
21408
16552



17526
4434
6675
10332
16527
11695
11834
4106
21315
20826
18978
17269



22168
21255
19151
14377
2774
19782
12983
9784
9962
16564
850
18492



20740
21582
7445
1779
10977
17687
11634
18743
18450
11001
19140
11439



11743
13008
6064
17602
13592
13552
15418
11134
16033
20106
17363
12458



12874
6513
15906
16223
1784
874
1125
9165
17183
4284
8998
583



17287
963
5591
19466
4320
9188
20258
21605
17648
19691
3335
6816



19609
22222
3733
6330
8114
19196
6605
22558
3373
5980
4992
10542



21937
2186
4838
2923
20053
20960
4335
13352
2747
5898
8059
18499



19914
7292
2255
21251
19565
10896
14138
1474
1803
22420
2054
18604



4980
12543
19544
7953
17754
18426
21889
21464
7125
21785
14725
10578



7357
2460
2281
4747
13666
6881
21446
19660
14216
1004
17442
21195



9433
21820
7213
8549
21471
8344
17607
6026
13716
12371
18944
19208



20867
13887
15334
8749
16682
14433
12986
7761
12475
15833
15505
8725



627
20270
7644
15253
15959
13593
13483
12343
18196
3966
1640
20730



3351
10803
1315
1603
19482
6421
12053
1137
8850
12068
12197
8734



19465
2535
2409
22297
7627
19726
1553
13474
15450
3620
5719
11963



3815
19768
758
13039
16269
11639
7621
5670
7361
8209
17309
14298



22309
19799
6584
3893
10041
3814
2858
7122
20012
8232
12230
820



11268
7920
3024
942
16980
14141
2812
19328
19907
16962
16151
12559



3658
994
16669
2146
14691
14451
2080
3146
18837
7883
935
4402



21465
15552
7472
6823
18275
16984
17026
7620
5223
8813
21417
1708



22055
15118
13508
19764
22214
16335
1874
6227
19122
5567
20812
16276



19961
11837
11145
14544
15166
9889
16056
20845
14946
6840
72213
349



14215
14913
12175
2181
19628
13753
5595
13445
8711


 


452:
21725
4108
17790
10816
10072
11215
7845
21938
14000
12290
9072
2188



13197


 


453:
17069
19235
13531
17697
11206
10541
21126
13101
21378
21802
1329
11808



2880
22230
1218
9626
19047
3040
17741
3001
15767
17052
3724
7152



16952
20547
5794
17379
9932
18642
18433
2347
8182
3667
10010
9258



4330
16872
13931
10125
15345
15750
14295
6095
14904
11788
22535
2662



5270
2833
2917
19197
11031
8241
18241
12357
932
9546
3545
5011



5922
15762
21458
11169
19093
2950
4496
8786
17325
12345
8819
14838



11101
7574
21973
11052
5177
10577
9162
18537
19268
11571
9690
8586



17826
10897
21454
12803
2767
20158
11415
6066
16018
13857
10790
8706



12567
13232
21790
2221
16327
22364
9930
11940
14381
12027
5625
20421



6213
18883
15885
991
14231
1117
8576
18603
5865
1558
1668
7347



20793
5291
16647
5611


 


454:
10637
22601
3315
5883
11574
14796
9991
4552
7533
6653
8525
1420



15386
21880
6261
12607
8500
22030
1956


 


455:
7653
1606
10840
3715
10418
14035
4843
2101
19615
10922
3554
1735



3155
13503
6907
7784
20054
21733
1169
1452
5818
22385


 


456:
0


 


457:
3034
22132
9120
13729
6791
11897
5149
22382
7782
1951


 


458:
21696
15323
6711
794
10202
21441
6649
17320
15012
6988
16064
7702



2108
7337
13972
18351
2384
18596
11446
5769
20712
818
9873
11398



20352
19777
18363
16843
5030
6560
11056
12761
9908
4279
12906
8596



16833
6392
2335
20071
16868
4580
21828
17448
14949
21452
5673
10050



5093
22411
1705
11285
10329
5612


 


459:
655
11975
8069
20491
17684
1394
19340
17701
5908
684
14348
21852



5434
14730
2715
13227
21854
8282
16106
10308
21982
7567
4049
15967



16174
14809
13372
20868
798
20016
19024
1342
3575
14227


 


460:
19142
17102
15893
22335
12299
15442
11496
10074
5733
14018
4497
12860



17771
17375
8161
11868
317
11262
6451
9249
12173
13520
2381
4681



6996
6490
9579
1276
5404
8032
6363
10026
7316
14084
8908
17837



8863
19169
750
18053
13467
9093
12920
8746
301
4625
12849
8975



1460
8614
20285
11049
15351
15199
22494
14045
10173
6414
13682
21841



1623
17211
18365
4519
21203
2011
22000
10060
950
10516
18657
11078



6908
20626
18762
9992
11321
16068
13071
20575
9335
10629
4241
17420



13570
10156
14609
12058
7665
21534
18040
5706
574
11168
21837
3354



20317
6124
1526
12392
16093
22474
21550
2099
15300
14848
1309
13170



19435
2632
6374
14407
14649
12249
8309
550
7797
3769
17901
16142



2063
6092
19535
13070
9812
1776
3443
17767
15918
12414
2144
9885



3591
9545
21263
14344
19754
19431
11670
6760
19223
12846
21920
21081



22381
17475
18403
21905
12709
15904
5736
14499
13058
11572
10692
7183



16133
15670
12332
1756
16939
22104
4806
15031
18332
20634
7994
12919



9064
5045
14968
9551
2414
1646
4753
1805
18175
18991
9750
1618



17712
22081
22029
14969
2458
1011
14504
3779
21507
6619
4824
5015



20098
2353
13906
16938
1847
13819
11957
12498
17391
1983
2927
13991



20531
20599
1625
606
8646
7497
11561
11796
8562
3676
16567
11565



15599
13705
12415
13948
1711
2922
7520
21985
563
3562
1939
5754



19800
6341
9042
15748
16577
12974
4827
4371
1092
14881
16895
20528



1513
6790
19711
13821
5656
6520
2463
9526
17821
1888
18587
10549



18855
4858
7028
14908
22205
5964
4933
4340
5271
927
8374
3316



20014
21527
5008
9914
10822
13989
15266
13163
17347
7197
21212
6338



1401
3445
561
14444
20426
12281
9349
20565
7453
19524
4093
17235



8735
14800
9217
16762
10080
19195
11057
11977
8958
21372
19693
20196



6610
14503
22133
19181


 


461:
21226
10722
9090
9917
17608
3307
17248
19659
12888
16315
15869
13541



11628
14989
5547
13844
21236
9910
11202
10862
3786
7783
20043
16159



21123
21149
1353
5976
20875
12245
2040
1496
18804
8260
2945
3653



21684
4427
20639
22232
20700
626
868
17999
20858
2479
14206
10988



22292
12471
7299
12462
21503
15092
2732
4462
16160
13957
17698
19014


 


462:
10915
15022
4450
11366
20814
8548
7447
2554
10246
14947
16967
18188



8578
3396
12439
21412
21085
10559
15980
13299
6199
5121
3610
8985



5265
4663
6420
19456
9646
7090
9324
14666
21020
13611
3580
3309



3503
10431
15493
1658
4414
10484
19347
18475
3196
10873
7062
5347



14932
697
8959
9970
13746
17123
19066
3791
6974
21237
4075
21728



13369
5804
15214
3905
7104
15534
15425
15645
14841
13863
17523
14583



13266
16008
19678
809
17661


 


463:
15610
19505
3188
13558
22207
789
18180
4508
22145
6608
12144
14753



3301
17632
6183
18032
8172
21197
9377
13065
15119
10305
14673
12954



12468
8469


 


464:
7246
2525
8937
1249
20791
2442
13757
2102
8484
20391
22436
8284



6769
10971
8004
22023
4837
6659
8039
542
11456
2562
13478
12526



20405
19784
19698
17561
14237
21200
20356
7320
10248
1737
18419
11847



10624
18917
21915
21121
18416
17053
8529
2009
1335
2989
17827
5789



7577
14095
21524
13803
19598
11276
12421
21337
2916
21144
20276
15766



10716
15947
19883
13961
9267
13637
16960
15005
18795
2343
10609
15000



4509
3285
1871
22443
9056
14293
11451
17137
11684
5432
21943
5119



17025
20216
6625
22445
4609
21217
19722
18262
17059
19393
8393
18989



14155
18379
19003
4754
6752
7557
21304
14324
2929
15268
11094
6025



8713
14222
2187
1975
2856
8286
16534
8056
9369
15871
22373
5414



5457
8512
14839
13626
13511
18521
18560
2002
4104
11038
15162
19653



9129


 


465:
6459
6394
20221
2332
21782
13055
10580
2279
21367
19818
15567
13294



11815
16237
17819
16570
1622
15858
19159
18654
9082
10360
8884


 


466:
17835
13628
20663


 


467:
20037
7346
1314
12511
16115
17491
18854
2329


 


468:
17297
12034
9186
19531
5960
3320
6107
989
17365
1448
5740
10535



13789
18153
3130
2668
16090
6274
11423
19113
19718


 


469:
8693
16592
5005
19306
6604
16732
9754
13116
8230
21139
4079
5166



18517
6465
2795
16685
16688
21959
6043
13767
6173
22310
3259
13685



20252
7455
5044
3534
9389
10900
11649
8773
10993
11006
9330
11087



5753
19387
21478
15236
21689
1435
21991
913
7484
14285
18519
20798



16122
2721
770
15723
20967
12158
21702
18984
12751
12455
5999
11116



11824
9843
12418
7936
7869
5363
4568
3972
16205
22566
18031
20763



17285
18882
7781
5471
19353
4312
4716
18663
9603
15043
11217
14246



16326
9289
1155
13216
13122
13400
3648
8238
13201
897
19164
9520



12035
8212
12560
22169
3281
12562
11339
17667
12309
8806
22280
18666



22125
9307
6640
20092
2145
19241
15914
1213
13453
5332
6133
21968



9060
20524
10171
20916
20091
19282
10648
21566
4242
19533
11739
14917



21058
17627
7451
13855
20246
872
19965
10868
11173
2843
8856
6802



6353
5160
6251
13175
12961
12248
21646
18733
1820
15077
6061
2722



19163
13367
10128
22117
17264
16640
22485
19560
8034
1100
17788
13010



7758
19682
1515
12783
21145
7575
3152
16168
2508
13380
8293
1473



9684
17719
8565
4032
17866
18951
14592
14121
19857
5090
19600
22439



21996
16150
952
14805
7722
18382
14204
12365
18343
18356
17329
19831



6021
4198
17799
3839
721
9477
11433
18772
8387
1287
8906
17051



17649
19824
20739
20085
3542
5975
20850
9850
9401
8873
15065
7512



19446
6993
10543
8853
18552
12124
18137
16070
1895
21102
9226
7191



4365
12533
4665
5929
15384
20652
15768
3859


 


470:
4094
4249
6447
417
9699
4266
21799
19998
8507
4118
4930
21755



7422
16272


 


471:
5778
13042
20693
6337
16080
1704
20548
2346
20737
5152
6633
17650



2524
19716
3465
3548
16517
9459
12501
10090
1759
3273
12472
21412



14456
8135
18797
3137
17061
3402
9770
1269
2404
4759
7648
8568



8662
21076
11318
18004
13312
14804
6000
10393
14666
21987
3309
18931



14379
6524
14185
6945
8382
7629
16509
1933
4728
21025
17218
17638



1752
16078
3430
16885
19841
13228
577
3035
15647
12625
14586
21237



4075
7439
13720
15834
12529
21705
3905
17339
10133
5868
20567
18269



22275
13814
5994
10239
18618
20112
19325
13266
2962
2905
3571


 


472:
1296
2585
13077
15698
8567
10322
16480
13708
15389
17974
17451
15792



19280
10142
3469
11718
8642
15847
3638
2801
9326
3742
10221
13816



4884
3280
9923
15873
20214
10492
13528
8475
3713
15933
21666
11740



1633
11990
9134
17965
1788
6764
20492
17131
19212
13533
16793
11371



8594
20347
3728
17461
13823
8134
22260
7419
19185
22026
3272
1884



8730
8990
19977
1859
10999
4586
16348
19281
19991
11567
1008
20028



7658
20830
19756
20878
3207
20070
2980
18632
2087
6612
5122
7489



19025
20408
5867
8896
19056
19348
18818
3699
779
22456
11541
15161



11135
20058
2201
6739
16612
14992
19392
15341
8872
4204
21922
22540



19457
14075
4186
15353
17073
5661
13025
14487
6849
20671
6103
10343



4142
17366
14159
18164
2380
8153
15839
19958
6670
2506
8947
15249



9712
1377
18798
8620
19625
10515
9429
5493
7939
18930
8714
19761



3572
20073
19642
9602
11161
7180
10987
2971
8438
4675
8709
2938



4636
3485
9435
18162
15411
20078
14563
15262
14349
19829
9555
8928



16342
19361
1362
12060
11259
1940
18358
7454
21475
16972
20517
14458



5311
4246
3026
9206
21394
17932
5647
18756
632
3100
13204
18856



14390
13351
12707
15711
10265
18751
14958
5221
17138
17198
808
10940



11642
19473
3009
13298
15678
3551
14990
9133
19864
6205
1024
9670



22172
21447
19672
2996
6377
6123
21676
2672
19700
21984
15674
9974



14920
2725
11780
7537
15508
9998
18115
3670
16824
20178
4972
18635



8472
13466
21265
2831
8951
14511
7701
18486
7554
17622
11381
6309



22198
20143
15854
21658
12398
12681
11609
16625
12161
6162
3022
5109



7314
14439
8366
13945
12198
11746
13801
5301
15197
3210
11365
15206



17546
19669
2720
10926
1742
6963
11828
6972
1267
14550
8450
11591



19644
11212
14447
15125
18385
14497
17159
5903
832
17245
7432
3663



3061
9406
7511
4860
10188
13056
4196
11132
3684
3623
8675
8119



10193
16296
17100
11852
16004
17566
10552


 


