# Patent

## Title
Genetic loci associated with mechanical stalk strength in maize

## Abstract

The invention relates to methods and compositions for identifying and for selecting maize plants with mechanical stalk strength characteristics. The methods use molecular markers to identify and select plants with increased mechanical stalk strength or to identify and counter-select plants with decreased mechanical stalk strength. Maize plants generated by the methods of the invention are also a feature of the invention.

## Background
CROSS-REFERENCE TO RELATED APPLICATION
This application is a Continuation of U.S. application Ser. No. 12/502,365, filed Jul. 14, 2009, which claims the benefit of U.S. Provisional Application No. 61/080,783, filed Jul. 15, 2008, which is incorporated by reference in its entirety.


FIELD OF THE INVENTION
The present disclosure relates to compositions and methods useful in enhancing mechanical stalk strength in maize plants.
BACKGROUND OF THE INVENTION
In maize, stalk lodging, or stalk breakage, accounts for significant annual yield losses in the United States. During a maize plant's vegetative growth phase, rapid growth weakens cell walls, making stalk tissue brittle and increasing the propensity for stalks to snap when exposed to strong, sudden winds and/or other weather conditions. This type of stalk lodging, called green snap or brittle snap, typically occurs at the V5 to V8 stage, when the growing point of a maize plant is emerging from the soil line, or at the V12 to R1 stage, about two weeks prior to tasseling and until just after silking. Another type of stalk lodging, late season stalk lodging occurs near harvest when the stalk cannot support the weight of the ear. Factors that weaken the stalk during late season include insect attack, such as the European corn borer tunneling into stalk and ear shanks, and infection by pathogens such as Colletotrichum graminicola, the causative agent in Anthracnose stalk rot. Adverse fall weather conditions also contribute to late season stalk lodging.
The mechanical strength of the maize stalk plays a major role in a plant's resistance to all types of stalk lodging, and therefore, is of great value to the farmer. Enhancing overall mechanical stalk strength in maize will make stalks stronger during both vegetative development and late season, thereby reducing yield and grain quality losses. Moreover, maize plants with increased mechanical stalk strength can remain in the field for longer periods of time, allowing farmers to delay harvest, if necessary.
Selection through the use of molecular markers associated with mechanical stalk strength has the advantage of permitting at least some selection based solely on the genetic composition of the progeny, and selections can be made very early on in the plant life cycle, even as early as the seed stage. The increased rate of selection that can be obtained through the use of molecular markers associated with mechanical stalk strength means that plant breeding for increased mechanical stalk strength can occur more rapidly.
It is desirable to provide compositions and methods for identifying and selecting maize plants that display overall increased mechanical stalk strength.
SUMMARY
Compositions and methods for selecting maize plants with mechanical stalk strength characteristics are provided herein, including compositions and methods for identifying and selecting maize plants with increased mechanical stalk strength and compositions and methods for identifying and counter-selecting maize plants that have decreased mechanical stalk strength.
In one embodiment, methods of selecting a maize plant or germplasm with mechanical stalk strength characteristics are provided. In these methods, DNA is obtained, and the presence of at least one marker allele is detected. The marker allele can include any marker allele that is linked to and associated with any of the following marker alleles: a “G” at PHM3468.1, a “T” at PHM3468.4, a “G” at PHM3468.18, a “T” at PHM12521.12, a “C” at PHM10840.105, an “A” at PHM10840.118, a “C” at PHM10840.130, a “C” at PHM16736.6, an “A” at PHM16736.14, a “C” at PHM14053.7, a “C” at PHM14053.8, a “C” at PHM14053.14, a “T” at PHM405.35, a “C” at PHM12025.26, a “T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C” at PHM10786-5-U, a “T” at PHM10786-6-U, a “G” at PHM8057-801-U, a “C” at PHM201-16-U, a “C” or a “G” at PHM201-17-U, a “T” or a “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a “G” at PHM5421-5-V, a “G” or a “T” at PHM4115-35-U, a “T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a “C” at C00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, and a “T” at PHM11186-16-V. A maize plant or germplasm that has the marker allele linked to and associated with any of the marker alleles listed above is then selected.
In other embodiments, the marker allele can be linked to any of the following marker alleles: a “G” at PHM3468.1, a “T” at PHM3468.4, a “G” at PHM3468.18, a “T” at PHM12521.12, a “C” at PHM10840.105, an “A” at PHM10840.118, a “C” at PHM10840.130, a “C” at PHM16736.6, an “A” at PHM16736.14, a “C” at PHM14053.7, a “C” at PHM14053.8, a “C” at PHM14053.14, a “T” at PHM405.35, a “C” at PHM12025.26, a “T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C” at PHM10786-5-U, a “T” at PHM10786-6-U, a “G” at PHM8057-801-U, a “C” at PHM201-16-U, a “C” or a “G” at PHM201-17-U, a “T” or a “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a “G” at PHM5421-5-V, a “G” or a “T” at PHM4115-35-U, a “T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a “C” at C00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, and a “T” at PHM11186-16-V by 30 cM, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 cM.
In other embodiments, the marker allele can be any of the following marker alleles: a “G” at PHM3468.1, a “T” at PHM3468.4, a “G” at PHM3468.18, a “T” at PHM12521.12, a “C” at PHM10840.105, an “A” at PHM10840.118, a “C” at PHM10840.130, a “C” at PHM16736.6, an “A” at PHM16736.14, a “C” at PHM14053.7, a “C” at PHM14053.8, a “C” at PHM14053.14, a “T” at PHM405.35, a “C” at PHM12025.26, a “T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C” at PHM10786-5-U, a “T” at PHM10786-6-U, a “G” at PHM8057-801-U, a “C” at PHM201-16-U, a “C” or a “G” at PHM201-17-U, a “T” or a “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a “G” at PHM5421-5-V, a “G” or a “T” at PHM4115-35-U, a “T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a “C” at C00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, and a “T” at PHM11186-16-V.
In another embodiment, methods of selecting a maize plant or germplasm with mechanical stalk strength characteristics are provided. In these methods, DNA is obtained, and the absence of at least one marker allele is detected. The marker allele can include any marker allele that is linked to and associated with any of the following marker alleles: a “T” at PHM2130.24, an “A” at PHM2130.29, a “C” at PHM2130.30, a “G” at PHM2130.33, a “G” at PHM15089.13, a “C” at PHM12706.14, a “C” at PHM201.10, an “A” at PHM201.18, a “T” at PHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G” at PHM9364-6-U. A maize plant or germplasm that does not have the marker allele linked to and associated with any of the marker alleles listed above is then selected.
In other embodiments, the marker allele can be linked to any of the following marker alleles: a “T” at PHM2130.24, an “A” at PHM2130.29, a “C” at PHM2130.30, a “G” at PHM2130.33, a “G” at PHM15089.13, a “C” at PHM12706.14, a “C” at PHM201.10, an “A” at PHM201.18, a “T” at PHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G” at PHM9364-6-U, by 30 cM, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 cM.
In other embodiments, the marker allele can be any of the following marker alleles: a “T” at PHM2130.24, an “A” at PHM2130.29, a “C” at PHM2130.30, a “G” at PHM2130.33, a “G” at PHM15089.13, a “C” at PHM12706.14, a “C” at PHM201.10, an “A” at PHM201.18, a “T” at PHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G” at PHM9364-6-U.
In another embodiment, methods for identifying maize plants with increased or decreased mechanical stalk strength by detecting at least one marker allele associated with increased or decreased mechanical stalk strength in the germplasm of a maize plant are provided. The marker locus can be selected from any of the marker loci provided in Table 3 or Table 7, as well as any other marker that is linked to these markers. The marker locus can be found within any of the following chromosomal intervals on linkage group 5, comprising and flanked by:

    
    
        (i) PHM654 and PHM6727;
        (ii) PHM12632 and PHM3323;
        (iii) PHM201 and PHM3323; and
        (iv) PHM201 and PHM3468.

More than one marker locus can be selected in the same plant with no limitation as to which markers are selected in combination. The markers used in combinations can be any of the markers in Table 3 or Table 7, any other marker linked to the markers in Table 3 or Table 7 (e.g., the linked markers determined from theMaizeGDB resource), or any marker within the intervals described herein.

    
    


In another embodiment, methods of selecting maize plants with increased mechanical stalk strength are provided. In one aspect, a first maize plant is obtained that has at least one allele of a marker locus wherein the allele is associated with increased mechanical stalk strength. The marker locus can be found within any of the following chromosomal intervals on linkage group 5, comprising and flanked by:

    
    
        (i) PHM654 and PHM6727;
        (ii) PHM12632 and PHM3323;
        (iii) PHM201 and PHM3323; or
        (iv) PHM201 and PHM3468.

The first maize plant is crossed to a second maize plant, and the progeny plants resulting from the cross are evaluated for the allele of the first maize plant. Progeny plants that possess the allele from the first maize plant can then be selected as having increased mechanical stalk strength.

    
    


In another embodiment, methods for not selecting plants with decreased mechanical stalk strength are provided. In one aspect, a first maize plant is obtained that has at least one allele of a marker locus wherein the allele is associated with decreased mechanical stalk strength. The marker locus can be found within any of the following chromosomal intervals on linkage group 5, comprising and flanked by:

    
    
        (i) PHM654 and PHM6727;
        (ii) PHM12632 and PHM3323;
        (iii) PHM201 and PHM3323; and
        (iv) PHM201 and PHM3468.

The first maize plant is crossed to a second maize plant, and the progeny plants resulting from the cross are evaluated for the allele of the first maize plant. Progeny plants that possess the allele from the first maize plant can be identified as having decreased mechanical stalk strength and can be removed from a breeding program or planting.

    
    


In another embodiment, methods for selecting a maize plant with mechanical stalk strength characteristics are provided in which at least one marker locus is assayed within the maize plant. The marker locus can be located:
a. on chromosome 1, within the interval comprising and flanked by PHM7844 and PHM8029;
b. on chromosome 1, within the interval comprising and flanked by PHM7844 and PHM574;
c. on chromosome 1, within the interval comprising and flanked by PHM11754 and PHM1481;
d. on chromosome 1, within the interval comprising and flanked by PHM6427 and PHM1481;
e. on chromosome 1, within the interval comprising and flanked by PHM11125 and PHM13958;
f. on chromosome 1, within the interval comprising and flanked by PHM10468 and PHM13958;
g. on chromosome 9, within the interval comprising and flanked by PHM4578 and PHM11186; or
h. on chromosome 9, within the interval comprising and flanked by PHM14053 and PHM16736; and is associated with mechanical stalk strength. The maize plant is then selected if it possesses a favorable allele at the marker locus.
In another embodiment, methods for selecting a maize plant with mechanical stalk strength characteristics are provided in which at least one marker locus is assayed within the maize plant. The marker locus can be located:
a. on chromosome 1, within the interval comprising and flanked by PHM7844 and PHM8029;
b. on chromosome 1, within the interval comprising and flanked by PHM7844 and PHM574;
c. on chromosome 1, within the interval comprising and flanked by PHM11754 and PHM1481;
d. on chromosome 1, within the interval comprising and flanked by PHM6427 and PHM1481;
e. on chromosome 1, within the interval comprising and flanked by PHM11125 and PHM13958;
f. on chromosome 1, within the interval comprising and flanked by PHM10468 and PHM13958;
g. on chromosome 9, within the interval comprising and flanked by PHM4578 and PHM11186; or
h. on chromosome 9, within the interval comprising and flanked by PHM14053 and PHM16736; and is associated with mechanical stalk strength. The maize plant is then selected if it does not possess an unfavorable allele at the marker locus.
Maize plants identified or selected by any of the methods described herein are also included.



