# Patent

## Title
Tomato plants exhibiting tolerance to continuous light

## Abstract

The present invention relates to a method of selecting a tomato plant comprising exposing a tomato plant and determining whether said plant exhibits tolerance to exposure to continuous light, wherein said tolerance is indicated by decreased level of chlorosis upon said exposure, and further determining whether said plant exhibits a significant increase in yield when exposed to continuous light. The invention further relates to a method for increasing the yield of a tomato plant line, said method comprising selecting a tomato plant by the method of the invention; crossing the thus selected plant with a plant of a said tomato plant line to produce offspring plants; optionally backcrossing or selfing said offspring plant to produce further offspring plants, and selecting from said offspring plants a plant having tolerance to continuous light and having increased yield as compared to said plant of the original tomato plant line.

## Background
RELATED APPLICATION DATA
This application is a National Stage Application under 35 U.S.C. 371 of PCT application PCT/NL2010/050562 designating the United States and filed Sep. 6, 2010; which claims the benefit of EP patent application number 09169555.1.2 and filed Sep. 4, 2009 each of which are hereby incorporated by reference in their entireties.
FIELD OF THE INVENTION
The present invention relates to plant breeding and molecular biology. More specifically, the present invention relates to a method for detecting a gene associated with tolerance to continuous light in tomato, to a method of producing a continuous light-tolerant tomato plant therewith and to continuous light-tolerant tomato plants thus obtained and parts thereof.
BACKGROUND OF THE INVENTION
Tomato (Solanum lycopersicum, formerly known as Lycopersicon esculentum) is a plant of the Solanaceae or nightshade family. It is a short-lived perennial plant, grown as an annual plant, and a close relative of the potato. The fruit (i.e. the tomato) is an edible, brightly coloured (usually red, from the pigment lycopene) berry, 1-2 cm diameter in wild plants, commonly much larger in cultivated forms. The plant is now grown worldwide for its edible fruits, which are major fresh market vegetables worldwide.
During the last decades, tomato breeding was mainly focused on yield, disease tolerance, and fruit quality aspects such as uniform ripening and taste. Yield improvements have been achieved due to new production methods, improved pest management and varieties that are better suited for new production methods, but the gains in yield become smaller. New varieties with 5 or 15 fruits more per plant gave a yield increase of 2-4%.
As the world population continues to grow, the demand for fresh vegetables, such as tomatoes, is ever increasing worldwide. Thus, a continuing need exists for means and methods for improving yield of tomatoes.
SUMMARY OF THE INVENTION
The inventors have surprisingly found that the yield of some tomato plants can be increased by the use of longer photoperiods or even continuous photoperiods. This is surprising, because commercially used varieties, when grown in continuous light, show a strong increase in chloroplastic starch, leaf chlorosis, necrosis and consequently very poor biomass production. In effect, continuous light is often lethal for the crop. However, the inventors have found that tomato plants exist which are not only tolerant to continuous light, but in fact display an increase in yield when exposed to continuous light when compared to 16 hours of light.
The present inventors have successfully identified a quantitative trait locus (gene) which is responsible for tolerance to continuous light present in the genome of several wild relatives of tomato, i.e. in S. neorickii, S. habrochaites, S. pennellii, S. peruvianum, S. chilense and S. chemielewskii, and in S. lycopersicum, e.g. var. Sub Arctic Plenty.
The inventors were subsequently able to produce continuous light tolerant tomato plants by crossing these continuous light tolerant wild (donor) tomato lines with non-tolerant recipient tomato plants. These plants thus produced exhibited more tolerance to continuous light than any cultivated tomato plant produced thus far. In addition, several plants also produced more biomass when exposed to continuous light than plants from the original recipient line when exposed to 16 hours of light. Therefore, the present invention enables more efficient culturing and higher yields.
The present invention provides in a first aspect a method of selecting a tomato plant comprising exposing a tomato plant and determining whether said plant exhibits tolerance to continuous light, wherein said tolerance is indicated by decreased level of chlorosis upon said exposure, and further determining whether said plant exhibits a significant increase in yield.
The invention further provides a method for producing a first tomato plant by a method according to the invention, crossing said selected plant with a second tomato plant to produce offspring plants and selecting from said offspring plants a plant having tolerance to continuous light and having increased yield as compared to said second tomato plant.
The invention further provides a method of producing a tomato plant, tolerant to exposure to continuous light, said method comprising steps of:
a) providing a tomato donor plant tolerant to exposure to continuous light, preferably of the species S. neorickii, S. habrochaites, S. pennellii, S. lycopersicum, S. peruvianum, S. chilense or S. chemielewskii; 
b) transferring nucleic acid from said donor plant to at least one recipient tomato plant which is not tolerant to exposure to continuous light, wherein said transfer results in the introduction of genomic material from said donor plant in the corresponding region of the genome of said at least one recipient plant;
c) selecting from said at least one recipient plant a plant that comprises within its genome a gene for continuous light tolerance derived from said donor plant, wherein said selection comprises detecting in chromosome 7 of said recipient tomato plant at least one genetic marker linked to said gene for light tolerance.
In preferred embodiments, the location of said gene on chromosome 7 of said plant is indicated by a genomic region comprising the genetic markers “7-20-1”, “7-20-2” on chromosome 7 of S. neorickii, “7-10”, “7-25” on chromosome 7 of S. neorickii, “7-17”, “7-25” on chromosome 7 of S. chilense, “7-19”, “7-25” on chromosome 7 of S. pennellii. 
In another preferred embodiment, said transfer of nucleic acid comprising said gene for tolerance to continuous light, or a light tolerance conferring part thereof, is performed by crossing said donor tomato plant tolerant to exposure to continuous light with a recipient tomato plant which is not tolerant to exposure to continuous light, to produce offspring plants comprising said gene as an introgression, and wherein step c) is performed on at least one offspring plant.
In another preferred embodiment, said step c) is performed by detecting said genetic marker in DNA isolated from said at least one recipient tomato plant.
In another preferred embodiment, said step c) further comprises subjecting said plant to bioassay for measuring the capability of growing in continuous light of said plant.
In another aspect the invention provides a continuous light tolerant tomato plant or a part thereof, obtainable by a method according to the invention.
The invention further provides a gene for continuous light tolerance, wherein said gene is associated with tolerance to continuous light and wherein the location of said gene on chromosome 7 of said plant is indicated by a genomic region comprising the genetic markers “7-20-1”, “7-20-2” on chromosome 7 of S. neorickii, “7-10”, “7-25” on chromosome 7 of S. neorickii, “7-17”, “7-25” on chromosome 7 of S. chilense, “7-19”, “7-25” on chromosome 7 of S. pennellii. The alleles on the positions of the genome indicated by this gene is an aspect of the present invention.
A gene of the present invention may be in the form of an isolated, preferably double stranded nucleic acid sequence comprising said gene or a tolerance-conferring part thereof. Very suitably, the size of the nucleic acid sequence, which may for instance be isolated from the chromosome of a suitable donor plant, may represent a genetic distance of 1-100 cM, preferably 10-80 cM on said chromosome. Preferably said genetic distance is between 64 and 78 cM on said chromosome, even more preferably between 72 and 75 cM. Said nucleic acid may comprise at least 50, more preferably at least 500, even more preferably at least 1000, still more preferably at least 5000 base pairs. One or more nucleic acid sequences comprising a gene or a tolerance-conferring part thereof according to the invention may in turn be comprised in a nucleic acid construct, said construct may further comprise regions that flank said one or more nucleic acid sequences and which regions are capable of being integrated into a suitable vector for transfer of said one or more nucleic acid sequences into a suitable continuous light intolerant recipient tomato plant. The vector may further comprise suitable promoter regions or other regulatory sequences. Said gene may also be in a form present within the genome of a tomato plant. The gene of the present invention preferably comprises at least one marker, preferably two markers associated with continuous light-tolerance selected from the group consisting of the markers of FIG. 10 linked to said gene.
The invention further provides a method for detecting a gene for tolerance to continuous light, comprising detecting at least one genetic marker linked to a gene for tolerance to continuous light derived on chromosome 7 of a suspected continuous light tolerant tomato plant, wherein the location of said gene on chromosome 7 of said plant is indicated by a genomic region comprising the genetic markers “7-20-1”, “7-20-2” on chromosome 7 of S. neorickii, “7-10”, “7-25” on chromosome 7 of S. neorickii, “7-17”, “7-25” on chromosome 7 of S. chilense, “7-19”, “7-25” on chromosome 7 of S. pennellii. 
In another aspect the invention provides a continuous light-tolerant tomato plant, or a part thereof, comprising within its genome a gene for continuous light tolerance, or a continuous light-tolerance-conferring part thereof, wherein the location of said gene on chromosome 7 of said plant is indicated by a genomic region comprising the genetic markers “7-20-1”, “7-20-2” on chromosome 7 of S. neorickii, “7-10”, “7-25” on chromosome 7 of S. neorickii, “7-17”, “7-25” on chromosome 7 of S. chilense, “7-19”, “7-25” on chromosome 7 of S. pennellii, wherein said gene or said continuous light-tolerance-conferring part thereof is not in its natural genetic background.
In another aspect the invention provides a method of producing a continuous light-tolerant inbred tomato plant, comprising

    
    
        a) producing a continuous light-tolerant tomato plant according to the invention;
        b) crossing said continuous light-tolerant tomato plant with itself or another tomato plant to yield progeny tomato seed;
        c) growing said progeny tomato seed of step b) to yield an additional continuous light-tolerant tomato plant;
        d) repeating the crossing and growing steps from 0 to 7 times to generate a continuous light-tolerant inbred tomato plant.
    
    


Preferably said step c) further comprises the step of identifying plants that exhibit continuous light tolerance and possess commercially desirable characteristics.
In another preferred embodiment said method further comprises the step of selecting homozygote inbred tomato plants, preferably wherein said inbred plants are homozygous for said continuous light allele.
In a further aspect, the invention provides a continuous light-tolerant inbred tomato plant, or parts thereof, obtainable the method according to the invention.
In a further aspect, the invention provides a hybrid tomato plant, or a part thereof, that exhibits tolerance to continuous light, wherein said hybrid tomato plant is obtainable by crossing a continuous light-tolerant inbred tomato plant obtainable by a method according to the invention with an inbred tomato plant that exhibits commercially desirable characteristics. Such characteristics include inter alia a high yield (in excess of 50, 60 kg/m2 of tomatoes and/or resistance to tobacco mosaic virus (TMV), blight and/or Botrytis. 
In another aspect, the invention further provides the use of a genetic marker selected from the group consisting of the genetic markers of FIG. 10 for the detection of genes for continuous light-tolerance, and/or for the detection of continuous light-tolerant tomato plants.
In another aspect, the invention provides an indeterminate tomato plant, tolerant to exposure to continuous light or a part thereof.
In a further aspect, the invention further provides a tissue culture of regenerable cells of the tomato plants according to the invention, preferably said regenerable cells comprise cells or protoplasts isolated from a tissue selected from the group consisting of leaves, pollen, embryos, roots, root tips, anthers, flowers, fruits, and stems and seeds.
The continuous light-tolerant donor tomato plant used in methods of the present invention is preferably selected from the group consisting of S. neorickii, S. habrochaites, S. pennellii, S. lycopersicum, S. peruvianum, S. chemielewskii, more preferably, a wild tomato accession is used as the donor plant. Highly preferred donor plants are S. pennellii LA716, S. peruvianum LA1708, S. habrochaites LA1777, S. habrochaites G1560, S. habrochaites Lyc4/78, S. chilense LA1959, S. chmielewskii LA1840, S. neorickii LA2133 and S. lycopersicum variety “Sub arctic plenty”.
The continuous light-tolerant recipient tomato plant used in methods of the present invention is preferably a plant of the species Solanum lycopersicum, more preferably an S. lycopersicum cultivar that possess commercially desirable characteristics, or another commercial tomato line.



DESCRIPTION OF THE DRAWINGS
FIG. 1: Species and varieties tolerant to continuous light. Plants shown on the left were exposed to 16 hr and plants on the right are exposed to continuous light. Clearly the typical symptoms of light intolerance (leaf chlorosis, necrosis and poor biomass production) are not expressed in these tolerant lines, while the symptoms are clearly visible in non tolerant lines under the same conditions.
FIG. 2: typical symptoms caused by exposure to continuous light in non-tolerant lines. The first visible symptoms of non tolerant lines are light green leaves with inter-vine chlorosis, followed by severe chlorosis. In severe non tolerant plants chlorosis is followed by necrosis, decreased leaf development decreased stem development and even plant mortally.
FIG. 3: Degreening and chlorosis. Quantification of a part of the visible symptoms (degreening and chlorosis) by measuring the chlorophyll concentration by a SPAD® meter was performed with plants grown at 131 μmol m−2 s−1 (ML) or 230 μmol m−2 s−1 (HL). The resolution to detect differences between continuous and non-continuous light was very difficult or impossible at very low light intensities (16 mol m−2 S−1=LL). The visible symptoms developed earlier in time at 230 mol m−2 s−1 compared to 131 μmol m2 s−1. Left: averages with standard deviations, number of replicates is 20 to 40. Right: Data extracted from the left graph, plotted is the differences between 16 and 24 hours of light per light intensity. Significance level is shown by ns=not significant, *=p<0.05, **=p<0.01, ***=p<0.001 and ****=p<0.00001.
FIG. 4: Stem diameter and plant height is decreased in non tolerant lines. Therefore stem diameter and plant height are also parameters to be able to quantify continuous light symptoms. Averages with standard errors, number of replicates is 4 to 8. Significance level is shown by +=p<0.1, *=p<0.05, **=p<0.01, ***=p<0.001.
FIG. 5: Chlorophyll concentration of IL lines from a neorickii*a sensitive esculentum (e.g. Moneyberg) population where 9 days old seedlings exposed for 33 days to continuous light. Temperature 21° C. for 24 hours and, with an RH of 70% and a light intensity of 110-120 μmol m−2 s−1 (PAR) from fluorescent tubes. Averages with standard errors, number of replicates was 3 to 6 plants per line.
FIG. 6: One highly tolerant line (42) from a neorickii*a sensitive esculentum (left). Tolerant lines are exposed to continuous light for 90 days. The first 33 days under fluorescent tubes, followed by illumination under High pressure sodium lamp (SON-T). The leaves stay green, leaf and stem development is normal compared to non tolerant lines.
FIG. 7: Chlorophyll concentration of IL lines from a chilense LA1959*Moneyberg GT population where 15 days old seedlings exposed for 35 (CLT1), 42 (CLT2), 49 (CLT3) and 60 (CLT4) days to continuous light. Temperature 21° C./20° C. for 16/8 hours and, with an RH of 70% and a light intensity of 110-120 μmol m−2 s−1 (PAR) from fluorescent tubes during the first 15 days followed by illumination under High pressure sodium lamp (SON-T) for 24 Hr. Averages with standard errors, number of replicates was 3 to 6 plants per line.
FIG. 8: One highly tolerant line (5) from a chilense LA1959*a sensitive Moneyberg GT. Tolerant lines are exposed to continuous light for 90 days. The leaves stay green, leaf and stem development is normal compared to non tolerant lines.
FIG. 9: Chlorophyll concentration of IL lines from a pennellii*M82 population with an introgression on chromosome 7. Chlorophyll concentration of IL lines from a pennellii*M82 population where 14 days old seedlings were exposed for 21 (CLT1), 28 (CLT2), 35 (CLT3), 42 (CLT4) and 53 (CLT5) days to continuous light. Temperature 21° C./20° C. for 16/8 hours and, with an RH of 70% and a light intensity of 110-120 μmol m−2 s−1 (PAR) from fluorescent tubes the first 14 days after that under SON-T for 24 Hr. Averages with standard errors, number of replicates was 4 to 6 plants per line. Two tolerant lines were found (4 and 5), which have been in continuous light for 53 days.
FIG. 10: Genotypic information of the different mapping populations, based on Illumina SNP markers. Combining the phenotypic and genotypic data from the three populations, the continuous light tolerant loci is located on chromosome 7 between marker “7-19” and “7-23” that is between 64 and 99.6 cM based on the EXPEN2000 linkage map. Wild donor alleles are shown in light grey, recurrent parent alleles in dark grey, different alleles in black and no call in white. Some SNPs are non polymorphic in some wild donor x recurrent parent combinations, in such cases a gap is present. SNPs markers were genotyped using GoldenGate assays (Illumina).
FIG. 11: Sequences of genetic markers. The markers indicate the polymorphic sequences between the tolerant and sensitive genotype. The polymorphic positions as indicated in these sequences are SNPs. An example for use of these SNPs as markers is provided in Example 7 below. The skilled artisan will understand that the markers as indicated in this figure refer specifically to the polymorphic positions in the sequences given. Marker 1: Sequence of the marker “7-1” derived from S. neorickii LA2133 located at 0.4 cM of chromosome 7 ([T/G] indicates the polymorphic position) (SEQ ID NO:1). Marker 2: Sequence of the marker “7-2” derived from S. pennellii 0716 located at 1.2 cM of chromosome 7 ([T/G] indicates the polymorphic position) (SEQ ID NO:2). Marker 3: Sequence of the marker “7-3” derived from S. neorickii LA2133 located at 2.0 cM of chromosome 7 ([A/T] indicates the polymorphic position) (SEQ ID NO:3). Marker 4: Sequence of the marker “7-4” derived from S. neorickii LA2133 located at 3.5 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:4). Marker 5: Sequence of the marker “7-5” derived from S. neorickii LA2133 located at 21.4 cM of chromosome 7 ([A/T] indicates the polymorphic position) (SEQ ID NO:5). Marker 6: Sequence of the marker “7-6” derived from S. neorickii LA2133 located at 22.3 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:6). Marker 7: Sequence of the marker “7-7” derived from S. neorickii LA2133 located at 24.8 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:7). Marker 8: Sequence of the marker “7-8” derived from S. neorickii LA2133 located at 27.5 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:8). Marker 9: Sequence of the marker “7-9” derived from S. pennellii 0716 located at 28.1 cM of chromosome 7 ([A/G] indicates the polymorphic position) (SEQ ID NO:9). Marker 10: Sequence of the marker “7-10” derived from S. neorickii LA2133 located at 38.0 cM of chromosome 7 ([A/G] indicates the polymorphic position) (SEQ ID NO:10). Marker 11: Sequence of the marker “7-11” derived from S. neorickii LA2133 located at 38.8 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:11). Marker 12: Sequence of the marker “7-12” derived from S. pennellii 0716 located at 40.0 cM of chromosome 7 ([T/G] indicates the polymorphic position) (SEQ ID NO:12). Marker 13: Sequence of the marker “7-13” derived from S. neorickii LA2133 located at 43.0 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:13). Marker 14: Sequence of the marker “7-14” derived from S. neorickii LA2133 located at 45.6 cM of chromosome 7 ([A/G] indicates the polymorphic position) (SEQ ID NO:14). Marker 15: Sequence of the marker “7-15” derived from S. neorickii LA2133 located at 48.1 cM of chromosome 7 ([A/T] indicates the polymorphic position) (SEQ ID NO:15). Marker 16: Sequence of the marker “7-16” derived from S. neorickii LA2133 located at 49.2 cM of chromosome 7 ([A/T] indicates the polymorphic position) (SEQ ID NO:16). Marker 17: Sequence of the marker “7-17” derived from S. neorickii LA2133 located at 54.0 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:17). Marker 18: Sequence of the marker “7-18” derived from S. neorickii LA2133 located at 61.0 cM of chromosome 7 ([T/G] indicates the polymorphic position) (SEQ ID NO:18). Marker 19: Sequence of the marker “7-19” derived from S. pennellii 0716 located at 64.0 cM of chromosome 7 ([A/T] indicates the polymorphic position) (SEQ ID NO:19). Marker 20: Sequence of the marker “7-20” derived from S. neorickii LA2133 located at 46.18 cM of chromosome 7 based on mapping in the 2133-42 population and at 73.0 cM based on mapping using the EXPEN 2000 linkage map of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:20). Marker 21: Sequence of the marker “7-21” derived from S. neorickii LA2133 located 46.74 cM of chromosome 7 based on mapping in the 2133-42 population and at at 78.0 cM based on mapping using the EXPEN 2000 linkage map of chromosome 7 ([A/G] indicates the polymorphic position) (SEQ ID NO:21). Marker 22: Sequence of the marker “7-22” derived from S. neorickii LA2133 located at 80.0 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:22). Marker 23: Sequence of the marker “7-23” derived from S. neorickii LA2133 located at 99.6 cM of chromosome 7 ([T/C] indicates the polymorphic position) (SEQ ID NO:23). Marker 24: Sequence of the marker “7-24” derived from S. pennellii 0716 located at 100.4 cM of chromosome 7 ([A/T] indicates the polymorphic position) (SEQ ID NO:24). Marker 25: Sequence of the marker “7-25” derived from S. neorickii LA2133 located at 104.0 cM of chromosome 7 ([A/G] indicates the polymorphic position) (SEQ ID NO:25). Marker 26 Sequence of the marker “7-20-1” derived from S. neorickii LA2133 located at 43.68 cM of chromosome 7 based on mapping in the LA2133-42 population and at 75 cM based on mapping using the EXPEN 2000 linkage map ([T/G] indicates the polymorphic position) (SEQ ID NO:26). Marker 27 Sequence of the marker “7-20-2” derived from S. neorickii LA2133 located at 44.35 cM of chromosome 7 based on mapping in the LA2133-42 population and at 72 cM based on mapping using the EXPEN 2000 linkage map ([C/A] indicates the polymorphic position) (SEQ ID NO:27). Marker 28 Sequence of the marker “7-20-3” derived from S. neorickii LA2133 located at 44.46 cM of chromosome 7 based on mapping in the LA2133-42 population and at 73 cM based on mapping using the EXPEN 2000 linkage map ([G/C] indicates the polymorphic position) (SEQ ID NO:28).
FIG. 12: The CLT locus. The CLT locus is positioned between markers 7-17 and 7-20. Presented genotypic data (the genotypes include “homozygous for donor” or “recurrent parent” and “heterozygous”) is generated from F2 plants, while phenotypic scoring (the phenotypes include “Tolerant to continuous light”, “susceptible for continuous light” and “segregating”) is performed on F3 plants. Light grey blocks indicate genomic segments originating from the tolerant parent. Dark grey blocks indicate segments that are originating from the sensitive parent. White blocks indicate heterozygous segments. Markers are ordered according to map LA2133-42.
FIG. 13. The CLT locus is positioned between markers 7-20-1 and 7-20-2. Presented genotypic data (the genotypes include “homozygous for donor” or “recurrent parent” and “heterozygous”) is generated from F2 plants, while phenotypic scoring (the phenotypes include “tolerant to continuous light”, “susceptible for continuous light” and “segregating”) is performed on F3 plants. Dark grey blocks indicate genomic segments originating from the sensitive parent. Light grey blocks indicate segments that are originating from the tolerant parent. White blocks indicate heterozygous segments. Markers are ordered according to map LA2133-42.