473:
2421
20836
18507
17807
18922
14668
9172
2056
648
16255
7978
2322



11200


 


474:
17916
15231
15741
4645
21977
15829
10291
21573
1806
2663
8036
9618



16693
3960
15864
14578
17125
15924
21826
13440
17249
8650
20159
15742



1986
19706
22092
8766
6813
17830
10853
21281
13394
5285
8139
21004



14220
17563
2086
2488
1597
4698
13233
4654
1250
15737
2907
1469



9957
13288
6516
22526
16496
14873
10471
18290
3086
11953
18592
3185



9418
17135
8081
9593
19180
4673
7979
1300
13933
16544
16782
15551



8460
15960
13997
3405
1566
21046
8636
17134
6596
6512
13346
15639



21591
15042
12093
14396
9252
18637
22523
6953
16784
6262
16933
22448



4612
19863
6076
19303
12192
16828
17089
4133
19601
6118
3344
15088



14986
21070
2153
771
3291
10644
20638
4377
21183
9519
21234
11970



21215
18173
18301
17764
13810
10948
3781
21029
11162
16613
18091
22003



3801
21866
22213
6526
5846
20765
21771
14860
5007
861
6743
5529



14267
14880
21391
10210
5693
5970
3793
15855
1007
13001
6878
9875



16912
19329
13614
10333
13714
8204
21112
6903
1133
21262
21547
15703



21338
6248
15242
13567
16788
11020
18655
19496
10528
22414
8142
17440



20388
7760
17480
18357
14595
12914
2829
16249
2569
7096
6689
12534



6105
16041
9145
9242
1552
1379
10313
9596
10872
11771
3268
15445



593
5820
18410
6984
14744
9713
10053
1383
8837
676
4305
10421



2944
20363
19120
7463
16753
20969
18430
10227
12905
11066
6057
13677



18640
4083
1527
285
19285
5385
17557
20851
15693
17304
1683
14391



15965
19854
8425
14916


 


475:
17494
14507
18062
7991
15677
2025
16965
15154
22007
1299
4864
1869



18259
5076
18894
10621
1194
21241
4280
3561
13154
8656
12338
18120



3266
19426
6888
17781
1697
1251
15687
7276
7126
4960
7248
3598



8316
21056
7036
15143
13926
18581
16569
4999
20923
5501
4338
10604



10287
9635
11127
8702
12055
20545
19675
16542
11075
17224
15872
11732



6843
1680
5509
10865
16067
3406
18908
8719
4022
6395
4623
22346



12095
17333
5395
17305
10611
7631
18432
21902
7759
3032
10224
17508



1337
3062
10847
4797
20901
3679
21087
9348
17993
3743
10092
14394



9088
16620
9528
17353
12317
17070
22147
22431
10652
9220
12697
20558



11610
11793
17146
2355
4084
3480
10028
2838
20595
20642
8186
16379



10383
4591
4288
8591
15333
9201
15765
5294
3608
4038
2297
7144



1916
16736
3319
11341
10440
4734
20772
4271
19954
7624
2955
17939



21353
11932
19797
2289
9733
10875
12426
18048
20434
18464
630
8403



6184
18819
5523
17228
16302
10762
9202
16022
17350
11159
4597
17792



15612
4183
5466
17341
10729
21044
9980
4892
1108
1406
18198
1264



712
13091
8047
14712
22157
7745
6934
7635


 


476:
3284
22524
15910
13589
1006
11575
20349
10911
18523
22545
13067
2372



10217
9609
535
306
507
533
5233
478
4966
9015
21390
314



1764
12941
18461
9866
21665
7323
5049
9997
7294
5240
7571
6367



19001
13429
2814
21443
20459
1880
1396
3615
13867
7787
22538
13411



13344
8482
1205
18398
13083
12473
14941
17485
1550
757
11281
11029



21967
827
1873
21174
14091
5281
885
7332
8229
10933
12672
21103



4400
1012
15883
7812
7121
22179
16424
1339
1866
3474
11617
19918



16996
14296
1352
17699
9135
12367
19265
15111
3388
20896
20167
6697



6564
10047
19989
9358
22350
21749
15375
4618
531
16283
300
534



286
6575
7081
11468
2641
13543
15224
3097
15540
19380
19031
14287



6260
5051
12104
13901
11086
12459
12824
919
13271
15196
6303
4194



14638
1182
15803
1842
19299
7287
2857
17664
21589
11961
19154
12506



21814
4986
5343
21106
6334
21437
20936
11195
21506
2582
22069
5476



3533
7949
19583
22113
4677
18511
4730
696
9569
17797
18687
7097



20259
15252
13221
13305
450
22502
14915
21157
19855
11089
1744
10324



19917
6730
2195
20371
3563
12591
16308
7626
4212
19798
19458
5691



18292
8005
20912
21422
6721
10358
13601
6637
1272
17775
16130
21296



2832
830
14553
6434
18295
17956
829
12043
2457
5731
4395
10908



3209
13959
12384
5220
21843
19341
16539
19751
20779
1281
13763
17745



16586
7955
12705
15449
988
20859
1768
11129
6547
22518
5552
9854



11376
6669
5359
9132
10964
14922
1879
22087
13137
17298
3333
13752



20301
19370
2425
14659
6946
19538
14273
15340
6427
17165
15603
7257



12419
10809
8003
13658
14064
4221
7387
13284
20981
2316
3116
8418



3386
6508
18217
3578
22249
992
13442
3471
14855
19949
5277
19885



2816
10917
9436
18538
18659
12816
12308
15943
2515
20364
14701
8193



17579
22076
8890
2649
9329
21073
8376
15177
10882
859
5990
9814



16053
5209
7718
3144
13848
22428
19076
5454
17056
14918
4088
11270



5837
9604
22455
19840
12234
1149
801
953
9960
21301
1656
3755



12278
22119
13793
21098
1005
10264
13037
11702
20954
5581
5063
1118



6454
20632
1696
6301
9792
5396
19474
14911
20315
9381
4111
11600



7691
13374
2932
3071
18551
22362
1431
8710
12923
6509
19229
14012



5372
12362
17380
14108
20272
16391
19694
13395
5132
901
14568
17630



2371
4694
12396
15369
13051
16597
3697
10741
16309
4927
13396
21621



8841
16662
11120
14081
21259
19449
8692
9484
15204
6565
18317
14304



2769
14320
2978
9294
7482
9493
1277
4952
7813
7306
12296
21386



12021
6645
16505
9286
22489
3622
13745
16653
19069
7780
15219
16969



14762
10802
10479
16663
3678
16713
7751
13703
3630
4691
9472
10709



8542
7060
6112
22457
21974
20476
7333
6482
14526
7151
2644
835



10655
12264
9315
2786
16253
9488
5634
17372
3836
18362
11559
7637



4946
18143
12059
6995
17942
3772
3625
788
20809
16318
18862
7143



13134
9280
22095
18903
3706
15256
18593
5764
11115
12583
11568
13613



2331
5136
8073
15998
5630
11304
19137
5817
5580
18341
8588
12540



2454
4970
17445
2401
11869
6193
2434
3951
10889
5190
13207
16465



5825
17346
3323
13177
5354
2210
9333
13864
21502
19147
15973
9673



11863
9771
9608
11716
16811
18575
9584
12794
21399
20485
9218
18691



3350
18263
4846
544
19667
9933
4140
1318
20418
11128
20105
16734



2376
15699
7061
4232
15357
14036
5339
7107
19030
7165
21370
12103



4848
13211
22530
15360
12863
9975
6398
14067
16683
21170
1924
890



8155
15866
10131
7187
4332
1235
20330
12927
7088
5099
2302
12424



8303
17466
14322
11383
2282
15956
3414
12982
18548
15665
10961
21084



10824
18440
16819
3730
13940
18821
14864
1818
19607
7969
6546
16771



5441
8459
18266
5000
15749
13014
14274
8444
4707
13097
15930
11872



2621
7158
6942
18502
5408
10837
21928
13800
5188
19614
16117
4719



1833
13499
3107
21492
12503
21929
7921
1429
14398
22189
6439
12993



12307
19714
6650
21398
18994
747
5932
18630
6683
1921
15651
5594



6958
13597
19763
10097
14124
14687
1094
5780
7770
7688
15110
5797



7907
21169
3329
12627
4065
19882
22067
19211
2061
7038
8909
16914



13129
19881
16317
1551
13235
14888
1201
20264
7800
7526
2209
7384



5887
10798
15317
10223
11351
10105
10661
15014
2908
16412
22277
19851



12003
19616
11003
7768
6166
4620
8674
20541
3458
1240
15787
20099



9282
11480
4994
19545
802
17144
13420
13439
14301
1357
14011
7026



1145
6281
5258
6013
2805
18662
7992
9661
19875
11156
3619
14086



20948
16946
3456
6143
786
6709
5954
17926
20839
22351
21072
13277



15307
11701
15113
22057
6368
7704
18694
3644
12154
3794
21257
21638



18386
18111
21498
10731
13776
5539
14530
20282
7762
9529
17675
15191



12380
14865
15825
2818
5956
16442
6255
5901
8220
6918
18578
17465



13297
2495
21913
4526
16085
10965
15558
22565
22251
810
14127
7570



2692
13168
10912
15666
20574
4894
2526
10115
18982
19796
4020
2984



11522
11245
21570
8695
427
16749
17392
7325
10184
2132
20312
4655



16568
21093
13882
5730
7976
7079
13035
2800
16658
18348
16408
4157



5264
8112
509
307
305
12545
2037
11704
9571
21379
3712
7685



4483
18456
22341
3397
11197
13107
675
16006
532
20604
18536


 


477:
14477
7991
15716
18037
2783
11158
22007
11719
9318
11530
1246
21214



10621
8796
4280
19646
1037
3266
19426
6571
6888
17781
1697
10045



1251
14694
7276
7126
12814
14355
17583
1180
17847
8316
20573
7140



18820
7036
21056
15143
13926
18581
4999
20923
5501
1142
3253
7339



4338
16547
15305
10287
11127
20511
19012
13060
4347
12691
12055
3704



15743
19675
16542
21229
11524
9274
11075
8804
17224
3133
3607
10123



11459
11620
2973
15872
11732
19109
740
1359
17419
10051
6843
1680



16067
3406
4041
4022
9511
2595
4623
2369
19292
17333
5395
17305



10611
1428
7631
15733
18432
1959
21902
6814
13183
20320
12959
3032



4738
13205
22294
3286
19123
13158
20759
22152
18157
16447
10847
14538



4797
21087
7452
19143
16659
16620
11651
17070
10166
6601
15463
22431



10652
13842
11862
19136
1897
9220
12697
22065
20558
17946
13996
16162



7667
10376
2355
4084
14756
8726
10960
18769
3480
2838
20595
20275



1292
4853
20642
11502
11759
11992
9201
12553
14724
15765
17537
5294



9934
3608
15894
19202
4614
3319
11943
7304
4298
9147
11434
11341



10440
4734
13768
7695
7039
10493
13737
14607
16005
5151
2008
20772



4271
19954
4883
11303
15488
5027
17894
16991
7608
5609
16228
8198



18306
2556
6626
5829
13676
20616
12968
22378
16437
7624
20184
6039



10274
13230
16495
10978
13806
5631
6912
2955
21137
12918
16985
16430



8847
22400
13954
1905
19874
18505
9524
7297
20802
5035
20321
12454



9019
13953
2462
4887
16352
1889
17058
15457
8559
7962
11126
15081



22027
2791
7622
8042
4393
8437
1128
12634
21033
21247
5138
1952



10420
4794
21353
11932
13160
19797
13358
2289
10875
9733
12426
14341



7723
5120
12064
10942
15784
8171
17196
20434
21075
20432
4222
8716



18464
12196
630
8403
8582
6184
18819
9392
16256
4056
17228
16302



10762
13412
17350
12703
17792
4597
11159
7291
1072
4183
5466
17341



4892
1108
22046
1630
17859
11923
5617
18198
20318
1264
22123
2225



1997
13091
5701
8047
6109
22157
7745
16695
6934
1574
18516


 


478:
19887
10135
21002
14580
12123
15919
12579
5844
2744
4525
1601
15195



13066
547
20326
18377
314
8202
4733
7841
16690
17977
2012
6367



10918
3637
18964
16866
17780
7398
19262
8152
16337
16385
15522
10127



11277
18934
3901
22130
7812
16808
7324
19680
2408
19428
11882
19522



16434
9215
10784
19210
9199
9487
12925
15096
6578
16140
12862
976



11288
12658
20505
12973
11420
19873
8358
17414
300
8602
17310
6259



15328
15736
16123
1022
4666
6077
8815
10323
18236
1980
4532
17009



7605
19668
1127
2857
17664
21589
11961
19154
6093
3017
11359
3533



21439
16651
4677
18511
4730
696
9569
17797
18687
7097
20259
450



9399
14915
21157
20002
20371
3563
12591
16308
15626
7639
2807
13480



8017
8740
16746
716
21161
1662
7626
4212
19798
19458
5691
18292



16130
21296
2832
830
14553
6434
18295
22345
20848
19451
4148
6755



20779
1281
17745
16586
7955
12705
15449
988
20859
1768
11129
6547



22518
5552
9854
11376
6669
5359
9132
10964
1879
22087
13137
17298



3333
13752
20301
19370
2425
6946
14568
4694
21621
8841
16662
11120



14081
7280
15204
6565
1345
14320
2978
9294
7482
9493
1277
7813



7306
12296
12021
6645
9286
22489
13745
16653
19069
7780
16663
20476



7333
6482
14526
7151
14429
6992
788
4653
2925
17755
9673
7253



9771
3362
11716
21088
4458
18575
9584
12794
21399
14605
15265
10699



6281
18662
7992
9661
19875
11156
3619
14086
20948
16946
3456
6143



16455
786
6709
5954
17926
20839
22351
19605
13277
16667
9507
1392



9772
15558
21992
22438
5600
18371
10363
19255
11144
11875
3318
21951



4300
19052
16019
10566
17447
7523
21878
18982
5589
4804
15183
17109



19009
17084
20892
22180
11635
13124
4102
12865
2079
3506
509
6351



4815
532
372
6844
9299
5904
14428


 