BRIEF DESCRIPTION OF FIGURES AND SEQUENCE LISTINGS
The invention can be more fully understood from the following detailed description and the accompanying drawings and Sequence Listing which form a part of this application. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Research 13:3021-3030 (1985) and in the Biochemical Journal 219 (No. 2): 345-373 (1984), which are herein incorporated by reference in their entirety. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
FIGS. 1A-I show the physical map arrangement of sequenced BACs (obtained from the Maize Genome Browser, which is publicly available on the internet; http://www.maizesequence.org) that assemble to the chromosome 5 region defined by and including BACs c0216105 and c0117h02. The positions of the markers listed in Table 7 are also indicated.
FIG. 2A shows a structured association analysis, wherein chromosome 5 markers were tested for significance of association with mechanical stalk strength. Mechanical stalk strength values were obtained using an InstronTM machine and a three-point bend test, on a set of 189 lines. Stalk strength values were acquired from plants in late season. X axis: Distance expressed in cM on Chr. 5. Y axis: probability value.
FIG. 2B shows an association analysis of an NSS subpopulation, wherein chromosome 5 markers were tested for significance of association with mechanical stalk strength. The NSS subpopulation consisted of 60 lines, which varied in maturity from a CRM (comparative relative maturity) of 105 to a CRM of 110. Mechanical stalk strength values were obtained using an InstronTM machine and a three-point bend test and were acquired from plants in late season. X axis: Distance expressed in cM on Chr. 5. Y axis: probability value.
FIGS. 2C-E show the clusters of markers on chromosome 5 that co-segregate with mechanical stalk strength in the NSS subpopulation at the following p-levels: C) a p-level of ≦0.01 (white data points represent the region defined by and including PHM654 and PHM6727), D) a p-level of ≦0.001 (white data points represent the region defined by and including PHM12632 and PHM3323, and E) a p-level of ≦0.0001 (white data points represent the region defined by and including PHM201 and PHM3323). Black dots represent associated markers that do not fall within each respective cluster.
FIG. 3 shows associations between chromosome 9 marker loci and mechanical stalk strength in the NSS subpopulation. The NSS subpopulation consisted of 60 lines, which varied in maturity from a CRM (comparative relative maturity) of 105 to a CRM of 110. Mechanical stalk strength values were obtained using an InstronTM machine and a three-point bend test and were acquired from plants in late season. X axis: Distance expressed in cM on Chr. 9. Y axis: probability value.
FIG. 4 shows associations between chromosome 1 marker loci and mechanical stalk strength in the NSS subpopulation. The NSS subpopulation consisted of 60 lines, which varied in maturity from a CRM (comparative relative maturity) of 105 to a CRM of 110. Mechanical stalk strength values were obtained using an InstronTM machine and a three-point bend test and were acquired from plants in late season. X axis: Distance expressed in cM on Chr. 1. Y axis: probability value.
FIG. 5 shows the PHM marker alleles for A) PHM201, B) PHM5421, and C) PHM3468. The positions of the polymorphisms relative to the reference sequence are represented in the numbers at the top of each table.
FIG. 6 depicts a plot of the average mechanical stalk strength values for: A) haplotypes constituting marker alleles at PHM201 and at PHM5421 and B) PHM3468 marker alleles.
FIG. 7 shows the composite interval mapping results for data set 1 (portable InstronTM data) using the B73×Mo17 (IBM) syn4 population. A peak of significance was identified on chromosome 5. Marker positions on the x-axis correspond to the modified IBM2 genetic map. The y-axis represents the LOD score.
FIG. 8 shows the composite interval mapping results for data set 2 (device and method described in patent application US2007/0125155 (published Jun. 6, 2007)) using the B73×Mo17 (IBM) syn4 population. A peak of significance was identified on chromosome 5. Marker positions on the x-axis correspond to the modified IBM2 genetic map. The y-axis represents the LOD score.



The sequence descriptions and Sequence Listing attached hereto comply with the rules governing nucleotide and/or amino acid sequence disclosures in patent applications as set forth in 37 C.F.R. §1.821-1.825. The Sequence Listing contains the one letter code for nucleotide sequence characters and the three letter codes for amino acids as defined in conformity with the IUPAC-IUBMB standards described in Nucleic Acids Res. 13:3021-3030 (1985) and in the Biochemical J. 219 (2):345-373 (1984) which are herein incorporated by reference. The symbols and format used for nucleotide and amino acid sequence data comply with the rules set forth in 37 C.F.R. §1.822.
Table 1 lists the sequences described herein that are associated with the PHM markers, along with the corresponding identifiers (SEQ ID NO:) as used in the attached Sequence Listing.







TABLE 1







PHM Marker Sequences












Reference





sequence














Marker
(SEQ ID

Forward
Reverse












Locus
NO:)

(SEQ ID NO:)

















PHM654
1
Internal
40
41





External
42
43



PHM111
2
Internal
44
45





External
46
47



PHM10100
3
Internal
48
49





External
50
51



PHM7357
4
Internal
52
53





External
54
55



PHM5349
5
Internal
56
57





External
58
59



PHM4167
6
Internal
60
61





External
62
63



PHM14947
7
Internal
64
65





External
66
67



PHM5266
8
Internal
68
69





External 
70
71



PHM12632
9
Internal
72
73





External
74
75



PHM201
10
Internal
76
77





External
78
79



PHM4861
11
Internal
80
81





External
82
83



PHM5421
12
Internal
84
85





External
86
87



PHM4115
13
Internal
88
89





External
90
91



PHM12521
14
Internal
92
93





External
94
95



PHM3468
15
Internal
96
97





External
98
99



PHM10840
16
Internal
100
101





External
102
103



PHM12755
17
Internal
104
105





External
106
107



PHM13879
18
Internal
108
109





External
110
111



PHM4103
19
Internal
112
113





External
114
115



PHM5363
20
Internal
116
117





External
118
119



PHM14751
21
Internal
120
121





External
122
123



PHM16138
22
Internal
124
125





External
126
127



PHM7877
23
Internal
128
129





External
130
131



PHM9518
24
Internal
132
133





External
134
135



PHM7802
25
Internal
136
137





External
138
139



PHM2134
26
Internal
140
141





External
142
143



PHM7808
27
Internal
144
145





External
146
147



PHM9627
28
Internal
148
149





External
150
151



PHM13716
29
Internal
152
153





External
154
155



PHM18731
30
Internal
156
157





External
158
159



PHM2189
31
Internal
160
161





External
162
163



PHM7734
32
Internal
164
165





External
166
167



PHM3323
33
Internal
168
169





External
170
171



PHM4736
34
Internal
172
173





External
174
175



PHM6441
35
Internal
176
177





External
178
179



PHM430
36
Internal
180
181





External
182
183



PHM12224
37
Internal
184
185





External
186
187



PHM11904
38
Internal
188
189





External
190
191



PHM6727
39
Internal
192
193





External
194
195



PHM4578
196
Internal
215
216





External
214
217



PHM11186
197
Internal
219
220





External
218
221



PHM14053
198
Internal
223
224





External
222
225



PHM16736
199
Internal
227
228





External
226
229



PHM7844
200
Internal
231
232





External
230
233



PHM8029
201
Internal
235
236





External
234
237



PHM2130
202
Internal
239
240





External
238
241



PHM11754
203
Internal
243
244





External
242
245



PHM1481
204
Internal
247
248





External
246
249



PHM15089
205
Internal
251
252





External
250
253



PHM574
206
Internal
255
256





External
254
257



PHM6427
207
Internal
259
260





External
258
261



PHM11125
208
Internal
263
264





External
262
265



PHM13958
209
Internal
267
268





External
266
269



PHM10468
210
Internal
271
272





External
270
273



PHM12706
211
Internal
275
276





External
274
277



PHM405
212
Internal
279
280





External
278
281



PHM12025
213
Internal
283
284





External
282
285










SEQ ID NOs:1-285 (See Table 1).
SEQ ID NOs:286-389 (See Table 2).







TABLE 2







Production Markers and Their Sequences

















Forward
Reverse





Probe
Probe



Primer
Primer

Allele
Allele
Dye
Dye
1
2














Marker Name
SEQ ID NO:
Sense
1
2
1
2
SEQ ID NO:



















PHM18693-9-U
286
287
Anti-
T
C
Red
Fam
288
289





Sense








 


PHM10786-11-
290
291
Sense
T
G
Red
Fam
292
293


U











 


PHM10786-5-U
294
295
Anti-
C
A
Red
Fam
296
297





Sense








 


PHM10786-6-U
298
299
Anti-
T
C
Red
Fam
300
301





Sense








 


PHM8057-801-
302
303
Anti-
T
G
Red
Fam
304
305


U


Sense








 


PHM4044-11-U
306
307
Sense
T
A
Red
Fam
308
309


 


PHM14080-16-
310
311
Sense
G
A
Red
Fam
312
313


V











 


PHM15089-10-
314
315
Sense
G
C
Red
Fam
316
317


U











 


PHM9364-6-U
318
319
Sense
G
A
Red
Fam
320
321


 


PHM201-16-U
322
323
Anti-
T
C
Red
Fam
324
325





Sense








 


PHM201-17-U
326
327
Sense
C
G
Red
Fam
328
329


 


PHM4861-20-U
330
331
Sense
T
G
Red
Fam
332
333


 


PHM4861-21-U
334
335
Anti-
G
A
Red
Fam
336
337





Sense








 


PHM5421-5-V
338
339
Sense
G
T
Red
Fam
340
341


 


PHM4115-35-U
342
343
Anti-
G
T
Red
Fam
344
345





Sense








 


PHM12521-18-
346
347
Sense
T
G
Red
Fam
348
349


U











 


PHM12521-19-
350
351
Sense
G
A
Red
Fam
352
353


U











 


PHM12521-29-
354
355
Sense
G
A
Red
Fam
356
357


U











 


C00386-397-U
358
359
Sense
T
C
Red
Fam
360
361


 


PHM13418-18-
362
363
Sense
T
C
Fam
Red
364
365


U











 


PHM13418-10-
366
367
Sense
C
T
Fam
Red
368
369


U











 


PHM113-7-U
370
371
Anti-
C
T
Fam
Red
372
373





Sense








 


PHM10337-11-
374
375
Anti-
T
C
Red
Fam
376
377


U


Sense








 


PHM16736-8-V
378
379
Sense
T
A
Red
Fam
380
381


 


PHM12025-48-
382
383
Anti-
T
C
Red
Fam
384
385


U


Sense








 