DETAILED DESCRIPTION OF THE INVENTION
Definitions
The term “light” as used herein means light of a suitable wavelength for growing plants at an average light intensity which is higher than the light energy known as the “light compensation point”. This is the light intensity at which the amount of carbon dioxide released in respiration equals the amount used in photosynthesis, and the amount of oxygen released in photosynthesis equals the amount used in respiration.
The compensation point is the amount of light intensity on the light curve where the rate of photosynthesis exactly matches the rate of respiration. At this point, the uptake of CO2 through photosynthetic pathways is exactly matched to the respiratory release of carbon dioxide, and the uptake of O2 by respiration is exactly matched to the photosynthetic release of oxygen. Methods to establish the light compensation point of a plant are well known in the art. Preferably, said light has a light intensity of at least 20 μmol m−2 s−1, more preferably at least 100 μmol m−2 s−1, still more preferably at least 250 μmol m−2 s−1 PAR (photosynthetic active radiation). Preferably said light has a light intensity of less than 2000 μmol m−2 s−1, preferably less than 1000 μmol m−2 s−1.
As used herein, the term “continuous light” means a photoperiod of more than 20 hours light per day, preferably more than 21 hours, preferably more than 22 hours, more preferably more than 23 hours and most preferably 24 hours. Short interruptions of a few minutes of this photoperiod may be comprised in continuous lighting. The total duration of the exposure to said light per day must be more than 20 hours.
As used herein, the term “continuous light tolerance” refers to a plant which, when grown under continuous light, exhibits a reduction in severity of at least one symptom of the complex of symptoms typically associated with continuous light intolerance as observed in normal non tolerant (tomato) plants of for instance the variety Moneyberg within 20 days after the first exposure to continuous light. Preferably, a plant which is tolerant to continuous light does not exhibit said at least one symptom after at least 3 weeks of exposure to continuous light. Even more preferably, said at least 3 weeks is at least four weeks. More preferably, it is at least 35, 42, 63 or 90 days. Preferably, said plant which is tolerant to continuous light has a higher chlorophyll concentration in its chlorophyll containing cells when exposed to continuous light than a non tolerant control plant which is exposed to the same conditions. Preferably, said plant which is tolerant to continuous light has a chlorophyll concentration of at least 35 (relative scale from 0-100, 100 is deep green), more preferably 36, 37, 38, 39, 40, 41, 42, 43, 44, 45, 46, 47, 48, 49, 50, 51, 52, 53, 54, 55, 56, 57, 58, 59, 60 as determined by a SPAD-502® (Konica Minolta) meter, wherein said plant has been exposed for at least, 33, 35, 42, 49, 53, 90 days to continuous light with a light intensity of 130 μmol m−2 s−1 from fluorescent tubes and grown at a temperature of 21° C. and a relative humidity between 60-80%, preferably 70%. In another preferred embodiment, said plant which is tolerant to continuous light has a chlorophyll concentration which is at least 5%, more preferably 10%, 15%, 20%, 25%, 30%, 35%, 40%, 50% higher than its sensitive parent plant exposed to the same conditions, as determined by using a SPAD-502® (Konica Minolta) meter, wherein said plant has been exposed for at least, 33, 35, 42, 49, 53, 90 days to continuous light with a light intensity of 130 μmol m2 s−1 from fluorescent tubes and grown at a temperature of 21° C. and a relative humidity between 60-80%, preferably 70%.
Said complex of symptoms includes but is not limited to leaf chlorosis, necrosis, decreased leaf development, increase in chloroplastic starch, decreased stem development, reduced photosynthesis, degreening and poor biomass production. Preferably all symptoms are absent in a plant tolerant to continuous light.
The term “leaf chlorosis” as used herein refers to a yellowing of leaf tissue due to absence of chlorophyll. The absence of chlorophyll can be visually observed as yellow spots on the leaf tissue.
The term “necrosis” as used herein refers to the premature death of cells and living tissue due to external factors. Necrosis can be visually observed as light to dark brown spots or absence of cells in tissue, preferably leaf tissue.
The term “decreased leaf development” as used herein refers to a decreased total surface area of the combined leaves of a plant which has been exposed to continuous light compared to the total surface area of the combined leaves of a comparable control plant which has not been exposed to continuous light. Total surface area of the combined leaves of a plant can be determined by measuring the total leaf surface area of a plant and/or by weighing the mass of the leaves of a plant.
The term “increase in chloroplastic starch” refers to an increase in the amount of starch in a sample comprising chloroplasts from a plant which has been exposed to continuous light compared to the amount of starch from a comparable sample of a comparable control plant which has not been exposed to continuous light. Alternatively, the increase of chloroplastic starch is determined by comparing the chloroplastic starch content of a plant or part thereof which contains chloroplasts with the chloroplastic starch content of a comparable control plant or chloroplast containing part thereof which has not been exposed to continuous light. The amount of starch of a plant is preferably determined by allowing a sample from said plant or part thereof which contains chloroplasts to gelatinize, incubating said sample with amyloglucosidase and subsequently determining the amount of glucose released. Preferably, said sample comprises isolated chloroplasts from said plant. The chloroplastic starch content is determined by calculating the amount of chloroplastic starch per kg of the plant.
The term “decreased stem development” as used herein refers to a decreased length and/or diameter of a stem of a plant or a part thereof which has been exposed to continuous light compared to the length and/or diameter of a comparable stem of a comparable control plant which has not been exposed to continuous light.
The term “reduced photosynthesis” as used herein refers to a reduced level of activity of photosynthesis in a plant or in a part thereof which has been exposed to continuous light compared to level of activity of photosynthesis in a comparable control plant which has not been exposed to continuous light. The term “photosynthesis” as used herein refers to the process in which the plant converts carbon dioxide into organic compounds using sunlight or artificial light as energy. The level of activity of photosynthesis can be determined by measuring the carbon dioxide uptake or oxygen production. A decrease in the carbon oxide uptake of a plant and/or in the oxygen production in comparison to a control plant indicates a decrease in the activity of photosynthesis.
The term “degreening” as used herein refers to the decrease in the content of chlorophyll in leaf tissue of a plant or in a part thereof which has been exposed to continuous light compared to a comparable control plant which has not been exposed to continuous light. Degreening can be visually observed as light green or whitened leaves. Alternatively, the content of chlorophyll in leaf tissue can be determined by performing a SPAD measurement well known in the art and compare the chlorophyll measurements performed in leaf tissue of said control plant and establish whether the content of chlorophyll is decreased.
The term “poor biomass production” as used herein refers to a lower (e.g. at least 5%, 10%, 15% lower) amount of biomass of a plant which has been exposed to continuous light compared to a comparable control plant which has not been exposed to continuous light. The biomass of a plant can be determined by measuring the fresh and/or dry weight of a plant.
As used herein, the term “allele(s)” means any of one or more alternative forms of a gene, all of which alleles relate to at least one trait or characteristic. In a diploid cell or organism, the two alleles of a given gene occupy corresponding loci on a pair of homologous chromosomes. Since the present invention relates to genes, i.e. genomic regions that may comprise one or more genes, but also regulatory sequences, it is in some instances more accurate to refer to “haplotype” (i.e. an allele of a chromosomal segment) in stead of “allele”, however, in those instances, the term “allele” should be understood to comprise the term “haplotype”.
A “gene” is defined herein as a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristics or trait in an organism.
A “locus” is defined herein as the position that a given gene occupies on a chromosome of a given species.
As used herein, the term “heterozygous” means a genetic condition existing when different alleles reside at corresponding loci on homologous chromosomes.
As used herein, the term “homozygous” means a genetic condition existing when identical alleles reside at corresponding loci on homologous chromosomes.
As used herein, the term “hybrid” means any offspring of a cross between two genetically unlike individuals, including but not limited to the cross between two inbred lines.
As used herein, the term “inbred” means a substantially homozygous individual or line
In this application a “recombination event” is understood to mean a meiotic crossing-over.
As used herein, the terms “introgression”, “introgressed” and “introgressing” refer to both a natural and artificial process whereby genes of one species, variety or cultivar are moved into the genome of another species, variety or cultivar, by crossing those species, varieties or cultivars. The process may optionally be completed by backcrossing to the recurrent parent.
“Genetic engineering”, “transformation” and “genetic modification” are all used herein as synonyms for the transfer of isolated and cloned genes into the DNA, usually the chromosomal DNA or genome, of another organism.
As used herein, the term “plant part” indicates a part of the tomato plant, including single cells and cell tissues such as plant cells that are intact in plants, cell clumps and tissue cultures from which tomato plants can be regenerated. Examples of plant parts include, but are not limited to, single cells and tissues from pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems shoots, and seeds; as well as pollen, ovules, leaves, embryos, roots, root tips, anthers, flowers, fruits, stems, shoots, scions, rootstocks, seeds, protoplasts, calli, and the like.
As used herein, the term “population” means a genetically heterogeneous collection of plants sharing a common genetic derivation.
As used herein, the term “tomato” means any plant, line or population formerly known under the genus name of Lycopersicon, including but not limited to Lycopersicon cerasiforme, Lycopersicon cheesmanii, Lycopersicon chilense, Lycopersicon chmielewskii, Lycopersicon esculentum (now Solanum lycopersicum), Lycopersicon hirsutum, Lycopersicon parviflorum, Lycopersicon pennellii, Lycopersicon peruvianum, Lycopersicon pimpinellifolium, or Solanum lycopersicoides. The newly proposed scientific name for Lycopersicon esculentum is Solanum lycopersicum. Similarly, the names of the wild species has been altered. L. pennellii has become Solanum pennellii, L. hirsutum has become S. habrochaites, L. peruvianum has be split into S. ‘N. peruvianum’ and S. ‘Callejon de Huayles’, S. peruvianum, and S. corneliomuelleri, L. parviflorum has become S. neorickii, L. chmielewskii has become S. chmielewskii, L. chilense has become S. chilense, L. cheesmaniae has become S. cheesmaniae or S. galapagense, and L. pimpinellifolium has become S. pimpinellifolium (Solanacea Genome Network (2005) Spooner and Knapp).
The term “S. lycopersicum”, as used herein, refers to any variety or cultivar of the garden tomato.
It is especially noted that S. habrochaites can be defined as a tomato species that carries hairy fruits, while S. lycopersicum is a tomato species carrying hairless fruits.
As used herein, the term “variety” or “cultivar” means a group of similar plants that by structural or genetic features and/or performance can be distinguished from other varieties within the same species.
The term “gene associated with tolerance to continuous light” as well as the shorter term “gene for continuous light tolerance” refer to a region located on a particular chromosome of tomato that is associated with at least one gene that encodes for continuous light-tolerance or at least a regulatory region, i.e. a region of a chromosome that controls the expression of one or more genes involved in tolerance to continuous light. The phenotypic expression of that gene may for instance be observed as a reduced degree of chlorosis or an improved biomass production upon exposure to continuous light. A gene may for instance comprise one or more genes of which the products confer the genetic continuous light tolerance. Alternatively, a gene may for instance comprise regulatory genes or sequences of which the products influence the expression of genes on other loci in the genome of the plant thereby conferring the tolerance to continuous light. The gene of the present invention may be defined by indicating their genetic location in the genome of the respective wild tomato accession using one or more molecular genomic markers. One or more markers, in turn, indicate a specific locus. Distances between loci are usually measured by frequency of crossing-over between loci on the same chromosome. The farther apart two loci are, the more likely that a crossover will occur between them. Conversely, if two loci are close together, a crossover is less likely to occur between them. As a rule, one centimorgan (cM) is equal to 1% recombination between loci (markers). When a gene can be indicated by multiple markers the genetic distance between the end-point markers is indicative of the size of the gene. It is to be understood that the genes identified herein as residing in the CLT locus (see Tables 2 and 3) may be used as markers for the continuous light resistance trait in aspects of this invention. In particular, polymorphisms in these genes between the tolerant and non-tolerant phenotype are envisioned.
The term “natural genetic background” is used herein to indicate the original genetic background of a gene. Such a background may for instance be the genome of a continuous light-tolerance wild accession of tomato. For instance, the gene of the present invention was found at specific locations on chromosomes 7 of Solanum pennellii 0716. As an example, the Solanum pennellii 0716 represents the natural genetic background of the genes on chromosomes 7 of Solanum pennellii 0716. Conversely, a method that involves the transfer of DNA comprising the gene, or a tolerance-conferring part thereof, from chromosome 7 of Solanum pennellii 0716 to the same position on chromosome 7 of another tomato species, will result in that gene, or said tolerance-conferring part thereof, not being in its natural genetic background.
The terms “determinate” and “indeterminate” as used herein refers to the type of growth habit of tomato plants, which is commonly classified as determinate or indeterminate. This classification depends preferably on the capacity of the shoot system for continued sympodial growth. The terms are used in their art-recognized meaning.
The term “standard practice conditions”, “standard greenhouse conditions” and “standard conditions” refer to the conditions of light, humidity, temperature, etc. where under plants are grown or incubated, for instance for the purpose of phenotypic characterization, as being standard. For greenhouses or climate chambers for instance, this refers to 8-16-h day, 15° C.-25° C. More in general, the terms refer to standard and reference growth conditions with a photoperiod of 8 to 24 h (photosynthetic photon flux (PPF) 25 to 1000 μmol m−2 s−1), preferably a light regime of 16 hours light and 8 hours dark, an air temperature of about 21° C. during the day and 18° C. at night, a water vapour pressure deficit of about 4.4 g m−3 corresponding to a relative humidity (RH) of about 60%-85%, at 400-900 ppm CO2 and atmospheric O2 concentration and at atmospheric air pressure (generally 1008 hPa). Water and nutrients may be given drop wise near the stem, or in the form of spray or mist or by ebb and flow.
With the term “bioassay for measuring the capability of growing in continuous light” as used herein, is meant any bioassay which is suitable for determining differences in said symptoms. Preferably, chlorosis is determined using fluorescence measurements of the chlorophyll concentration using a SPAD® measuring device Marquard and Tipton 1987, HortScience 22: 1327. Standard bioassay experimentation conditions, such as stem diameter and plant height measurements, chlorophyll concentration measurements and assays for determining tolerance to continuous light are further specified in the Examples below.
The term “marker-assisted selection”, as used herein, refers to the diagnostic process of identifying, optionally followed by selecting a plant from a group of plants using the presence of a molecular marker as the diagnostic characteristic or selection criterion. The process usually involves detecting the presence of a certain nucleic acid sequence or polymorphism in the genome of a plant.
The term “marker”, as used herein, refers to an indicator that is used in methods for visualizing differences in characteristics of nucleic acid sequences. Examples of such indicators are restriction fragment length polymorphism (RFLP) markers, amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), microsatellite markers (e.g. SSRs), sequence-characterized amplified region (SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of the markers described herein which defines a specific genetic and chromosomal location.
The term “linked”, as used herein, with reference to markers linked to a trait, refers to a marker the presence of which in the genome of the plant coincides with the presence of the trait. Usually the term refers to a genetic marker that falls within the physical boundaries of a genomic region spanned by at least two markers. For RIL (recombinant inbred lines), such markers having established LOD scores above a certain threshold thereby indicating that no or very little recombination between these markers and the trait locus occurs in crosses; as well as any marker in linkage disequilibrium to the trait locus; as well as markers that represent the actual causal mutations within the trait locus. The term “linked” is used in its broadest sense and indicates that the marker and the gene are located within a continuous DNA sequence of several centiMorgan. The term is used herein with reference to the linkage between markers and phenotype and refers to a distance of preferably less than 20 cM, preferably less than 10 cM, still more preferably less than 6, 5, 4, 3, 2, or 1 cM.
The term “gene”, as used herein, refers to a hereditary unit consisting of a sequence of DNA that occupies a specific location on a chromosome and that contains the genetic instruction for a particular characteristics or trait in an organism. The term “gene” thus refers to a nucleic acid (for example, DNA or RNA) sequence that comprises coding sequences necessary for the production of an RNA, or a polypeptide or its precursor. A functional polypeptide can be encoded by a full length coding sequence or by any portion of the coding sequence as long as the desired activity or functional properties (for example, enzymatic activity, ligand binding, signal transduction, etc.) of the polypeptide are retained. The term “gene” encompasses both cDNA and genomic forms of a gene.
The term ‘yield’ as used herein, refers to the biomass of a tomato plant. Any parameter which is related to the biomass of said plant can be used to determine the yield of a plant. Preferably, said parameter comprises the total leaf area, the shoot dry matter, the shoot fresh mass, the dry mass or the shoot dry mass of said plant.
For optimal growth, fruit vegetables like tomato need 6 hours of darkness per day. This implies that no photosynthesis occurs for 33% per day. If these vegetables could be grown under continuous light, a substantial increase in production would be expected. However, continuous light causes severe problems in these crops: tomato plants, grown in continuous light show a strong increase in chloroplastic starch, leaf chlorosis, necrosis and consequently very poor biomass production; continuous light can even be lethal for the crop.
The present inventors have now discovered tolerant lines in several wild species and in an old cultivar. However, tolerant lines could not be found in any of the modern tomato varieties like Encore, Campari, DRS540, Tourance, Westland, etc.
There are no published reports of tomato varieties exhibiting tolerance to continuous light, save a congress abstract of a miniature tomato cultivar. There is no report on the sensitivity of other tomato species to continuous light, except for the paper of Daskaloff and Ognjanova (Zeitschrift für Pflanzenzuchtung 1965 54: 169-181) where it is claimed that S. habrochaites, S. peruvianum and S. racemigerum (=pimpinellifolium) are tolerant to continuous light. It is remarkable that in this publication the focus is on experiments at low light intensities as it is described that the continuous light symptoms are more profound at these lower light intensities. S. pimpinellifolium was found to be tolerant by Daskaloff and Ognjanova. In contrast, the present inventors have now found that it is just the opposite and that the continuous light symptoms are more profound at higher light intensities, and S. pimpinellifolium was not found to be tolerant.
In the experiments as conducted natural variation in the continuous light symptoms was sought. Several tomato species are clearly tolerant. The symptoms are quantified by measuring the chlorophyll concentration (using e.g., a SPAD® meter). For some symptomless lines biomass production increased linearly with the increase of available light. Moreover, in addition to a linear increase some lines showed even a further increase in biomass production relative to the light available, i.e. higher than was predicted. The wild tomato donors pennellii LA716, S. peruvianum LA1708, S. habrochaites LA1777, G1560, Lyc4, S. chmielewskii LA1840, S. neorickii LA2133, S. chilense LA1959 are tolerant to continuous light the same is true for an old variety “Sub arctic plenty”. Tolerance lines do not show deleterious symptoms.
The IL lines, 4 and 5 S. pennellii*M82, 42 and 43 S. neorickii (LA2133), 5 S. chilense (LA1959) are tolerant to continuous light.