479:
21743
1760
3495
6194
14521
17761
15144
21403
16214
6891
10614
7713



1057
3816
17150
12489
10225
5157
8783


 


480:
21754
6794
12450
9614
4412
5824
9276
21813
1463
11059
4808
13206



9578
13428
3072
15596
4328
3690
8876
7086
15462
6444
12263
8993



5420
6407
12246
1242
4404
4259
22020
4611
9408
14680
3680
10238



9797
6568
8934
7500


 


481:
17081
17400
17733
17377
12323
9631
5203
18639
16889
16035
17467
19493



3726
14585
14637


 


482:
6638
7562
10477
1852
4661
1068
13778
18473
9233
12209
5474
831



3220
16416
19485
21657
17736
2773
6559
19274
11791
2362
1290


 


483:
15521
9926
9372
3535
14815
17378
15342
16992
22270
6694
8651
10442



9501
20248
14291
19051
3324
8926
19254
5893
16393
10149
19420
11920



8831
8248
4517
12007
9386
15721
21801
17774
10932
11965
11946


 


484:
3750
6233
2713
11380
12023
5512
8397
9963
4401
17914
21042
20938



15212
2563
21028
4455
18141
21707
15335
2976
12163


 


485:
12915
8353
10649
15559
21532
1766
2706
8964
20052
20655
17361
14417



5510
14469
5001
8855
14600
20649
14614
15591
20286
2606
9471
1305



12051
13261
12077
17015
15608
2231
9209
13625
14471
16566
18336
20911



10270
15117
22496
11939
2250
12212
10593
15979
5988


 


486:
21080
8598
20902
564
3747
14584
4202
15248
1415
21487
2776
8440



6441
6856
4639
13249
14411
6300
7237
7568
8497
20720
5330
2288



3218
10413
16585
19827
2466
13415
19277
19479
4274
5139
8341
13715


 


487:
6455
20477
1176
8864
15945
3915
15571
16266
6450
1211
18350
6737



21409
1653
14641
22433
16158
5655
11725


 


488:
13633
11464
17818
4487
22079
19811
14672
20311
5875
1804
5805
8760



3617
12276
17631
6644
1838
1416
13999
2846
1536
21067
951
9346



9547
21436
4596
13884
12781
4010
10761
12933
16680
14715
12582
16262



4172
17700
6985
11107
17731
21246
3488
4816
11798
4381
9680
2173



8661
11802
13722
4211
7971
6750
14655
2765
2588
17564
9533
14335



13862
22447
12417
16557
9483
20169
6497
2050
3881
20747
3124
21130



11454
11582
14837
2614
4779
6358
16323
14350
7275
16234
10634
8984



18673
3036
12657
921
5211
10884
8362
20529
8627
7103
8149
15778



6773
11819
1672
12073
7102
20447
2872
7433
19593
19806
21911
5516



15286
11661
8992
14562
3219
17911
15808
5506
20741
21530
15240
20344



18214
15575
12427
3217
20435
7796
2375
11017
10944
13834
12971
20581



6080
16884
1900
7505
3937
11188
811
9207
2830
20690
12613
5666



4380
15312
18349
17639
10145
13494
664
6567
13052
6979
13432
17364



2224
22187
6961
13798
7944
4112
9995
15431
15530
10764
1676
8678



7407
22255
22276
8963
4880
11857
15344
7420
20168
20601
21712
7009



15363
8126
3045
7044
2098
12067
13898
7690


 


489:
21080
8598
20111
19932
20211
6441
6856
13249
4639
14411
6300
15103



8497
20720
5330
2288
19827
16585
13715


 


490:
18170
1316
6082
19848
9272
9871
15911
5917
19405
7429
21327
20596



8045
10178
4720
16346
7401
11417
5176
9427
22532
19611
6616
10826



2235
7529
22399
668
22163
10888
10947
17345
20889
1019
13964
12858



12745
21803
19731
15370
19455


 


491:
14629


 


492:
9026
18203
9034
17176
19316
18425
10191
3057
4291
19993
19117
633



19344
14181
6581
17129
12690
10130
10179
8823
14022
7424
4363


 


493:
11676
10500
20010
22061
13770
5046
17871
10828
15052
1942
11251
10057



14213


 


494:
2867
20983
17787
12926
13702
2680
4336
1898
7033
4692
1931
11547



4004
17355
7966
21453
6141
17522
20009
20155
3861
20641
13805 9004



5518
9100
6706
6460
9101
19945
14203
18615
8818
2078
21644
13350



9869
8593


 


495:
10049
5921
9432
4046
3753
21873
9833
14362
8333
13672
19390 1216



3131
18992
9486
11253


 


496:
3127
1607
15003
2751
9086
18256
7201
3526
20063
8184
14575
14132



3886
15754
11997
10334
8002
19176
6325
12719
15443
14454
10921
14581



14745
9990
9911
1762
10972
16402
1520
16638
1856
10874
3967
20026



4426
20686
9163
20462
20942
17529
11988
17803
8476
6783
21168
19191



2426
12096
15664
7561
11891
12826
17289
3387
19858
12743
98531
9481



615
8569
14610
1310
14283
3903
16792
22556
12950
6759
5113
5803



20040
13866
7531
12876
9534
7024
13251
663
2377
20537
22479
20413



17045
11984
22225
3341
1812
7226
19298
20768
6766
10544
12090
22407



18361
8222
5162
22240
11887
8295
15781
14468
2137
9880
12861
16260



21244
21191
22495


 


497:
4536
12720
13909
16764
14068
5500
2174
17809
20213


 


498:
7246
13566
4922
7189
11908
21432
19713
9021
8001
4169
9591
18319



12836
14209
15108
20572
4881
2021
2629
13362
21200
14093
18917
5199



21121
8529
17053
2009
2989
6906
19492
5789
6127
7577
22482
6916



4185
8063
10716
15947
17616
22376
714
9711
8995
19804
16484
10432



16415
18397
17120
5711
20348
11410
22426
5459
17025
20374
5057
3996



1399
16384
8557
3401
18399
9470
18719
2158
3928
19925
15780
1172



19003
4754
6752
7557
15694
2929
15268
8286
16534
8056
2719
1955



18895
5457
22161
17041
8512
21678
7854
18098
13647
15831
11038
15162



8205


 


499:
14500
1413
1282
1702
21840
19911
17917
16929
17820
4161
16375
12532



18092
1532
5902
19438
13718
6024
19735
13044
2787
19276
8082
9213



13875
2729
19946
19636
13441
11906
5009
21052
8166
12467
5788
10596



21948
8953
11461
18890
14782
22256
22200
7502
22419
22028
9688
19279



20234
20394
3662
13624
5037
17822
19294


 


500:
17384
19962
11922
12514
975
11652
22218
7145
9489
16374
595
10439



11174
6276
2673
2273
5345
20790
14336
21369
16362
13555
12890
20920



16827
21155
6409
16703
4160
18249
5053
1371
16010
3265
21504
10158



7203
22449
12631
15261
19335
9198
7318
16504
7195
4503
21207
10641



8201
15388
10167
17571
2611
3434
12571
13501
9308
8098
13986
4294



6690
17338
11438
16124
14788
9510
20668
22077
11494
16723
5320
3714



18672
19620
11228
1183
17223
1503
20710
7345
17503
10327
18402
18415



3756
1040
19384
13970
1810
20021
12766
10244
7980
16489
3481
1266



2213
5652
5230
6678
19037
22307
5389
18546
3014
22259


 


501:
16649
18243
16414
4207
20589
9444
16449


 


502:
22464
13448
15971
16611
16582
2337
16931
10640
13818
19924
15868
13017



5333
7271
18106
6613
20999
5327
19529
19566
15187
19703
13925
6927



17013
11388
20074
15701
3553
10267
14357


 


503:
10199
16886
2241
18245
1886
9716
7985
8744
20562
11084
13088
19819



22302
17049
990
2306
15235
2417
1433
2115
19923
19064
1002
16264



8492
2272
70913
39912
601
4956
19182
6191
7426
9434
14994
11105



6247
10698
8645


 


504:
8253
556
21865
11532
13731
17389
13431
10078
1271
12610
10733
21490



10485
18060
14669
11177
20124
7109
12716
1462
14936
5986
8097
5080



6289
3372
19867
2939
10924
773
11608
16138
4495
6375
3829
10354



13565
9492
7772
17813
19072
19386
6691
804
12615
3475
9031
16143



1291
12201
12820
1857
14937
19224
20789
10619
4585
1580
8663
12262



20460
1321
17853
6723
5237
11503
749
5651
8384
14280
17145
5419



15332
13013
5297
12732
19655
7901
7020
16059
19539
4387
1475
21505



2671
5714
1191
2608
21672
3296
15495
21919
1209
12295
15820
14193



14631
6254
7207
20109
13220
15146


 


505:
15140
1261
13853
2474
14749
2848
6699
13353
5174
10742
4290
12981



5167
19956
3782
14080
3433


 


506:
4704
7409
3027
1782
17403
2728
12116
22243
18039
19464
20608
21816



20161
9586
9832
1168
20257
8215
2055
2915
21159
8994
19097
4800



10831
3258
3511
5759
8010
5683
18509
3517
13649
8892
8848
13892



15978
11205
19106
17806
10914
8163
22263
5036
9958
17199
17195
12100



14343
7851
2637
16675
11823


 


507:
1698
1006
429
10217
7363
6232
18461
5240
6367
19001
18925
7778



526
3438
7812
20057
286
2857
17664
21589
11961
19154
21437
11195



20933
21506
3533
4677
18511
4730
696
9569
17797
18687
7097
20259



15252
13221
450
11335
20777
12713
22502
14915
21157
20371
3563
12591



16308
7626
4212
19798
19458
5691
18292
17864
4456
16130
21296
2832



830
14553
6434
18295
17956
829
476
12043
20779
1281
17745
16586



7955
12705
15449
988
20859
1768
11129
6547
22518
5552
9854
11376



6669
5359
9132
10964
1879
22087
13137
884
17298
3333
13752
20301



19370
2425
6946
19538
14273
15340
6427
17165
15603
12419
10809
8003



13658
14064
4221
7387
13284
20981
2316
3116
8418
3386
6508
18217



3578
22249
992
13442
3471
14855
19949
5277
19885
2816
10917
9436



18538
18659
12816
12308
15943
2515
20364
14701
8193
17579
22076
8890



2649
9329
21073
8376
15177
10882
859
5990
9814
16053
5209
3144



13848
22428
19076
5454
17056
14918
4088
11270
5837
9604
22455
19840



12234
1149
801
953
9960
21301
1656
3755
12278
22119
21098
1005



10264
13037
11702
20954
5581
5063
1118
6454
20632
1696
19474
14911



9381
4111
13374
2932
3071
18551
22362
1431
12923
6509
19229
14012



5372
12362
17380
20272
16391
13395
5132
901
9540
19228
14568
17630



2371
4694
12396
15369
16597
3697
10741
16309
4927
13396
21621
8841



16662
11120
14081
21135
8692
9484
15204
6565
14320
2978
9294
7482



9493
1277
4952
7813
7306
12296
12021
6645
9286
22489
13745
16653



19069
7780
15219
16969
14762
18330
10802
10479
16663
3678
16713
7751



13703
3630
4691
9472
10709
8542
7060
6112
22457
21974
20476
7333



6482
14526
7151
2644
835
10655
12264
9315
2786
16253
9488
5634



17372
788
11307
11198
9280
22095
18903
3706
15256
18593
5764
11115



12583
11568
13613
2331
5136
8073
15998
5630
11304
19137
5817
5580



14145
18341
8588
12540
2454
4970
17445
2401
11869
6193
21516
10889



5190
13207
16465
9673
9771
11716
22279
18575
9584
12794
21399
20485



9218
18691
3350
18263
4846
544
19667
9933
4140
1318
20418
11128



20105
16734
2376
15699
7061
4232
15357
14036
5339
7107
19030
7165



21370
12103
4848
13211
22530
15360
12863
9975
6398
14067
16683
21170



1924
890
8155
15866
10131
7187
4332
1235
20330
12927
7088
5099



2302
12424
8303
17466
14322
11383
2282
15956
3414
12982
18548
15665



10961
21084
10824
18440
16819
3730
13940
18821
14864
1818
19607
7969



6546
16771
5441
8459
18266
5000
15749
13014
14274
8444
4707
13097



15930
11872
2621
7158
6942
18502
5408
10837
21928
13800
5188
19614



16117
4719
1833
13499
3107
21492
12503
21929
7921
1429
14398
22189



6439
12993
12307
19714
6650
18994
747
5932
18630
6683
1921
15651



5594
6958
13597
19763
10097
14124
14687
1094
5780
7770
7688
15110



5797
7907
21169
3329
4065
19882
12627
22067
19211
2061
7038
8909



16914
13129
19881
16317
1551
13235
14888
1201
20264
7800
7526
2209



5887
10798
15317
10223
11351
10105
10661
15014
2908
16412
22277
19851



12003
19616
11003
7768
6166
4620
13850
16231
7016
2717
20541
3458



1240
15787
20099
9282
11480
4994
6281
18662
7992
9661
19875
11156



3619
14086
20948
16946
3456
6143
786
6709
5954
17926
20839
22351



13277
21057
15113
6368
7704
18694
3644
12154
3794
21257
21638
18386



18111
21498
10731
13776
5539
14530
20282
7762
9529
17675
15191
12380



14865
15825
2818
16442
5901
8220
18578
13297
2495
21913
4526
16085



10965
15558
18982
2984
11245
20659
477
304
427
12456
2132
20312



4655
16568
21093
13882
5730
7976
7079
13035
2800
19890
12545
2037



11704
9571
21379
3712
7685
4483
18456
22341
11197
13107
12449
16006



532


 