PHM11186-16-
386
387
Sense
T
C
Red
Fam
388
389


V









DETAILED DESCRIPTION
The present invention provides allelic compositions in maize and methods for identifying and for selecting maize plants with favorable mechanical stalk strength. Also within the scope of this invention are allelic compositions and methods used to identify and to counter-select maize plants that have decreased mechanical stalk strength. The following definitions are provided as an aid to understand this invention.
The term “allele” refers to one of two or more different nucleotide sequences that occur at a specific locus.
The term “altered mechanical stalk strength” refers to an increase or a decrease in the ability of maize plants to resist breakage as a result of having a particular allele at a marker locus or a combination of alleles at multiple marker loci.
An “amplicon” is a DNA fragment generated using the polymerase chain reaction.
The term “amplifying” in the context of nucleic acid amplification is any process whereby additional copies of a selected nucleic acid (or a transcribed form thereof) are produced. Typical amplification methods include various polymerase based replication methods, including the polymerase chain reaction (PCR), ligase mediated methods such as the ligase chain reaction (LCR) and RNA polymerase based amplification (e.g., by transcription) methods.
The term “assemble” applies to BACs and their propensities for coming together to form contiguous stretches of DNA. A BAC “assembles” to a contig based on sequence alignment, if the BAC is sequenced, or via the alignment of its BAC fingerprint to the fingerprints of other BACs. The assemblies can be found using the Maize Genome Browser, which is publicly available on the internet.
An allele is “associated with” a trait when it is linked to it and when the presence of the allele is an indicator that the desired trait or trait form will occur in a plant comprising the allele.
A “BAC”, or bacterial artificial chromosome, is a cloning vector derived from the naturally occurring F factor of Escherichia coli. BACs can accept large inserts of DNA sequence. In maize, a number of BACs, or bacterial artificial chromosomes, each containing a large insert of maize genomic DNA, have been assembled into contigs (overlapping contiguous genetic fragments, or ‘contiguous DNA”).
“Backcrossing” refers to the process whereby hybrid progeny are repeatedly crossed back to one of the parents. In a backcrossing scheme, the “donor” parent refers to the parental plant with the desired gene or locus to be introgressed. The “recipient” parent (used one or more times) or “recurrent” parent (used two or more times) refers to the parental plant into which the gene or locus is being introgressed. For example, see Ragot, M. et al. (1995) Marker-assisted backcrossing: a practical example, in Techniques et Utilisations des Marqueurs Moleculaires Les Colloques, Vol. 72, pp. 45-56, and Openshaw et al., (1994) Marker-assisted Selection in Backcross Breeding, Analysis of Molecular Marker Data, pp. 41-43. The initial cross gives rise to the F1 generation; the term “BC1” then refers to the second use of the recurrent parent, “BC2” refers to the third use of the recurrent parent, and so on.
A centimorgan (“cM”) is a unit of measure of recombination frequency. One cM is equal to a 1% chance that a marker at one genetic locus will be separated from a marker at a second locus due to crossing over in a single generation.
As used herein, the term “chromosomal interval” designates a contiguous linear span of genomic DNA that resides in planta on a single chromosome. The genetic elements or genes located on a single chromosomal interval are physically linked. The size of a chromosomal interval is not particularly limited. In some aspects, the genetic elements located within a single chromosomal interval are genetically linked, typically with a genetic recombination distance of, for example, less than or equal to 20 cM, or alternatively, less than or equal to 10 cM. That is, two genetic elements within a single chromosomal interval undergo recombination at a frequency of less than or equal to 20% or 10%.
A “chromosome” can also be referred to as a “linkage group”.
The term “complement” refers to a nucleotide sequence that is complementary to a given nucleotide sequence, i.e. the sequences are related by the base-pairing rules.
The term “contiguous DNA” refers to overlapping contiguous genetic fragments.
The term “crossed” or “cross” means the fusion of gametes via pollination to produce progeny (e.g., cells, seeds or plants). The term encompasses both sexual crosses (the pollination of one plant by another) and selfing (self-pollination, e.g., when the pollen and ovule are from the same plant). The term “crossing” refers to the act of fusing gametes via pollination to produce progeny.
Maize plants with “decreased mechanical stalk strength” are more prone to stalk lodging and have mechanically weaker stalks. The term “decreased” relates to the degree of physical strength and/or the degree of resistance to breakage and is used to describe the effect on mechanical stalk strength when a particular allele is present or absent.
A plant referred to herein as “diploid” has two sets (genomes) of chromosomes.
A plant referred to herein as a “doubled haploid” is developed by doubling the haploid set of chromosomes. A doubled haploid plant is considered a homozygous plant.
An “elite line” is any line that has resulted from breeding and selection for superior agronomic performance.
A “favorable allele” is the allele at a particular locus that confers, or contributes to an agronomically desirable phenotype and that allows the identification of plants with that agronomically desirable phenotype e.g., increased mechanical stalk strength. A “favorable allele” of a marker is a marker allele that segregates with the favorable phenotype.
Maize plants with “favorable” mechanical stalk strength characteristics have higher than average mechanical stalk strength and are less prone to stalk lodging. Maize plants with “unfavorable” mechanical stalk strength characteristics have lower than average mechanical stalk strength and are more prone to stalk lodging.
An “unfavorable allele” of a marker is a marker allele that segregates with the unfavorable plant phenotype, therefore providing the benefit of identifying plants that can be removed from a breeding program or planting.
“Fragment” is intended to mean a portion of a nucleotide sequence. Fragments can be used as hybridization probes or PCR primers using methods disclosed herein.
A “genetic map” is a description of genetic linkage relationships among loci on one or more chromosomes (or linkage groups) within a given species, generally depicted in a diagrammatic or tabular form. For each genetic map, distances between loci are measured by the recombination frequencies between them, and recombinations between loci can be detected using a variety of markers. A genetic map is a product of the mapping population, types of markers used, and the polymorphic potential of each marker between different populations. The order and genetic distances between loci (e.g. markers) can differ from one genetic map to another. For example, 10 cM on the internally derived genetic map (also referred to herein as “PHB” for Pioneer Hi-Bred) is roughly equivalent to 25-30 cM on the IBM2 2005 neighbors frame map (a high resolution map available on maizeGDB). However, information can be correlated from one map to another using a general framework of common markers. One of ordinary skill in the art can use the framework of common markers to identify the positions of markers and loci of interest on each individual genetic map. A comparison of marker positions between the internally derived genetic map and the IBM2 neighbors genetic map for the chromosome 5 QTL can be seen in Table 7.
“Genetic recombination frequency” is the frequency of a crossing over event (recombination) between two genetic loci. Recombination frequency can be observed by following the segregation of markers and/or traits following meiosis.
“Genome” refers to the total DNA, or the entire set of genes, carried by a chromosome or chromosome set.
The term “genotype” is the genetic constitution of an individual (or group of individuals) at one or more genetic loci, as contrasted with the observable trait (the phenotype). Genotype is defined by the allele(s) of one or more known loci that the individual has inherited from its parents. The term genotype can be used to refer to an individual's genetic constitution at a single locus, at multiple loci, or, more generally, the term genotype can be used to refer to an individual's genetic make-up for all the genes in its genome.
“Germplasm” refers to genetic material of or from an individual (e.g., a plant), a group of individuals (e.g., a plant line, variety or family), or a clone derived from a line, variety, species, or culture. The germplasm can be part of an organism or cell, or can be separate from the organism or cell. In general, germplasm provides genetic material with a specific molecular makeup that provides a physical foundation for some or all of the hereditary qualities of an organism or cell culture. As used herein, germplasm includes cells, seed or tissues from which new plants may be grown, or plant parts, such as leafs, stems, pollen, or cells that can be cultured into a whole plant.
A plant referred to as “haploid” has a single set (genome) of chromosomes.
A “haplotype” is the genotype of an individual at a plurality of genetic loci, i.e. a combination of alleles. Typically, the genetic loci described by a haplotype are physically and genetically linked, i.e., on the same chromosome segment. The term “haplotype” can refer to polymorphisms at a particular locus, such as a single marker locus, or polymorphisms at multiple loci along a chromosomal segment.
A “heterotic group” comprises a set of genotypes that perform well when crossed with genotypes from a different heterotic group (Hallauer et al. (1998) Corn breeding, p. 463-564. In G. F. Sprague and J. W. Dudley (ed.) Corn and corn improvement). Inbred lines are classified into heterotic groups, and are further subdivided into families within a heterotic group, based on several criteria such as pedigree, molecular marker-based associations, and performance in hybrid combinations (Smith et al. (1990) Theor. Appl. Gen. 80:833-840). The two most widely used heterotic groups in the United States are referred to as “Iowa Stiff Stalk Synthetic” (BSSS) and “Lancaster” or “Lancaster Sure Crop” (sometimes referred to as NSS, or non-Stiff Stalk).
The term “heterozygous” means a genetic condition wherein different alleles reside at corresponding loci on homologous chromosomes.
The term “homozygous” means a genetic condition wherein identical alleles reside at corresponding loci on homologous chromosomes.
The term “hybrid” refers to the progeny obtained between the crossing of at least two genetically dissimilar parents.
“Hybridization” or “nucleic acid hybridization” refers to the pairing of complementary RNA and DNA strands as well as the pairing of complementary DNA single strands.
The term “hybridize” means to form base pairs between complementary regions of nucleic acid strands.
An “IBM genetic map” refers to any of following maps: IBM, IBM2, IBM2 neighbors, IBM2 FPC0507, IBM2 2004 neighbors, IBM2 2005 neighbors, or IBM2 2005 neighbors frame. IBM genetic maps are based on a B73×Mo17 population in which the progeny from the initial cross were random-mated for multiple generations prior to constructing recombinant inbred lines for mapping. Newer versions reflect the addition of genetic and BAC mapped loci as well as enhanced map refinement due to the incorporation of information obtained from other genetic maps.
The term “inbred” refers to a line that has been bred for genetic homogeneity.
Maize plants with “increased mechanical stalk strength” are resistant to stalk lodging and have mechanically stronger stalks. The term “increased” relates to the degree of physical strength and/or the degree of resistance to breakage and is used to describe the effect on mechanical stalk strength when a particular allele is present or absent.
The term “indel” refers to an insertion or deletion, wherein one line may be referred to as having an insertion relative to a second line, or the second line may be referred to as having a deletion relative to the first line.
The term “introgression” refers to the transmission of a desired allele of a genetic locus from one genetic background to another. For example, introgression of a desired allele at a specified locus can be transmitted to at least one progeny via a sexual cross between two parents of the same species, where at least one of the parents has the desired allele in its genome. Alternatively, for example, transmission of an allele can occur by recombination between two donor genomes, e.g., in a fused protoplast, where at least one of the donor protoplasts has the desired allele in its genome. The desired allele can be, e.g., a selected allele of a marker, a QTL, a transgene, or the like. In any case, offspring comprising the desired allele can be repeatedly backcrossed to a line having a desired genetic background and selected for the desired allele, to result in the allele becoming fixed in a selected genetic background.
The process of “introgressing” is often referred to as “backcrossing” when the process is repeated two or more times.
As used herein, the term “linkage” is used to describe the degree with which one marker locus is associated with another marker locus or some other locus (for example, a mechanical stalk strength locus). The linkage relationship between a molecular marker and a phenotype is given as a “probability” or “adjusted probability”. Linkage can be expressed as a desired limit or range. For example, in some embodiments, any marker is linked (genetically and physically) to any other marker when the markers are separated by less than 50, 40, 30, 25, 20, or 15 map units (or cM). In some aspects, it is advantageous to define a bracketed range of linkage, for example, between 10 and 20 cM, between 10 and 30 cM, or between 10 and 40 cM. The more closely a marker is linked to a second locus, the better an indicator for the second locus that marker becomes. Thus, “closely linked loci” such as a marker locus and a second locus display an inter-locus recombination frequency of 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are also said to be “proximal to” each other. Since one cM is the distance between two markers that show a 1% recombination frequency, any marker is closely linked (genetically and physically) to any other marker that is in close proximity, e.g., at or less than 10 cM distant. Two closely linked markers on the same chromosome can be positioned 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.75, 0.5 or 0.25 cM or less from each other.
The term “linkage disequilibrium” refers to a non-random segregation of genetic loci or traits (or both). In either case, linkage disequilibrium implies that the relevant loci are within sufficient physical proximity along a length of a chromosome so that they segregate together with greater than random (i.e., non-random) frequency (in the case of co-segregating traits, the loci that underlie the traits are in sufficient proximity to each other). Markers that show linkage disequilibrium are considered linked. Linked loci co-segregate more than 50% of the time, e.g., from about 51% to about 100% of the time. In other words, two markers that co-segregate have a recombination frequency of less than 50% (and by definition, are separated by less than 50 cM on the same linkage group.) As used herein, linkage can be between two markers, or alternatively between a marker and a phenotype. A marker locus can be “associated with” (linked to) a trait, e.g., mechanical stalk strength. The degree of linkage of a molecular marker to a phenotypic trait is measured, e.g., as a statistical probability of co-segregation of that molecular marker with the phenotype.
Linkage disequilibrium is most commonly assessed using the measure r2, which is calculated using the formula described by Hill, W. G. and Robertson, A, Theor. Appl. Genet. 38:226-231 (1968). When r2=1, complete LD exists between the two marker loci, meaning that the markers have not been separated by recombination and have the same allele frequency. Values for r2 above 1⁄3 indicate sufficiently strong LD to be useful for mapping (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)). Hence, alleles are in linkage disequilibrium when r2 values between pairwise marker loci are greater than or equal to 0.33, 0.4, 0.5, 0.6, 0.7, 0.8, 0.9, or 1.0.
As used herein, “linkage equilibrium” describes a situation where two markers independently segregate, i.e., sort among progeny randomly. Markers that show linkage equilibrium are considered unlinked (whether or not they lie on the same chromosome).
A “locus” is a position on a chromosome, e.g. where a gene or marker is located.
The term “lodge” is synonymous with break. Hence, stalks that lodge are those that break at a position along the stalk.
The “logarithm of odds (LOD) value” or “LOD score” (Risch, Science 255:803-804 (1992)) is used in interval mapping to describe the degree of linkage between two marker loci. A LOD score of three between two markers indicates that linkage is 1000 times more likely than no linkage, while a LOD score of two indicates that linkage is 100 times more likely than no linkage. LOD scores greater than or equal to two may be used to detect linkage.
“Maize” refers to a plant of the Zea mays L. ssp. mays and is also known as corn.
The term “maize plant” includes: whole maize plants, maize plant cells, maize plant protoplast, maize plant cell or maize tissue cultures from which maize plants can be regenerated, maize plant calli, and maize plant cells that are intact in maize plants or parts of maize plants, such as maize seeds, maize cobs, maize flowers, maize cotyledons, maize leaves, maize stems, maize buds, maize roots, maize root tips, and the like.
A “marker” is a nucleotide sequence or encoded product thereof (e.g., a protein) used as a point of reference. A marker can be derived from genomic nucleotide sequence or from expressed nucleotide sequences (e.g., from a spliced RNA or a cDNA), or from an encoded polypeptide. The term also refers to nucleic acid sequences complementary to or flanking the marker sequences, such as nucleic acids used as probes or primer pairs capable of amplifying the marker sequence.
Markers corresponding to genetic polymorphisms between members of a population can be detected by methods well-established in the art. These include, e.g., DNA sequencing, PCR-based sequence specific amplification methods, detection of restriction fragment length polymorphisms (RFLP), detection of isozyme markers, detection of polynucleotide polymorphisms by allele specific hybridization (ASH), detection of amplified variable sequences of the plant genome, detection of self-sustained sequence replication, detection of simple sequence repeats (SSRs), detection of single nucleotide polymorphisms (SNPs), or detection of amplified fragment length polymorphisms (AFLPs). Well established methods are also known for the detection of expressed sequence tags (ESTs) and SSR markers derived from EST sequences and randomly amplified polymorphic DNA (RAPD).
A “marker allele”, alternatively an “allele of a marker locus”, can refer to one of a plurality of polymorphic nucleotide sequences found at a marker locus in a population that is polymorphic for the marker locus. Marker alleles designated with a number, such as e.g. PHM3468 allele 2, represent the specific combination of alleles, also referred to as a “marker haplotype”, at that specific marker locus.
“Marker assisted selection” (of MAS) is a process by which individual plants are selected based on marker genotypes.
“Marker assisted counter-selection” is a process by which marker genotypes are used to identify plants that will not be selected, allowing them to be removed from a breeding program or planting.
A “marker locus” is a specific chromosome location in the genome of a species where a specific marker can be found. A marker locus can be used to track the presence of a second linked locus, e.g., a linked locus that encodes or contributes to expression of a phenotypic trait. For example, a marker locus can be used to monitor segregation of alleles at a locus, such as a QTL, that are genetically or physically linked to the marker locus.
A “marker probe” is a nucleic acid sequence or molecule that can be used to identify the presence of a marker locus, e.g., a nucleic acid probe that is complementary to a marker locus sequence, through nucleic acid hybridization. Marker probes comprising 30 or more contiguous nucleotides of the marker locus (“all or a portion” of the marker locus sequence) may be used for nucleic acid hybridization. Alternatively, in some aspects, a marker probe refers to a probe of any type that is able to distinguish (i.e., genotype) the particular allele that is present at a marker locus. Nucleic acids are “complementary” when they specifically “hybridize”, or pair, in solution, e.g., according to Watson-Crick base pairing rules.
“Mechanical stalk strength” refers to the physical strength of a maize stalk and its resistance to breakage (also known as “lodging”).
“Nucleotide sequence”, “polynucleotide”, “nucleic acid sequence”, and “nucleic acid fragment” are used interchangeably and refer to a polymer of RNA or DNA that is single- or double-stranded, optionally containing synthetic, non-natural or altered nucleotide bases. A “nucleotide” is a monomeric unit from which DNA or RNA polymers are constructed, and consists of a purine or pyrimidine base, a pentose, and a phosphoric acid group. Nucleotides (usually found in their 5′-monophosphate form) are referred to by their single letter designation as follows: “A” for adenylate or deoxyadenylate (for RNA or DNA, respectively), “C” for cytidylate or deoxycytidylate, “G” for guanylate or deoxyguanylate, “U” for uridylate, “T” for deoxythymidylate, “R” for purines (A or G), “Y” for pyrimidines (C or T), “K” for G or T, “H” for A or C or T, “I” for inosine, and “N” for any nucleotide.
The terms “phenotype”, or “phenotypic trait” or “trait” refers to one or more trait of an organism. The phenotype can be observable to the naked eye, or by any other means of evaluation known in the art, e.g., microscopy, biochemical analysis, or an electromechanical assay. In some cases, a phenotype is directly controlled by a single gene or genetic locus, i.e., a “single gene trait”. In other cases, a phenotype is the result of several genes.
Each “PHM” marker represents two sets of primers that when used in a nested PCR, amplify a specific piece of DNA. The external set is used in the first round of PCR, after which the internal sequences are used for a second round of PCR on the products of the first round. This increases the specificity of the reaction. A “physical map” of the genome is a map showing the linear order of identifiable landmarks (including genes, markers, etc.) on chromosome DNA. However, in contrast to genetic maps, the distances between landmarks are absolute (for example, measured in base pairs or isolated and overlapping contiguous genetic fragments) and not based on genetic recombination.
A “plant” can be a whole plant, any part thereof, or a cell or tissue culture derived from a plant. Thus, the term “plant” can refer to any of: whole plants, plant components or organs (e.g., leaves, stems, roots, etc.), plant tissues, seeds, plant cells, and/or progeny of the same. A plant cell is a cell of a plant, taken from a plant, or derived through culture from a cell taken from a plant.
A “polymorphism” is a variation in the DNA that is too common to be due merely to new mutation. A polymorphism preferably has a frequency of at least 1% in a population. A polymorphism can include a single nucleotide polymorphism (SNP), a simple sequence repeat (SSR), or an insertion/deletion polymorphism, also referred to herein as an “indel”.
The “probability value” or “p-value” is the statistical likelihood that the particular combination of a phenotype and the presence or absence of a particular marker allele is random. Thus, the lower the probability score, the greater the likelihood that a phenotype and a particular marker will co-segregate. In some aspects, the probability score is considered “significant” or “nonsignificant”. In some embodiments, a probability score of 0.05 (p=0.05, or a 5% probability) of random assortment is considered a significant indication of co-segregation. However, an acceptable probability can be any probability of less than 50% (p=0.5). For example, a significant probability can be less than 0.25, less than 0.20, less than 0.15, less than 0.1, less than 0.05, less than 0.01, or less than 0.001.
A “production marker” or “production SNP marker” is a marker that has been developed for high-throughput purposes. Production SNP markers were developed for specific polymorphisms identified using PHM markers and the nested PCR analysis. These production SNP markers were specifically designed for use with the Invader Plus® (Third Wave Technologies) platform.
The term “progeny” refers to the offspring generated from a cross.
A “progeny plant” is generated from a cross between two plants.
The term “quantitative trait locus” or “QTL” refers to a region of DNA that is associated with the differential expression of a phenotypic trait in at least one genetic background, e.g., in at least one breeding population. QTLs are closely linked to the gene or genes that underlie the trait in question.