The chromosomal location of the tolerance is chromosome 7 for the S. pennellii, S. neorickii, and S. chilense source.
Producing Plants with Tolerance to Continuous Light
Plant breeders and in particular seed companies employ elite breeding lines, generally referred to as “elite lines” to provide a constant quality product. The elite lines are the result of many years of inbreeding and combine multiple superior characteristics such as high yield, fruit quality, and resistance to pests, disease, or tolerance to abiotic stress. The average yield of these elite lines is generally much higher than the original wild (landrace) accessions from which many of the modern tomato varieties are descendants. The elite lines can be used directly as crop plant, but are typically used to produce so-called F1 or single-cross hybrids, produced by a cross between two (homozygous or inbred) elite lines. The F1 hybrids thus combine the genetic properties of the two parents into a single plant. An add-on benefit of hybrids is that they express hybrid vigour or heterosis, the poorly understood phenomenon that hybrid plants grow better than either (inbred) parent and show higher yields.
Backcross or pedigree selection is one method by which breeders add desirable agronomic traits to their elite breeding lines. The method involves crossing the breeding line with a line that expresses the desirable trait followed by backcrossing offspring plants expressing the trait to the recurrent parent. As a result, the selection of an individual as a parent in a breeding program is based on the performance of its forebears. Such methods are most effective in breeding for qualitatively-inherited traits, i.e traits which are present or absent.
Recurrent selection is an alternative breeding method for improving breeding lines and involves systematic testing and selection of desirable progeny followed by recombination of the selected individuals to form a new population. Recurrent selection has proven effective for improving quantitative traits in crop plants. Recurrent selection, however, decreases the rate of broadening genetic basis underlying the various traits in a breeding program, and its potential is therefore limited.
The present inventors discovered that the yield of a tomato plant may be increased by introgressing into an elite breeding line the trait of tolerance to continuous light.
A first method would comprise introgressing the trait from a tomato plant exhibiting tolerance to continuous light, such as a plant of the wild tomato species S. pennellii, such as S. pennellii LA716, or an offspring plant thereof having said tolerance to continuous light, into a plant of a tomato line of interest. This may for instance be achieved by crossing a plant of a recipient breeding line of S. lycopersicum capable of producing commercially valuable fruits, with a plant of a donor line of a tomato species, having tolerance to continuous light. This will result in a situation wherein the gene for tolerance to continuous light is in the genetic background of the tomato line of interest. The establishment of the proper introgression in offspring plants may be monitored by using specific markers as defined herein.
Recombination is the exchange of information between two homologous chromosomes during meiosis. In a recombinant plant, DNA that is originally present on a specific location within the chromosome is exchanged for DNA from another plant (i.e. maternal for paternal or vice versa). In order to exchange only the required material, and maintain the valuable original information on the chromosome as much as possible, will usually require two crossover events. The normal way to find such a recombinant is to screen a population of F2-plants. This population must be of sufficient size in order to detect the rare (low frequency) double recombinants. The frequency of recombination can be expressed in a genetic distance. For instance, a single recombinant in a 10 cM area can be found with a frequency of 10% (1 centimorgan is defined as 1% recombinant progeny in a testcross).
The present invention also provides methods of producing the plants of the invention using marker assisted selection (MAS). The invention therefore relates to methods of plant breeding and to methods to select plants, in particular tomato plants, particularly cultivated tomato plants as breeder plants for use in breeding programs or cultivated tomato plants for having desired genotypic or potential phenotypic properties, in particular related to producing quantities of valuable tomato fruits, also referred herein to as agronomically desirable plants. Herein, a cultivated plant is defined as a plant being purposely selected or having been derived from a plant having been purposely selected in agricultural or horticultural practice for having desired genotypic or potential phenotypic properties, in particular a plant obtained by inbreeding.
Since the gene can only be properly identified phenotypically when the plant remains healthy when subjected to continuous light, it is of particular advantage that the establishment of the proper introgression in offspring plants may be monitored by using the gene-specific markers as provided herein, either in cis or in trans coupling as explained below. By using marker assisted selection (MAS) or marker assisted breeding (MAB) methods, the skilled person is therefore provided with methods for selecting plants carrying the desired genotype loci and discarding plants lacking the potential of producing progeny tolerant to continuous light.
The present invention thus also provides methods for selecting a tomato plant exhibiting tolerance to continuous light, comprising detecting in said plant the presence of the gene for tolerance to continuous light as defined herein. In a preferred method of the invention for selecting such a plant the method comprises:
a) providing a sample of genomic DNA from a tomato plant;
b) detecting in said sample of genomic DNA at least one molecular marker linked to the gene for tolerance to continuous light.
The step of providing a sample of genomic DNA from a tomato plant may be performed by standard DNA isolation methods well known in the art.
The step of detecting a molecular marker (step b) may, in a preferred embodiment, comprise the use of CAPS markers, which constitute a set of bi-directional primers in combination with a restriction enzyme. This allows for the detection of specific SNPs linked to the trait. Bi-directional means that the orientation of the primers is such that one functions as the forward and one as the reverse primer in an amplification reaction of nucleic acid.
Alternatively, the step of detecting a molecular marker (step b) may in another preferred embodiment, comprise the use of a nucleic acid probe having a base sequence which is substantially complementary to the nucleic acid sequence defining said molecular marker (e.g. said SNP) and which nucleic acid probe specifically hybridizes under stringent conditions with a nucleic acid sequence defining said molecular marker. A suitable nucleic acid probe may for instance be a single strand oligonucleotide of the amplification product corresponding to the marker.
The step of detecting a molecular marker (step b) may also comprise the performance of a unique nucleic acid amplification reaction on said genomic DNA to detect said gene. This can suitably be done by performing a PCR reaction using a pair of marker-specific primers based on the internal or adjacent (up to 500 kilo base) sequence. In a preferred embodiment, said step b) comprises the use of at least one pair of primers defining a marker for said gene (e.g. being complementary to said marker or hybridizing specifically to said marker or allowing polymerase chain extension to occur when bound to said marker), or a pair of primers which specifically hybridize under stringent conditions with the nucleic acid sequence of a marker for said gene.
The step of detecting an amplified DNA fragment having a certain predicted length or a certain predicted nucleic acid sequence may be performed such that the amplified DNA fragment has a length that corresponds (plus or minus a few bases, e.g. a length of one, two or three bases more or less) to the expected length as based on the nucleotide sequence of the genes and markers identified herein. The skilled person is aware that markers that are absent in plants having the introgression as defined herein (donor plants), while they are present in the plants receiving the introgression (recipient plants) (so-called trans-markers), may also be useful in assays for detecting the introgression among offspring plants, although detecting the presence of a specific introgression is not optimally demonstrated by the absence of a marker.
The step of detecting an amplified DNA fragment having the predicted length or the predicted nucleic acid sequence may be performed by standard gelelectrophoresis techniques, real time PCR, or by using DNA sequencers. The methods need not be described here as they are well known to the skilled person. It should be noted that a marker is usually defined based on its nucleotide sequences in combination with its position relative to other markers on a linkage map.
Molecular Markers and Genes
Molecular markers are used for the visualisation of differences in nucleic acid sequences. This visualisation is possible due to DNA-DNA hybridisation techniques after digestion with a restriction enzyme (RFLP) and/or due to techniques using the polymerase chain reaction (e.g. STS, microsatellites, AFLP). All differences between two parental genotypes will segregate in a mapping population (e.g., BC1, F2) based on the cross of these parental genotypes. The segregation of the different markers may be compared and recombination frequencies can be calculated. The recombination frequencies of molecular markers on different chromosomes is generally 50%. Between molecular markers located on the same chromosome the recombination frequency depends on the distance between the markers. A low recombination frequency corresponds to a short genetic distance between markers on a chromosome. Comparing all recombination frequencies will result in the most logical order of the molecular markers on the chromosomes. This most logical order can be depicted in a linkage map. A group of adjacent or contiguous markers on the linkage map that is associated with tolerance to continuous light, pinpoints the position of a gene associated with tolerance to continuous light.
The markers identified herein may be used in various aspects of the invention as will now be illustrated. Aspects of the invention are not limited to the use of the markers identified herein. It is stressed that the aspects may also make use of markers not explicitly disclosed herein or even yet to be identified.
In the present invention amplified fragment length polymorphism (AFLP) markers, single nucleotide polymorphisms (SNPs), and insertion deletions (INDELs), microsatellite markers, restriction fragment length polymorphism (RFLP) markers, sequence-characterized amplified region (SCAR) markers, cleaved amplified polymorphic sequence (CAPS) markers or isozyme markers or combinations of these markers might be used.
In general, a gene may span a region of several hundreds to thousands of bases. The plants that have the genetic potential for exhibiting a particular phenotypic trait (tolerance to continuous light) may be traced amongst a population of offspring plants through the observed correlation between the presence of a (string of contiguous) genomic marker(s) and the presence of the phenotypic trait. By providing a non-limiting list of markers, the present invention thus provides for the effective utility of the gene in a breeding program.
It is further important to note that the contiguous genomic markers can also be used to indicate the presence of the gene (and thus of the phenotype) in an individual plant, i.e. they can be used in marker assisted selection (MAS) procedures. In principle, the number of potentially useful markers is limited but may be very large, and the skilled person may easily identify additional markers to those mentioned in the present application. Any marker that is linked to the gene, e.g. falling within the physical boundaries of the genomic region spanned by the markers, wherein between said markers and the gene no or very little recombination occurs in crosses; as well as any marker in linkage disequilibrium to the gene; as well as markers that represent the actual causal mutations within the gene, may be used in MAS procedures.
This means that the markers identified herein, are mere examples of markers suitable for use in MAS procedures. Moreover, when the gene, or the specific trait-conferring part thereof, is introgressed into another genetic background (i.e. into the genome of another plant line), then some markers may no longer be found in the offspring although the trait is present therein, indicating that such markers are outside the genomic region that represents the specific trait-conferring part of the gene in the original parent line only and that the new genetic background has a different genomic organisation. Such markers of which the absence indicates the successful introduction of the genetic element in the offspring are called “trans markers” and may be equally suitable in MAS procedures under the present invention.
Upon the identification of the gene, the gene effect (tolerance to continuous light) is confirmed by determining the tolerance to continuous light of progenies respectively recombinant or segregating for the genes under investigation. Preferably, detecting the presence of a gene of the invention is performed with at least one of the markers for a gene as defined herein. The present invention therefore also relates to a method for detecting the presence of a gene for tolerance to continuous light as defined herein in tomato by the use of the said markers.
The nucleotide sequence of the genes of the present invention may be resolved by determining the nucleotide sequence of one or more markers associated with said gene and designing internal primers for said marker sequences that may then be used to further determine the sequence the gene adjacent to said marker sequences. For instance the nucleotide sequence of CAPS markers may be obtained by isolating said markers from the electrophoresis gel used in the determination of the presence of said markers in the genome of a subject plant, and determining the nucleotide sequence of said markers by for instance Sanger or pyro sequencing methods, well known in the art.
In embodiments of methods for detecting the presence of a gene in a tomato plant, the method may also comprise the steps of providing an oligonucleotide or polynucleotide capable of hybridizing under stringent hybridization conditions to a nucleic acid sequence of a marker linked to said gene, contacting said oligonucleotide or polynucleotide with nucleic acid of a tomato plant, and determining the presence of specific hybridization of said oligonucleotide or polynucleotide to said nucleic acid.
Preferably said method is performed on a nucleic acid sample obtained (isolated) from said tomato plant, although in situ hybridization methods may also be employed. Alternatively, and in a more preferred embodiment, the skilled person may, once the nucleotide sequence of the gene has been determined, design specific hybridization probes or oligonucleotides capable of hybridizing under stringent hybridization conditions to the nucleic acid sequence of said gene and may use such hybridization probes in methods for detecting the presence of a gene of the invention in a tomato plant.
Production of Tomato Plants Exhibiting Tolerance to Continuous Light by Transgenic Methods
According to another aspect of the present invention, a nucleic acid (preferably DNA) sequence comprising one or more of the genes as defined herein may be used for the production of a tomato plant exhibiting tolerance to continuous light. In this aspect, the invention provides for the use of genes as defined herein or tolerance to continuous light-conferring parts thereof, for producing a tomato plant tolerant to continuous light as defined herein, which use involves the introduction of a nucleic acid sequence comprising said gene in a suitable recipient plant. As stated, said nucleic acid sequence may be derived from a suitable donor plant. A suitable source according to the present invention for the gene for tolerance to continuous light is tomato line S. pennellii LA716 (PI 246502 available from the Agricultural Research Service (ARS-GRIN) of the US Department of Agriculture, Washington D.C., USA), S. neorickii, S habrochaites, S. lycopersicum, S peruvianum or S. chemielewskii. 
Preferably, said gene is selected from the genes present listed in Table 2. The gene sequences thereof are provided in for Table 3.
Preferably said gene is SL1.00sc07408_313.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_312.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_311.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_310.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_309.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_308.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_307.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_306.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_305.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_304.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_303.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_302.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_301.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_300.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_299.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_298.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_297.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_296.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_295.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_294.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_293.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_292.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_291.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_290.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_289.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_288.1.1.
In an alternative or additional preferred embodiment, said gene is SL1.00sc07408_287.1.1
In a preferred embodiment, said gene is a gene located between markers 7-20-1 and 7-20-2 as listed in Table 2. Most preferably, said gene is SL1.00sc07408_301.1.1.
The nucleic acid sequence that comprises a gene for tolerance to continuous light, or a tolerance to continuous light-conferring part thereof, may be transferred to a suitable recipient plant by any method available. For instance, the said nucleic acid sequence may be transferred by crossing a plant of line PI 246502 with a selected breeding line which is not tolerant to continuous light or of which the tolerance is to be improved, i.e. by introgression, by transformation, by protoplast fusion, by a doubled haploid technique or by embryo rescue or by any other nucleic acid transfer system, optionally followed by selection of offspring plants comprising the gene for tolerance to continuous light (as assessed by markers) and/or exhibiting tolerance to continuous light. For transgenic methods of transfer a nucleic acid sequence comprising a gene for tolerance to continuous light may be isolated from said donor plant by using methods known in the art and the thus isolated nucleic acid sequence may be transferred to the recipient plant by transgenic methods for plant transformation, for instance by means of a vector, in a gamete, or in any other suitable transfer element, such as a bombardment with a particle coated with said nucleic acid sequence.
Plant transformation generally involves the construction of a vector with an expression cassette that will function in plant cells. In the present invention, such a vector consists of a nucleic acid sequence that comprises a gene for tolerance to continuous light, which vector may comprise such a gene that is under control of or operatively linked to a regulatory element, such as a promoter. The expression vector may contain one or more such operably linked gene/regulatory element combinations, provided that at least one of the genes contained in the combinations confers tolerance to continuous light. The vector(s) may be in the form of a plasmid, and can be used, alone or in combination with other plasmids, to provide transgenic plants that exhibit tolerance to continuous light, using transformation methods known in the art, such as the Agrobacterium transformation system.
Expression vectors can include at least one marker gene, operably linked to a regulatory element (such as a promoter) that allows transformed cells containing the marker to be either recovered by negative selection (by inhibiting the growth of cells that do not contain the selectable marker gene), or by positive selection (by screening for the product encoded by the marker gene). Many commonly used selectable marker genes for plant transformation are known in the art, and include, for example, genes that code for enzymes that metabolically detoxify a selective chemical agent which may be an antibiotic or a herbicide, or genes that encode an altered target which is insensitive to the inhibitor. Several positive selection methods are known in the art, such as mannose selection. Alternatively, marker-less transformation can be used to obtain plants without mentioned marker genes, the techniques for which are known in the art.
One method for introducing an expression vector into a plant is based on the natural transformation system of Agrobacterium (See e.g. Horsch et al., 1985). A. tumefaciens and A. rhizogenes are plant pathogenic soil bacteria that genetically transform plant cells. The Ti and Ri plasmids of A. tumefaciens and A. rhizogenes, respectively, carry genes responsible for genetic transformation of the plant. Methods of introducing expression vectors into plant tissue include the direct infection or co-cultivation of plant cells with Agrobacterium tumefaciens. Descriptions of Agrobacterium vectors systems and methods for Agrobacterium-mediated gene transfer are provided in U.S. Pat. No. 5,591,616. General descriptions of plant expression vectors and reporter genes and transformation protocols and descriptions of Agrobacterium vector systems and methods for Agrobacterium-mediated gene transfer can be found in Gruber and Crosby, 1993. General methods of culturing plant tissues are provided for example by Miki et al., 1993 and by Phillips, et al., 1988. A proper reference handbook for molecular cloning techniques and suitable expression vectors is Sambrook and Russell, 2001.