508:
19543
4932
20425
19108
19812
10140
21350
7766
14738
13289
13966
559



10501
18670
21326
12614
16425
9945
1996
21651
12806
13430
17362
21330


 


509:
5942
14707
1006
429
20069
10217
6051
533
535
478
10352
18305



21211
21561
20755
17549
15547
13386
8817
2523
18461
15773
19497
5240



22508
3614
6367
19001
3539
12446
15801
15469
13944
13012
20409
13620



15377
4732
4983
11962
10129
873
20622
7812
18242
8809
14606
18228



2931
3322
9023
6902
3558
3304
13684
21120
3950
12291
4958
21675



7657
4473
17017
4318
22410
19999
2415
22330
534
6329
4819
10093



8054
966
16945
14639
6901
14032
21397
2354
2669
10194
5075
2367



11025
6751
20059
21308
14038
2857
17664
21589
11961
19154
2340
7408



6244
17569
13074
21437
14633
11195
20933
21506
4167
3533
18759
4677



18511
4730
696
9569
17797
18687
7097
20259
15252
13221
450
10725



22502
14915
21157
18816
20371
3563
12591
16308
7626
4212
19798
19458



5691
18292
16130
21296
2832
830
14553
6434
18295
17956
829
476



12043
2658
7437
19427
14568
17630
2371
4694
12396
15369
16597
3697



10741
16309
4927
13396
21621
8841
16662
11120
14081
8692
9484
15204



6565
14320
2978
9294
7482
9493
1277
4952
7813
7306
12296
12021



6645
9286
22489
13745
16653
19069
7780
15219
16969
14762
18330
10802



10479
16663
3678
16713
7751
13703
3630
4691
9472
10709
8542
7060



6112
22457
21974
20476
7333
6482
14526
7151
2644
835
10655
12264



9315
2786
16253
9488
5634
17372
21009
788
5630
10889
5190
13207



16465
3748
4100
8138
14088
9673
20541
3458
1240
15787
20099
9282



11480
4994
5496
4783
20946
10965
15558
12797
4801
18251
14926
14677



6310
18248
15246
7925
15740
18982
2984
5765
4060
11245
20786
4379



2132
20312
4655
16568
21093
13882
5730
7976
7079
13035
2360
2800



16617
17577
19315
17164
15726
12545
2037
11704
9571
21379
3712
7685



4483
18456
22341
11197
13107
2227
16006
17470
10632
372
16331
4975



18567
13264


 


510:
2585
13077
1296
15698
1085
3129
8567
7012
16480
1063
15389
13708



17974
17451
15792
19280
10142
3469
4227
11718
15847
3638
2801
9326



10221
3742
13816
3280
4884
9923
11360
17722
15873
1490
20214
10492



13528
3713
8475
15933
21666
11740
9134
1633
11990
17965
20492
19212



13533
17131
16793
11371
3728
8594
17461
20347
8134
13823
22260
9279



7419
22025
5521
19185
8730
10999
4586
16348
19281
11567
19991
17098



20028
1067
6210
7658
19756
20830
10712
16037
2865
20878
3207
18632



20070
2980
6612
2087
5122
7489
19025
20408
5867
9692
13244
17130



19056
19348
18818
779
3699
15218
22456
11541
11135
2201
20058
15161



16612
6739
10651
14992
19392
15341
4204
21922
22540
19457
14075
17880



4186
15353
17073
5661
13025
14487
20671
6849
10343
6103
4142
17366



14159
2380
18164
13366
8153
19958
15839
6670
8947
2506
15249
9712



22234
1377
18798
8620
19625
10515
15887
9429
5493
7939
18930
19761



8714
3572
20073
19642
9602
11161
19552
7180
10987
2971
8438
4675



2938
8709
9900
4636
9435
3485
18162
15411
20078
15262
14563
3415



19829
9555
8928
16342
19361
1362
1940
12060
11259
18358
21475
7454



16972
20517
14458
5311
9206
3026
4246
21394
11663
5647
21680
18756



632
3100
18856
13204
14390
13351
12707
15711
10265
5221
14958
18751



17138
17198
2504
8197
10940
11642
7410
19473
13298
3551
3009
15678



14990
9133
18124
6205
1024
9670
14097
21447
2996
6377
19672
22172



21975
4551
21676
6123
2672
21984
2725
19700
14920
15674
7537
9974



11780
5242
13895
9998
3670
18115
16824
20178
18010
7493
13466
21265



8951
14511
18486
7701
6098
7554
17622
11381
6309
22198
19059
21658



15854
16625
12681
12161
11609
12398
6162
3022
5109
7314
14439
8366



13945
12198
11746
19463
8918
13801
5301
15197
3210
11365
15206
19669



17546
2720
10926
1742
6963
11828
6972
8450
14550
1267
11591
19644



11212
14447
15125
21242
13309
14497
17159
832
17245
5903
7432
18321



3663
3061
4860
9406
7511
10188
13056
4196
11132
3684
3623
8675



14569
21430
19252
10739
16443
8119
10193
17100
16296
16004
11852
17566



10552


 


511:
1655
3326
8875
4957
8311
19832
4954
10256
13907
20915
2896
5571



12394
6016
4944
12546
7522
2423
2681
8552
10982
7116
5314
12119



21099
18607
12329
4674
13459
21805
3969
14769
20266
22203
3114
336



12480
7283
9746
8207
7204
14337
11300
20397
20746
1530
5147
14828



21034
3906
2359
21177
8537
10139
16166
2594
13784
16485
20871
14720



1825
16147
17234
8065
17167
16501
11902
18924
5861
14539
14230
2788



2072
18571
2501
4192
13524
4842
3095
7769
10089
16575
6956
16293



11570
10846
19759
20187
21448
19904
10891
15038
1868
12675
6957
653



16915
12203
12409
8067
4947
19061
2619
13364
4888
5566
10650
17254



18049
16316
7313
11076
14440
1765
10770
13783
5839
16261
12061
9852



10237
10732
19814
4893
6882
6805
4262
4557
3971
638
11527
13153



22347
12730
10796
3084
18028
9061
7136
21153
925
8750
18082
12774



8584
13536
12518
6788
979
8900
18133
5696
15970
16063
14470
16701



5862
12183
3705
6658
16350
9557
8971
3717
1724
22354
16718
3854



3536
4599
16869
11895
16555
16258
4214
20552
21819
7135
16259
15280



4762
13775
4531
3367
13517
21549
8105
18643
4066
13282
14115
5130



10429
5920
4306
3192
5443
21499
6557
11929
20598
20903
4176
9050



15849
2723
15314
5993
12041
21869
14163
7930
9081
20569
9795
20396



11150
12443
4358
9475
10811
2552
11877
17685
21729
20974
12598
14790



20989
15004
17240
6237
17208
21930
8580
11818
5442
17259
20256
16046



5065
2308
2128
12182
17118
2251
7155
2892
13229
9845
20431
4311



16583
21037
18490
9763
6826
11713
15211
21554
16813
7056
18009
3039



19296
6673
11484
17841
21732
6456
2378
9054
19996
15355
21362
7381


 


512:
11789
20550
12924
14464
4011
19063
17858
148251
17933
18215
18506
8574



11311
14714
10870
5173
18503
12871
11792
6319
5991
16072
5679
7664



7997
7724
13724
15843
5179
21486
13257
20843
5253
15239
8745
4317



11829
5762
17201


 


513:
5799
17656
10980
3449
13335
20959
11238
9736
21154
8759
1349
18282



16665
17907
1107
9170
6492
20516
7727
5847
9065
19902
9931
8677



21320
19055
9044
19475
21629
4209
3873
10407
3831
21694
7261
11569



1480
1407
5128
14542
607
2784
18463
12009
8535
4917
22501
18918



12152
19362
6874
3988
10827
20140
18080
14167
1710
4124
3650
20877



6031
11044
12822
11665
16321
4669
19156
10011
15957
10312
16955
17469



9415
12205
21650
12324
10985
4763
17086
19498
18609
9205
19889
13760



11234
3128
3060
17868
7045
10721
18775
8340
16607
22199
8687
3652



8147
19720
4090
22058
13973
15250
5813
3005
6442
5423
4181
11396



20254
11686
18648
22257
3514
5054
17718
19944
22383
13148
10185
9304



9674
13580
20000
5106
14024
15026
15535
16386
22442
21036
12653
8647



894
4170
18619
16370
14161
13064
13337
2689


 


514:
19430
2217
13653
7909
3447
18614
2248
3672
3914
9037
2661
14057


 


515:
21829
10688
1896
2695
15238
6923
13321
678
19909
21886
6620
4554



4937
9312
1954
4679
2233
18331
21411


 


516:
12014
16420
2750
15538
18414
1466
5013
18876
17795
12046
9951
6600



5934
11750
5835
2034
4693
13740
10307
909
4231
1436
13779
16112



13219
4984
13214
18231
18179
2811
4814
18531
554
6050
18257
2903



3484
19943
17271
7129
1721
20263
20319
16055
20881
9942
13994
18838



11636
7029
16119
5257
1043
19478
20619
13417
11422
10160
20613
5029



8196
19429
9805
1861
17540
5288
14683
10272
7443
2656
16627
2315



772
9104
9410
4096
4548
9824
8485
17962
16475
22107
21011
15087



8840
15594
20392
9440
6125
9757
11164
17673
20924
4254
8176
16401



14437
18646
12057
12830
10159
17174
8040


 


517:
13062
19774
2825
8829
8332
18558
21964
19050
1627
16157
11534
19742



19647
13280
1517
9695
19320
10835
10293
13202
9174
14643
15719


 


518:
3195
21607
10760
14223
22323
21616
11305
21976
16169
8132
2667
11216



15637
4652
2270
8808
10309
18374
9291
7790
12784
7569
1295
5525



20767
17464
15920
7877
6705
4550
14008
15544
7616
1791
14358
15686



13182
14560
16722
8357
1472
4000
4261
10693
20144
4624
13338
17893



7464
11142
13491
15676
17655
17116
17984
9925
18333
10247
16818
4579



2385
20648
983
3200
6435
14763
13495
12036
16066
6740
14948
2307



14063
6851
10240
1728
16533
12629
10196
17078
16048
17609
11181
20629



575
18286
21518
5309
13744
15880
19484
16840
4959
15622
1288
18892



15176
815
7329
1175
20218
8827
7588
658
8704
16710
12907
17705



21364
16998
4945
15440
14372
12879
20119
17194
19468
10410
10451
11618



12006
12191
17175
20965
12916
6796
9139
17057
11008
18605
3353
4650



12741
1237
7988
20088
8231
4522
18497
11952
2866
15466
10449
5850



8504
22473
14806
5364
12620
20699
20034
4409
917
9156
629
12665



12908
7530


 


519:
11427
19017
6602
1864
10522
13385
3923
15070
3821
6415
1572
5050



15882
10935
5721
20535
12645
22402
8448
1350
7112
8649
5148
21245



11236
6476
14907
12714
20807
15311
9469
4739
13575
9732
8564
14799



12721
13373
4868
12893
19038
13333
8973
17817
15414
18146
11390
22248



13609
10100
15109
10007
5125
12633
6880
5388
20060
21032
8509
17168



19622
20280
21442
12066
17299
20123
12902
2062
656
6457
19453
20032



10850
8420
3813
21339
19516
16171
21635
2450
21079
19226
22202
17856



19847
1215
16964
11846
9253
18683
16927
3145
2877
4684
8416
4696



4925
14172
18178
10122
20134
11437
19509
20038
17265
10957
11114
11985



13382
4424
21277
6642
11395
19354
3216
17000
10584
4383
608
11099



3606
4061
6926
11774
3799
5172
3696
17840
9961
17641
22548
13507



6182
1731
2207
5353
5367
13860
8912
20922
11967
3241
16963
17734



10073
13903
22521
12149
4120
617
13506
13612
8031
6763
7546
6218



16311
10707
1223
21269
9414
8360
4885
3852
955
17284
19833
11354



2803
14827
1301
19175
10717
10910
12224
11516
13340
5262
5684
2178



13200
5913
9157
7959
17542
20440
18229
4805
8038
13587
16238
15602



18860
3544
7650
12674
17624
7074
576
17060
7413
22374
15047
21681



651
17560
6085
12160
12805
10775
4709
11401
6838
18364
17014
10752



17012
8980
17510
10711
8219
16107
16692
19401
17191
18405
15984
5384



19201
4362
883
6527
6410
867
12321
17889
17964
11941
14152
6591



4578
7461
4633
14845
14113
9478
16306
10792
5394
8423
16472
5538



5726
15272
22541
13274
19969
1344
17982
3299
6241
17488
6970
16632



2724
14874
2212
22392
12804
12135
15618
11858
552
17686
5585
19912



21050
1222
14554
13068
15732
8905
5854


 


520:
12932
18316
8070
15251
8216
15182
21583
18345
17227
19404
5489
17421



19679
6636
1134
14441
20247
13276
19767
5072


 