A “reference sequence” is a defined sequence used as a basis for sequence comparison. The reference sequence for a PHM marker is obtained by genotyping a number of lines at the locus, aligning the nucleotide sequences in a sequence alignment program (e.g. Sequencher), and then obtaining the consensus sequence of the alignment. Hence, a reference sequence identifies the polymorphisms in alleles at a locus. A reference sequence may not be a copy of an actual DNA sequence; however, it is useful for designing primers and probes for actual polymorphisms in the locus.
“Stalk lodging” refers to the breakage of the stalk. Stalk lodging typically occurs at or below the ear, but can occur at any position along the stalk.
A “three-point bend test” is an electromechanical system for evaluating mechanical stalk strength. In this test, load can be applied to internodes below the ear using an InstronTM machine, such as Model 4411 (Instron Corporation, 100 Royall Street, Canton, Mass. 02021), or other crushing device. The load needed to break the internode is used as a measure of mechanical strength. The mechanical stalk strength values obtained from the three-point bend test have shown to be highly correlated to lodging scores that have been assigned based on field observations.
A “topcross test” is a test performed on progeny derived by crossing each parent with the same tester, usually a homozygous line. The parent being tested can be an open-pollinated variety, a cross, or an inbred line.
The phrase “under stringent conditions” refers to conditions under which a probe or polynucleotide will hybridize to a specific nucleic acid sequence, typically in a complex mixture of nucleic acids, but to essentially no other sequences. Stringent conditions are sequence-dependent and will be different in different circumstances.
Longer sequences hybridize specifically at higher temperatures. Generally, stringent conditions are selected to be about 5-10° C. lower than the thermal melting point (Tm) for the specific sequence at a defined ionic strength pH. The Tm is the temperature (under defined ionic strength, pH, and nucleic acid concentration) at which 50% of the probes complementary to the target hybridize to the target sequence at equilibrium (as the target sequences are present in excess, at Tm, 50% of the probes are occupied at equilibrium). Stringent conditions will be those in which the salt concentration is less than about 1.0 M sodium ion, typically about 0.01 to 1.0 M sodium ion concentration (or other salts) at pH 7.0 to 8.3, and the temperature is at least about 30° C. for short probes (e.g., 10 to 50 nucleotides) and at least about 60° C. for long probes (e.g., greater than 50 nucleotides). Stringent conditions may also be achieved with the addition of destabilizing agents such as formamide. For selective or specific hybridization, a positive signal is at least two times background, preferably 10 times background hybridization. Exemplary stringent hybridization conditions are often: 50% formamide, 5×SSC, and 1% SDS, incubating at 42° C., or, 5×SSC, 1% SDS, incubating at 65° C., with wash in 0.2×SSC, and 0.1% SDS at 65° C. For PCR, a temperature of about 36° C. is typical for low stringency amplification, although annealing temperatures may vary between about 32° C. and 48° C., depending on primer length. Additional guidelines for determining hybridization parameters are provided in numerous references.
Sequence alignments and percent identity calculations may be determined using a variety of comparison methods designed to detect homologous sequences including, but not limited to, the MEGALIGN® program of the LASERGENE® bioinformatics computing suite (DNASTAR® Inc., Madison, Wis.). Unless stated otherwise, multiple alignment of the sequences provided herein were performed using the Clustal V method of alignment (Higgins and Sharp, CABIOS. 5:151-153 (1989)) with the default parameters (GAP PENALTY=10, GAP LENGTH PENALTY=10). Default parameters for pairwise alignments and calculation of percent identity of protein sequences using the Clustal V method are KTUPLE=1, GAP PENALTY=3, WINDOW=5 and DIAGONALS SAVED=5. For nucleic acids these parameters are KTUPLE=2, GAP PENALTY=5, WINDOW=4 and DIAGONALS SAVED=4. After alignment of the sequences, using the Clustal V program, it is possible to obtain “percent identity” and “divergence” values by viewing the “sequence distances” table on the same program; unless stated otherwise, percent identities and divergences provided and claimed herein were calculated in this manner.
Standard recombinant DNA and molecular cloning techniques used herein are well known in the art and are described more fully in Sambrook, J., Fritsch, E. F. and Maniatis, T. Molecular Cloning: A Laboratory Manual; Cold Spring Harbor Laboratory Press: Cold Spring Harbor, 1989 (hereinafter “Sambrook”).
Before describing the present invention in detail, it should be understood that this invention is not limited to particular embodiments. It also should be understood that the terminology used herein is for the purpose of describing particular embodiments, and is not intended to be limiting. As used herein and in the appended claims, terms in the singular and the singular forms “a”, “an” and “the”, for example, include plural referents unless the content clearly dictates otherwise. Thus, for example, reference to “plant”, “the plant” or “a plant” also includes a plurality of plants. Depending on the context, use of the term “plant” can also include genetically similar or identical progeny of that plant. The use of the term “a nucleic acid” optionally includes many copies of that nucleic acid molecule.
Turning now to the embodiments:
Stalk Lodging and Mechanical Stalk Strength
Methods for identifying and for selecting maize plants with favorable or unfavorable mechanical stalk strength characteristics through the genotyping of associated marker loci are provided. Mechanical stalk strength in maize is an agronomically important trait, as increased mechanical stalk strength enhances resistance to stalk lodging.
Stalk lodging, or stalk breakage, can occur at various developmental stages. During vegetative growth, internodes are rapidly elongating, causing cell walls to thin, thereby making stalks more prone to breakage. This type of breakage is called green snap, or brittle snap, and most often occurs between the vegetative stages of V5 and V8 and between the vegetative V12 stage and the reproductive R1 stage. Plants that lodge during the V5 to V8 stage usually do not recover, since breakage typically occurs below the growing point. Between the vegetative V12 stage and the reproductive R1 stage, stalks typically break at nodes just below or above the ear. If the site of breakage is below the ear, ear development is severely impeded, resulting in no grain production. If the site of breakage is above the ear, limited or no grain production may still result, due to the lack of photosynthetic surface area, which is required for supplying the developing ear(s) with nutrients.
Stalk lodging can also occur late season. As a maize plant matures, ear weight increases, as does the load imposed on the stalk. The increased load can cause the maize stalk to break, especially when additional mechanical stresses, either biotic or abiotic, are imposed on the plant. If stalk breakage or lodging does occur, the ear may fall to the ground or to a height where harvest machinery cannot access the ear, thereby reducing yield. Alternatively, the proximity of the fallen ear to the ground increases the probability of fungal spores being splashed on to the ear, resulting in a loss of grain quality.
Maize plants with increased mechanical stalk strength, however, have a greater capacity to bear the weight of the ear and any applied mechanical force. It is therefore desirable to identify and select maize plants with increased mechanical stalk strength to prevent yield and grain quality losses due to stalk lodging. It is also desirable to eliminate maize plants with decreased mechanical stalk strength from maize breeding programs for the same purpose.
QTL Mapping
It has been recognized for quite some time that specific chromosomal loci (or intervals) in an organism's genome that correlate with particular quantitative phenotypes, such as mechanical stalk strength, can be mapped genetically using markers. Such loci are termed quantitative trait loci, or QTL. The plant breeder can advantageously use molecular markers to identify desired individuals by identifying marker alleles that show a statistically significant probability of co-segregation with a desired phenotype, manifested as linkage disequilibrium. By identifying a molecular marker or clusters of molecular markers that co-segregate with a quantitative trait, the breeder is thus identifying a QTL. By identifying and selecting a marker allele (or desired alleles from multiple markers) that associates with the desired phenotype, the plant breeder is able to rapidly select a desired phenotype by selecting for the proper molecular marker allele (a process called marker-assisted selection, or MAS). Such markers could also be used by breeders to design genotypes in silico and to practice whole genome selection.
A variety of methods well known in the art are available for detecting molecular markers or clusters of molecular markers that co-segregate with a quantitative trait such as mechanical stalk strength. The basic idea underlying these methods is the detection of markers, for which alternative genotypes (or alleles) have significantly different average phenotypes. Thus, one makes a comparison among marker loci of the magnitude of difference among alternative genotypes (or alleles) or the level of significance of that difference. Trait genes are inferred to be located nearest the marker(s) that have the greatest associated genotypic difference.
Two such methods used to detect QTLs are: 1) Population-based structured association analysis and 2) Pedigree-based association analysis. In a population-based structured association analysis, lines are obtained from pre-existing populations with multiple founders, e.g. elite breeding lines. Population-based association analyses rely on the decay of linkage disequilibrium (LD) and the idea that in an unstructured population, only correlations between QTL and markers closely linked to the QTL will remain after so many generations of random mating. In reality, most pre-existing populations have population substructure. Thus, the use of a structured association approach helps to control population structure by allocating individuals to populations using data obtained from markers randomly distributed across the genome, thereby minimizing disequilibrium due to population structure within the individual populations (also called subpopulations). The phenotypic values are compared to the genotypes (alleles) at each marker locus for each line in the subpopulation. A significant marker-trait association indicates the close proximity between the marker locus and one or more genetic loci that are involved in the expression of that trait.
The same principles underlie traditional linkage analysis; however, LD is generated by creating a population from a small number of founders. The founders are selected to maximize the level of polymorphism within the constructed population, and polymorphic sites are assessed for their level of cosegregation with a given phenotype. A number of statistical methods have been used to identify significant marker-trait associations. One such method is an interval mapping approach (Lander and Botstein, Genetics 121:185-199 (1989), in which each of many positions along a genetic map (say at 1 cM intervals) is tested for the likelihood that a gene controlling a trait of interest is located at that position. The genotype/phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio). When the LOD score exceeds a threshold value, there is significant evidence for the location of a QTL at that position on the genetic map (which will fall between two particular marker loci).
The present invention provides QTLs that demonstrate statistically significant co-segregation with mechanical stalk strength, as determined by association analyses. Detection of these loci or additional linked loci can be used in marker assisted maize breeding programs to produce plants with a favorable mechanical stalk strength phenotype or to eliminate plants with an unfavorable mechanical stalk strength phenotype from breeding programs or planting.
Markers Associated with Mechanical Stalk Strength
Markers associated with mechanical stalk strength are identified herein.
For the QTL identified on chromosome 5 (referred to herein as QTL5), the marker locus can be selected from any of the marker loci provided in Table 3 or Table 7, including the PHM markers, PHM201, PHM5421, PHM3468, PHM12521, and PHM10840, and the production SNP markers PHM201-16-U, PHM201-17-U, PHM4861-20-U, PHM4861-21-U, PHM5421-5-V, PHM4115-35-U, PHM12521-18-U, PHM12521-19-U, PHM12521-29-U, and C00386-397-U, as well as any other marker linked to these QTL markers (linked markers can be determined from the MaizeGDB resource; see framework of markers in Table 7).
For the QTL identified on chromosome 9 (referred to herein as QTL9), the marker locus can be selected from any of the marker loci provided in Example 1C, Table 5, or Table 9, including the PHM markers, PHM4578, PHM11186, PHM12025, PHM14053, PHM405, and PHM16736, and the production SNP markers, PHM13418-18, PHM13418-10, PHM113-7, PHM10337-11-U, PHM16736-8-V, PHM12025-48, and PHM11186-16-V, as well as any other marker linked to these QTL markers.
For one of the QTLs identified on chromosome 1 (referred to herein as QTL1A), the marker locus can be selected from any of the marker loci provided in Example 1D, Table 5, or Table 9, including the PHM markers, PHM7844, PHM8029, PHM2130, and PHM574, and the production SNP markers, PHM18693-9-U, PHM10786-11-U, PHM10786-5-U, PHM10786-6-U, and PHM8057-801-U, as well as any other marker linked to these QTL markers.
For one of the QTLs identified on chromosome 1 (referred to herein as QTL1B), the marker locus can be selected from any of the QTL marker loci provided in Example 1D, Table 5, or Table 9, including the PHM markers, PHM11754, PHM1481, PHM6427, and PHM15089, and the production SNP markers, PHM4044-11-U, PHM14080-16-V, PHM15089-10-U, and PHM9364-6-U, as well as any other marker linked to these QTL markers.
For one of the QTLs identified on chromosome 1 (referred to herein as QTL1C), the marker locus can be selected from any of the QTL marker loci provided in Example 1D, Table 5, or Table 9, including the PHM markers, PHM11125, PHM13958, PHM10468, and PHM12706, as well as any other marker linked to these QTL markers.
Physical Map Locations of QTLs
The genetic elements or genes located on a contiguous linear span of genomic DNA on a single chromosome are physically linked.
For the QTL5 region, the two markers with the largest physical distance between them that still remain associated with the phenotype of interest, mechanical stalk strength, are PHM654 (reference sequence=SEQ ID NO:1) and PHM6727 (reference sequence=SEQ ID NO:39). PHM654 is located on BAC c0216105, and PHM6727 is located on BAC c0117h02. Hence, these two BACs delineate the mechanical stalk strength QTL on the maize physical map. Any BAC that assembles to the contiguous DNA between and including BAC c0216l05 and BAC c0117h02 can house marker loci that are associated with the mechanical stalk strength trait. FIGS. 1A-I show the physical map arrangement of the sequenced BACs that make up the contiguous stretch of DNA between and including BAC c0216l05 and BAC c0117h02. The gaps (represented by dotted lines) are not gaps in the contiguous stretch of DNA per se but are areas where BACs that have not been sequenced assemble to the physical map.
An area on chromosome 9 defined by and including PHM4578 and PHM11186 delineates the QTL9 region. Any polynucleotide that can hybridize to the contiguous DNA between and including SEQ ID NO:196 (the reference sequence for PHM4578), or a nucleotide sequence that is 95% identical to SEQ ID NO:196 based on the Clustal V method of alignment, and SEQ ID NO:197 (the reference sequence for PHM11186), or a nucleotide sequence that is 95% identical to SEQ ID NO:197 based on the Clustal V method of alignment, and that is associated with mechanical stalk strength can be used as a marker for mechanical stalk strength. On the current B73 physical map, PHM4578 is located on BACs c0478c20, c0414c21, and b0505j22, while PHM11186 is located on BACs c0475 m02 and b0197d12.
An area on chromosome 1 defined by and including PHM7844 and PHM8029 delineates the QTL1A region. Any polynucleotide that can hybridize to the contiguous DNA between and including SEQ ID NO:200 (the reference sequence for PHM7844), or a nucleotide sequence that is 95% identical to SEQ ID NO:200 based on the Clustal V method of alignment, and SEQ ID NO:201 (the reference sequence for PHM8029), or a nucleotide sequence that is 95% identical to SEQ ID NO:201 based on the Clustal V method of alignment, and that is associated with mechanical stalk strength can be used as a marker for mechanical stalk strength. On the current B73 physical map, PHM7844 is located on BAC b0109 m14, while PHM8029 is located on BAC c0230j20.
An area on chromosome 1 defined by and including PHM11754 and PHM1481 delineates the QTL1B region. Any polynucleotide that can hybridize to the contiguous DNA between and including SEQ ID NO:203 (the reference sequence for PHM11754), or a nucleotide sequence that is 95% identical to SEQ ID NO:203 based on the Clustal V method of alignment, and SEQ ID NO:204 (the reference sequence for PHM1481), or a nucleotide sequence that is 95% identical to SEQ ID NO:204 based on the Clustal V method of alignment, and that is associated with mechanical stalk strength can be used as a marker for mechanical stalk strength. On the current B73 physical map, PHM11754 is not located on a sequenced BAC, while PHM1481 is located on BAC c0347b01.
An area on chromosome 1 defined by and including PHM11125 and PHM13958 delineates the QTL1C region. Any polynucleotide that can hybridize to the contiguous DNA between and including SEQ ID NO:208 (the reference sequence for PHM11125), or a nucleotide sequence that is 95% identical to SEQ ID NO:208 based on the Clustal V method of alignment, and SEQ ID NO:209 (the reference sequence for PHM13958), or a nucleotide sequence that is 95% identical to SEQ ID NO:209 based on the Clustal V method of alignment, and that is associated with mechanical stalk strength can be used as a marker for mechanical stalk strength. On the current B73 physical map, PHM11125 is located on BAC c0042p07, while PHM13958 is located on BAC c0188c22.
Linkage Relationships
A common measure of linkage is the frequency with which traits cosegregate. This can be expressed as a percentage of cosegregation (recombination frequency) or in centiMorgans (cM). The cM is a unit of measure of genetic recombination frequency. One cM is equal to a 1% chance that a trait at one genetic locus will be separated from a trait at another locus due to crossing over in a single generation (meaning the traits segregate together 99% of the time). Because chromosomal distance is approximately proportional to the frequency of crossing over events between traits, there is an approximate physical distance that correlates with recombination frequency.
Marker loci are themselves traits and can be assessed according to standard linkage analysis by tracking the marker loci during segregation. Thus, one cM is equal to a 1% chance that a marker locus will be separated from another locus, due to crossing over in a single generation.
The closer a marker is to a QTL marker, the more effective and advantageous that marker is as an indicator for the desired trait. Closely linked loci display an inter-locus cross-over frequency of about 10% or less, preferably about 9% or less, still more preferably about 8% or less, yet more preferably about 7% or less, still more preferably about 6% or less, yet more preferably about 5% or less, still more preferably about 4% or less, yet more preferably about 3% or less, and still more preferably about 2% or less. In highly preferred embodiments, the relevant loci (e.g., a marker locus and a target locus such as a QTL) display a recombination frequency of about 1% or less, e.g., about 0.75% or less, more preferably about 0.5% or less, or yet more preferably about 0.25% or less. Thus, the loci are about 10 cM, 9 cM, 8 cM, 7 cM, 6 cM, 5 cM, 4 cM, 3 cM, 2 cM, 1 cM, 0.75 cM, 0.5 cM or 0.25 cM or less apart. Put another way, two loci that are localized to the same chromosome, and at such a distance that recombination between the two loci occurs at a frequency of less than 10% (e.g., about 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, 1%, 0.75%, 0.5%, 0.25%, or less) are said to be “proximal to” each other.
Although particular marker alleles can show co-segregation with the mechanical stalk strength phenotype, it is important to note that the marker locus is not necessarily part of the QTL locus responsible for the expression of the mechanical stalk strength phenotype. For example, it is not a requirement that the marker polynucleotide sequence be part of a gene that imparts mechanical stalk strength (for example, be part of the gene open reading frame). The association between a specific marker allele with either a favorable or unfavorable mechanical stalk strength phenotype is due to the original “coupling” linkage phase between the marker allele and the QTL allele in the ancestral maize line from which the QTL allele originated. Eventually, with repeated recombination, crossing over events between the marker and QTL locus can change this orientation. For this reason, the favorable marker allele may change depending on the linkage phase that exists within the resistant parent used to create segregating populations. This does not change the fact that the marker can be used to monitor segregation of the phenotype. It only changes which marker allele is considered favorable in a given segregating population.
For QTL5, markers identified in Table 3 or Table 7, as well as any marker within 50 cM of the markers identified in Table 3 or Table 7, can be used to predict mechanical stalk strength trait in a maize plant. This includes any marker within 50 cM of the PHM markers, PHM201, PHM5421, PHM3468, PHM12521, and PHM10840, and the production SNP markers PHM201-16-U, PHM201-17-U, PHM4861-20-U, PHM4861-21-U, PHM5421-5-V, PHM4115-35-U, PHM12521-18-U, PHM12521-19-U, PHM12521-29-U, and C00386-397-U.
For QTL9, markers identified in Example 1C, Table 5, or Table 9, as well as any marker within 50 cM of the markers identified in Example 1C, Table 5, or Table 9, can be used to predict mechanical stalk strength in a maize plant. This includes any marker within 50 cM of the PHM markers, PHM4578, PHM11186, PHM12025, PHM14053, PHM405, and PHM16736, and of the production SNP markers, PHM13418-18, PHM13418-10, PHM113-7, PHM10337-11-U, PHM16736-8-V, PHM12025-48, and PHM11186-16-V.
For QTL1A, markers identified in Example 1D, Table 5, or Table 9, as well as any marker within 50 cM of the markers identified in Example 1D, Table 5, or Table 9, can be used to predict mechanical stalk strength in a maize plant. This includes any marker within 50 cM of PHM7844, PHM8029, PHM2130, or PHM574, and of the production SNP markers, PHM18693-9-U, PHM10786-11-U, PHM10786-5-U, PHM10786-6-U, and PHM8057-801-U.
For QTL1B, markers identified in Example 1D, Table 5, or Table 9, as well as any marker within 50 cM of the markers identified in Example 1D, Table 5, or Table 9, can be used to predict mechanical stalk strength in a maize plant. This includes any marker within 50 cM of the PHM markers, PHM11754, PHM1481, PHM6427, and PHM15089, and of the production SNP markers, PHM4044-11-U, PHM14080-16-V, PHM15089-10-U, and PHM9364-6-U.
For QTL1C, markers identified in Example 1D, Table 5, or Table 9, as well as any marker within 50 cM of the markers identified in Example 1D, Table 5, or Table 9, can be used to predict mechanical stalk strength in a maize plant. This includes any marker within 50 cM of the PHM markers, PHM11125, PHM13958, PHM10468, and PHM12706.
Chromosomal Intervals
Chromosomal intervals that correlate with mechanical stalk strength are provided. A variety of methods well known in the art are available for identifying chromosomal intervals. The boundaries of such chromosomal intervals are drawn to encompass markers that will be linked to one or more QTL. In other words, the chromosomal interval is drawn such that any marker that lies within that interval (including the terminal markers that define the boundaries of the interval) can be used as a marker for mechanical stalk strength. Each interval comprises at least one QTL, and furthermore, may indeed comprise more than one QTL. Close proximity of multiple QTL in the same interval may obfuscate the correlation of a particular marker with a particular QTL, as one marker may demonstrate linkage to more than one QTL. Conversely, e.g., if two markers in close proximity show co-segregation with the desired phenotypic trait, it is sometimes unclear if each of those markers identify the same QTL or two different QTL. Regardless, knowledge of how many QTL are in a particular interval is not necessary to make or practice the invention.
Each of the intervals described shows a clustering of markers that co-segregate with mechanical stalk strength. This clustering of markers occurs in relatively small domains on the linkage groups, indicating the presence of one or more QTL in those chromosome regions. QTL intervals were drawn to encompass the markers that co-segregate with mechanical stalk strength. The intervals are defined by the markers on their termini, where the interval encompasses markers that map within the interval, whether known or unknown, as well as the markers that define the termini.
For QTL5, any marker located within any of the following intervals finds use as a marker for mechanical stalk strength. These intervals include:

    
    
        (i) PHM654 and PHM6727;
        (ii) PHM12632 and PHM3323;
        (iii) PHM201 and PHM3323; and
        (iv) PHM201 and PHM3468.
    
    


PHM654 and PHM6727 are separated by  ̃25 cM on the internally-derived genetic map and define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.01 (FIG. 2C). PHM12632 and PHM3323, separated by  ̃9 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.001 (FIG. 2D). PHM201 and PHM3323, separated by  ̃9 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.0001 (FIG. 2E).
For QTL9, any marker located within any of the following intervals finds use as a marker for mechanical stalk strength:

    
    
        (i) PHM4578 and PHM11186 and
        (ii) PHM14053 and PHM16736.
    
    


PHM4578 and PHM11186, separated by  ̃3.5 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.01. PHM14053 and PHM16736, separated by  ̃1.5 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.001.
For QTL1A, any marker located within any of the following intervals finds use as a marker for mechanical stalk strength:

    
    
        (i) PHM7844 and PHM8029 and
        (ii) PHM7844 and PHM574.
    
    


PHM7844 and PHM8029, separated by  ̃16 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.01. PHM7844 and PHM574, separated by  ̃6.5 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.001.
For QTL1B, any marker located within any of the following intervals finds use as a marker for mechanical stalk strength:

    
    
        (i) PHM11754 and PHM1481 and
        (ii) PHM6427 and PHM1481.
    