Another method for introducing an expression vector into a plant is based on microprojectile-mediated transformation (particle bombardment) wherein DNA is carried on the surface of microprojectiles. The expression vector is introduced into plant tissues with a biolistic device that accelerates the microprojectiles to speeds of 300 to 600 m/s which is sufficient to penetrate plant cell walls and membranes. Another method for introducing DNA to plants is via the sonication of target cells. Alternatively, liposome or spheroplast fusion has been used to introduce expression vectors into plants. Direct uptake of DNA into protoplasts using CaCl2 precipitation, polyvinyl alcohol or poly-L-ornithine has also been reported. Electroporation of protoplasts and whole cells and tissues has also been described.
Other well known techniques such as the use of BACs, wherein parts of the tomato genome are introduced into bacterial artificial chromosomes (BACs), i.e. vectors used to clone DNA fragments (100- to 300-kb insert size; average, 150 kb) in Escherichia coli cells, based on naturally occurring F-factor plasmid found in the bacterium E. coli may for instance be employed in combination with the BIBAC system to produce transgenic plants.
Following transformation of tomato target tissues, expression of the above described selectable marker genes allows for preferential selection of transformed cells, tissues and/or plants, using regeneration and selection methods now well known in the art.
Production of Tomato Plants Exhibiting Tolerance to Continuous Light by Non-Transgenic Methods
In an alternative embodiment for producing a tomato plant exhibiting tolerance to continuous light, protoplast fusion can be used for the transfer of nucleic acids from a donor plant to a recipient plant. Protoplast fusion is an induced or spontaneous union, such as a somatic hybridization, between two or more protoplasts (cells of which the cell walls are removed by enzymatic treatment) to produce a single bi- or multi-nucleate cell. The fused cell, that may even be obtained with plant species that cannot be interbreeded in nature, is tissue cultured into a hybrid plant exhibiting the desirable combination of traits. More specifically, a first protoplast can be obtained from a tomato plant of accession PI 246502. A second protoplast can be obtained from a second tomato plant variety, preferably a tomato line that comprises commercially valuable characteristics, such as, but not limited to disease resistance, insect resistance, valuable fruit characteristics, etc. The protoplasts are then fused using traditional protoplast fusion procedures, which are known in the art.
Alternatively, embryo rescue may be employed in the transfer of a nucleic acid comprising the gene as described herein from a donor plant to a recipient plant. Embryo rescue can be used as a procedure to isolate embryo's from crosses wherein plants fail to produce viable seed. In this process, the fertilized ovary or immature seed of a plant is tissue cultured to create new plants.
The present invention also relates to a method for improving the tolerance to continuous light of a plant of a tomato breeding line, comprising the steps of:
a) crossing a plant of a tomato breeding line with a plant of tomato line PI 246502 or an offspring plant thereof harbouring the gene for tolerance to continuous light as described herein;
b) selecting a progeny tomato plant resulting from said crossing having an introgression from tomato accession PI 246502 or an offspring plant thereof associated with tolerance to continuous light;
c) selfing and/or backcrossing said progeny tomato plant selected in step (b) using said tomato breeding line as a recurrent parent;
d) selecting a progeny tomato plant resulting from the selfing or backcrossing in step (c) having an introgression from tomato accession PI 246502 or an offspring plant thereof associated with tolerance to continuous light,
e) repeating said steps of selfing and/or backcrossing and selection of steps (c) and (d) to provide a plant of a tomato breeding line essentially homozygous for said introgression,
wherein preferably at least one selection as performed in steps (b) or (d) is performed by marker-assisted selection.
In a preferred embodiment of such a method, said tomato breeding line is an elite line.
In an alternative preferred embodiment of the above method, the marker-assisted selection procedure comprises the selection for at least one marker as exemplified in the Examples below.
The introgression of the nucleic acid sequence comprising the gene for tolerance to continuous light as described herein may suitably be accomplished by using traditional breeding techniques. The gene is preferably introgressed into commercial tomato varieties by using marker-assisted selection (MAS) or marker-assisted breeding (MAB). MAS and MAB involves the use of one or more of the molecular markers for the identification and selection of those offspring plants that contain one or more of the genes that encode for the desired trait. In the present instance, such identification and selection is based on selection of the gene of the present invention or markers associated therewith. MAS can also be used to develop near-isogenic lines (NIL) harboring the gene of interest, or the generation of gene isogenic recombinants (QIRs), allowing a more detailed study of each gene effect and is also an effective method for development of backcross inbred line (BIL) populations. Tomato plants developed according to this embodiment can advantageously derive a majority of their traits from the recipient plant, and derive tolerance to continuous light from the donor plant.
Crossing can be achieved by mechanically pollinating the female flower of one parent plant with pollen obtained from male flowers of another parent plant.
As discussed briefly above, traditional breeding techniques can be used to introgress a nucleic acid sequence encoding a gene for tolerance to continuous light into a recipient tomato plant requiring tolerance to continuous light. In one method, which is referred to as pedigree breeding, a donor tomato plant that exhibits tolerance to continuous light and comprising a nucleic acid sequence encoding for the gene associated with tolerance to continuous light as defined herein is crossed with a recipient tomato plant (preferably a plant of an elite line) that exhibits agronomically desirable characteristics, such as, but not limited to, disease (e.g. TMV) resistance, insect resistance, valuable fruit characteristics, etc., but which is not tolerance to continuous light, or which requires improvement of such tolerance. The resulting plant population (representing the F1 hybrids) is then self-pollinated and set seeds (F2 seeds). The F2 plants grown from the F2 seeds are then screened for tolerance to continuous light. The population can be screened in a number of different ways.
First, the population can be screened using a bioassay wherein the plant is grown under continuous light for a prolonged period of time and its health or productivity (phenotype) is inspected. Second, marker-assisted selection can be performed using one or more of the hereinbefore-described molecular markers to identify those progeny that comprise a nucleic acid sequence encoding a gene for tolerance to continuous light as defined herein. Other methods, described above by methods for detecting the presence of a gene may be used. Also, marker-assisted selection can be used to confirm the results obtained from the phenotype scores, and therefore, several methods may also be used in combination.
Inbred tomato plant lines exhibiting tolerance to continuous light can be developed using the techniques of recurrent selection and backcrossing, selfing and/or dihaploids or any other technique used to make parental lines. In a method of recurrent selection and backcrossing, the tolerance to continuous light-conferring genetic element as disclosed herein can be introgressed into a target recipient plant (the recurrent parent) by crossing the recurrent parent with a first donor plant, which differs from the recurrent parent and is referred to herein as the “non-recurrent parent”. The recurrent parent is a plant of which the tolerance is to be improved and possesses agronomically desirable characteristics, such as, but not limited to disease resistance, insect resistance, valuable fruit characteristics, etc. The non-recurrent, or donor, parent may suitably be a plant of suitable donor line as described herein which comprises a nucleic acid sequence that encodes for a gene that confers tolerance to continuous light. Alternatively, the donor parent can be any plant variety or inbred line that is cross-fertile with the recurrent parent and has acquired the gene for tolerance to continuous light in an earlier cross with a plant of the said donor line. The progeny resulting from a cross between the recurrent parent and non-recurrent parent is backcrossed to the recurrent parent. The resulting plant population is then screened for the desired characteristics, which screening may occur in a number of different ways. For instance, the population can be screened using phenotypic screens as described herein. As an alternative to phenotypic assays, marker-assisted selection (MAS) can be performed using one or more of the hereinbefore described molecular markers, hybridization probes or polynucleotides to identify progeny that comprise a nucleic acid sequence encoding the gene responsible for tolerance to continuous light.
Following screening, the F1 hybrid plants that exhibit a tolerant phenotype or, more preferably, genotype and thus comprise the requisite nucleic acid sequence encoding for a gene conferring tolerance to continuous light are then selected and backcrossed to the recurrent parent for a number of generations in order to allow for the tomato plant to become increasingly elite. This process can be performed for two to five or more, such as 6, 7 or 8 generations. In principle the progeny resulting from the process of crossing the recurrent parent with the non-recurrent parent are heterozygous for one or more genes that encode for tolerance to continuous light.
In a preferred embodiment, a method of introducing a desired trait into a hybrid tomato variety comprises the steps of:
(a) crossing an inbred tomato parent with another tomato plant that comprises one or more desired traits, to produce F1 progeny plants, wherein the desired trait is tolerance to continuous light as conferred by the gene from any suitable donor plant of the said trait as defined herein, or an offspring plant thereof;
(b) selecting said F1 progeny plants that have the desired trait to produce selected F1 progeny plants, preferably using molecular markers as defined herein;
(c) backcrossing the selected progeny plants with said inbred tomato parent plant to produce backcross progeny plants;
(d) selecting for backcross progeny plants that have the desired trait (tolerance to continuous light) and morphological and physiological characteristics of said inbred tomato parent plant, wherein said selection preferably comprises the isolation of genomic DNA and testing said DNA for the presence of at least one molecular marker for the gene as defined above;
(e) repeating steps (c) and (d) two or more times in succession to produce selected third or higher backcross progeny plants;
(f) optionally selfing selected backcross progeny in order to identify homozygous plants;
(g) crossing at least one of said backcross progeny and/or selfed plants with another inbred tomato parent plant to generate a hybrid tomato variety with the desired trait and all of the morphological and physiological characteristics of hybrid tomato variety when grown in the same environmental conditions.
As indicated, the last backcross generation may be selfed in order to provide for homozygous pure breeding (inbred) progeny exhibiting tolerance to continuous light. Thus, the result of recurrent selection, backcrossing and selfing is the generation of lines that are genetically homozygous for the genes associated with tolerance to continuous light as well as other genes associated with traits of commercial interest.
It should be noted that heterozygous plants having the gene for tolerance to continuous light may also be of interest as intermediate products, and such plants are therefore also an aspect of the present invention.
Tomato Plants and Seeds
The goal of plant breeding is to combine various desirable traits in a single variety or hybrid. For commercial crops, these traits may include resistance to diseases and insects, tolerance to heat and drought, reducing the time to crop maturity, greater yield, and better agronomic quality. Uniformity of plant characteristics such as germination, growth rate, maturity, and plant height may also be of importance.
Commercial crops are bred through techniques that take advantage of the plant's method of pollination. A plant is self-pollinated if pollen from one flower is transferred to the same or another flower of the same plant. A plant is sibling mated when individuals within the same family or line are used for pollination. A plant is cross-pollinated if the pollen comes from a flower on a different plant from a different family or line.
Plants that have been self-pollinated and selected for type for many generations become homozygous at almost all gene loci and produce a uniform population of true-bred progeny. A cross between two different homozygous lines produces a uniform population of hybrid plants that may be heterozygous for many gene loci. A cross of two plants each heterozygous at a number of gene loci will produce a population of heterogeneous plants that differ genetically and will not be uniform.
The development of a hybrid tomato variety in a tomato plant breeding program involves three steps: (1) the selection of plants from various germplasm pools for initial breeding crosses; (2) the selfing of the selected plants from the breeding crosses for several generations to produce a series of inbred lines, which, individually breed true and are highly uniform; and (3) crossing a selected inbred line with an unrelated inbred line to produce the hybrid progeny (F1). After a sufficient amount of inbreeding successive filial generations will merely serve to increase seed of the developed inbred. Preferably, an inbred line should comprise homozygous alleles at about 80% or more of its loci.
An important consequence of the homozygosity and homogeneity of the inbred lines is that the hybrid created by crossing a defined pair of inbreds will always be the same. Once the inbreds that create a superior hybrid have been identified, a continual supply of the hybrid seed can be produced using these inbred parents and the hybrid tomato plants can then be generated from this hybrid seed supply.
Using the methods as described above, the skilled person will be able to produce the required inbred lines and from those produce the commercial (F1) hybrid seeds by crossing said inbred lines.
The present invention will now be explained in more detail by way of the following non-limiting Examples.
EXAMPLES
Example 1 Tolerant Donors
Different accessions of wild species (lines) were sown in a growth chamber with 16 hours of light per day. Two weeks after sowing, the seedlings of each line were randomly divided in two groups, one group continued to grow in 16 hours of light per day while the other was grown in another growth chamber under continuous light. After 3-4 weeks symptoms of the sensitive lines were clearly visible, while absent in the tolerant lines (FIG. 1). The typical symptoms of a sensitive line are leaf chlorosis, necrosis and poor biomass production which was absent in the tolerant genotypes shown in FIG. 1.
Example 2 Quantifying the Symptoms and Optimise Protocol
In the same experiment as experiment 1 the sensitive lines showed at first light green leaves with inter-vine chlorosis, followed by severe chlorosis (FIG. 2). In severe non tolerant plants chlorosis is followed by necrosis, decreased leaf development decreased stem development and even plant mortally.
Besides the clear visible symptoms of the sensitive line in continuous light the symptoms can also be quantified by measuring the chlorophyll concentration. The chlorophyll concentration can be measured chemically but also spectroscopically with a SPAD meter (SPAD-502, Konica Minolta, Nieuwegein, Netherlands). Two sensitive lines (Moneyberg and Westland) were grown as described in experiment 1, except that also the light intensity was a experimental factor from two weeks after sowing. The plants were placed in low light intensities (16 μmol m−2 s−1=LL) moderate light 131 μmol m−2 s−1 (ML) or relative high light 230 μmol m−2 s−1 (HL). Quantification of a part of the visible symptoms (degreening and chlorosis) by measuring the chlorophyll concentration by a SPAD meter was performed with plants grown at 131 μmol m−2 s−1 (ML) or 230 μmol m−2 s−1 (HL). The resolution to detect differences between continuous and non-continuous light was very difficult or impossible at very low light intensities (16 μmol m−2 s−1=LL) (FIG. 3). The visible symptoms developed earlier in time at 230 μmol m−2 s−1 compared to 131 μmol m−2 s−1. In the same experiment also the stem diameter and plant height was decreased in these non tolerant lines (FIG. 4). Not only a higher light intensity resulted in a faster induction of symptoms also a prolonged exposure to continuous light did increase the symptoms in sensitive lines (FIG. 7, 9). In several lines the symptoms did appear later than after 3 to 4 weeks in continuous light.
In conclusion the continuous light symptoms can be quantified by measuring the chlorophyll concentration in the leaves but also by measuring the stem diameter or height. The quantitative tolerance trait can be measured qualitative by increasing the light intensity or increasing the duration of the continuous light. This makes it easier to map the tolerance.
Example 3 Tolerant Lines from Neorickii (LA2133)
A segregating population between neorickii and a sensitive esculentum (e.g. Moneyberg) showed large natural variation in the chlorophyll concentration as measured with a SPAD meter (FIG. 5). In FIG. 6 two tolerant lines (42 and 43) are shown, which have been in continuous light for 90 days.
Example 4 Tolerant Lines from Chilense (LA1959)
A segregating population between chilense and a sensitive esculentum (e.g. Moneyberg GT) showed large natural variation in the chlorophyll concentration as measured with a SPAD meter (FIG. 7). In FIG. 8 one tolerant line (5) is shown, which have been in continuous light for 60 days.
Example 5 Tolerant Lines from Pennellii (LA716*M82)
A segregating population between pennellii and M82 was used and only lines with a chromosomal introgression from pennellii into M82 on chromosome 7 were used. Natural variation in the chlorophyll concentration as measured with a SPAD meter was identified (FIG. 9). Two tolerant lines (4 and 5) are found, which have been in continuous light for 53 days.
Example 6 Continuous Light Tolerance Loci on Chromosome 7
The genetic information from the 6 different mapping population it is clear that the loci for continuous light tolerance is located on chromosome 7 (FIG. 10). Moreover, by combining the information of all populations it is shown that the loci is located between marker “7-19” and “7-23” that is between 64 and 99.6 cM. The sequence in formation from these 2 markers and 23 additional ones which are also linked to the trait are shown in FIG. 11.
Example 7 Detection of Marker 7-20-1
To detect the G/T polymorphism of marker 7-20-1, a TaqMan assay (Applied biosystems) was used. The assay employs two universal primers (a forward primer having the sequence 5′-GAATTGGCTTTATGTATTTGGAATCCTTGT-3′ (SEQ ID NO:29) and a reverse primer having the sequence 5′-CCCATCTAGGACCCCTGCAT-3′ (SEQ ID NO:30)) to amplify a fragment of 76 bp and two allele specific probes. A probe having the sequence (5′-TTTACTGTATTTTTCTTTTTCTCCA-3′ (SEQ ID NO:31) (S. lycopersicum (recurrent)) specifically detects the S. lycopersicum allele (The “T” SNP) and carries a VIC reporter fluorescent dye. A second probe having the sequence 5′-TTTACTGTATTTTTCTTTTGCTCCA-3′ (SEQ ID NO:32) (S. neorickii LA2133 (donor)) detects the allele from S. neorickii LA 2133 (the “G” SNP) and carries a 6-carboxyfluorescein (FAM) dye. Amplification of one of the two or both alleles can be monitored using a real-time PCR (polymerase chain reaction) machine which measures the level of fluorescence of the respective dyes. DNA samples that only contain the S. lycopersicum allele will emit a VIC signal while samples that contain the S. neorickii allele will emit the FAM signal. Samples that contain both alleles will emit both signals.
The assay was validated on DNA samples of elite sensitive lines and CLT donor LA2133. All other CLT donor lines had the same allele. The assay was also validated on the 46 introgression lines that were derived from LA2133 and “Moneyberg”. The marker showed presence of the “G” allele in LA2133 and introgression lines LA2133-42 and LA2133-43. All other tested lines showed presence of the “T” allele
Example 8 Fine Mapping of Continuous Light Tolerance Trait
To identify closer linked markers for the continuous light tolerance trait fine mapping in the LA2133 background was performed. Lines LA2133-42 and LA2133-43 were backcrossed with their sensitive recurrent parent. Both F1s were selfed and the F2 offspring was genotyped for recombination with flanking markers 7-10 and 7-25. In total 770 and 616 plants were genotyped for LA2133-42 and LA2133-43 respectively. In total 40 lines were selected to generate a F3 from which 15 lines were controls. 22 Seeds per F3 family were sown in 2 blocks of 11 seeds. A total of 18 F3 families were screened. From several F3 families the germination was too low to yield sufficient (>=13) plants for phenotyping the continuous light tolerance trait.
Mapping indicated that the position of the continuous light locus is located between markers 7-17 and 7-20 (FIG. 12). Marker 7-19 was monomorphic in the tested LA2133 populations.
New markers were developed between marker 7-17 and 7-20 and are named 7-20-1, 7-20-2 and 7-20-3 (FIG. 11). Using the new markers on the same phenotyped populations revealed that the position of the continuous light locus is located between markers 7-20-1 and 7-20-2 (FIG. 13).
The three newly developed markers were tested on DNA samples of complete F2 families to generate a new linkage map of the CLT locus (continuous light trait locus). The linkage map EXPEN2000 and the generated linkage map of LA2133-42 showed some differences on the location of the new SNP markers (See Table 1 and FIG. 10). The LA2133-42 map shows the preferred order.