521:
14618
7172
4577
10111
4971
9921
11310
2304
19058
4919
2407
21318



6019
13841
12405
6079
22166
21222
11447
3834
2957
2876
3847
10030



3657
8737
2532
19313
13162
19695
11283
21477
13270
11090
20277
5569



11224
1524
7689
5785
1443
1174
5378
3463
7893
2512
20456
11012



5357
17062
17203
17936
14894
10285
19219
19980
15015
19783
22068
1918



19153
5369
14302
9654
16742
15180
4073
14772
14901
3954
13470
8012



15898
12241
11155
18311
11712
3109
18530
17791
1700
6065
18830
8838



7741
20399
7063
8515
13562
2024
8969
7647
12054
19078
2184
16655



8540
7852
16491
13169
20514
20202
7535
18320
11710
7747
15987
17730



7302
1993
2766
4031
5562
21493
10046
1799
4731
18131
8100
10005



7123
2520
10396
22156
9867
7052
11207
4505
21049
11504
21868
11614



5889
12118
13000
22505
2411
10176
6296
9619
18566
20017
11928
3767



11046
7551
2528
11302
21596
6991
7083
2350
736
10023
5402
21313



14934
14110
14248
11521
15498
3429
17829
15576
19218
22393
10506
19723



20354
6720
17694
8466
4273
4916
22208
20427
9107
15921
4439
10197



2338
16397
13942
15715
20725
18142
3311
2926
10795
3889
17244
10288



19160
8477
12011
3565
21062
9658
16357
6172
2617
1734
15245
22324



7974
4678
3698
3992
10876
20303
19258
17048
4272
20359
11598
8412



6693
19116
5690
8276
912
4024
10472
12647
6733
5738
15800
21800



3295
8590
20011
17717
4177
7227
20102
5504
13132
11762
15477
18479



9793
17968
11404
21844
17913
17281
14938
1225
7663
13582
11349
16247



19951
3778
8781
7268
10618
18594
14849
15471
22337
22454
4787
4244



12088
1992
9332
15917
17106
13790
19542
19573
2089
1592
7878
12361



19383
14253
17356
4238
9481
1439


 


522:
14431
6239
11377
22466
13606
11763
4871
19242
19036
9779
970
4882



15417
18050
2958
13661
18314
19970
8044
8672
8262
4077
12369
12815



15194
7431
19112
585
5289
17307
6089
5537
5935
21766
1304
15168



4356
6485
15400
19439
706
14453
1031
5411
11160
12131
10020
11249



8415
21643
14449
14017
1963
10261
1703
1878
4023
12040
9720
17260



21578
15343
11117
16514
14299
20222
12107
18114
13664
4184
14261
16823



13551
22313
3921
1998
666
4628
20928
18508
9588
8794
5200
619



4115
20171
1555
3070
5376
7843
10040
19791
19447
20093
16957
19231


 


523:
5718
10805
6298
2166
20556
21748
9437
8027
3472
10338
7212
20501



11491
4341
11669
9303
16917
8868
14506
2160
21476
17596
18688
9966



15557
11767
2124
18956
7553
18747
18697
17873
7757
16320
22134
13292



7913
9948
6980
17735
634
12444
16435
5350
17955
19423
15565
11353



20128
13016
5239
860
22552
2379
6033
16520
21986
17573
5222
14074



2603
19569
20484
2875
1313
5161
7288
21179
5370
19621
980
10087



20381
3366
6986
21533
16164
20883
3426
1157
16729
6898
20593
17576



9698
12169
10986
8048
17173
5809
21767
582
9317
13750
3110
18945



19532
13594
14859
19926
14149
16203
18916
2694
2259
3885
16893
6833



6096
16248
7576
1925
6278
10533
5649
20618
13912
17331
9831
9223



1851
20107
18714


 


524:
5146
21711
9443
19434
17798
8271
15157
6680
1121
22072
17654
19028



13218
6405
9244
5217
7137
8194
21221
12019
13471
17578
11153
9140



11727
17633
3462
17166
6295
21753
828
13617
9059
2402
16458
7601



13675
21744
12228
3556
9011
10297
8699
1058
17727
3732
888
19098



19007
20328
22435
9743
8151
9884
5912
13874
1793
20963
14276
11662



13913
18489
16461
9898
8731
15745
3973
880
2296
9092
18765
17370



18888
13547
17231
13680
4367
14754
17832
2159
8925
6054
4836
21950



16483
17585
20898
15188
9783
19045
2088
7805
5781
7540
21423
10626



3232
3412
16951
12593
7133
7230
17422
10172
19629
20061
15226
16396



13106
16858
12395
8064
18608
10913
18148
14559
11640
22231
949
3806



922
6047
14424
20294
21512
2844
21750
10411
1285
598
9413
13278



5979
15078
9781
11382
15932
16934
17171
3994
21427
3887
3113
10974



15635
18526
5503
9904
16377
17644
12020
15507
18396
5802
16207
16206



6951
13409
14980
12042
9197
20762
11969
14942
19111
1373
9467
797



8518
2947
13489
6590
10901
7185
7646
6204
6548
8825
7264
13425



13837
10201
11983
19803
21421
11553
9049
3356
19634
16334
3944
7749



20163
662
8190
6651
20724
22285
8240
12389
5598
11233
14315
15485



11282
20624
12145
12327
13242
17891
1217
8203
12727
22111
8826
9668



20863
11483
21587


 


525:
6387
20127
15922
6468
22478
5164
14961
7042
16305
2182
16677
7733



8221
13250
4225
10787
11039
2823
13281
6380
6824
6360
20082
1398



14175
14977
21194
5933
9986
10799
2049
7444
4924
15193
2703
12215



13851
836
5401
12967
18193
18801
1103
3308
20749
18446
18404
15606



19308
7496
14462
10044
21187
16900
8754
16101
22544
1746
5089
13111



9288
15179
4423
14999
11584
15673
5028
18076
21294
12622
21553
14776



12325
6837
10013
20597
20543
11805
18067
18468
4428
3795
8921
3661



8624
4476
13323
11313
7963
22062
20636
18513
1030
8805
22083
5807



3830
19568
5055
4025
10497
21824
10898
19772
590
10473
8226
20982



6273
21435
16209
9607
18881
10143
5700
11596
10735
19808
17961
19021



1837
21662
20927
1365
7598
21243
15848
17757
8577
12564
9707
7488



9892
1675
13967
1620
3282
2358
18278
15275
15531
18220
21540
22120



14812
13263
6574
14679
2574
16215
1945
10259
15499
11627
12347
2605



6895
7286
10483
2487
10289
3346
7293
9601
10220
7996
4547
4237



7808
7341
5250
14483


 


526:
8075
16099
8242
429
13036
10217
18174
535
533
507
306
11062



15313
16399
14594
314
3820
5717
20464
15374
18461
18690
9504
8137



11945
3327
4030
1064
15390
4708
6367
19001
8581
6859
18172
3305



18107
12112
13896
2465
12130
19906
4949
17359
16220
13607
10705
15889



16978
8246
16554
18467
9002
9780
17882
21655
20242
9063
5705
9204



9915
11991
16148
15840
14589
6171
20389
6007
14898
1840
4491
7812



8977
13027
6371
15170
21310
12535
9755
2819
3052
12267
8377
3804



7983
22167
21674
22450
891
13549
7113
14532
9354
7858
8394
2044



9345
2341
6836
19148
17763
286
18665
2645
14240
13516
9032
15489



8304
7822
9993
17083
7310
5481
11010
1093
7789
21306
21699
11113



8956
18128
7880
3416
6279
2857
11961
19154
10481
11542
21609
21506



3533
6663
3391
4677
18511
4730
696
9569
17797
18687
7097
20259



15252
13221
3261
14692
22502
14915
21157
20371
3563
12591
16308
7626



16130
21296
2832
830
14553
6434
18295
17956
20230
10392
12043
21065



20779
1281
17745
3289
3754
5487
8587
11548
12578
2882
5552
16586



7955
1768
22518
12705
15449
988
20859
11129
6547
9854
11376
6669



5359
9132
10964
19776
1879
22087
13137
13752
17298
3333
20301
19370



15830
2425
6946
19538
14273
15340
6427
17165
15603
12419
10809
8003



13658
14568
12544
17630
2371
4694
12396
15369
16597
3697
10741
13396



21621
16662
8841
11120
14081
13456
21472
7487
12898
12787
8692
9484



15204
6565
11203
14320
2978
9294
7482
9493
1277
4952
7813
7306



12296
12021
6645
9286
22489
13745
16653
19069
7780
15219
16969
19357



14762
10802
10479
16663
3678
16713
7751
13703
3630
4691
9472
10709



8542
7060
6112
22457
21974
20476
7333
6482
14526
7151
2644
835



10655
12264
17372
10889
6281
10965
15558
5973
1482
22476
19408
9637



5773
5016
20819
18982
19997
4572
640
19168
8691
17905
2970
2132



20312
4655
16568
21093
13882
5730
7976
7079
13035
3505
2800
12522



8939
2155
3206
2017
15587
6277
1297
1423
18728
305
509
12545



2037
11704
9571
21379
13089
11197
13107
22367
17943
2420
7599
5997



372
1801
21374
16528
8722
16672


 


527:
3642
15482
8461
18796
11646
4433
18911
12512
19895
14727
7891
5229



8941
8192
671
3621
19293
20133
7775
16444
5601
9047
7456
1332



15241
20870
3570
13126
6335
14830
17816
4052
1048
11934
7415
16817



11466
15006
17149
16740
13546
20225
18675
12360
11085
4464
12955
7636



18831
17524
21731
2244
8988
9012
18649
8617
6285
18612
1053
1455



4829
16779
15889
18827
14953
12980
10447
1034
15656
2581
22412
4354



10756
21138
3468
6872
6990
15371
16865
14170
14485
16553
7703
21199



6847
729
14001
18779
7182
2836
8312
3038
18069
16737
4357
10422



3567
6722
22039
3264
5085
20829
6719
2934
10564
14973
13027
2205



9393
19576
7148
2665
6959
16605
4997
17039
7857
4310
11556
19590



12715
6725
8377
3489
3770
22024
3546
20586
5189
20926
2245
1604



7700
18947
5485
22116
21537
11706
18715
21536
17691
17398
2949
3079



6290
961
6042
4750
8302
20882
5852
13136
16392
2643
14077
9240



11996
3702
14090
19402
7233
5546
10148
20680
8638
3269
15282
9663



15009
15604
8648
14392
18782
10570
6318
19862
13514
13217
3980
5231



11578
4812
18337
3004
3073
8249
18941
9036
3233
864
15080
19930



6746
1476
10866
20483
19942
7450
762
21276
3235
15320
22462
11955



1562
13099
7710
9136
7251
16129
19467
10892
10630
13600
12857
14732



4581
18096
3518
12368
1009
17283
4039
6864
5773
6063
15842
16851



18149
7977
14608
4572
10553
4514
11848
18068
17157
12223
18597
15178



10550
11835
16242
11778
20884
16907
14445
5068
7404
1516
6277
17663



17662
21513
5526
14789
7565
21347
1238
10660
18496
16381
20827
19639


 


528:
22368
2262
4500
18087
11171
3257
5531
17538
11849
9017
9287
9644



19677
20195
3911
3945
21129
6446


 


529:
18280
2326
4826
11407
11186
16051
1917
13113
10636
10206
19238
18366



6941
11986
7683
16587
14340
18850
6892
3404
17954
20223
11450
10435



19074
13384
7518
15985
8336
5383
3907
13379
12855
14509
12217
3398


 


530:
1719
15257
12828
16494
8849
18495
10061
1831
11005
20446
21892
17075



9052
1023
10386
2348
6364
14482
15814
7746
9263
3529
9935
10786



20919
12407
11705
8493
5095
8481
6104
3104
14770
19267
1032
17399



8085
17728
7344
673
15714
3361
5103
21218
15350
7539
761
19758



16925
17515
8371
9594
13769
16744
17772
4420
8605
6819
6628
9541



19100
13982
7360
20665
9903
5639
8487
13488
9666
5511
11106
17709



5021
19088
20007
12811
14758
15329
7823
21543
20508
1178
5558
16935



1252
7312
20192
18963
5305
19575
17193
1262
3577
20951
2711
18829



9159
6009
6176
17751
9694
17992
12429
15448
4598
18452
7077
10597



16197
705
8820
11971
19192
1370
996
1578
21971
6312
17349
2736



3936
10254
10398
22265
20083
15853
14066
5564
9113
3087
1078
7435



14028
16372
9321
18704
8983
21564
9660
11558
17282
12695
19057
13847



17094
11400
7613
2232
17213
17103
19378
6072
14868
8879
6825
12193



1427
3275
9458
15007
6271
4411
4190
12430
16366
20438
15680
10929



4035
20030
4556
17229
4660
597
1587
15707
4334
22484
21035
8211



16251
13133
11295
2966
14058
5665
6592
8738
6102
13087
3436
12074



14546
13772
20692
10970
19345
13509
5329
19187
16282
9968
21671
9048



8976
7492
21462
19662
8457
8317
18846
12864
10746
4141
8673
21010



10417
4329
7895
998
10132
8852
14795
5620
6695
13781
19165
7258



20895
4154
7359
12259
11737
12704
21606
3865
5520
6932
12901
20940



15027
3236
4907
20734
15492
10607
2472
15046
8506
18724
11671
15259



15936
7889
5596
2053
6905
8323
16351
10263
15826
21160
19461
17456



3953
3584
4314
6428
14221
4758
5800
22365
17678
16252
14893
17154



19437
16453
21224
9572
16225
9367
1899
8751
8426
12483
4441
20592



6867
2168
16928
13225
22460
20208
12970
2591
5304
13498
21581
4699



21894
1461
5860
21622
18703
2500
7014
15116
596
14962
5517
13693



21739
19681
11533
3338
3956
11364
17988


 