    


PHM11754 and PHM1481, separated by  ̃21 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.01. PHM6427 and PHM1481, separated by  ̃21 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.001.
For QTL1C, any marker located within any of the following intervals finds use as a marker for mechanical stalk strength:

    
    
        (i) PHM11125 and PHM13958 and
        (ii) PHM10468 and PHM13958.
    
    


PHM11125 and PHM13958, separated by  ̃15 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.01. PHM10468 and PHM13958, separated by  ̃12 cM on the internally-derived genetic map, define a chromosomal interval encompassing a cluster of markers that co-segregate with mechanical stalk strength in the NSS subpopulation at a p-level of ≦0.001.
Chromosomal intervals can also be defined by markers that are in linkage disequilibrium with a known QTL marker, and r2 is a common measure of linkage disequilibrium (LD) in the context of association studies. For example, if the r2 value of LD between a chromosome 5 marker locus lying within the interval of PHM654 and PHM6727 and any of the chromosome 5 QTL markers identified in Table 3 or Table 7 is greater than 1⁄3 (Ardlie et al., Nature Reviews Genetics 3:299-309 (2002)), the loci are in linkage disequilibrium.
Marker Alleles and Haplotypic Combinations
A marker of the invention can also be a combination of particular alleles at one or more marker loci, otherwise known as a haplotype. The alleles described below could be used in combination to identify and select for maize plants with mechanical stalk characteristics.
Favorable SNP alleles at QTL5 marker loci have been identified herein and include: a “G” at position 314 in SEQ ID NO:12 (PHM5421), a “G” at position 93 in SEQ ID NO:15 (PHM3468), a “T” at position 101 in SEQ ID NO:15 (PHM3468), a “G” at position 245 in SEQ ID NO:15 (PHM3468), a “T” at position 101 in SEQ ID NO:14 (PHM12521), an “A” at position 186 in SEQ ID NO:14 (PHM12521), a “C” at position 37 in SEQ ID NO:16 (PHM10840), an “A” at position 240 in SEQ ID NO:16 (PHM10840, and a “C” at position 315 in SEQ ID NO:16 (PHM10840), a “C” at PHM201-16-U, a “C” or “G” at PHM201-17-U, a “T” or “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a ‘G” at PHM5421-5-V, a “G” or “T” at PHM4115-35-U, a “T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, and a “C” at C00386-397-U.
Favorable SNP alleles at QTL9 marker loci have been identified herein and include: a “C” at position 225 in SEQ ID NO:199 (PHM16736), a “T” at position 326 in SEQ ID NO:199 (PHM16736), an “A” at position 422 in SEQ ID NO:199 (PHM16736), a “C” at position 193 in SEQ ID NO:198 (PHM14053), a “C” at position 341 in SEQ ID NO:198 (PHM14053), a “C” at position 386 in SEQ ID NO:198 (PHM14053), a “T” at position 374 in SEQ ID NO:212 (PHM405), a “C” at position 216 in SEQ ID NO:213 (PHM12025), a “C” at PHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, and a “T” at PHM11186-16-V.
Favorable SNP alleles at QTL1A marker loci have been identified herein and include: a “T” at position 75 of SEQ ID NO:202 (PHM2130), an “A” at position 170 of SEQ ID NO:202 (PHM2130), a “C” at position 179 of SEQ ID NO:202 (PHM2130), a “G” at position 358 of SEQ ID NO:202 (PHM2130), a “T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C’ at PHM10786-5-U, a “T” at PHM10786-6-U, and a “G” at PHM8057-801-U.
Unfavorable SNP alleles at QTL5 marker loci have been identified herein and include: a “C” at position 132 in SEQ ID NO:10 (PHM201) and an “A” at position 230 in SEQ ID NO:10 (PHM201).
Unfavorable SNP alleles at QTL1B marker loci have been identified herein and include: a “G” at position 284 in SEQ ID NO:205 (PHM15089), a “T” at PHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, and a “G” at PHM9364-6-U.
An unfavorable SNP allele at a QTL1C marker locus has been identified herein, a “C” at position 322 of SEQ ID NO:210.
The skilled artisan would expect that there might be additional polymorphic sites at marker loci in and around the QTL markers identified herein, wherein one or more polymorphic sites is in high linkage disequilibrium (LD) with an allele at one or more of the polymorphic sites in the haplotype. Two particular alleles at different polymorphic sites are said to be in LD if the presence of the allele at one of the sites tends to predict the presence of the allele at the other site on the same chromosome (Stevens, Mol. Diag. 4:309-17 (1999)).
Marker Assisted Selection
Methods for marker assisted selection (MAS), in which phenotypes are selected based on marker genotypes, are also provided. To perform MAS, a nucleic acid corresponding to the marker nucleic acid allele is detected in a biological sample from a plant to be selected. This detection can take the form of hybridization of a probe nucleic acid to a marker allele or amplicon thereof, e.g., using allele-specific hybridization, Southern analysis, northern analysis, in situ hybridization, hybridization of primers followed by PCR amplification of a region of the marker, DNA sequencing of a PCR amplification product, or the like. The procedures used to detect marker alleles are known to one of ordinary skill in the art. After the presence (or absence) of a particular marker allele in the biological sample is verified, the plant is selected and is crossed to a second plant, e.g. a maize plant from an elite line. The progeny plants produced by the cross can be evaluated for that specific marker allele, and only those progeny plants that have the desired marker allele will be chosen.
Maize plant breeders desire combinations of desired genetic loci, such as those marker alleles associated with increased mechanical stalk strength, with genes for high yield and other desirable traits to develop improved maize varieties. Screening large numbers of samples by non-molecular methods (e.g., trait evaluation in maize plants) can be expensive, time consuming, and unreliable. Use of the polymorphic markers described herein, when genetically-linked to mechanical stalk strength loci, provide an effective method for selecting varieties with increased mechanical stalk strength in breeding programs. For example, one advantage of marker-assisted selection over field evaluations for mechanical stalk strength is that MAS can be done at any time of year, regardless of the growing season. Moreover, environmental effects are largely irrelevant to marker-assisted selection.
Another use of MAS in plant breeding is to assist the recovery of the recurrent parent genotype by backcross breeding. Backcross breeding is the process of crossing a progeny back to one of its parents or parent lines. Backcrossing is usually done for the purpose of introgressing one or a few loci from a donor parent (e.g., a parent comprising desirable mechanical stalk strength marker loci) into an otherwise desirable genetic background from the recurrent parent (e.g., an otherwise high yielding maize line). The more cycles of backcrossing that are done, the greater the genetic contribution of the recurrent parent to the resulting introgressed variety. This is often necessary, because plants may be otherwise undesirable, e.g., due to low yield, low fecundity, or the like. In contrast, strains which are the result of intensive breeding programs may have excellent yield, fecundity or the like, merely being deficient in one desired trait such as mechanical stalk strength.
One application of MAS is to use the markers to increase the efficiency of an introgression or backcrossing effort aimed at introducing an increased mechanical stalk strength QTL into a desired (typically high yielding) background. In marker assisted backcrossing of specific markers (and associated QTL) from a donor source, e.g., to an elite or exotic genetic background, one selects among backcross progeny for the donor trait and then uses repeated backcrossing to the elite or exotic line to reconstitute as much of the elite/exotic background's genome as possible.
In general, MAS uses polymorphic markers that have been identified as having a significant likelihood of co-segregation with the mechanical stalk strength trait. Such markers are presumed to map near a gene or genes that give the plant its mechanical stalk strength phenotype, and are considered indicators for the desired trait, and hence, are termed QTL markers. Plants are tested for the presence of a desired allele in the QTL marker, and plants containing a desired genotype at one or more loci are expected to transfer the desired genotype, along with a desired phenotype, to their progeny.
The markers and QTL intervals presented herein find use in MAS to select maize plants or germplasm for mechanical stalk strength characteristics. Methods for selection involve obtaining DNA accessible for analysis, detecting the presence or absence of either an identified marker allele or an unknown marker allele that is linked to and associated with an identified marker allele, and then selecting the maize plant or germplasm based on the allele detected.
Marker alleles that can be detected include: a “G” at PHM3468.1, a “T” at PHM3468.4, a “G” at PHM3468.18, a “T” at PHM12521.12, a “C” at PHM10840.105, an “A” at PHM10840.118, a “C” at PHM10840.130, a “C” at PHM16736.6, an “A” at PHM16736.14, a “C” at PHM14053.7, a “C” at PHM14053.8, a “C” at PHM14053.14, a “T” at PHM405.35, a “C” at PHM12025.26, a “T” at PHM18693-9-U, a “G” at PHM10786-11-U, a “C” at PHM10786-5-U, a “T” at PHM10786-6-U, a “G” at PHM8057-801-U, a “C” at PHM201-16-U, a “C” at PHM201-17-U, a “G” at PHM201-17-U, a “T” at PHM4861-20-U, a “G” at PHM4861-20-U, an “A” at PHM4861-21-U, a “G” at PHM5421-5-V, a “G” at PHM4115-35-U, a “T” at PHM4115-35-U, a “T” at PHM12521-18-U, an “A” at PHM12521-19-U, a “G” at PHM12521-29-U, a “C” at C00386-397-U, a “C” at PHM13418-18, a “C” at PHM13418-10, a “T” at PHM113-7, a “T” at PHM10337-11-U, an “A” at PHM16736-8-V, a “C” at PHM12025-48, a “T” at PHM11186-16-V, and any marker allele associated with and linked to any of the marker alleles listed above by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1 cM. The maize plant or germplasm that has any of these marker alleles can be selected.
Marker alleles that can be detected can also include: a “T” at PHM2130.24, an “A” at PHM2130.29, a “C” at PHM2130.30, a “G” at PHM2130.33, a “G” at PHM15089.13, a “C” at PHM12706.14, a “C” at PHM201.10, an “A” at PHM201.18, a “T” at PHM4044-11-U, an “A” at PHM14080-16-V, a “C” at PHM15089-10-U, a “G” at PHM9364-6-U, and any marker allele associated with and linked to any of the marker alleles listed above by 30, 25, 20, 15, 10, 9, 8, 7, 6, 5, 4, 3, 2, 1, 0.9, 0.8, 0.7, 0.6, 0.5, 0.4, 0.3, 0.2, or 0.1. The maize plant or germplasm that does not have any of these marker alleles can be selected.
Methods for selecting maize plants with mechanical stalk strength characteristics can also involve detecting alleles at one or more marker loci lying within specific chromosomal intervals or assaying at least one marker locus within a specific interval in which the marker locus is associated with mechanical stalk strength.
For instance, a maize plant having at least one allele of a marker locus lying within any of the following QTL5 intervals:
(i) PHM654 and PHM6727;
(ii) PHM12632 and PHM3323;
(iii) PHM201 and PHM3323; and
(iv) PHM201 and PHM3468, wherein the allele is associated with increased mechanical stalk strength, can be crossed to another maize plant, and a progeny plant arising from the cross can be evaluated for the marker allele that is associated with increased mechanical stalk strength and then selected if it possesses the marker allele.
Methods can also include assaying at least one marker locus in the maize plant. The marker can lie within any of the following chromosomal intervals comprising and flanked by:
(1) PHM7844 and PHM8029;
(2) PHM7844 and PHM574;
(3) PHM11754 and PHM1481;
(4) PHM6427 and PHM1481;
(5) PHM11125 and PHM13958;
(6) PHM10468 and PHM13958;
(7) PHM4578 and PHM11186; or
(8) PHM14053 and PHM16736; and the marker locus is associated with mechanical stalk strength. Either a maize plant that possesses a favorable allele or a maize plant that does not possess an unfavorable allele could then be selected.
Phenotypic Assessment of Mechanical Stalk Strength
Any method known in the art can be used to evaluate mechanical stalk strength. Some methods involve the measurement of stalk diameter or dry weight per plant, while others can utilize an InstronTM machine or other similar crushing device to assess the load needed to break a stalk. The three point bend test is often used in conjunction with an InstronTM machine or other similar crushing device, and mechanical stalk strength values obtained from the three-point bend test have shown to be highly correlated to lodging scores assigned based on field observations. Still another method can involve the use of a stalk-penetrating device.
In addition, any method that uses a device to accurately reproduce wind forces, in order to select plants with increased mechanical stalk strength in the field, can be utilized for the characterization of mechanical stalk strength in maize plants and for the identification of favorable and/or undesirable quantitative trait loci (QTLs) associated with mechanical stalk strength. A device and method used to screen for selected wind-resistance traits in maize, including stalk strength, are described in patent application US2007/0125155 (published Jun. 6, 2007). When this device and method are used, the unit of measure is the number or percentage of plants that have lodged, or broken, stalks (or, alternatively, the number or percentage of plants that do not lodge).
EXAMPLES
The following examples are offered to illustrate, but not to limit, the appended claims. It is understood that the examples and embodiments described herein are for illustrative purposes only and that persons skilled in the art will recognize various reagents or parameters that can be altered without departing from the spirit of the invention or the scope of the appended claims.
Example 1A
QTL Detection: Association Mapping Analysis
An association mapping strategy was undertaken to identify markers associated with mechanical stalk strength in maize. In this association analysis, a collection of 599 maize lines was analyzed by DNA sequencing at 4000-10000 genes (genetic loci). The lines encompassed elite germplasm, commercially released cultivars, and other public varieties.
Phenotypic scores were obtained from 189 individuals in the collection. Mechanical stalk strength was measured using an InstronTM machine, Model 4411 (Instron Corporation, 100 Royall Street, Canton, Mass. 02021), and a three point bend test, with the force applied between nodes 3 and 4 below the ear. The score was the load applied to break the internode, or the weight in kg applied to the internode at the yielding, or breaking, point. Data collection was typically done in one scoring after flowering time (at end of season), and an average score for each line was assigned based on data accumulated over multiple locations. Mechanical stalk strength values for the 189 lines varied from 4.208 to 19.569 kg at yield point. Plants with high scores have greater relative mechanical stalk strength.
The phenotypic scores and marker information for each of the 189 lines was input into the association analysis. A structure-based association analysis was conducted using standard association mapping methods, where the population structure is controlled using marker data. The model-based cluster analysis software, Structure, developed by Pritchard et al., (Genetics 155:945-959 (2000)) was used with haplotype data for 880 elite maize inbreds at two hundred markers to estimate admixture coefficients and assign the inbreds to seven subpopulations. This reduces the occurrence of false positives that can arise due to the effect of population structure on association mapping statistics. Kuiper's statistic for testing whether two distributions are the same was used to test a given marker for association between haplotype and phenotype in a given subpopulation (Press et al., Numerical Recipes in C, second edition, Cambridge University Press, NY (2002)).
QTLs of interest were identified based on the results of the association analyses. These QTLs are described in examples 1B-D and are shown in FIGS. 2-4. In FIGS. 2-4, positions are given in cM, with position zero being the first (most distal from the centromere) marker known at the beginning of the chromosome. These map positions are not absolute and represent an estimate of map position based on the internally derived genetic map (PHB).
Example 1B
Identification of QTL5
A peak of significant marker-trait associations was identified on chromosome 5 (FIG. 2A). Further analysis at this peak revealed its occurrence in one of the seven subpopulations (FIG. 2B), a non-stiff stalk (NSS) group. Table 3 provides a listing of the chromosome 5 markers significantly associated with the mechanical stalk strength phenotype at the p≦0.01 level in this NSS subpopulation, representing an interval of  ̃25 cM on the internally derived genetic map. This chromosomal interval is delineated by and includes markers PHM654 at position 64.21 (p=0.007) and PHM6727 at position 89.62 (p=0.0031) (FIG. 2C). Multiple markers on chromosome 5 are significantly associated with mechanical stalk strength at the p≦0.001 level, identifying an interval delineated by and including markers PHM12632 at position 71.5 (p=0.0003) and PHM3323 at position 80.89 (p=0.001) (FIG. 2D); these markers are noted with an asterisk in Table 3. In addition, multiple markers on chromosome 5 are significantly associated with mechanical stalk strength at the p≦0.0001 level, identifying an interval delineated by and including markers PHM201 at position 71.5 (p=0.0003) and PHM3323 at position 80.89 (p=0.001) (FIG. 2E). The most associated markers are PHM201 at position 71.57 (p=0.00001), PHM5421 at position 72.15 (p=0.0000052), and PHM3468 at position 76.53 (p=0.000064).