TABLE 1







Marker
LA2133-42
EXPEN2000









7-20-1
43.68 cM
75 cM



7-20-2
44.35 cM
72 cM



7-20-3
44.46 cM
73 cM



7-20
46.18 cM
73 cM



7-21
46.74 cM
78 cM










Based on the mapping in LA2133-42 highly preferred markers linked to the continuous light trait, span a locus of about 0.67 cM.

Example 9 Genes at the CLT Locus
A BLAST® search to the tomato genome (version 1.0; released December 2009) revealed that the markers of the CLT locus arc preferably located on scaffold 07408 (SL1.00sc07408 position:1975288 through 2058583). The region between the markers 7-20-1 and 7-20-2 spans 83296 bp and contains 15 genes (ITAG1 annotation). Table 2 lists all genes at this locus and the available TaqMan markers. Table 3 lists all sequences of these genes. One of the genes (SL1.00sc07408_301.1.1 is the LHCB3 Light-Harvesting Chlorophyll B-Binding Protein 3 structural molecule”. This gene encodes a protein that—in one preferred embodiment of the invention—plays a crucial role in the continuous light trait. Plants of the present invention therefore in a preferred embodiment of the invention comprise this gene. The other genes in this locus can also be used in aspects of this invention.







TABLE 2







Genes in the CLT locus













Name
Type
Description
Size
Start
Stop
Strand
















7-18


100
2878606
2878726
forward


SL1.00sc07408_313.1.1
transcript_ITAG
late embryogenesis
573
2093160
2093732
forward




abundant protein-related/




LEA protein-related (AHRD




V1 ***NG AT5G54370.1);




contains Interpro domain(s)




IPR009646 Root cap


SL1.00sc07408_312.1.1
transcript_ITAG
LDL2 LSD1-LIKE2 amine
6585
2093055
2086471
reverse




oxidase/electron carrier/




oxidoreductase (AHRD V1




**-NG AT3G13682.1);




contains Interpro domain(s)




IPR002937 Amine oxidase


SL1.00sc07408_311.1.1
transcript_ITAG
Unknown Protein (AHRD
7561
2082437
2074877
reverse




V1); contains Interpro




domain(s) IPR009651




Aluminium resistance


SL1.00sc07408_310.1.1
transcript_ITAG
Transmembrane protein
3624
2073719
2070096
reverse




184C (AHRD V1 ***NG




Q5RET6); contains Interpro




domain(s) IPR005178




Protein of unknown




function DUF300


SL1.00sc07408_309.1.1
transcript_ITAG
Unknown Protein (AHRD
4836
2069193
2064358
reverse




V1)


SL1.00sc07408_308.1.1
transcript_ITAG
Unknown Protein (AHRD
2919
2061327
2064245
forward




V1)


SL1.00sc07408_307.1.1
transcript_ITAG
AGD5 ARF-GAP domain 5
6889
2060607
2053719
reverse




ARF GTPase activator/




DNA binding/zinc ion




binding (AHRD V1 *-*G




AT5G54310.1); contains




Interpro domain(s)




IPR001164 Arf GTPase




activating protein


7-20-1

Flanking marker CLT
100
2057927
2058583
reverse


SL1.00sc07408_306.1.1
transcript_ITAG
Unknown Protein (AHRD
1950
2050327
2048378
reverse




V1); contains Interpro




domain(s) IPR008480




Protein of unknown




function DUF761, plant


SL1.00sc07408_305.1.1
transcript_ITAG
cytochrome c biogenesis
6176
2040973
2047148
forward




protein family (AHRD V1 *-




*NG AT5G54290.1);




contains Interpro domain(s)




IPR003834 Cytochrome c




assembly protein,




transmembrane region


SL1.00sc07408_304.1.1
transcript_ITAG
Unknown Protein (AHRD
2874
2034960
2037833
forward




V1)


SL1.00sc07408_303.1.1
transcript_ITAG
Myosin-Va (AHRD V1
8186
2030449
2022264
reverse




***NG Q02440); contains




Interpro domain(s)




IPR001609 Myosin head,




motor region


SL1.00sc07408_302.1.1
transcript_ITAG
ATM2 ARABIDOPSIS
1832
2022005
2020174
reverse




THALIANA MYOSIN 2




motor (AHRD V1 ***NG




AT5G54280.2); contains




Interpro domain(s)




IPR000048 IQ calmodulin-




binding region


SL1.00sc07408_301.1.1
transcript_ITAG
LHCB3 LIGHT-
1633
2018017
2019649
forward




HARVESTING




CHLOROPHYLL B-




BINDING PROTEIN 3




structural molecule (AHRD




V1 ***G AT5G54270.1);




contains Interpro domain(s)




IPR001344 Chlorophyll A-B




binding protein


SL1.00sc07408_300.1.1
transcript_ITAG
Dynein light chain 1
1195
2015289
2016483
forward




cytoplasmic-like protein




(AHRD V1 *-*NG




B0X7V2_CULQU); contains




Interpro domain(s)




IPR001372 Dynein light




chain, type 1 and 2


SL1.00sc07408_299.1.1
transcript_ITAG
Unknown Protein (AHRD
2477
2013512
2011036
reverse




V1); contains Interpro




domain(s) IPR010634




Protein of unknown




function DUF1223


SL1.00sc07408_298.1.1
transcript_ITAG
ATMAMI ARABIDOPSIS
3392
2006924
2010315
forward




THALIANA MEMBRANE-




ASSOCIATED




MANNITOL-INDUCED




structural molecule (AHRD




V1 *-*G AT5G54110.1);




contains Interpro domain(s)




IPR008962 PapD-like


SL1.00sc07408_297.1.1
transcript_ITAG
Wound induced protein
461
2001724
2002184
forward




(AHRD V1 ***NG




B6SKC8_MAIZE)


SL1.00sc07408_296.1.1
transcript_ITAG
Ubiquitin carboxyl-terminal
9792
1988575
1998366
forward




hydrolase (AHRD V1 ***G




A7P1D3_VITVI); contains




Interpro domain(s)




IPR016652 Ubiquitinyl




hydrolase


SL1.00sc07408_295.1.1
transcript_ITAG
Unknown Protein (AHRD
1254
1987084
1985831
reverse




V1)


SL1.00sc07408_294.1.1
transcript_ITAG
Unknown Protein (AHRD
1308
1979129
1977822
reverse




V1)


SL1.00sc07408_293.1.1
transcript_ITAG
universal stress protein
3278
1975876
1972599
reverse




USP family protein (AHRD




V1 *-*NG AT4G27320.1);




contains Interpro domain(s)




IPR006016 UspA


7-20-2

Flanking marker CLT
100
1975288
1975845
forward


SL1.00sc07408_292.1.1
transcript_ITAG
PBP1 PINOID-BINDING
668
1969824
1970491
forward




PROTEIN 1 calcium ion




binding/protein binding




(AHRD V1 **-G




AT5G54490.1); contains




Interpro domain(s)




IPR011992 EF-Hand type


SL1.00sc07408_291.1.1
transcript_ITAG
Serine/threonine kinase-like
3701
1962690
1966390
forward




protein (AHRD V1 ***G




Q0WPA4_ARATH);




contains Interpro domain(s)




IPR002290 Serine/threonine




protein kinase


SL1.00sc07408_290.1.1
transcript_ITAG
ARK3 A. THALIANA
3742
1957638
1961379
forward




RECEPTOR KINASE inase/




transmembrane receptor




protein serine/threonine




kinase (AHRD V1 *-*G




AT4G21380.1); contains




Interpro domain(s)




IPR002290 Serine/threonine




protein kinase


SL1.00sc07408_289.1.1
transcript_ITAG
protein kinase family
7051
1953626
1946576
reverse




protein (AHRD V1 *-*NG




AT4G11470.1); contains




Interpro domain(s)




IPR002290 Serine/threonine




protein kinase


SL1.00sc07408_288.1.1
transcript_ITAG
ferredoxin-related (AHRD
2040
1945028
1942989
reverse




V1 ***NG AT1G02180.1)


7-20-3


100
1943149
1943396
reverse


SL1.00sc07408_287.1.1
transcript_ITAG
protein kinase family
1459
1940509
1941967
forward




protein (AHRD V1 ***G




AT4G23150.1); contains




Interpro domain(s)




IPR002290 Serine/threonine




protein kinase


7-20


100
1686186
1686306
reverse


7-21


100
1480300
1480420
reverse


7-25


100
464589
464709
reverse
















TABLE 3







SL1.00sc07408_313.1.1 (SEQ ID NO: 33)


TGCATGCTT TCTCAGTGGA GGCTACCAAG GCAGAGTCTT GGGACGACGA AGTTGATCAT CTGAAATTCT


 


ATTATAATGG AAAAGAGTTA GGCTTACCAG AAGGATATCC ATCCATATGG GAATCTTCTG AAAGCGGCAT


 


CAAGGTAGAA AGAACTGCAA ACAAGAACGG TGCTTTTATC ACACTACCAG AAGTAGCAGA AATTTCAGTA


 


AATGTAGTAC CTATTACCAA GGAAGATGAC AGGATCCACA ACTATCAGTT ACCTTCTGAT GACTGCTTCG


 


CTCACCTGGA CGTGCAGTTC AGATTCTATG GCCTCTCAAC GAAGGTTGAA GGCGTTCTTG GCCGGACGTA


 


CCAGCCAGAC TTCAAAAATC CAGCAAAACC AGGTGTTGCA GTGCCTGTAG TGGGAGGTGA AAAGATGTAC


 


AGAACTTCAA CCCTTTTGTC TTCCAAATGT AACTCCTGTA TCTTCTCTCC AGCTGGAGTT TCTGAAGAAT


 


CAGACCCCCT AGTCATGGAT TATGGGACTT TAGATTGTAC TGGAGGATCA AGCGGTGGCC ACGGAATAAT


 


TTGCAGAAAA TGA


 


SL1.00sc07408_312.1.1 (SEQ ID NO: 34)


ATGGAAATCC CAAATTCAGG GGGTTCAGTC TCAAGGAGAC CTGTGAGAAG GAGGTTCGCT TCTAGGAATT


 


ATGATGAGAC TGTGATGGAT AAAATCATAG ATGAGCAGTT GGGTAGTCCG GTGGGGAAGA AGATTAGAAC


 


GAAGAAAGAT TTGGAGAAAG AAACTGAAAA AGAGGCCTTG ATTGCTCTTT CTTTGGGCTT CCCAATTGAT


 


GACCTTCTTG AAGAAGAAAA AAAGGCTGGA GTTGTAAGTG AATTGGATGG GAAAGAGCAA AACGATTACA


 


TCGTTGTGAG AAATCATATT CTCGCAAAAT GGAGGGAGAA TGTGCATATT TGGCTGAACA AAGGAAGGAT


 


AAGGGAAATT ATAAGTGTCG AGTATGAACA TTTGGTAGCC ATAGCATATG ATTTTCTTTT AAGTAACGGG


 


TATATAAATT TCGGGGTTTC ATCATCATTT GAATCTAATC TTCCTGAGGA ACCTAGAGAA GGGTCTGTAA


 


TTATCATTGG TGCTGGACTC GCTGGTTTGG CTGCAGCAAG GCAACTGATG GCTTTTGGAT TTAAGGTAAC


 


TGTCCTTGAA GGTAGGAACC GACCTGGAGG GAGAGTTTAT TCTGAGAAAA TGGGATGGAA GGGAAAGTTT


 


GCTGCTGTGG ATCTTGGTGG CAGCGTTATA ACTGGTATCC ATGCGAATCC TTTGGGAGTT TTGGCTAGAC


 


AACTTTCCAT TCCGCTTCAC AGTGTTAGAG ATAAGTGTCC TTTATACAAG CCTGATGGTG CTCCTGTTGA


 


TTCAGTAGTT GATTCCACAG TTGAACTCAT TTTCAATAAG CTACTAGACA AAGTTGCTGA ACTACGAAAA


 


ATCGTAAGTG GATTGGCTAC TGATGTCTCG TTAGGCTCCG TTTTGGAGAC ACTTAGACGA TTATATTGTG


 


TGGCTAAAAC TAAAGAGGAG AAGCAACTTC TGCATTGGCA TTTTGCAAAC TTGGAATATG CAAATGCTGG


 


ATGCCTCTCG GAACTCTCTG CTGCCTACTG GGATCAGGAC GATCCTTATG AAATGGATGG TGATCATTGT


 


TTTCTTGTTG GTGGAAATCG AGCTATGATC AGGGCATTGT GTAAAGGAGT TCCTATATTC TATGGAAAGA


 


CTGTTCAGAC AATTAAGTAT GGAAATGAAG ACTAAAAAGA AGATCAATTA GATTTGAACC AGAGTTACCT


 


GAGAAGAAGC TTGAAGCTAT TGATAGGCTA GGATTTGGGT TGCTGAATAA AGTTGCCATG GTATTTCCTC


 


ATATTTTTTG GGGCGAAGAC TTGGATACCT TCGGTTGCCT CAACCATCAT AGCCACAGAC GAGGAGAGTA


 


CTTCTTATTT TACAGTTACC ATACTGTTTC TGGGGGTCCA GTACTTATTG CACTTGTTGC TGGTGACGCT


 


GCTCAACTTT TCGAAAGCAC AGATCCGTCC ACTTTAATTA ATCGAGTGAT TAACATTCTC AAAGGCATTT


 


ATGAGCAAAA GGGAATAAGC GTGCCTGATC CTATACAATC CATATGCACA AAATGGGGAA GTGATCCCTT


 


TTCGTTTGGC TCATATTCAC ATGTTCGTGT TCAGTCATCT GGCAGTGATT ATGACATACT TGCAGAAAAT


 


CTCGGAGGTC GGTTGTTTTT TGCTGGAGAG GCTACGATTC GACAACATCC AGCCACCATG CATGGAGCCT


 