531:
15910
22524
13589
1006
11575
20349
18523
10911
13067
10217
9609
5233



4966
9015
21390
18461
9866
21665
7323
5049
9997
5240
6367
19001



2814
21443
1880
1396
3615
13867
7787
22538
13411
13344
1205
18398



13083
17485
1550
757
11281
11029
21967
827
1873
21174
14091
5281



885
7332
8229
21103
4400
1012
15883
7812
7121
16424
1339
1866



3474
11617
19918
14296
1352
17699
12367
3388
20896
20167
6697
19989



9358
15375
4618
11468
13543
3097
15540
19380
14287
5051
11086
12824



13271
15196
6303
4194
1182
15803
1842
19299
2857
17664
21589
11961



19154
12506
21814
6334
21437
20936
11195
20933
21506
22069
5476
3533



19583
7949
4677
18511
4730
696
9569
17797
18687
7097
20259
15252



13221
13305
450
22502
14915
21157
6730
2195
19855
10324
11089
1744



19917
16130
21296
2832
830
14553
6434
18295
17956
829
476
12043



2457
13959
12384
5220
3209
19341
5731
10908
4395
21843
14568
17630



2371
4694
12396
15369
13051
16597
3697
10741
16309
4927
13396
21621



8841
16662
11120
14081
19449
21259
15204
6565
18317
14304
4952
7813



7306
12296
12021
6645
21386
16505
9286
22489
3622
14762
18330
10802



10479
16663
3678
16713
7751
13703
3630
4691
9472
10709
8542
7060



6112
22457
21974
20476
7333
6482
14526
7151
2644
835
10655
12264



9315
2786
16253
9488
5634
17372
3836
7637
4946
18143
11559
18362



12059
10889
5190
13207
16465
17346
3323
5354
5825
2210
19147
9333



13177
20541
3458
1240
15787
20099
9282
11480
4994
14011
1145
19545



802
17144
13420
13439
14301
1357
10965
15558
22251
22565
810
7570



2692
15666
13168
10912
20574
4894
2526
18982
4020
19796
2984
11522



11245
21570
8695
16749
17392
2132
20312
4655
16568
21093
13882
5730



7976
7079
13035
2800
16658
18348
16408
4157
8112
5264
305
12545



2037
11704
9571
21379
3712
7685
4483
18456
22341
3397
11197
13107



675
16006
20604
18536


 


532:
15542
3190
1006
429
7236
21108
10856
10217
1694
1170
507
478



7946
4963
10952
8465
18461
9739
1850
5240
2382
6367
19001
19407



14319
21808
15520
1498
14212
14282
19450
526
3987
18034
12939
14517



22533
14740
7812
18725
15422
12515
6665
8294
20640
9749
7933
18742



10601
1528
14184
8297
11430
21253
7897
4901
7308
16574
19527
9882



8330
6186
16730
3076
15579
16073
19400
17335
28571
76642
15891
1961



19154
6997
3197
21437
4440
11195
20933
21506
3533
4677
18511
4730



696
9569
17797
18687
7097
20259
2920
15252
13221
3452
14930
450



11335
20777
12713
22502
14915
21157
14054
10864
5059
21184
8816
9146



14653
14579
20371
3563
12591
16308
7626
4212
19798
19458
5691
18292



7706
5100
4540
16130
21296
2832
830
14553
6434
18295
17956
829



476
12043
13241
11023
4301
9298
2269
1306
13291
17064
20779
5613



1281
17745
11486
9723
16586
7955
12705
15449
988
20859
1768
11129



6547
22518
5552
9854
11376
6669
5359
9132
10964
1879
22087
13137



17298
3333
13752
20301
19370
2425
20461
6946
19538
14273
15340
6427



17165
15603
12419
10809
8003
13658
14064
14568
17630
2371
4694
12396



15369
16597
3697
10741
12482
16309
4927
13396
21621
8841
16662
11120



14081
15751
4903
21508
18755
14599
6384
8692
9484
15204
6565
14320



2978
9294
7482
9493
1277
4952
7813
7306
12296
12021
6645
9286



22489
13745
16653
19069
7780
15219
16969
14762
18330
10802
10479
16663



3678
16713
7751
13703
3630
4691
9472
10709
8542
7060
6112
22457



21974
20476
7333
6482
14526
7151
2644
835
10655
12264
9315
2786



16253
9488
5634
17372
1733
20453
21787
16624
7001
20828
13302
10357



4549
788
10490
19133
12318
5515
9280
22095
18903
3706
15256
18593



5764
11115
12583
11568
13613
2331
5136
8073
15998
5630
11304
19137



5817
5580
3044
18341
8588
5857
12540
2454
4970
16919
17445
2401



11869
6193
21516
10889
5190
13207
16465
9673
11625
9771
2283
11716



19554
905
7481
12401
19686
13656
22135
9553
18575
9584
12794
5644



21399
20485
9218
18691
3350
18263
4846
544
19667
9933
4140
1318



20418
11128
20105
16734
2376
15699
7061
4232
15357
14036
5339
7107



19030
7165
21370
12103
4848
13211
22530
15360
12863
9975
6398
14067



16683
21170
1924
890
8155
15866
10131
7187
4332
1235
20330
12927



7088
5099
2302
12424
8303
17466
14322
11383
2282
15956
3414
12982



18548
15665 10961
21084
10824
18440
16819
3730
13940
18821
14864
1818



19607
7969
6546
16771
5441
8459
18266
5000
15749
13014
14274
8444



4707
13097
15930
11872
2621
7158
6942
18502
5408
10837
21928
13800



5188
19614
16117
4719
1833
13499
3107
21492
12503
21929
7921
1429



14398
22189
6439
12993
12307
19714
6650
18994
747
5932
18630
6683



1921
15651
5594
6958
13597
19763
10097
14124
14687
1094
5780
7770



7688
15110
5797
7907
21169
3329
12627
4065
19882
22067
19211
2061



7038
8909
16914
13129
19881
11720
16317
1551
13235
14888
1201
20264



7800
7526
2209
5887
15285
10798
15317
10223
11351
10105
10661
15014



2908
16412
22277
19851
12003
19616
11003
7768
6166
21613
4620
13850



16231
7016
2717
20541
3458
1240
15787
20099
9282
11480
4994
937



16775
18459
14476
9221
17450
6281
18662
7992
9661
19875
11156
3619



14086
20948
16946
3456
6143
786
6709
5954
17926
20839
22351
13277



11255
5140
4283
9455
4818
14459
5187
16668
10965
15558
17215
14289



11685
4388
14119
18982
2984
11245
304
477
427
18112
965
2900



2132
20312
4655
16568
21093
13882
5730
7976
7079
13035
5498
2800



19337
9600
307
12545
2037
11704
9571
21379
3712
7685
4483
18456



22341
11197
13107
16006
7623


 


533:
6090
1006
429
10217
10250
19910
18933
314
18461
5240
6367
19001



16913
19119
526
3693
7812
13179
534
300
531
2857
17664
21589



11961
19154
21437
11195
20933
21506
3533
4677
18511
4730
696
9569



17797
18687
7097
20259
15252
13221
450
11335
20777
12713
22502
14915



21157
20371
3563
12591
16308
7626
4212
19798
19458
5691
18292
16130



21296
2832
830
14553
6434
18295
17956
829
476
12043
20779
1281



17745
16586
7955
12705
15449
988
20859
1768
11129
6547
22518
5552



9854
11376
6669
5359
9132
10964
1879
22087
13137
17298
3333
13752



20301
19370
2425
6946
19538
14273
15603
15340
6427
17165
12419
10809



8003
13658
14064
4221
7387
13284
20981
2316
3116
8418
3386
6508



18217
3578
22249
992
13442
34711
48551
9949
5277
19885
2816
10917



9436
18538
18659
12816
12308
15943
2515
20364
14701
8193
17579
22076



8890
2649
9329
21073
8376
15177
10882
859
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9814
16053
5209



3144
13848
22428
19076
5454
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14918
4088
11270
5837
9604
22455



19840
12234
1149
801
953
9960
21301
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3755
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21098



1005
10264
13037
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14911
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14012
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17380
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901
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11589



13306
3179
14568
17630
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12396
15369
11784
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3697
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16309
4927
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21621
8841
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11120
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8692
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6565



14320
2978
9294
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4952
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12021
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9286
22489
13745
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19069
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15219
16969
14762
18330
10280
10802



10479
16663
3678
16713
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3630
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8542
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6112
22457
21974
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7333
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14526
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10655
12264



9315
2786
16253
9488
5634
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9280
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5764
11115
12583
11568
13613
2331
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8073
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11304



19137
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8588
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17445
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11869
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21516
2447
10889
5190
13207
16465
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11716
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21399
20485
9218
18691
3350
18263
4846
544
19667
9933
4140
1318



20418
11128
20105
16734
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15699
7061
4232
15357
14036
5339
7107



19030
7165
21370
12103
4848
13211
22530
15360
12863
9975
6398
14067



16683
21170
1924
890
8155
15866
10131
7187
4332
1235
20330
12927



7088
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12424
8303
17466
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11383
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12982



18548
15665
10961
21084
10824
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16819
3730
13940
18821
14864
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19607
7969
6546
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15749
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14274
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4707
13097
15930
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6942
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5408
10837
21928
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5188
19614
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13499
3107
21492
12503
21929
7921
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14398
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747
6439
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12307
19714
6650
18994
5932
18630
6683



1921
15651
5594
6958
13597
19763
10097
4065
14124
14687
1094
5780



7770
7688
15110
5797
7907
21169
3329
12627
19882
22067
19211
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7038
8909
16914
13129
19881
16317
1551
13235
14888
1201
20264
7800



7526
2209
5887
10223
10798
15317
11351
10105
10661
15014
2908
16412



22277
19851
12003
19616
11003
7768
6166
4620
16231
13850
7016
2717



20886
14522
5254
15519
20541
3458
1240
15787
20099
9282
11480
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6281
18662
7992
9661
19875
11156
3619
14086
20948
16946
3456
6143



786
6709
5954
17926
20839
22351
13277
15113
6368
7704
18694
3644



12154
3794
21257
21638
18386
18111
21498
10731
13776
5539
14530
20282



7762
9529
17675
15191
12380
14865
15825
2818
16442
5901
8220
18578



13297
2495
21913
4526
16085
10965
15558
18982
2984
11245
21040
477



11140
2132
20312
4655
16568
21093
13882
5730
7976
7079
13035
2800



15563
509
12545
2037
11704
9571
21379
3712
7685
4483
18456
22341



11197
13107
13802
16006
428


 


534:
6383
10450
1006
8266
17986
13269
9010
9822
18387
3735
10217
533



535
1774
16191
18891
13849
14706
571
8356
3340
18461
20284
20753



10192
13836
5240
2653
20833
3564
1870
11250
6367
19001
20137
15672



13176
20207
8314
4015
1887
8795
21466
7023
18171
6876
8560
16477



13630
10793
4136
5249
17099
9152
2730
4724
8684
5821
3710
16562



22562
9500
21952
12129
1151
5863
2635
13579
5947
9543
6219
4851



7812
10216
19130
12258
1950
8843
1084
2519
12495
7141
1845
6128



9067
2618
1308
1202
16045
2887
3970
22176
2992
14189
2020
3687



18669
17082
7279
10946
10754
10583
4481
915
16903
18701
14442
11182



12710
8108
4737
8174
21990
14565
20445
20022
9861
2888
17080
6216



3441
9629
4034
4019
17401
1207
22219
1481
5143
2857
17664
21589



11961
19154
12425
21437
17489
11195
20933
21506
35331
2843
4677
18511



4730
696
9569
17797
18687
7097
20259
20966
15252
13221
11335
20777



12713
22502
14915
21157
13596
7735
6111
14062
20371
3563
12591
16308



7626
21483
4212
19798
19458
5691
18292
16130
21296
2832
830
14553



4632
6434
18295
17956
829
12043
1189
3932
18731
6845
21360
6772



19039
21673
20779
1281
16128
14568
17630
2371
4694
12396
15369
14260



13396
21621
8841
16662
11120
14081
6657
8692
9484
14065
2540
15204



6565
14320
2978
9294
7482
9493
1277
4952
7813
7306
12296
12021



6645
9286
22489
19908
893
14762
18330
10802
10479
16663
3678
16713



7751
13703
3630
4691
9472
10709
8542
7060
6112
22457
21974
20476



7333
6482
14526
7151
2644
835
10655
12264
9315
2786
16253
9488



5634
17372
2081
12643
15099
7425
15838
10094
788
3348
9280
22095



18903
3706
15256
18593
5764
11115
12583
5630
11568
13613
2331
5136



8073
15998
11304
19137
5817
4430
5580
8588
18341
12540
2454
4970



17445
2401
10889
5190
13207
16465
11291
16479
21467
4263
2705
9673



20541
3458
1240
15787
20099
9282
11480
4994
2798
5475
1814
2683



8435
4345
3627
15897
9366
6281
20716
18662
7992
9661
19875
11156



21818
3619
14086
20948
16946
3456
6143
786
6709
5954
17926
20839



13891
10965
15558
15276
3513
17600
11426
1123
16786
5326
17407
18982



2984
19817
11245
5838
21039
15310
2132
20312
4655
16568
21093
13882



5730
7976
7079
13035
2800
16353
4982
22032
3731
7388
13436
8698



509
12545
2037
11704
9571
21379
3712
7685
4483
18456
22341
15886



11197
13107
5444
16006
9573
20805
372
19913
9499
18057
7973
22459



21704
8464


 