TABLE 3







Chromosome 5 markers significantly associated with mechanical stalk


strength at p ≦ 0.01 in the NSS subpopulation









Marker
Relative PHB map



Locus
position (cM)
P-Value












 PHM654
64.21
0.007


 PHM111
65.11
0.003


 PHM10100
66.69
0.0023


 PHM7357
67.42
0.0056


 PHM5349
67.59
0.0018


 PHM4167
69.19
0.0023


 PHM14947
70.29
0.0018


*PHM12632
71.5
3.00E−04


*PHM201
71.57
1.00E−05


*PHM4861
71.57
1.00E−04


*PHM5421
72.15
5.20E−06


*PHM4115
73.37
5.00E−04


*PHM12521
74.86
1.00E−04


*PHM3468
76.53
6.40E−05


*PHM10840
76.53
1.00E−04


*PHM12755
76.82
2.00E−04


 PHM13879
77.01
0.0016


 PHM4103
77.29
0.0079


*PHM5363
77.66
9.00E−04


 PHM14751
77.84
0.0039


 PHM16138
78.02
0.0012


 PHM7877
78.86
0.0029


 PHM9518
78.89
0.005


 PHM7802
79.48
0.0047


*PHM2134
79.53
3.00E−04


*PHM7808
79.6
4.00E−04


*PHM9627
79.6
0.001


 PHM13716
79.6
0.0073


 PHM18731
79.72
0.0019


 PHM2189
80.36
0.0018


*PHM7734
80.78
1.20E−04


*PHM3323
80.89
1.00E−04


 PHM12224
89.17
0.0026


 PHM11904
89.45
0.0096


 PHM6727
89.62
0.0031





*Markers associated with mechanical stalk strength at p ≦ 0.001






There were 60 lines assigned by the model-based cluster analysis software, Structure, to the NSS subpopulation in which the QTL for mechanical stalk strength were detected. The lines can be sorted by phenotype and can be assessed at the following marker loci: PHM201, PHM5421, and PHM3468. The phenotype and marker allele data for all 60 lines at the following marker loci: PHM201, PHM5421, and PHM3468, are shown in Table 4. (For the PHM marker alleles, see FIG. 5.)







TABLE 4







Phenotype and marker allele data (PHM201, PHM5421, and 


PHM3468) for lines in NSS subpopulation











Line
Phenotype
PHM201
PHM5421
PHM3468














PH07H
19.569
2
2
8


PH14E
15.29
2
2
8


PH891
14.863
2
2
8


PHRF5
14.854
1
3
2


PH1AA
13.738
1
3
2


PH2V7
13.667
2
2
8


PHAP9
13.536
2
2
8


PH2FT
13.355
2
2
8


PH1GC
12.48
4
2
8


PH589
12.432
1
3
2


PHDG1
12.421
4
2
8


PHG44
12.335
2
2
NA


PH2T6
11.993
2
2
8


PH1TB
11.983
2
2
8


PH806
11.783
2
2
8


PHW89
11.683
2
2
8


PHM10
11.629
1
3
2


PHK42
11.492
1
3
2


PH7JD
11.311
2
2
8


PH8CW
11.22
1
3
2


PH81B
11.164
1
NA
2


PH8KF
10.871
2
2
NA


PHJ90
10.869
5
2
8


PH1CP
10.799
2
NA
8


PH8KG
10.531
2
2
8


PHR31
10.447
1
3
2


PH3KP
10.206
5
2
8


PH1B5
10.148
2
2
8


PH0HR
10.142
2
NA
8


PHNG2
10.069
2
2
8


PHRF1
9.956
2
2
6


PHH93
9.919
1
3
2


PH7C8
9.88
1
3
2


PH24E
9.675
1
3
2


PH7DD
9.632
1
3
2


PH5HP
9.474
1
3
2


PH7CP
9.345
1
3
2


PHTE7
9.297
1
3
2


PH1N8
9.286
1
3
8


PHDP0
9.204
1
3
2


PH1W0
8.985
1
3
2


PHPP8
8.895
1
3
2


PH16M
8.855
1
3
2


PHK74
8.597
1
3
2


PH1G5S
8.468
1
3
2


PHG29
8.432
1
3
2


PHP55
8.317
1
3
2


PHN82
8.224
1
NA
2


PH0N7
8.182
1
3
2


PH3MW
8.167
1
3
6


PH05N
8.156
1
3
2


PH1G5R
8.014
2
2
8


PH23D
7.913
1
3
2


PH51K
7.629
1
3
2


PH7CM
7.596
1
3
6


PH1B8
7.503
1
3
6


PHG50
7.435
4
NA
8


PHKW3
7.143
1
3
6


PHACJ
6.764
1
3
6


PH3N0
6.629
1
3
6









Further analyses can be performed using marker data from PHM201 and PHM5421. Five lines have missing marker data, and four lines had a haplotype that did not occur with a frequency of at least 10%. Thus, thirty three lines had the haplotype consisting of allele 1 at PHM201 and allele 3 at PHM5421 (designated as “1-3”). The average phenotypic score for the thirty three lines with haplotype 1-3 was 9.326 kg at yield point with a standard error of 0.107. The remaining eighteen lines had the haplotype consisting of allele 2 at PHM201 and allele 2 at PHM5421 (designated as “2-2”). The average phenotypic score for the eighteen lines with haplotype 2-2 was 12.275 kg at yield point with a standard error of 0.370. A depiction of these results can be seen in FIG. 6A.
The allelic variation at PHM3468 can also be an indicator of phenotype. Of the 60 lines, two had missing marker data. Seven lines had marker allele 6 at PHM3468 and an average phenotypic score of 7.680 kg at yield point with a standard error of 0.183. Twenty eight lines had marker allele 2 at PHM3468 and an average phenotypic score of 9.788 kg at yield point with a standard error of 0.113. The remaining twenty three lines had marker allele 8 at PHM3468 and an average phenotypic score of 11.801 kg at yield point with a standard error of 0.289. A depiction of these results can be seen in FIG. 6B.
Thus, allele 1 at PHM201, allele 3 at PHM5421, allele 6 at PHM3468, and the haplotype consisting of allele 1 at PHM201, allele 3 at PHM5421, and allele 6 at PHM3468 are associated with decreased mechanical stalk strength, while allele 2 at PHM201, allele 2 at PHM5421, allele 8 at PHM3468, and the haplotype consisting of allele 2 at PHM201, allele 2 at PHM5421, and allele 8 at PHM3468 are associated with increased mechanical stalk strength.
Example 1C
Identification of QTL9
A peak of significant marker-trait associations was identified on chromosome 9 (FIG. 3) in the same non-stiff stalk (NSS) group in which the chromosome 5 QTL was identified. Chromosome 9 markers associated with mechanical stalk strength at p≦0.01 lie within a  ̃3.5 cM chromosomal interval delineated by and including markers PHM4578 at position 178.04 (p=0.0034) and PHM11186 at position 181.5 (p=0.0074), while markers associated with mechanical stalk strength at p≦0.001 lie within a  ̃1.5 cM chromosomal interval delineated by and including markers PHM14053 at position 179.85 (p=0.001) and PHM16736 at position 181.19 (p=4.00E-04). The top associated markers also include: PHM12025 at position 179.78 (p=0.0015) and PHM405 at position 180.29 (p=0.0024).
Example 1D
Identification of QTL1A, QTL1B, and QTL1C
Three peaks of significant marker-trait associations were identified on chromosome 1 (FIG. 4) in the same non-stiff stalk (NSS) group in which the chromosome 5 and 9 QTLs were identified.
In the first chromosome 1 interval, denoted as QTL1A, markers associated with mechanical stalk strength at p≦0.01 lie within a  ̃16 cM chromosomal interval delineated by and including markers PHM7844 at position 104.55 (p=0.001) and PHM8029 at position 120.44 (p=0.0087), while markers associated with mechanical stalk strength at p≦0.001 lie within a  ̃6.5 cM chromosomal interval delineated by and including markers PHM7844 at position 104.55 (p=0.001) and PHM574 at position 111.02 (p=6.00E-04). The marker most significantly associated with mechanical stalk strength is PHM2130 at position 107.69 (p=1.00E-04).
In the second chromosome 1 interval, denoted as QTL1B, markers associated with mechanical stalk strength at p≦0.01 lie within a  ̃21 cM chromosomal interval delineated by and including markers PHM11754 at position 125.26 (p=0.0089) and PHM1481 at position 146.41 (p=2.00E-04), while markers associated with mechanical stalk strength at p≦0.001 lie within a  ̃21 cM chromosomal interval delineated by and including markers PHM6427 at position 125.63 (p=5.00E-04) and PHM1481 at position 146.41 (p=2.00E-04). The marker most significantly associated with mechanical stalk strength is PHM15089 at position 133.73 (p=4.00E-05).
In the third chromosome 1 interval, denoted as QTL1C, markers associated with mechanical stalk strength at p≦0.01 lie within a  ̃15 cM chromosomal interval delineated by and including markers PHM11125 at position 184.03 (p=0.0019) and PHM13958 at position 198.8 (p=6.00E-04), while markers associated with mechanical stalk strength at p≦0.001 lie within a  ̃12 cM chromosomal interval delineated by and including markers PHM10468 at position 187.27 (p=4.00E-04) and PHM13958 at position 198.8 (p=6.00E-04). One marker significantly associated with mechanical stalk strength is PHM12706 at position 191.11 (p=1.40E-04).
Example 1E
Identification of Favorable and Unfavorable Marker Alleles
There were 60 lines assigned by the model-based cluster analysis software, Structure, to the NSS subpopulation in which the QTL for mechanical stalk strength were detected. The lines were sorted by phenotype and assessed at the following marker loci: PHM2130, PHM15089, PHM12706, PHM5421, PHM201, PHM12521, PHM10840, PHM3468, PHM16736, PHM14053, PHM405, and PHM12025. Table 5 shows the individual polymorphisms associated with increased mechanical stalk strength (“favorable”; select for) or decreased mechanical stalk strength (“unfavorable”; select against).