ATTTGAGTGG CTTAAGAGAA GCTTCTCACA TTTCCCAATC CATGAAAGCG AGGCAAAACA ATCCAAGGAG


 


AACTGTATCA AAGAATGTTC GACCAAGCAA TGATACATTG GAAGAGTTGT TTGAAAAGCC AGATCTAGCA


 


TTCGGGAAGT TGTTATTTGT ATTTGATCCC CTCACTTGTG ATTCTAATTC TTTAGGACTG ATGAGAGTTA


 


CTTTTGGAAA ATCCAACGAC GAGCTTAATT CAGAAGAGGT AGACAATATG CCTCAACATT TATTAAATCC


 


ATCACTGCAG CTTTATACAG TTGTGTCTCG TGAGCAAGCA CATGAGCTGC AGTTGGTGAA GGAGGGAAAC


 


AATTGCAAAT TGTTAGATTT GCTTGAAGGT CTTGGGTTAA AGTTAGTGGG AGCGAATGGA CTAGGAGTTC


 


AAGGCCATAC TTTGGCTGCT AAAATTGTTA AAGGTAGAAG GAGTCGAAGC TATACTGCCA AGCAGAAGGC


 


AGGCGAAAGT AGCAGTCAGT ATATATTGTG CATCTGGCAG CAGAGTGTGA AAAAAGGATC GATGAACGCG


 


CGTTTTGCAT CTGGTTTGGG AGTTTCAAGC CTTCCTACTC TGTGCTGTTG TTTATGTTGT GGATATTTTT


 


CTATATGCTT GTGCTTTGTA CTCCTAGATA GTGCAAACTT TCTTGTAGGC TAA


 


SL1.00sc07408_311.1.1 (SEQ ID NO: 35)


ATGGCGGTCT TATGCTGCGC CACCTCTGCT TATCCTACGC ATACTCTGAG AGTAACTTCT GCTAAAGCTG


 


CAGTGCGTTC GAGCTCTCGA GTTTCAGTTC CTCAGCTGCA TCATTCCGAT TCCCCGTTTG TTCCCGAGGT


 


TAATAAGGCT GTTGATTCTT TGTCAAAAGA GTTCAGAGAA GTTGACAATT TAGTGGCACG CAATACTGCG


 


CGAGTTTTGA GAGCTTTCCA AAGGGTCAAG GTTGGGTCTC ATCACTTTGG TGGTAGCACT GGCTATGGTC


 


ATGAAGAAGC TGGTGGGCGT GAAGCCTTGG ACCAGGCTTT TGCAGAAATT GTTGGTGCGG AGTCTGCAAT


 


TGTTCGATCA CAGTTCTTCT CAGGTACTCA TGCAATCACT TGTGCCTTAT TCGCTTTCTT AAGGCCAGGG


 


GATGAGTTGT TAGCGATAGC TGGTGCACCT TATGATACTC TGGAGGAAGT AATTGGAAAA AGGGACTCTG


 


GTGGATTCGG TTCCTTGAAA GATTTTGGAG TAGAATACCG GGAAGTCCCA CTTGCAGAGG ATGGCGGGCT


 


TGATTGGGAT GCACTTAAAA CCTCTATAAG ACCTCACACT AAGTGTGCAC TCATACAGAG ATCATGTGGT


 


TATTCTTGGC GTCGCAGTTT GAGTGTCACT GAGATAGGTC GAGCAATTGA TATAATCAAG ATGCAGAACC


 


CAGGCTGCAT GGTCATGGTA GATAACTGCT ATGGTGAATT TGTTGACGAC ATCGAACCTC CTATGGTGGG


 


TGCTGACCTA ATTGCCGGAA GTTTGATTAA AAATCCGGGT GGAACGATTG CACCATGTGG TGGATATGTT


 


GCGGGAAGGA AAAAATGGGT AGAAGCAGCA GCTGCCCGTC TCTCGGCCCC AGGACTTGGA GTTGATTGCG


 


GTTCTACCCC TGGTGATATA ATGAGAACTT TATTTCAGGG TTTATTCCTC TCACCTCAAA TGGTTGGTGA


 


AGCAATAAAG GGAAGCTTCC TGATAGCTGA AGTCATGGCG GCTAAAGGTT ATAAAGTGCA GCCACTTTGC


 


CGGATCAAGC GTCATGACAC AGTGCAGGCT GTACAACTTG GAAATCGTGA GAATCTACTT TCCTTTTGCG


 


AGGCTGTTCA GAGAAGTTCT CCTGTCAGCT CTTTTATCAG GCCTGTTGCA GGTGCAACTG CTGGCTATGC


 


ATCTGAGGTA ATTTTTGCTG ATGGAACCTT CATTGATGGA AGTACTAGCG AGCTCTCATG TGATGGACCA


 


CTAAGAGAGC CTTTCTCTGT TTTCTGTCAG GGTGGCACTC ATTGGACGCA GTGGGGACTA GTTCTGGGGG


 


AGAAACTGGG GCCGGCAACT GTGGGCCCTC CGAGTAAGTC GGATCGGCAC GTGCATGAGC ATGTGGTTCG


 


GTGCCACGTG TTAAATGTTC TTCTAGATAT TGGGCCCACT ATCTCCAGGC TCAGCCAATA G


 


SL1.00sc07408_310.1.1 (SEQ ID NO: 36)


ATGGAATTGG ATCTGGATCG TGGGCAAGTC ACACTGATAG CATCCAGTAT ATGTCTTATG CTGACTTTAC


 


ATTTCAGCAT ACAGCTAGTG ACAGAACATT TTACGTCATG GAAGAAGCCT AAAGAGCAAA AGGCCATAAT


 


TATTATCGTC CTCATGGCAC CTTTGTATGC TATTGTCTCC TTCATTGGGT TGGTCGATTT CATGGGAAGC


 


AAACCCTTTT TCACTTTCTT GGAATCTGTC AAAGAATGTT ATGAAGCAAT TGTGATGGCT AAGTTCCTGG


 


GGTTGATGTA CACTTACTTG AATATATCCA TAAGCAAAAA CATAGTCCCT GATGAAATTA AGGGAAGACA


 


GATTCACCAC TCATTCCCAA TGACACTCTT CCAGCCTCAC ACTGCTCATT TGAACCATCA TACATTGAAG


 


CTTCTCAAGA ACTGGACATG GCAGTTTGTT GTGATTCGCC CTGTATGCTC TATTTTAATG ATTGTTTTAC


 


AAATGTTTGG AGTGTACCCT AGTTGGGTTA GCTGGACCTT TACCATCATT TTAAACATAT CCGTTTCACT


 


GGCATTGTAC TCTCTTGTGG TTTTCTACCA CGTGTTTGCG AAGGAGTTGG CGCCTCACAA GCCACTAGCC


 


AAGTTCCTGT GTGTCAAAGG AATTGTCTTT TTCGTCTTCT GGCAGGGCAT TCTGCTTAGT GTTCTGGTAT


 


CACTAGGCAT AATAAAATCT CACTATTTCT GGCTTGAGGT GGAGCGCCTT CAGGAAGGTA TGCAGAATGA


 


ACTAGTGATC CTGGAGATGG TTTTCTTCGC TATCCTTATG CGTCATGCAT ACAGTGCAGC GCCATATCGC


 


GCAGAAGCTG TTACAACTAC TTCAGAAGAT GCTACTTCTG GAGATAAAAA GAATGAGTGA


 


SL1.00sc07408_309.1.1 (SEQ ID NO: 37)


ATGACAGCAC TCGCTCAAAT TCTCATTAAT TTATCACCCT TAAAAACACC ACATGATGCT TTGTATAGAA


 


AACCGAACTT TCTTGGGCTG AAATTACAGC GACCCAGTTG TTGTTTCACT AATTTAAGGG CAAGAAAAGT


 


GTCAATTTGT AGTAGTTGGT ATAAATTAGG TGCTTTTAAG GAGAAAAACT CAATCTTGAC AGATAAAAAT


 


GGGATTTTTA TGAAGGAGGA GAGATGGGGT TGTGAGAAAA GAATGGTTTT TGTGAAATTT AAACAAGGTT


 


TTGGATTGGA TGGGATTGGT GATGGTGGGG GTGGTGGGAG GGATAATAGT GAGACTGTGA GAGTGTTGAG


 


TAATCTTGTT TTAGCAATTG GGCTTACTTA TCTTACTATG ACTGGTCAGC TAGGTTGGAT CTTGGATGCT


 


ATTGTTTCTG TTTGGCTCCT TGCAGTTCTA CTTCCAATTT TAGGTTTGGG AGCTTTTATT TGGTGGGCTG


 


GACGAGATAT TGTTCAAAGC GCTTGCCCAA ACTGCGGGAA TGAATTTCAA ATTTTCAAGT CTACTTTAAA


 


TGATGAGGTT CAGCTTTGCC CTTTCTGTAC TCAGCCATTC TCAGTTGTTG GCAATAAGTT CGTGAGAGAC


 


CCAGTAACGT TCTCCAACCA GTCAACTACA TTTGGTCAAG CATTTGGTGA TTTCAGTACT CGTTCTAAGA


 


AAGGTAAGAA TTCCTCTGTC GGAATTGTTG ATATAGAAGC AGAAGTTAAG GACGCGGACT AG


 


SL1.00sc07408_308.1.1 (SEQ ID NO: 38)


ATGGCGGCGT GGACGGCGGC GGCGAGGCAA GCAGCGAACC TATATCGATT CTCAGCTTCT AAATCAGTTA


 


GCTCAACGAA GCAAGGCGCT TTACTTATCC AGCGGCGCGG CCTTGCCGGC GGTGGTGATC ATCATGGACC


 


TCCAAAGGTG AATTTTTGGC AGGATCCGAT GAGTCCGTCT AAATGGAAAG AAGAGCATTT TGTGATCGTC


 


TCTCTTACTG GTTGGGGGTT GGCCTTCTAT GGAGGTTATA AGCTCTTCAC AAAGGGAAAG AAAGAGGAGA


 


AGGAAGAGAA AGTTGGTGAA GGATCCCACT AA


 


SL1.00sc07408_307.1.1 (SEQ ID NO: 39)


ATGAATGAGA AGGCTAATGT TACTAAAGAG CTTAATGCGA AGCATAGAAA GATACTAGAA GGACTTCTTA


 


AGTTGCCCGA GAACAGGGAA TGTGCAGATT GCAAAGCCAA GGGTCCTAGA TGGGCAAGCG TGAACCTGGG


 


AATATTTATA TGCATGCAAT GCTCTGGGAT CCACAGAAGC CTGGGGGTGC ACATATCAAA GGTCAGATCA


 


GCTACACTAG ACACATGGCT TCCTGAACAA GTTGCATTTA TCCATTCAAT GGGAAATGAG AGGTCAAATA


 


GTTTTTGGGA AGCAGAGCTG CCTCCAAATT ATGATAGAGT TGGTATTGAG AATTTCATCC GAGCAAAATA


 


TGAAGAGAAG AGGTGGGTCC CTAAGGATGG GATTCAAAAA TCACCTTCCA GGGTTCAAGA AGAGAGGGCT


 


TCGGTGCAAT GGCAACAAAA CAATGATAGA AGTGGGCGTA TACATGCAGC TAGCTCAGGA TGTGCATCTG


 


ACGAGAGGAA AAATATTCAA GCCTCGAAAG TGAAGCAAGA TGTACCTGCT GCTAGAGTCA GTATACCAGT


 


GCCTCCTAGA GGACCAGAGC ATGTAACTTC AGGTCAGGTT GCTAACCAGA CAAGTCAGAA AGCAGAGCCA


 


GTTGCAGTCA CTGAACCAGC TAAGCAGGTT CCGGAAGCTG CCAGTCCTCC TAAAGTTGAT TATGCTACTG


 


ATCTATTTGA CATGCTTTCT ATGGATTGTC CAACTGACAA TGGCTCAGAA GCAGCTTCTA CCGATGATAA


 


CTCCTGGGCA GGCTTCCAGT CTGCTCAAGA AGCAACAAAA GCAGAAAATA CTGGGGTTAC AAACTCCGTT


 


GATCAGAAGA AGTCTCAATC TGCTGCTGCT TCTGGAATTG AAGATTTATT CAAGGATTTA CCATCAATTG


 


TGCCTTCTGC CTCATCGCAG AAGCCACAGA AAGATGCTAA AAACGATATA ATGAGCCTTT TCGAGAAGTC


 


CAATATTGTG TCACCTTTTG CTATGCATCA ACAACAACTT GCTATGCTGG CGCAACAACA GTCTTTACTG


 


ATGGCTGCGG CTGCTGCAGG TGGTGCTGTA AGACTTCCTG TAAATGCACA ACAAAGCACT AATGGCACCA


 


ATATGGTAAA TCAGAACTGG CCAAATTTAG GCTATCAGTT CCCTGGAGTG ATAATGCCAG CAGCTGGTAA


 


GACTGAGCTG GAGAAATATA TGCAGGTAGG CAATATGGGA CCAGCACATG TAGTTGGAAA CTCTGTGCCA


 


ATTCCAGCAT CCAGCTTCTA CTCAATGGGA CAGAACACTT CCAGCAACGG TATTGTGCCG CCAGGACCAA


 


GCAAGCAAGC AGCTACCCCG ATATCATCAA GCTCCACACA GTCAACAAAA GAATTCGACT TTTCATCCTT


 


GACACAGGGT ATGTTCACAA AACGCTGA


 


SL1.00sc07408_306.1.1 (SEQ ID NO: 40)


ATGGCGATTT TGTTTCAAAG TTCAAGTAGC TCCATGTTAT CTATCAAAGT TTTTCTGATT TCGACTACTG


 


TTTTATCTGC TGCTATTATG TTAAAGGTGT CTGCTCCTGT TGTTACTGAA TTCGCGGTCA GTGAAGTTCC


 


GTCGATCTGG AACGGTGTCG TTTCGTGGCT TAAACCTCCG TATCTATACC TTGTTATCAA CTGCATTATC


 


ATTACGATTG TAGCCTCTTC TAAGTTGCAG AACAAGCTTG ATGAGAACTC ATCTCCGGTG CCGGCGGTTG


 


TTTCGCCGGA GAACTCGTCC CAGTTTCACC CGATTAAGGA TGTAAGGCCG GTTACAGACT ATTATACACC


 


GGTCCTTCAT GACTTAAACG GCTCCGTGCT GAAGAATCAA GCAGTGGAGG CGGAGGCTAG ACCGATAGTT


 


TACGAGTATC CTACTGCTGG TGTTTATGAT GCAAAGGTCG AGAAACTTCC AGTAGTTAAT CCGTACATAT


 


CGGAGAAAGG TACATCGTTC AACACCTATC CGGAGCCTAA CGATGTTGTT GCTGAAAAGG ATGATTTCGT


 


AATCTCGAAA TCTTCTTGGG CGCCGGTGAT GAGACAGGAC TCTATTGATT ATTCCATTTC AGGCAACTCG


 


GCTGAGAAAC CTCCTGCTTC TGCCAGATTC GCTCACCGGA GAAATGTCAA ATCCACTCCT GAAGGTGGAA


 


AGGGAGCATT GAGAGTATCA AAACCTAAAC GGCAAGACAC GCTGGAGAGT ACATGGAAGA CGATAACGGA


 


GGGCCGTGCA ATGCCACTAA CGAGGCACCT GAGGAAATCG GACACGTGGG AGACATACGG TGGTCGGAAC


 


CCGGTTACAC CACCACCGCA GAAGATGAAG AAATCTGAGA CGTTCAATGA CAGAACTACC CCTGACTCCT


 


CGCCATTGCT GACTCCGTCC CCGGGTGGTT CAGGGAAACT TAAGAAAGAG CCATCGCTAA GTCAAGACGA


 


GCTGAACAGG CGAGTTGAAG CGTTTATTAA GAAGTTTAAT GAAGATATGA GGTTGCAGAG GCAGCAGTCG


 


ATGCAACAGT ACACTCAGAT GATCAATCGA GGCTCACATT AG


 


SL1.00sc07408_305.1.1 (SEQ ID NO: 41)


ATGGCGCTCC AGCACTCGCC TATTGGCACA ATTACTTCTT CAGCTCAAAT GAAGCTGGCT GTTAGCAGTT


 


TGAGGTGCTA CGAGCACCTT CGTCCTATCA CTATCTCAAG CTTCAAGCGT TTTCCACGAA GAATTCAGGG


 


TGCGCATAGT AGTGTGGAGC AAAGGAAGTT GCTTAGCAGA GGTATTTCGA CAAGCAAAGA AAAATCGAAG


 


GATCTCCATG AAGATGTTTT TTCTTTGCCA ATGGCTTGCA CTAGTGCCCC AATTAGATTC ACAATGCTCT


 


CAACTGCTGT CATAGCCACA AATTTGGTTG CAACACATAC CGCAAATGCT TTGACTATGG ATAACATGAT


 


GGATTTCTCT AGCGCTGTCT ATACATTAGC TGATGGAAGC ATTGGAGATC TTTTTGGTGG CCTTCTGTAT


 


TCTGCTGGTC AACAAGCTAA TGAAGCTGTT CAGGGCCAGT TGACTGCTCT TAGTTTTACT AGTTTGGCCA


 


CTATTTTTGG TGCCGGGCTT GTAACTAGTC TGTCGCCTTG TACACTCAGC GTACTGCCTC TGACCCTTGG


 


TTATATTGGG TGCCGGGCTT CTGGGAAAAG CCGAGTAGCG GTTGTTGGAG ATTCAATTGC ATTTGCACTG


 


GGATTGGCAA CCACACTAGC ATTATTGGGT ATTGCGGCTG CATTTGCTGG AAAGGCATAT GGACAAATAG


 


GACAAGGATT GCCCGTGGCT GCTTCCTTTT TAGCTATTGT TATGGGGCTA AACCTGTTAG AGGTAATAGA


 


GTTGCAACTT CCCTCATTTT TTGACAACTT TGATCCTCGC TCAGCCGCTG CTAGCTTTCC GTCCAGTGTT


 


CAAGCTTATT TGGCCGGTCT TACATTTGCA TTAGCTGCAT CACCATGCAG TACACCAGTC CTCGCAACCT


 