535:
9382
1006
429
10217
2438
9883
314
18461
5240
6367
19001
16674



12484
526
11896
7812
9819
2857
17664
21589
11961
19154
21437
11195



20933
2150
63533
4677
18511
4730
696
9569
17797
18687
7097
20259



15252
13221
450
11335
20777
12713
22502
14915
21157
20371
3563
12591



16308
7626
4212
19798
19458
5691
18292
16130
21296
2832
830
14553



6434
18295
17956
829
476
12043
20779
1281
17745
16586
7955
12705



15449
988
20859
1768
11129
6547
22518
5552
9854
11376
6669
5359



9132
10964
1879
22087
13137
17298
3333
13752
20301
19370
2425
6946



19538
14273
15340
6427
17165
15603
12419
10809
8003
13658
14064
4221



7387
13284
20981
2316
3116
8418
3386
6508
18217
3578
22249
992



14855
13442
3471
19949
5277
19885
2816
10917
9436
18538
18659
12816



12308
15943
2515
20364
14701
8193
17579
22076
8890
2649
9329
21073



8376
15177
10882
859
5990
9814
16053
5209
3144
13848
22428
19076



5454
17056
14918
4088
11270
5837
9604
22455
19840
12234
1149
801



953
9960
21301
1656
3755
12278
22119
21098
1005
10264
13037
11702



20954
5581
5063
1118
6454
20632
1696
19474
14911
9381
4111
13374



2932
3071
18551
22362
1431
12923
6509
19229
14012
5372
12362
17380



20272
16391
13395
5132
901
9540
19228
14568
17630
2371
4694
12396



15369
16597
3697
10741
16309
4927
13396
21621
8841
16662
11120
14081



8692
9484
15204
6565
14320
2978
9294
7482
9493
1277
4952
7813



7306
12296
12021
6645
9286
22489
13745
16653
19069
7780
15219
16969



14762
18330
10802
10479
16663
3678
16713
7751
13703
3630
4691
9472



10709
8542
7060
6112
22457
21974
20476
7333
6482
14526
7151
2644



835
10655
12264
9315
2786
16253
9488
5634
17372
788
9280
22095



18903
3706
15256
18593
5764
11115
12583
11568
13613
2331
5136
8073



15998
5630
11304
19137
5817
5580
8588
18341
12540
2454
4970
17445



2401
11869
6193
21516
10889
5190
13207
16465
9673
9771
11716
18575



9584
12794
21399
20485
9218
18691
3350
18263
4846
544
19667
9933



4140
1318
20418
11128
20105
16734
2376
15699
7061
4232
15357
14036



5339
7107
19030
7165
21370
12103
4848
13211
22530
15360
12863
9975



6398
14067
16683
21170
1924
890
8155
15866
10131
7187
4332
1235



20330
12927
7088
5099
2302
12424
8303
17466
14322
11383
2282
15956



3414
12982
18548
15665
10961
21084
10824
18440
16819
3730
13940
18821



14864
1818
19607
7969
6546
16771
5441
8459
18266
5000
15749
13014



14274
8444
4707
13097
15930
11872
2621
7158
6942
18502
5408
10837



21928
13800
5188
19614
16117
4719
1833
13499
3107
21492
12503
21929



7921
1429
14398
22189
6439
12993
12307
19714
6650
18994
747
5932



18630
6683
1921
15651
5594
6958
13597
19763
10097
14124
14687
1094



5780
7770
7688
15110
5797
7907
21169
3329
19882
12627
4065
22067



19211
2061
7038
8909
16914
13129
19881
16317
1551
13235
14888
1201



20264
7800
7526
2209
5887
10798
15317
10223
11351
10105
10661
15014



2908
16412
22277
19851
12003
19616
11003
7768
6166
4620
13850
16231



7016
2717
20541
3458
1240
15787
20099
9282
11480
4994
6281
18662



7992
9661
19875
11156
3619
14086
20948
16946
3456
6143
786
6709



5954
17926
20839
22351
13277
15113
6368
7704
18694
3644
12154
3794



21257
21638
18386
18111
21498
10731
13776
5539
14530
20282
7762
9529



17675
15191
12380
14865
15825
2818
16442
5901
8220
18578
13297
2495



21913
4526
16085
10965
15558
18982
2984
11245
16474
304
477
427



5067
2132
20312
4655
16568
21093
13882
5730
7976
7079
13035
2800



20416
509
307
305
12545
2037
11704
9571
21379
3712
7685
4483



18456
22341
11197
13107
13268
16006
428


 


536:
8250
5293
17519
10969
8775
16532
10286
11811
5689
2871
680
20415



14104
16888
17043
10273
6777
22396
602
4866
12333
9909
22327
4344



9005
16476
2573
10616
19638
15657
8950
4378
2657
3364
21708
985



6316
8776
2238
13856
1099
10466
19656
6879
9313
19604
12069
8122



21385
16087
20487
1424
14078
2427
4175
7418
9385
16892
15809
3499



16714
5768
6322
13627
19469
8252
19551
14976
12742
10499
11322
9597



3569
9336
20132
5283
22127
10381
3149
7067
13886
7478
3897
20869



19398
13086
4191
3669
20800
6799
8453
17539
21956
5724
13878
16687



1797


 


537:
12225
7991
19769
20707
6346
21228
18920
15677
22007
17216
1246
1299



3482
8428
4864
1869
18259
5076
18894
10621
1194
21241
4280
10372



9939
3461
22101
14388
9687
1934
3266
19426
6888
17781
1697
1251



15687
11467
11444
7248
4960
3598
18026
17460
7270
18737
20522
18581



16569
13403
4999
20923
3253
12847
6541
12065
13811
442
18246
10039



21176
20831
13532
19077
10287
9635
12724
898
4347
11482
12691
8702



12055
8006
14571
19675
16542
21229
14891
11459
15395
15872
9301
10051



6843
5509
11707
10865
18908
12031
9844
20469
4041
4623
22346
1849



12095
14281
3840
16733
15322
21786
17305
10611
1428
7631
22045
6676



5894
8015
21902
14896
17330
3090
15122
7759
6196
16678
475
20224



3032
21119
21396
1165
20759
22152
18157
1337
3062
12222
10847
14596



3679
21087
17626
3743
10092
21220
22333
4831
6340
2684
14394
17353



12317
2073
22147
10166
9664
11412
21185
7389
11862
11526
9220
2983



5429
13258
10529
12270
6868
5672
2355
11711
9314
17713
6624
12386



17898
3480
10028
20275
1292
12254
17594
20642
4853
10383
4591
8186



16379
15333
9201
15765
17720
5294
9934
7376
4038
2297
1916
7144



16736
10304
3319
3762
20614
11341
10440
4734
9677
12289
20772
4271



19954
22175
3020
19748
16083
8948
19737
22114
2955
21137
3274
1408



6780
10848
7124
14060
11449
17939
4794
16058
21761
10251
4961
19141



1661
9701
6223
5983
18048
10670
17196
1709
4904
12196
630
8403



8582
6184
18819
3380
9202
16022
17350
12703
15237
17792
4597
11159



1072
20441
15476
15612
17341
12261
21044
4892
1108
1406
22046
18198



6967
20318
12469
22123
7645
10452
1264
712
14602
6109
14712
22157



7745
16695
11050
22271
21574


 


538:
8633
21066
20486
15659
19893
20341
18625
7202
9210
15561
8243
19452



10486
9599
20104
5911
17836
3744
3313
22371
13393
8083
14598
8878



4121
11696
16024
6949
9760
18073
21021
1691
3955
13639
14076
5677



2739
10860
20215
14300
10639
13585
11393
3392
7331
2940
4631
3646



14037
5709
8396
7169
3234
9515
13640
21141
9848
13213
16854
10018



17683
4171
20607
15041
2557
19483
17558
19443
20760
13774
16065
5097



13747
713
8505
18788
18352
817
5302
8167
10335
22171
13497
18845



11474
8093
22332
17185
18392
15387
7019
17613
13932
22281
9232
21810



19046
14214
6221
10362
21188
12117
2909
9179
16519
21842
14950
5628



5467
18729
7240
8434
11000
2806
17205
1863
9645
18886
6782
20838



7708
6070
7788
22286
19842
16891
9527
18008
7791
11889
5849
2006



3823
12758
17857
15410
11552
11047
5550
18384
903
18870
12346
18758



1298
11723
11231
13187
11334
10943
11898
11754
4492
6688
19935
13184



13651
1487
20841
6884
6464
8803
16050
6362
1430
1363
10992
6297



3031
2620
18570
22021
21438
13674
22415
1440
6599
17348
16403
2064



5725
621
12956
13371
3689
813
8410
17547
5880
7069
7272
13965



9296
18273
16456
7801
2112
17834
8758
7378
19838
6388
10579
19671



16279
806
14835
2530
4277
795
12076
5622
13454
3935
4559
18529



18155
19209
15646
5247
6539
16369
5499
21300
14158
12126
17163
16184



993
21342
16623
17172
10687
11363
1456
21186
21282
21603
10623
6475



17459
18745
21602
9996
3042
16551
1505
5224
5300
3833
4119
10925



11838
21509
15409
13868
11753
16835
13881
7206
18880
6510
4201
3942



968
5155
13073
10765
5458
12673
3180
3108
15770
908
21482
5413



18381
14279
4351
15582
22158
16937
12412
13635
14711
5969
9888
21962



11801
16194
10513
4703
11781
15593
17383
18258
9184
7699
9109
1971



18780
3171
11419
10405
631
15583
21710
7483
5744
22254
12810
13894



20089
6909
5948
3120
15669
17412
19244
2016
16850
22002
7260
14202



3293
4461
2271
14582
18598
17872
1569
18470
5671
8965
4082
7327



22511
15167
1160
15734
5450
19432
14387
14587
3176
8359
18160
11357



15085
6948
5337
2567
10321
22182
10881
10168
18477
12537
19015
20260



2170
18422
2440
3225
5484
1260
1822
7532
19248
7693
4353
10441



2660
5734
10038
19976
3453
19019
1674
12312
16581
10232
3727
1523



2140
22432
20580
9947
19324
20180
1384
2325
18901
10198
20110
9744



9208
3182
14144
18875
14329
929
22054
2752
4524
986
977
21117



9567
9679
12352
20957
4772
12912
19652
11510
5608
21827
13765
8183



14331
13670
20503
12206
21116
22320
844
2226
21795
13733
11051
20995



2615
7715
4478
2290
12549
15082
2491
2301
14182
15076
1227
10071



3792
13186
4722
1595
10880
11269
8528
3213
14537
4257
8761
17108




4834
2651
19705
3355
7552
6681









Example 3
Consensus Sequence Build
ClustalW program was selected for multiple sequence alignments of the amino acid sequence of SEQ ID NO: 379 and 10 homologs. Three major factors affecting the sequence alignments dramatically are (1) protein weight matrices; (2) gap open penalty; (3) gap extension penalty. Protein weight matrices available for ClustalW program include Blosum, Pam and Gonnet series. Those parameters with gap open penalty and gap extension penalty were extensively tested. On the basis of the test results, Blosum weight matrix, gap open penalty of 10 and gap extension penalty of 1 were chosen for multiple sequence alignment. Attached are the sequences of SEQ ID NO: 379, its homologs and the consensus sequence SEQ ID NO: 22569 at the end. The symbols for consensus sequence are (1) uppercase letters for 100% identity in all positions of multiple sequence alignment output; (2) lowercase letters for >=70% identity; symbol; (3) “X” indicated <70% identity; (4) dashes “−” meaning that gaps were in >=70% sequences.














SEQ ID NO




 2406
--------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQQQQE-



15414
--------------------------MGSNGGSSNNNNNKVLEKPGQDQLVQQQQHPQE-


  587
------------------------------------MMGRVMEKPSQDLLQQQQQ-----


 9696
------------------------------------MMGRVMEKPSQDLLQQQQQ-----


 5895
------------------------------------MMGRVMEKPSQDLLQQQQQ-----


17251
MFGNGNCDVDNEKTIITSSKWTQSEIDDHKVSMASSTGNRVMEKPGQELLQQQQQ-----


19549
-----------------------------------------MEKQGQELLQQHHQQQQQQ


  379
-----------------------------------MQSKNMIVASSHQQQQQQQPQQPQP


21357
--------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE


17711
--------MGLSSKQVSSSGLDWKQTLLEAQNLELPKPNLMRKQQQQQQQQQQQTQPNSE


13715
-----------------------------------LTLTKCCMQRGSHFRSRSGSQEARS


consensus
--------------------------xxxxxxxxxxxxxxxxxxxxqxxxqqqqxxxxxx


22569


 








APKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRRNKRVKRP



APKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRRNKRVERP


ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP


ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP


ALKCPRCESSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRVKRP


ALRCPRCDSSNTKFCYYNNYSLTQPRHFCKACKRYWTRGGTLRNVPVGGGCRKNKRLKRP


ALKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGGYRRNNKRSTS


QLKCPRCDSSNTKFCYYNNYSLSQPRHFCKACKRYWTRGGTLRNVPVGGSYRKNKRVKRP


SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVKKS


SLKCPRCDSTNTKFCYYNNYNKSQPRHFCRACKRHWTKGGTLRNVPVGG-GRKNKRVRKS


GSSMSRCNSMDTKFCYYNNYNVNQPRHFCKNCQRYWTAGGSMRNVPVGAGRRKNKHTGSV


xlkcpRCxSsnTKFCYYNNYslsQPRHFCkaCkRyWTrGGtlRNVPVGggxRkNkrvxxx


 


LITTNPSSAAIDTAASNNSSN-SSSAPLQPPIDTASTS--------------NHINPLFY


ITSPCSAAIDTASNSSNSSSAPTAAASLQPQIDTASTS--------------NHINPLFY


TNHGDSSSSAANSPSSSNSNPPSQPHLDNIIASSSTTN------------HINNISPFFY


TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY


TNHGDSSSSAANSPSSSNSNPPSQPHIDNIIASSSTTN------------HINNISPFFY


TYPCSNNNNIDFSASPSSSTPSSVVANPNPPSQSQQQQQQQQHHSFDIAATSNHINTMLY


SSNGPTSTTTLIKRPISTIETATTSNSSSPSSTHSSTS--------------NHMNPMFY


------STATTTTASTVSTTNSSSPNNPHQISHFSSMN----------------HHPLFY


ITPITTSSTTTTPITTATSTCTATVTTSIGNNNNNMDAMLG----------CYSHMTIQT


ITTPITTSSTTTHQSQPPLQLALPQSQPQLATTTTTWMLCW----------VVIAT----


YRHTVITPDSLASLQVDGPDLVDHKPLSPFKVNGTILKFG--------------PDAPLC


xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx----------xxxxxxxxxy


 


GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGFSSGVLSNEASDNNGYR-


GLPSSS-SDVNLPLFSRFGSRISSS----GFDLQLNNALGLGFSSRVLSNEASDNNRYR-


GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN--


GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN--


GG-----DVMSSVPFPRFNLHSQLN------------ALGLGFSTGVSENGFSTSNNN--


GGNSCH-DVMNFPFSTRFNSTTRVSNPASGYDNLPQNGLGLGFSSGILMSAAGGEVNLNH


GLSSTNNPCDPNLPFSRFNITSRLSTSSGYDLQPQMNFFGLGFSSGFENNGYTNGFNTS-


GLSDHMSSCNNNLPMIPSRFSDSSK-----------TCSSSGLESEFLSSGFSSLSALG-


PLADDQKNMSSSLYQALIRPPPLLLQQQNLLNTRELEGKDFGIGIGNGNNGIFPSSTLAL


------------------------------------------------------------


ESMASILNLGEQNLSSQLDFTAGAE-----------NREETSCSSACKPVKKKDITQHN-


gxxxxxxxxxxxxxxxxxxxxxxxx----xxxxxxxxxxxxgxsxxxxxxxxxxxxxxx-


 


-------------NWFGSNNTLLSSYTSTTSTTTPAMSSLLSSSLLQQKFMTDGVD----


-------------SGFGSNNMLLSSYTSTT-TTTPAMSSLLLQQKFISGGLKNDAD----


------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQNLMGGGG---GG


------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSCTLLQQNFMGGGG---GG


------------SFFSAYNSMFGSSSSSTCAPSTPVMASLLSSTLLQQKLMSGGG---GG


HHHHHHDEGSYRNGFSTSNNNNYSSIFGSSSTTTPVMASLLSSTLLQQKFMGTGGGIKGG


----------------NNNYDSIFSSSTSASNNTSVMPSVLSSTLLQHKFFDDGLK----


--------LGLPHQMSHDHTINGSFINNSTTNKPFLLSGLFGSSMSSSSTLLQHP-----


P-------------IPHQSQSLLFPFSASSRSFDTNPCSVVSTSLRSSNVYNYGED----


------------------------------------------------------------


------------------------------------------------------------


------------xxxxxxxxxxxxxxxxxxxxxxxxxxsxxxxxxxxxxxxxxxxx--xx


 


--------STNTFQHGLGLTPLEQLQMASDHSSEAGMVALKDVKVELGQNNRLEWNNGAA


--------SSNTFQHGLSLTSLEQLQIASDHSSEAGMVALKDVKVELGQNNNRLEWNGGA


VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG


VKGRDHDQGDNTFHGLAPLQGLRVEGDSNNNIGSKEVKGEGQNRFEWSNNNNNNNNNGGG


EGEEVVIMIKVATLSMAWHRYKGCKWKGIIIIVIILAQKK--------------------


GGGGGGDDDPFHHHQEMDSKEVKLGEGLQNRLDQWNMNNLNGNGGAVFQNQMENMGLSDN


----------YGSDAGSNGAFQDLQFGSKMQNQMEHIGGFYDPASSIYLNATSSSAIGVW


----------HKPMNNGGDMLGQSHLQTLASLQDLHVGGNNEDMKYKEGKLDQISGNING


---------QFKAIEEPTINSTTATIVPSTGGTNNTHHPWEIAAATSGVGLGTSSNSNYW


------------------------------------------------------------


------------------------------------------------------------


xxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxxx


 


CQSQIQHVGLYDPSLYWNNSAATALGVWNDQAANIGSSVTSLI


FQSQIQHVGLYDPLLYWNN-SATALGVWNDQAANIGSSVTSLI


GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPNNIGPSVTSLI


GQNQMEHVGLSDPNSLYWN-TATGLGAWSDQPN-IGPSVTSLI


-------------------------------------------


NASLYWNNNHNNSNNNTSA-TATGLSSVWSTDQPGSNSVSSLI


NDQGANNIGSSVTSLI---------------------------


FMSSSSSLDPSNYNNMWNNASVVNGAWLDPTNNNVGSSLTSLI


NWEDFDSLVSTDLKDPWDDSDIKP-------------------


-------------------------------------------


-------------------------------------------


xxxxxxxxxxxxxxxxxxx-xxxxxxxxxxxxxxxxxxxxxxx









Example 4
Corn Transformation Construct
GATEWAYTM destination vectors (available from Invitrogen Life Technologies, Carlsbad, Calif.) are constructed for insertion of trait-improving DNA for corn transformation. The elements of each destination vector are summarized in Table 18 below and include a selectable marker transcription region and a DNA insertion transcription region. The selectable marker transcription region comprises a Cauliflower Mosaic Virus 35S promoter operably linked to a gene encoding neomycin phosphotransferase II (nptII) followed by both the 3′ region of the Agrobacterium tumefaciens nopaline synthase gene (nos) and the 3′ region of the potato proteinase inhibitor II (pinII) gene. The DNA insertion transcription region comprises a rice actin 1 promoter, a rice actin 1 exon 1 intron 1 enhancer, an att-flanked insertion site and the 3′ region of the potato pinII gene. Following standard procedures provided by Invitrogen the att-flanked insertion region is replaced by recombination with trait-improving DNA, in a sense orientation for expression of a trait-improving protein and in a gene suppression orientation (i.e., either anti-sense orientation or in a sense- and anti-sense orientation) for a trait-improving suppression of a protein. Although the vector with trait-improving DNA inserted at the att-flanked insertion region is useful for plant transformation by direct DNA delivery, such as microprojectile bombardment, it is preferable to bombard target plant tissue with tandem transcription units that have been cut from the vector. For Agrobacterium-mediated transformation of plants the vector also comprises T-DNA borders from Agrobacterium flanking the transcription units.
Vectors for Agrobacterium-mediated transformation are prepared with each of the trait-improving genes having a sequence of SEQ ID NO:1 through SEQ ID NO:269 with the DNA solely in sense orientation for expression of the encoded, cognate trait-improving protein and in a gene suppression orientation for suppression of the cognate protein. Each vector is transformed into corn callus which is propagated into a plant that is grown to produce transgenic seed for each transgenic event. Progeny plants are self-pollinated to produce seed which is selected for homozygous seed. Homozygous seed is used for producing inbred plants, for introgressing the trait into elite lines, and for crossing to make hybrid seed. The progeny transgenic plants comprising the trait-improving DNA with a sequence of SEQ ID NO: 1 through SEQ ID NO: 269 have one or more improved traits identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic corn including inbred and hybrids are also produced with DNA from each of the identified homologs of DNA of SEQ ID NO: 1 through SEQ ID NO: 269 to provide transgenic seeds and plants which are identified from total transgenic events by screening for the improved agronomic trait. Transgenic corn plants are also produced where the trait-improving DNA is transcribed by each of the promoters from the group selected from, a maize globulin 1 promoter, a maize oleosin promoter, a glutelin 1 promoter, an aldolase promoter, a zein Z27 promoter, a pyruvate orthophosphate dikinase (PPDK) promoter, a soybean 7S alpha promoter, a peroxiredoxin antioxidant (Per1) promoter and a CaMV 35S promoter.
Seed produced by the plants is provided to growers to enable production of corn crops with improved traits associated with the trait-improving DNA.









TABLE 18





FUNCTION
ELEMENT
REFERENCE







DNA insertion
Rice actin 1 promoter
U.S. Pat. No. 5,641,876


transcription region
Rice actin 1 exon 1, intron 1
U.S. Pat. No. 5,641,876



enhancer


DNA insertion
AttR1
GATEWAY TM Cloning Technology


transcription region

Instruction Manual


(att - flanked insertin
CmR gene
GATEWAY TM Cloning Technology


region)

Instruction Manual



ccdA, ccdB genes
GATEWAY TM Cloning Technology




Instruction Manual



attR2
GATEWAY TM Cloning Technology




Instruction Manual


DNA insertion
Potato pinII 3′ region
An et al., (1989) Plant Cell 1: 115-122


transcription region


selectable marker
CaMV 35S promoter
U.S. Pat. No. 5,858,742


transcription region
nptII selectable marker
U.S. Pat. No. 5,858,742



nos 3region
U.S. Pat. No. 5,858,742



PinII 3′ region
An et al., (1989) Plant Cell 1: 115-122


E. coli maintenance
ColE1 origin of replication


region
F1 origin of replication



Bla ampicillin resistance









Example 5
Soybean Transformation Construct
Constructs for use in transformation of soybean are prepared by restriction enzyme based cloning into a common expression vector. Elements of an exemplary common expression vector are shown in Table 19 below and include a selectable marker expression cassette and a gene of interest expression cassette. The selectable marker expression cassette comprises Arabidopsis act 7 gene (AtAct7) promoter with intron and 5′UTR, the transit peptide of Arabidopsis EPSPS, the synthetic CP4 coding region with dicot preferred codon usage and a 3′ UTR of the nopaline synthase gene. The gene of interest expression cassette comprises a Cauliflower Mosaic Virus 35S promoter operably linked to a trait-improving gene in a sense orientation for expression of a trait-improving protein and in a gene suppression orientation (i.e., either anti-sense orientation or in a sense- and anti-sense orientation for a trait-improving suppression of a protein.
Vectors similar to that described above are constructed for use in Agrobacterium mediated soybean transformation systems, with each of the trait-improving DNA having a sequence of SEQ ID NO:1 though SEQ ID NO:269 and the respective identified homologs with the DNA in sense orientation for expression of the encoded, cognate protein and in a gene suppression arrangement for suppression of the cognate protein. Each vector is transformed into soybean embryo tissue to produce transgenic events which are grown into plants that produce progeny transgenic plants and seed for screening to identify the transgenic soybean plants of this invention that exhibit the enhanced agronomic trait imparted by DNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respective homolog. The transgenic soybean plants of this invention are identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic soybean plants are also produced where the trait-improving DNA is transcribed by a napin promoter and Arabidopsis SSU promoter.
Seed produced by the plants is provided to growers to enable production of soybean crops with improved traits associated with the trait-improving DNA.









TABLE 19





Function
Element
Reference







Agro transformation
B-ARGtu.right border
Depicker, A. et al (1982) Mol




Appl Genet 1: 561-573


Antibiotic resistance
CR-Ec.aadA-SPC/STR


Repressor of primers from the ColE1 plasmid
CR-Ec.rop


Origin of replication
OR-Ec.oriV-RK2


Agro transformation
B-ARGtu.left border
Barker, R. F. et al (1983) Plant




Mol Biol 2: 335-350


Plant selectable marker expression cassette
Arabidopsis act 7 gene
McDowell et al., (1996) Plant



(AtAct7) promoter with
Physiol. 111: 699-711.



intron and 5′UTR



5′ UTR of Arabidopsis act 7 gene



Intron in 5′UTR of AtAct7



Transit peptide region of
Klee, H. J. et at (1987) MGG



Arabidopsis EPSPS
210: 437-442



Synthetic CP4 coding region with



dicot preferred codon usage



A 3′ UTR of the nopaline synthase
U.S. Pat. No. 5,858,742



gene of Agrobacterium



tumefaciens Ti plasmid


Plant gene of interest expression cassette
Promoter for 35S RNA from
U.S. Pat. No. 5,322,938



CaMV containing a duplication of



the −90 to −350 region



Gene of interest insertion site



Cotton E6 3′ end
GenBank accession U30508









Example 6
Cotton Transformation
Vectors similar to that described above for soybean transformation are constructed for use in 5 Agrobacterium mediated cotton transformation systems, with each of the trait-improving DNA having a sequence of SEQ ID NO:1 though SEQ ID NO:269 and the respective identified homologs with the DNA in sense orientation for expression of the encoded, cognate protein and in a gene suppression arrangement for suppression of the cognate protein. Each vector is transformed into cotton embryo tissue to produce transgenic events which are grown into plants that produce progeny transgenic plants and seed for screening to identify the transgenic soybean plants of this invention that exhibit the enhanced agronomic trait imparted by DNA with a sequence of SEQ ID NO:1 through SEQ ID NO:269 or a respective homolog. The transgenic cotton plants of this invention are identified by agronomic trait screening including, but not limited to, enhanced nitrogen use efficiency, increased yield, enhanced water use efficiency, growth under cold stress and enhanced oil, starch and protein levels. Transgenic cotton plants are also produced where the trait-improving DNA is transcribed by a napin promoter and Arabidopsis SSU promoter.
Seed produced by the plants is provided to growers to enable production of cotton crops with improved traits associated with the trait-improving DNA.

## Claims

1. A transgenic seed for a crop plant, wherein the genome of said transgenic seed comprises a recombinant DNA which expresses a protein having the amino acid sequence SEQ ID NO: 481, wherein transgenic plants grown from said seed exhibit increased yield as compared to control plants of the same species lacking said recombinant DNA.

2. The transgenic seed of claim 1, wherein the seed is corn, soybean, wheat or cotton seed.

3. A method of facilitating production of a crop comprising providing to a grower of said crop transgenic seeds, wherein the genome of said transgenic seeds comprises a recombinant DNA which expresses a protein having the amino acid sequence SEQ ID NO: 481, and wherein plants grown from said transgenic seeds exhibit increased yield as compared to control plants of the same species lacking said recombinant DNA; and obtaining from said transgenic seeds transgenic plants comprising said recombinant DNA, thereby facilitating production of said crop.

4. A method according to claim 3, wherein (a) said control plants are susceptible to a yield-limiting environment; and (b) transgenic plants grown from said transgenic seeds thrive in said yield-limiting environment.

5. The method of claim 3, wherein the transgenic seeds are corn, soybean, wheat or cotton seeds.

6. A method of producing a plant comprising planting transgenic seeds, wherein the genome of said transgenic seeds comprises recombinant DNA which expresses a protein having the amino acid sequence SEQ ID NO: 481, so that at least one plant grown from said transgenic seeds exhibits increased yield as compared to control plants of the same species lacking said recombinant DNA; and harvesting said at least one plant.