TABLE 5








In reference


Identifier
SNP
Position
sequence















Chromosome 1 - QTL1A


Select against:










PHM2130.24
T
 75
SEQ ID NO: 202


 


PHM2130.29
A
170
SEQ ID NO: 202


 


PHM2130.30
C
179
SEQ ID NO: 202


 


PHM2130.33
G
358
SEQ ID NO: 202


 







Chromosome 1 - QTL1B


Select against:










PHM15089.13
G
284
SEQ ID NO: 205


 







Chromosome 1 - QTL1C


Select against:










PHM12706.14
C
322
SEQ ID NO: 210


 







Chromosome 5


Select for:










PHM5421.5
G
314
SEQ ID NO: 12


 


PHM3468.1
G
 93
SEQ ID NO: 15


 


PHM3468.4
T
101
SEQ ID NO: 15


 


PHM3468.18
G
245
SEQ ID NO: 15


 


PHM12521.12
T
101
SEQ ID NO: 14


 


PHM12521.19
A
186
SEQ ID NO: 14


 


PHM10840.105
C
 37
SEQ ID NO: 16


 


PHM10840.118
A
240
SEQ ID NO: 16


 


PHM10840.130
C
315
SEQ ID NO: 16


 







Select against:










PHM201.10
C
132
SEQ ID NO: 10


 


PHM201.18
A
230
SEQ ID NO: 10


 







Chromosome 9


Select for:










PHM16736.6
C
225
SEQ ID NO: 199


 


PHM16736.8
T
326
SEQ ID NO: 199


 


PHM16736.14
A
422
SEQ ID NO: 199


 


PHM14053.7
C
193
SEQ ID NO: 198


 


PHM14053.8
C
341
SEQ ID NO: 198


 


PHM14053.14
C
386
SEQ ID NO: 198


 


PHM405.35
T
374
SEQ ID NO: 212


 


PHM12025.26
C
216
SEQ ID NO: 213









Example 2
QTL5 Detection: Composite Interval Mapping
A composite interval mapping approach that combines interval mapping with linear regression was undertaken to identify maize chromosomal intervals and markers associated with mechanical stalk strength. In an interval mapping approach (Lander and Botstein, Genetics 121:185-199 (1989)), each of many positions along the genetic map (say at 1 cM intervals) is tested for the likelihood that a QTL is located at that position. The genotype/phenotype data are used to calculate for each test position a LOD score (log of likelihood ratio). When the LOD score exceeds a threshold value (herein the threshold value is 2.5), there is significant evidence for the location of a QTL at that position on the genetic map (which will fall between two particular marker loci).
A high resolution genetic mapping population, the intermated B73×Mo17 (IBM) population, was created by Lee, M et al., Plant Mol Biol 48:453-461 (2002), and is a widely used resource for maize mapping. The B73 inbred represents the Iowa Stiff Stalk Synthetic (BSSS) heterotic group, whereas Mo17 represents a non-BSSS heterotic group. The population was developed by intermating the F2 for four generations and then deriving recombinant inbred lines.
Recombinant inbred lines from the IBM syn4 population were obtained for this study, and two sets of data were collected for each of the 272 individuals obtained. The first set of data was collected using a portable InstronTM machine, near flowering. One growing season with two field replications was used. Measurements were taken at nodes 3 and 4 below the ear and then averaged over replications. The second set of data was obtained using the device and method described in patent application US2007/0125155 (published Jun. 6, 2007). Data were collected near flowering, and the unit of measure was the number of plants that exhibited snapping at a node. Both sets of data were representative of mechanical stalk strength at the vegetative stage, or resistance to brittle snap.
The 272 individuals of the IBM Syn4 generation were genotyped using 324 markers, and the mean scores (averaged across replications; 2 replicates for the InstronTM data, 3 replicates for phenotypic analysis using artificial wind stimulus) were input into Windows QTL Cartographer. Windows QTL Cartographer (the most up-to-date version of this software was used at the date of QTL mapping) was used to perform the composite interval mapping. LOD scores (logarithm of the odds ratio) were estimated across the genome according to standard QTL mapping procedures.
For both sets of data, the composite interval mapping analysis showed one major significant QTL on chromosome 5 (FIG. 7 and FIG. 8), as defined by a significance LOD score threshold of 2.5. The linkage map used for composite interval mapping was a modified IBM2 map for which the genetic distances correspond to a single meiosis recombination fraction (this map was generated internally). The modified IBM2 map comprised the following PHM marker loci located within the chromosome 5 region of interest (with genetic map positions in parentheses): PHM5266 (70.56 cM), PHM4115 (73.37 cM), PHM12521 (74.86 cM), PHM10840 (76.53 cM), PHM4103 (77.29 cM), PHM9518 (78.89 cM), PHM4736 (81.94 cM), PHM6441 (82.51 cM), and PHM430 (86.77 cM). For data set 1 (portable InstronTM data; near flowering; referred to herein as CIM1 for “composite interval mapping data set 1”), the location of the QTL encompassed markers PHM12521, PHM10840, PHM4103, and PHM9518. For data set 2 (wind machine; near flowering; referred to herein as CIM2 for “composite interval mapping data set 2”), the location of the QTL encompassed markers PHM5266, PHM4115, PHM12521, PHM10840, PHM4103, PHM9518, PHM4736, PHM6441, and PHM430.
Example 3
Summary of QTL5 Studies
The results of the chromosome 5 analyses are summarized in Table 6 (the map position of each of the PHM markers on the internally derived genetic map is provided as a reference). All three studies identify the location of a QTL for mechanical stalk strength in the same region of chromosome 5.







TABLE 6







Summary of QTL5 Studies














Relative PHB






Marker
map position






Locus
(cM)
Association
CIM1
CIM2







PHM5266
70.56


X



PHM4115
73.37
**

X



PHM12521
74.86
**
X
X



PHM10840
76.53
**
X
X



PHM4103
77.29
*
X
X



PHM9518
78.89
*
X
X



PHM4736
81.94


X



PHM6441
82.51


X



PHM430
86.77


X







** = p < 0.001;



* = p < 0.01






A set of common markers can be used to establish a framework for identifying markers in the QTL interval (Table 7). In this table, the markers shaded in gray are public markers, while the unshaded markers are provided herein. All of the markers listed in this table are in a consistent position relative to one another on the PHB internally derived map, the IBM2 neighbors genetic map, and the current physical map (FIGS. 1A-I). In FIGS. 1A-I, PHM12521 and PHM3468 are not shown because the markers are currently on an “unknown” contig that is not assembled to the larger chromosome 5 contig.







TABLE 7





Molecular marker positions on the PHB map and the


IBM2 Neighbors map
















Markers shaded in gray are public markers.






Example 4
QTL Validation
Biparental crosses are typically created to validate QTLs. For example, near isogenic lines containing the individual QTL or combinations of QTL can be generated from a biparental cross of two lines (each with a distinct genotype at a QTL of interest), and the resulting plants can be evaluated for mechanical stalk strength. Phenotypes can be measured using an InstronTM machine to perform the three-point bend test during late season, a portable InstronTM to perform the three-point bend test at or near flowering, the device and method described in patent application US2007/0125155 (published Jun. 6, 2007), or any other method in the art that can be used to evaluate mechanical stalk strength. The lines are then sorted based on their phenotypic values.
Production markers can be developed in the QTL region. These markers will distinguish the parents from one another, preferably using a high throughput assay, and are used to genotype the segregating progeny plants. Production markers could be developed, for example, from individual SNPs that distinguished lines with increased mechanical stalk strength from lines with decreased mechanical stalk strength in the association study described in Examples 1A-E. For instance, PHM5421-5-V, PHM12521-19-U, and PHM16736-8-V were developed from SNPs PHM5421.5, PHM12521.19, and PHM16736.8, respectively.
The PHM markers could also be used to genotype the progeny via the sequencing of PCR products. The primers for each of the PHM marker loci and the corresponding reference sequence ID numbers are shown in Table 1. For PHM marker analysis, nested PCR reactions are performed, using the external and internal primers for each PHM marker. In the first PCR reaction, 0.90 μl of 10×PCR buffer, 0.18 μl of 10 mM dNTP mix, 0.27 μl of 50 mM MgCl2, 1.50 μl of 2.5 μM external forward primer, 1.50 μl of 2.5 μM external reverse primer, 0.04 μl of Platinum Taq, 1.61 μl of ddH2O, and 3 μl of 1.5 ng/μl DNA are used. The thermocycling conditions constitute: 95° C. at 5 minutes; 94° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, repeated for 24 cycles; 72° C. for 10 minutes; and a hold at 4° C. The DNA produced from the first round of PCR is then diluted 1:9 with TE for use in the second round of PCR. The reaction mix for the second round of PCR is the same except the internal sets of primers are used, and the DNA is the diluted DNA from the first round of PCR. The thermocycling conditions for the second round of PCR constitute: 95° C. at 5 minutes; 94° C. for 20 seconds, 55° C. for 30 seconds, and 72° C. for 2 minutes, repeated for 28 cycles; 72° C. for 10 minutes; and a hold at 4° C. The PCR products are then sequenced directly.
Each marker locus is examined, and standard statistical analysis, such as the student t-test or Kolmogorov-Smirnov test, can be used to determine if the trait distributions for each allele are significantly different from one another. If significant, the effect of this QTL on mechanical stalk strength would be validated.
Example 5
Validation of QTLS, QTL9, and QTL1A
A biparental population, PHO7H×PH7CM, was developed for the purpose of validating QTLS, QTL9, and QTL1A. PHO7H had a score of 19.569 in the association study and carries the favorable alleles at the QTL5 marker loci, PHM5421, PHM201, PHM12521, PHM10840, and PHM3468; at the QTL9 marker loci, PHM16736, PHM14053, PHM405, and PHM12025; and at the QTL1A marker locus PHM2130. PH7CM had a score of 7.596 and did not carry the favorable alleles at QTL5, QTL9, and QTL1A. F2 individuals were genotyped, and siblings carrying only the favorable alleles at either QTL5, QTL9, or QTL1A were selected. The selected siblings were selfed twice before phenotyping F4 individuals with an InstronTM machine after flowering. Plants carrying favorable alleles at QTL1A showed a greater positive effect on load at yield, dry weight, and diameter; however, favorable alleles at QTL5 increase stalk strength without a significant change in dry weight or diameter. No difference in stalk strength, dry weight, and diameter were seen amongst the siblings for the chromosome 9 segment. Data for stalk strength as measured by the InstronTM is shown in Table 8.












TABLE 8









median
median





(favorable 
(unfavorable




p-value
allele)
allele)





















QTL1A
3.81E−11
19.48
16.96



QTL5
6.49E−04
19.58
17.83



QTL9
1.66E−01
19.68
20.39










The apparent lack of association between QTL9 and mechanical stalk strength was resolved in another biparental population that was developed from a cross between PH891 and PH7CM. PH891 had a score of 14.863 in the association study and also carries the favorable alleles at QTL5, QTL9, and QTL1A. NILs (near isogenic lines) carrying only a subsegment of the chromosome 9 region were created. A subsegment of the chromosome 9 QTL from PH7CM, in the region between 174.3 and 179 cM, had an overall positive effect on mechanical stalk strength, while a sub-segment of the chromosome 9 QTL from PH7CM in the region between 179 and 181 had an overall negative effect on mechanical stalk strength. This explains why there was no net gain in observed stalk strength amongst the F4 siblings (described in the previous paragraph) that carried both segments together.
Example 6
Markers for Use in MAS of Plants with Increased Mechanical Stalk Strength
A set of production SNP markers specific for each chromosomal region has been developed (Table 9), and when used together, the markers can be used to identify the proper haplotype for the regions associated with mechanical stalk strength. The markers were developed by comparing the genotypes and phenotypes at a number of PHM marker loci in the intervals described herein (QTL1A, QTL1B, QTL5, and QTL9) for a parental panel of inbreds from NSS germplasm plus a few diverse lines. Markers were created using Invader PlusTM chemistry.







TABLE 9







Production Markers for Use In MAS













PHB map
Select
Select


Marker
QTL
position (cM)
For:
Against:














PHM18693-9-U
1A
106.7
T



PHM10786-11-U
1A
107.8
G



PHM10786-5-U
1A
107.8
C



PHM10786-6-U
1A
107.8
T



PHM8057-801-U
1A
110.2
G



PHM4044-11-U
1B
132.4

T


PHM14080-16-V
1B
133.2

A


PHM15089-10-U
1B
133.7

C


PHM9364-6-U
1B
135.5

G


PHM201-16-U
5
71.6
C



PHM201-17-U
5
71.6
C or G



PHM4861-20-U
5
71.6
T or G



PHM4861-21-U
5
71.6
A



PHM5421-5-V
5
72.2
G



PHM4115-35-U
5
73.4
G or T



PHM12521-18-U
5
74.9
T



PHM12521-19-U
5
74.9
A



PHM12521-29-U
5
74.9
G



C00386-397-U
5

C



PHM13418-18
9
174.37
C



PHM13418-10
9
174.37
C



PHM113-7
9
180.76
T



PHM10337-11-U
9
181.2
Ti



PHM16736-8-V
9
181.2
A



PHM12025-48
9
179.78
C



PHM11186-16-V
9
181.5
T

## Claims

1. A method of obtaining maize plants with increased mechanical stalk strength, the method comprising:
a. isolating nucleic acids from a maize plant;
b. analyzing the isolated nucleic acids for the presence of a QTL allele associated with the increased mechanical stalk strength, wherein the presence of said QTL allele is determined by detecting a haplotype within a chromosomal interval comprising and flanked by PHM14053 and PHM16736 wherein said haplotype comprises:
(a) a “C” at PHM16736.6;
(b) a “T” at PHM16736.8;
(c) an “A” at PHM16736.14;
(d) a “C” at PHM14053.7;
(e) a “C” at PHM14053.8;
(f) a “C” at PHM14053.14; and
(g) a “T” at PHM405.35; and

c. selecting a maize plant having said haplotype;
d. crossing said maize plant to a second maize plant;
e. evaluating progeny for the haplotype; and
f. selecting progeny plants that possess the haplotype.