TGCTCGGCTA TGTTGCTACT TCTCGGGATC CAGTTATTGG GGGCAGCTTG CTATTGACAT ACACAACTGG


 


CTATGTTACT CCCTTACTTC TTGCTGCTTC TTTTGCTGGA GCATTGCAGA GTATACTTTC ATTCCGCAAG


 


TTCTCAGCAT GGATCAATCC AATCAGTGGT GCGCTACTAC TAGGAGGGGG TGTCTATACC TTTCTCGACA


 


AGCTTTTTCC GGTGACGATG GCTATGTAG


 


SL1.00sc07408_304.1.1 (SEQ ID NO: 42)


ATGGCGAAGA ACAGAAACAA GAAGAAAAAC GGCCTAGCTG CCATGGATGT CTCCACTGAC CAGACGGTCA


 


TGGATGCCCA AGCGATGGAT ACTTCAGAAT CAGCTGCTCC AAAACCACAT ATAGGTGGAT CACTTAGAAA


 


GACGAAGGGA GTACAAATGA AAAGGACGAA GAATGTTAGG AAAAAGAAGG CCATGGCAAA GGCTATTTCA


 


AAAAGTGAGA AATTGGAGGA AAGAATCACT AGGAGTGAAA GCAAGATAGA GAGAACTAAA AATGCCAAAC


 


AGTTATACGA ATGA


 


SL1.00sc07408_303.1.1 (SEQ ID NO: 43)


ATGGATTTTG ATGTCCAGGG GGGAAAAATG TCTACTATAG TTCGTAGTTC GTTAGAGGAA ATGCTGGATT


 


CTCTCCGGCA AAGGGATGAA AATGAGAAGC CAAAAGACTT GCCGCCGGCG TTGCCTGCTC GGCCTAAGCT


 


TACATCGAGG ACTAGGCCTC CTTCACAGAG GCAACCGTTG AGCAAAAGGT TAAGCAAAGG TGATGTTGAA


 


TTGGAGAATG GTAAGAAGAA GGAGGAGTTG AAAGTGTTGA AAAGGAATGT TTTTGGTGCT ATGAAGGTGA


 


AAGGAATCGA AGATAGCGAA TCGCCATATG CAATGCCTTC AGTGAAGAAA AACAGCACAG GGAGATTGCG


 


GGAAGTAAAT GGTGGGAAGG TTGAGAAATG GCGTAGTGAA GCTGAATGGG ATGATCGGCT GGATTATTTT


 


GTTAAGAAGA AGCTTCGCAT CTGGTGTCGT CTGGGGAATG GGGCATGGGT ATCAGGACAT ATTCAATCAA


 


CTTCAGGAGG GAAAGCTATG GTGTTGCTTT CTGACGGCAG TGTTGTGCAA GGCAGGGCCT GTATTAATAG


 


ACCTTCTAAT CCGGATGTTC TTGAGGGTGT GGATGATCTC ATGCAGCTTA GTTATTTGAA CGAGCCATCT


 


GTTCTTCACA ACCTTCAACA CAGATATGCG CGAGATATGA TATATAGCAA GGCAGGGCCT GTATTAATAG


 


CAATCAATCC GTTCAAAGAT ATCCAATTGT ATGGAAACGA ATTTGTTACA GCTTACAGAC AGAAGCTCTT


 


GAGTGATCCT CATGTTTACT CTATTGCTGA TTCTGCCTAC GATCGAATGA TGGAAGATGA GATAAGTCAA


 


TCTATTATCA TAAGTGGGGA AAGTGGATCT GGGAAGACGG AAACAGCAAA ATTTGCAATG GAATACTTGG


 


CTATGCTTGG TGGAGGTAGT AATGGGATAG AGAAGGAGGT TTTGGAAACA AGCTACATAT TGGAGGCCTT


 


TGGGAATGCC AAAACTTCCA GGAACAATAA CTCCAGTCGA TTTGGAAAGT TGGTTGAAAT TCATTTTAGT


 


CCAGCAGGAA GAATATGTGG TGCTAAAATA CAAACCTGTA AGTGTAATTG TCCTTTGTCG AGAGTGGTTC


 


AGCTGCTTGA TGGAGAGAGG TCCTATCATA TTTTTTACCA ACTATGTGCC GGGGCTCCAC CTACTTTAGT


 


AGATAAACTT AAGTTAAAAG GTGCATCAGA ATACAAATAT CTCAACCAGA GTGGCTGCTT GGTGATCCAT


 


GATGTTGATG ATGCTGAGGA ATTTTGTAAG CTTATGGAAG CCTTAAATAC TGTTAGGATT TCTGAAAGGG


 


ATCAAGAGCA TGCTTTTCAG ATGATTGCTT CAGTTCTATG GCTGGGAAAC ATAACATTCC AAGTAATTGA


 


CGATGAAAGT CGTGCTGAAG TTGTGCAAAG TGAAGCTGTT ACAAATGCTG CTAGCTTGAT TGGCTGTACT


 


GTAAATGACC TCATACTAGC TTTGTCAACA TGCCAAATAC GAGCTGGCAA GGATAAGTAC GCCAAGAGTT


 


TAACTGTAGA GCAGGCAACT GATAGAAGAG ATGCATTGGC AAAGTTCATT TATGCAAACT TGTTTGACTG


 


GATAGTAGAT CAAATGAACA GAAACCTTGC AATGGACAAA GAACAGATGG GTAGATCCAT AAATATTCTA


 


GACATTTACG GTTTTGAATC ATTTCAGGGA AACTCATTTG AACAATTTCT GATAAACTAT GCAAATGAGA


 


GGCTCCAGCA GCATGCCAAC AGACATCTAT TGAAGCTCGA GCAAGAGGAA TATGAATTGG ATGGAATTGA


 


TTGGTCAAAA GTAGATTTCG AAGACAACCA AGAGTGCCTG AACCTTTTTG AAAAGGTATT CTTCCTTTTG


 


GTTCTTTTTC CTATTTATAT GGCGTTAGGA GCTTATATAA GCCGACAACT TTCTTCTGCC CCGGATAGCA


 


TGTTTACATT CAAGCCAATT GGCCTTATAT CTTTGTTGAA TGAAGAATCA AATTCCCTTA CAGCCACAGA


 


TTTGACCTTT GTATGTAAAC TTAAGCAGCA CATCAAATCT AGCCCTTGCT TTAAAAGTGA AAGAGAAGAA


 


TTTTGTATCC GTCATTATGC TGGAGAGGTC AGTTTCTCTT ATAAATGCAT CCTTTCTAGT TCTGTGCAAT


 


TTCTACGGAT ATTATTAGTA TTTTCACTAT TTTGGTTAAG AGACTTGATA GCTTTCACAT CCCCATGGTC


 


AGACTTACGC TCAATTGTTG GGAGAGTTTT TCTTTCTTAT CATGGTTTTA TTACAGTAAC TTATGATGCA


 


ACTGGCTTCT TAGCAAAGAA CAGAGATGTG TTGCATCCTG ACATTACTCA GCTACTCTCA TCAAGTGACA


 


GTCACCTGCC TGAAGATAAA AAATTATCAA TTCCATCAAC TGATGCAGGA GTGCTAGATT TTCAGAAGCA


 


AAGTGTTGCA ACTAAGTTTA AGGATAATTT GTTCAAATTG ATGCAGCAAT TGGAAAATAC CATACCACAT


 


TTCATATGTT GCATAAAACC AAATAATAAG CAGCTTCCTG GCATGTCTGA CAAAGATCTT GTCATAGAAC


 


AACTCAGATG CTGCGGTGTT CTTGAGGTGG TTAGAATATC AAGATCTGGC TATCCTACTA GGTTAACACA


 


TCAAGAATTC ACAAGCAGGT ACGGCTTCCT TCTGCCAAAG GATAGTGCAT GCCAAGATCC TTTAAGTATG


 


TCAGTTGCCA TTCTTCATCA ATTTGGTATT CTTCCGGAAC TGTACCAAGT TGGGTATACC AAGTTATATT


 


TCCGATCAGG ACAGATTGCT TCATTGGAGG ATGCAAGGAA CCAAGTTCTG CAAGGTACTC TTGAGTTGCA


 


GAAGTGCTTC CGTGGTCATC GTGCTCGTCG GCACTTCCAT GAACTGAAAG GAGGAGTAAT CATACTTCAA


 


TCATGTAAAG CAGATCCCTT TGCAACTCGT CTCTTTTCTT CCCTTTTTTC GTTGAATATA ATAGAAGATG


 


ATATTCATGA AGATTGTTGT GTCTATCAGG TTGGATGA


 


SL1.00sc07408_302.1.1 (SEQ ID NO: 44)


ATGTCTAAAG CGAAGGTTGC TCGTGAAGGA AGTGATGAGC AACTGGTGGC TGTCGTGCAG ATACAATCAG


 


CTATTCGTGG TTGGTTGGCT AGAAGGGGTC TTCGCAAACT GCGAAATTCA AAAATGTTAA ATGTAGACAA


 


ACGAAGATCA GGCAGAAAGA CGGAGGTCAA GGAGTTGCCT CGAGAAATCC TACCATCTGT TGTAGAAGAC


 


CTCGAAAGAC GGGTTGCAAA GGCCGAGGCA ACAACTGAAC AAAAGGAAAA GGAAAATGCT GCCCTGAAGG


 


AACAAGTAAA CCAATTCGAG ACCAGATGCT TAGAATATGA GGTCAAGATG AGGTCAATGG AGGAGATGTG


 


GCAAAAGCAA ATGACATCAT TGCAGGTTAG TCTAGCTGCA GCCAGGAACA GTCTTACCGC TGCTGACACT


 


ACTGGTCGAC CTGGAAAGCT TGAAGGTTCC CCATCTCCTC AGTATTATGA TTCTGATGAT GCAACATCTA


 


TGGACACTCC TGCGGGACGC ACTCCAGTTA GCTTTTCTAA CAACAGCTTG GGTGTTGTAG CTAATAGAGA


 


GGTTAATGGT GGTTTATCCT TAATCAGCCA CCTTGCAATG GAATTTGAGC AACGGAAGCA AAATTTTGAC


 


AACGAAGCCA TGGCAATTGT TCACTTGAAG CCAGGGCAGT TACAGTCTAC TAATAATCCT GCAGATGAGT


 


ATCGAAGACT GAAACACAGG TTTGAGGAAT GGAAAAAAGA GTACAAGGTT CGGTTAAAGG AGACAAAGTC


 


AAAAGTACAC AAGCTTGTTC ATTCTAAAGC AGGGAAGAGT CGTAGAAAAT GGTGGGGTAA GAAGAGCAAA


 


TGA


 


SL1.00sc07408_301.1.1 (SEQ ID NO: 45)


ATGGCAGCAA CAGGTAGCTC AGCCACAGTT GTTAGAGCAA CTCCATTTTT GGGCCAGACC AAATATGCTA


 


ACCCCCTAAG GGATATAGTT CCTATGGGCT CTGCCAGATT CACCATGAGT AATGATTTGT GGTATGGACC


 


TGACCGTGTC AAGTACTTGG GACCATTTTC TGCTCAAACT CCTTCATACT TGACTGGAGA ATTCCCTGGT


 


GATTACGGAT GGGATACTGC TGGTTTATCT GCTGATCCCG AGGCCTTTGC TAAGAACAGA GCTCTTGAGG


 


TTATCCATGG GAGATGGGCC ATGCTTGGAG CTTTTGGTTG CATTACACCA GAAGTTCTTG AAAAATGGGT


 


AAAAGTGGAC TTCAAAGAAC CAGTATGGTT CAAAGCTGGA GCCCAGATCT TCAGTGAAGG TGGGCTGGAC


 


TATTTGGGCA ACCCAAACCT TGTCCATGCT CAGAGCATTC TAGCAGTATT GGGCTTCCAA GTTGTACTAA


 


TGGGCCTTGT AGAAGGTTTC AGAATTAATG GGCTTCCTGG AGTTGGTGAA GGCAACAATC TCTACCCAGG


 


TGGACAGTAC TTTGACCCAT TGGGCCTAGC AGATGACCCA ACAACTTTCG CGGAACTCAA GGTCAAGGAA


 


ATCAAGAACG GAAGATTAGC TATGTTCTCC ATGTTTGGAT TCTTCGTTCA AGCTATTGTC ACCGGCAAAG


 


GCCCACTTGA AAATCTATTG GATCACCTTG ACAACCCTGT TGCTAACAAT GCATGGGTTT ACGCAACTAA


 


GTTTGTTCCT GGATCTTAA


 


SL1.00sc07408_300.1.1 (SEQ ID NO: 46)


ATGTTGGAAG GGAAAGCAGT AATTGGAGAT ACAGATATGT TGGGAACCAT GCAACAAGAT GCATTAGATT


 


TAGCTGCAAA GGCACTTGAC TTCTTTGATG TCACTGAGGC CACTGAAATT GCACGTTTTC TTAAAAAGGA


 


ATTTGATACA ATGTATGGAC CAGGGTGGCA ATGCATAGTA GGGACAGATT TTGGTTCATT TGTAACACAT


 


TGTTATGGTT GTTTCATCCA TTTCTACATT GGCAGCCTTG CTATTTTGCT CTTCAAGGGC TCTGCTGCCC


 


TAGAGGACCC GAAAGCCGAG GCCGAAGCTG ACCGATTTTC CACTCTGCAG GAAATAGCAT GA


 


SL1.00sc07408_299.1.1 (SEQ ID NO: 47)


ATGGGGCGCC GTCTCTTCAC CTGCTTCGGC AAAGGCGGTT CTTCTCATTC TTCTTCAAAA GATCCCGGCT


 


CGAATAACAA GGACAGTGCG ACGGCGGATT TGACGGCGGA GGAGCAAAAA CGGTGCGGGC CGGTGGTGGT


 


GGAGTTATTC TCATCGCAAG GCTGCGCCAC CTCACCTGAA GCGGAGCTGT TGTTTTCGAG GATTGGGAGA


 


GGCGATTTTA ACCTAGAAAT GCCGGTGATT TTGTTGGCTT ATCATGTGGA TTATTGGGAT TATATGGGTT


 


GGAAGGATCC GTTTGGGTCG AGTTTATGGA CGGTTAAACA AAAAGCGTAT GTGGAGACCT TAAATCTAGA


 


TACCATGTTT ACGCCTCAAA TTGTGGTTCA GGGAAGAGCT CAATGTGTTG GGAATGAACA AGATGCGGTT


 


TTCTCTTGTA TCAAATCTGC CCCCAGATTT GCTGCTCCTT CCTTCCAGGC AACATTCGAG AGGCCAACAC


 


CAGAGTCATT GCAAGTATCT CTATTAGGAT CTCTAAGGAG TAAGGTGGAC AATGATGGTC CCAACGTGAT


 


GATTGCTCTG TACGAAGGTG GTCTGGTGAC TGATATCGCT GCAGGAGAGA ACAAAGGAAA AATGCTTGCG


 


AATGACTATG TTGTCAGGAG GCTGGAAAAG CTTTGCTATG TAAAGGATAT TACTGCAAAG AAGACAATCT


 


CAGGAACTGT CAATTTCTCT CTTTGGGATG GCTTCAATAG CAGCAAATGT GGCGTAGCGC TCTTTGTGGA


 


ATCTGGCTCT CATCAAATAT GTGGATCACA AAACTTTAAA TTGCCAGAAA ATCTCTGA


 


SL1.00sc07408_298.1.1 (SEQ ID NO: 48)


ATGGCAATCG CCGACCACCA CAAGTCACAT AACTCAGACG GAAAGCTCTG GAAGCTTTGT CCTCTATGGC


 


AATCAGGAAC TACGTCTTCT TCTTCGTCGT CTACACAAAA TCTTCACTCT CAGAATCACA TGCACCAAAA


 


CGGCGTCGGA TCTAACAGCT CTCGTGCTTC TACGTCTGTT AGCTCCGTTG CTAGATCACT GCTTCCGGCT


 


AGACGTAGGC TTCGGCTCGA TCCAGCTAAC AGTCTCTACT TCCCTTATGA ACCGGGAAAG CAGGTGAAGA


 


GTGCTGTAAA GATTAAGAAC ACTAGCAAAT CTTATGTTGC ATTTAAGTTT CAAACGACTG CACCAAAGAG


 


CTGCTACATG CGACCTCCCG GAGGCATTCT CGAACCCGGT GAAAGTGTTA TTGCCACTGT CTTCAAGTTT


 


GTGGAGCACC CTGAGAACAA CGAAAAGCCT GTGGACCAAA AGAGCAAAGT TAAGTTCAAG ATCATAAGCT


 


TGAAGGTGAA AGAAGGTGTA GATTACGTAC CTGAGTTGTT TGAAGAACAA AAGGATCACG TGACTATTGA


 


ACGTATCCTA CGGGTGGTGT TCTTGGACCC AGAACGACCT TCTCCAGTGC TGGATAAACT AAAGCGTCAG


 


TTGGCTGAAG CTGAGGCAGC ATTAGAATCT CGCAAGAAAC CTCCAGTTGA AACTGGACCT AAAGTTGTAG


 


GAGAAGGTCT AGTAATAGAT GAATGGAAGG AACGAAGGGA GAAGTATCTC GCTCGGCAGC AGGTTGAGGC


 


TGTTGATTCA GTGTAA


 


SL1.00sc07408_297.1.1 (SEQ ID NO: 49)


ATGAATGCAG CAGCAGCAAA GGGATCGGCA TGGATCGTAG CAGCAAGTAT TGGAGCAGTA GAAGCATTAA


 


AAGATCAAGG ATTTGCCAGA TGGAATTACG CTTTAAGATC GATTCATCAC TATGCCAAAT CTAATTTAAT


 


TGCTTCTAGT AATACCTCGG CTCGGAGATT CTCGACGGCT TCGGCGCCGG CGGCGTCTTC TCCGGCAGTC


 


GTCTCCGGTG AGAAGCTGAG GAAAACGGAA GAGACGTTGA GTAAAGTTAT AGATCTGAAC TGTTGGGGTC


 


CAAGCACTGT CAGATTTTAG


 


SL1.00sc07408_296.1.1 (SEQ ID NO: 50)


ATGGAGCTTC TCCGATCAAA CCTTGCTCGT GTTCGGATTC CAGAGCCGAC TACTCGTATC TACAAGCACG


 


AGTGCTGCAT TTCTTTCGAT ACTCCGAAAT CCGATGGCGG GCTGTTTGTT GATATGAGTA CTTTTCTTGC


 


ATTTGGAAGG GATTGTGTTG ATTGGAACTA TGAGAAGACT GGGAACCCAG TTTATTTGCA TATAAAGCAG


 


ACAAAGAAGG CAGATGCTGA AGATAGACCG TCCAAAAAAC CCACTCTCTT GGCTATAGGT TTAGACGGAG


 


GGTTTGATAA CAGTGAACCC CAGTACGAAG AATTCTATGA AATAGTTATA TTGCCTGATA ATGTCACTCT


 


TCCTTTCCCA TCGGTGGAAT TGCCTGAGAA GGTTAGATTG GCTGTTGATG CTATTTTACT AGCTGAAGGT


 


GCTGAGAGGA AAGAGCAACT TGCTTCCTGG ACTGCTGACA AGAAGCTTGT CAGTAAATAT GCTACGGATC


 


TGCAACAGCT TGACAATGGT GTTGCTGTTC CACCTGTGGG TTGGAAATGT GCGAAATGTG ACAAGACTGA


 


CAATCTTTGG CTGAATCTAA CTGATGGAAC TATCCTATGT GGTAGGAAAA ATTGGGATGG AACTGGTGGT


 


AATGACCATG CAGTTAACCA TTACAAAGAA ACTGGTTATC CACTTGCTGT AAAGCTTGGG ACCGTAACTG


 


CTGATTTGGA GGGGGCAGAT GTTTACTCCT ATCCAGAGGA TGAAAGTGTT GTTGACCCAC TTTTAGCAGA


 


TCATCTGGCA CATTTTGGTA TTGACTTCTC ATCCTTGCAA AAGACTGAAA TGACGACTGC TGAGAGAGAA


 


CTAGACCAAA ATTTTAACTT TGATTGGAAC CGGATTCAAG AGACTGGTGA GGACGTTGAA CCACTTTTTG


 


GACCTGGTTA CACTGGATTA GTCAATCTTG GTAACAGTTG CTACTTGGCT GCTACAATGC AGGTTATGTT


 


CTCAACGCGT TCATTTTGTT CAAGATACTA CTTTGATCAA CGTCTGAAAG AAGCTTTTAC TACGGCTCCT


 


GCTGATCCGA CTGTAGACCT TAACATGCAG CTAACAAAGC TGGCTCATGG TTTGCTTTCT GGTAAATATT


 


CGGGTCCTGT TCTGGAGAAG GATAATACTG CTAATGCTGT AAGCTCACAG AAACAGGAGG GTATCCGTCC


 


TCGAATGTTC AAGTCAGTAA TAGCTGCTAG TCACCCTGAA TTTTCAACAA TGAGACAACA GGATGCGTTA


 


GAGTTCTTCC TGCATTTTAT TGATCAAGTT GAACGGATAA ACTCTGGGAC ACCTAATTTT GATCCATCAA


 


GGAGCTTCAA GTTTGGTATT GAAGAACGCC TCCAATGTTC CTCGGGCAAA GTCACTTACA ACAGAAGGAA


 


TGATTATATT CTGTCTCTTA ATATTCCTTT GGAGAGGGCT ATAAATAAAA AAGAGCTAGC AGAATTTCAA


 


AATTTGAAGG CTGAGAGAGC TGCAGGAGGA AAAGAACTGT CTGCTGATGA AATTGTTCGC CCTAGAGTAT


 


CATTGAAGGA TTGCCTAGAT TGCTTTTCAG CTCCTGAGGA GGTGCATGAT TTCTACAGCA CAGCTTTAAC


 


AGCTAGGACT ACAGCAATCA AAACTGCAGG TTTGACTTCT TTTCCAGATT ATCTGGTTTT GCACATGCGG


 


AAATTTGTTA TGGAGGAAGG TTGGGTGCCA AAGAAGCTCG ATGTCTACAT AGATGTCCCT GAAACCATTG


 


ACATAAGTAG CATGCGAAGT AATGGTATTC AACCAGGAGA AGAGCTGTTG CCTGACAGTG CTGCAGGGGA


 


TGGTGAGCAG TCAATAAAGC TTCTGGCTGA CGATGATATT GTTGCACAAC TTGTTTCAAT GGGATTTAAT


 


CTACTTCATT GTCAGAAGGC TGCTATCAAT ACTTCCAACA GTGGAGTAGA GGCAGCAATG GATTGGTTAC


 


TTAATCATAT GAATGATCCA GATATTGATG CTCCTATATC AGAAAACGTG CAAAATCCTG ATATTGATCA


 


ATCTAAAGTT GATACGCTGG TTTCATTTGG TTTCGAAGAG AAACTTGCCC ATAAGGCCCT GAAGGCATCG


 


GGAGGTGATG TTGAAAAAGC TACTGAATGG ATATTCAGCA GCCCTAGTGC CAGTACTGCA GCAGACATGG


 


ATGTTACTAC CAGTAGTGGA GCTGCAGTTG ATACCTTGAT GCCTGATGGA GGAGGAAAAT ACAGGCTCCT


 


GGGATTCGTG AGCCACATAG GCACATCTAC CCACTGTGGC CATTATGTCG CTCATATTAA CAAAGATGGC


 


CGGTGGGTGA TTTTCAACGA TGAAAAAGTT GGGGTCTCAA AAAACCCCCC TATGGATATG GGATACCTCT


 


ACTTTTTTGA AAGACTTGAG AGTTGA


 


SL1.00sc07408_295.1.1 (SEQ ID NO: 51)


ATGGCAAAAT TAGCGAAAAA TCGAGGAAGG AATAGATTTT TTAATAGTTG TTATAAGCCT CTACATTTCA


 


ACGATGATAT ATCTAATACA AATAAACGAG GTCATTTGTA TAATTCAACA GATATATCTA ATTTGAAGAG


 


AGTAAAATCA CAAGAAAAAT TGGATTCAAT GTTGGGAAAA GATTTGACCA AAAATTTTCG ACCAAATCGA


 


AAGGAGAATG CCTTTGGAAG GAATTTTTCT CATGCACTCA AAAATGTGTT TTTTGATACA TCATTGGGGA


 


AGAAAGGCCA GAGGAAAGAG CATAAGTATT CATTTGGATC ATGTAAAAAG TTATCAACAA AATTTGAGAA


 


AATATTTCAT TCTACAAAAG AAAAAAAGTT TTCTTCATCA AAGGATTTAC CAAAAATTAC AAGTAGAATG


 


TCCACAAGTG CAAGCATGGA TGATTTTTCC TTATTCACTA CTTCTACATC CTCTTTATTT TCTTCTTCTT


 


CTTCTTCTCG TTCGTCTTGT TCATCACAGA GAACGCAATT TTCATCTTTT CACAGGTCAA AATCAGAAAA


 


CAATATGCAA GTGTATGATA ACGTAAAAGA AAAAGATATC GCGCTATGTT ACAACAAGAA CGTTGGGACG


 


TATTCTCTTT TAATTTGTCT ATTGGTTATG GTTTTTTGTG GGAAAGTTTT TGCCATCATT TGCACTTCAA


 


CATGGTTCTA TTTTGCTCCT CATTGTTTCA AACGTATAGA CTCGGATGAG TACAAGAAAA ATGATGGATT


 


GTTATATTTA GTACAAGAAA AAAATCTAAC TTTTTAG


 


SL1.00sc07408_294.1.1 (SEQ ID NO: 52)


ATGGCTACTT CTCTTCAGTT TTCTTCAGAT CATCACCCTA TTCCTCAGGA AAATCATCAA ACAACGAACC


 


AGACCTCAAC GGGAGGACGT AGAAGGTCGA GTAAAAATGG ACAGAAGAAG AAGAAGCAAC CACAAAGAGG


 


AATGGGAGTT GAACAACTCG AACGTCTTCG AGTACAAGAT CAGATGAAAA ACAGTACTAT CCATGGCGTT


 


CATCATAATC ATCAGTACTA CTCTAATAAC AATTTCCCTA AATTAACTCC TGTTTCATCA TTTACCGGCG


 


GTGGTAGTGC TAGTGCTGAT CCTGGAAATT ATAGTAATTC TATTTTGAAC TCTTCACCAG TACTTCAGTT


 


CCCCAAATTG TGTGCAGTGA GCCCTAATGA TTTTTTTATG CAACAAAAAG TTGTGAATAC TGGGTTTATT


 


GGATCTAGTA GTACAAATCA GTTGATGATT TCTTCTCATG ATCATCATCA GTTTCAATCT CAGATGAATC


 


TCTACGGATT TGCAACTTCT AAGCCCAGTA CTGAGAAATC AAAGGAGCTG TATCCAATGC CAAATCTGTT


 


TAGCAGCAAC AACTCTTGTT TCTCCGATCG CTGCCGATCA TGCAACAAAA AGAAACGCAT GATCAATGGA


 


GAAGAAATTA GCGTTCATAT GGAGGACATG ATCAGAGAAA AGGAAGATTC TGGAACAAAG CCTTTGCTTC


 


ACTCATACAG TTTACCTAGC CATCAACAAA AGGGCGTAGA GATTGTGGCA ATCCATAGAA AGGGAAGTTC


 


ATCCGCGTTG TCATCCGATG AAGGAGCAGT AATGATGGAG TATGATTTTT TTCCAGAAAA AATCAGCAGC


 


AAAAGCACTA ATACTTACAA AAGTTGTTTC GAGAATGAAG CAACGATGAT GAGTGCTTAT AATTCACCAG


 


AATCTTCTTC ATTTGCTGCT GCAGCAGCAG CAGCTGGAAA TATTATTAAT GGTGAAGCTT CTTCTGTTAC


 


TACAATATCT TGGGCTGCAG ATACTACTAC TACTTCACCT ACCAGTTCCA TTGATCTTTC ACTGAAGCTT


 


TCTTGTTAG


 


SL1.00sc07408.293.1.1 (SEQ ID NO: 53)


ATGAGGGAGA GGTTGTGTCT TGAGGTTGAG AGGTTAGGGC TTAGTGCTGT TATTATGGGG AGTCGAGGAT


 


TTGGAGCTAC GAAGAGGGGG AGTGATGGAA GACTTGGGAG TGTTAGTGAT TATTGTGTTA GGCACTGTGT


 


GTGTCCTGTT GTGGTTGTTA GGTATCCCGA TGATAAGGAT GCTGGAAATG CTGTTGTAGA GCCCGTGGTT


 


TCTGTTGCTT CAGCTGCTGA AGAAGACGAG GAGGAAGCTG AGTAGGATGA TGCTTCTGAG GATCGAAAAG


 


ATTCATAA


 


SL1.00sc07408_292.1.1 (SEQ ID NO: 54)


ATGGAAGATC CTTATGGATT TGAAGATCAT TTCCCTTCAA TGATGGAGAG ATTAGGCGCG GAAGGATTCA


 


TGAGGGAGCT TTGCAATGGA TTTTGTTTAC TTATGGATGT GAGTATAGGG CTAATAACAT ATGAAAGTTT


 


GAAGAGAAAC ACTATGAATC TTGGTTTAAA TGATTTAAGA GATGATGAGT TGATTTACAT GTTGGCTGAA


 


GGAGATTTGG ATGGTGATGG AGCACTTAAT CAAATGGAAT TTTGTATTCT CATGTTTAGA TTGAGTCCTG


 


GTTTAATGGA TGGATCTAAG CAATACATGG ATGATGTGGG GCTTATTCAT TTCCAACCCT AA


 


SL1.00sc07408_291.1.1 (SEQ ID NO: 55)


ATGAAAGGCC TATTTTCTTT ATGCATTTGC TACCAATTTC TCTTCATTTT ACTAACTTCT GCAGCATTAG


 


ACACAATCAC TACAGATAAA TCCATTAGAG ATGGTGACAC AATTGTTTCA GCTGGAGGGG TTTATGAGCT


 


TGGATTTTTC AGCCCTGGAA ATTCGAAGAA TCATTACGTT GGCATATGGT ACAAGAAAAT ATCAAATGGA


 


ACTGTTGTCT GGGTTGCAAA CAGAAGCATT CCACTTAATG ACACTTCAGG AGTGTTAACA CTTAATCCCA


 


ATGGAATTCT TGTACTTGTT GATAAATCCA ATGTCTCAAT TTGGTCATCA AACTCATCAA GATTGTTAAA


 


GAATCCAAAA GCACGGTTAC TGGATTCAGG GAACCTTGTT GTCAGTGATG GAAATGATAG AGGCCTGGAA


 


AATAATTTCG CGTGGCAGAG TTTTGACTAT CCAGGAAATA CTTTGTTACC TGGTATGAGG CTAGGAAAAG


 


ATTTTGTCAC GGGAATGAAT TGGCATTTAA CGTCATGGAA GAGCACAGAT GATCCTACTC CTGGTGATTA


 


TGTAGATCGT GTTGATTCAC ATGGATATCC ACAATTGTTT GTGTGGAAAA ATTCATCTAT AGTATTTAGC


 


TCAGGGCCAT GGAATGGTAT TGCATTTAGT GGTAGTCCTA ATAATAAACC AAATACATAT TACAGTTTCG


 


AGTTTGTTAT TAATCAGCAG GAAATTTACT ACACATATAC AATTAAGAAT GACTCCATAC CCACCAGGGT


 


GGTGCTCAAT CCGTCTGGTG TGCTAGAACA CCTAACATGG ATAGAGCGCA GTCAGAGCTG GTTTCTCTAC


 


TTGACAGCAC AATTTGATAA TTGTGATCGT TTTGGTTTAT GTGGACCTTA TTCAAGTTGC AACATCAATA


 


ACTAAAATCC ATGTGACTGT TTGAAAGGTT TTGAGCCTAG GTATCCTCAA GATTCTGCAA CAGAGTGGTC


 


TAGTGGTTGC ATAAGGAGAA CTTCTTTGGA TTGTACCCAT GATGGTTTTC TTAAATTTTC AGGTATCAAA


 


ATGCCTGATT CTAGAAACTC CTGGTATAAT GACAGCATGA ACCTTGAAGA TTGCGAGAAA ATGTGCTTGG


 


CTGATTGCAA TTGTACAGCC TACTCAGATC TTGATGTTAG AAATGGCGGA AGTGGATGCT TACTATGGTT


 


TGGTGAACTC ATAGATATAC GCGGGTTCAG CCAAAATGAA CAAAACCTGT ATGTGAGAGT TACTATGGTT


 


GAATTAGACA GGAAGGGGAG GAGAAAGAGG GCAGCCCTGA TTGGCGTCAT TTCAGCAGTG GTAGCAACAT


 


TTATCCTCAG CTTTTTAGCT TGGTTTTACT TCCGAAGAAG GAAAAGAAGA AGAGGATTAG AAGTTGAAAA


 


TGAGGACATG GAGCTTCCAT TGTTTGATTT AGTTACTGTT ACTACTGCTA CTGATAACTT CTCTTCTGCT


 


AATGTGATTG GAGAGGGAGG CTTTGGACCG GTTTACAAGG GTATCCTACC AAATGGACAA GATATTGCAG


 


TAAAAAGACT ATCGAAGCAT TCTGGACAAG GCTTTCAAGA GTTAAAAAAT GAAATCGCTC TCATTTCCAA


 


GCTGCAACAT AGAAACCTTG TCAAGCTATT GGGTTGCTGC CTTGAAGGAG AAGAAAGGAT GCTAATCTAT


 


GAGTTCATGC CCAATGCTAG CTTGGACTAT TTCATTTTTG ATTCAAGTAG AAAAGCATCA CTTGCATGGA


 


AGAACCGTTT TGAAATTGCT ATGGGAATAT CTCGAGGTCT TCTTTACCTT CACCAGGATT CAAGATTACG


 


AATTATTCAC AGAGATCTCA AGACTAGCAA CATTTTATTA GATACTGACA TGAATGCCAA AATTTCGGAC


 


TTTGGCCTTG CCAAAATTTT TGGTGGAGAT CAAGTGGAAG GAAAAACTAA AAGAGTAATA GGGACATATG


 


GATATATGTC CCCGGAATAT GCTGTTGATG GGAAATATTC AGTAAAATCA GATGTATTCA GCATTGGCGT


 


AATTATTCTT GAAATAGTCA GTGGCAGAAA GAACAGAAAA TTTCGTCATT TGGAACATCA TCACAATCTC


 


TTGGGACATG TAAGCACAAA ACTTCATTCC CTTTATTCAC TCTTAGCTTC CTATTGTGAT TACAACTGTA


 


TGACTGTGTT GTTCTGTTTG TAA


 


SL1.00sc07408_290.1.1 (SEQ ID NO: 56)


ATGGTATTGA TTTTGTTTTT TGTGTCAGCT ATGTTGAGGC TCTTCATTTG CTGTCAATTT CTCTTCATGT


 


TACTAACTTC TGCTGCATTA GACACAATCA CTACAAATAA ATCTATTAGA GATGGTAATA CAATTGTTTC


 


AGCTGGAGGG GTTTATGAGC TTGGATTTTT CAGCCCTGGA AATTCGAAGA ATCGTTACGT TGGCATATGG


 


TACAAGAAGA TATCACCTAC AACTGTTGTC TGGGTTGCAA ACAGAGACAT TCCACTTAAT GACACTTCAG


 


GAGTGTTAAC ACTTAATCCC AATGGAATTC TTGTACTTGT TGATAAATCC AATGTCTCAA TTTGGTCATC


 


AAACTCATCA AGATTGTTAA AGAATCCAAA AGCAAGGCTC CTGGATACCG CAAACCTTGT TGTTAGTGAC


 


GGAAATGATA GAGATCAGGG AATTAATTTC GCGTGGCAGA GTTTTGATTA TCCAGGAAAT ACTTTATTAC


 


CTGGAATGAA GGTAGGAATA GATTTGGTTA CGGGGATGGA TAGGTATGTA ACGTCGTGGA AGAGCACAGA


 


TGATCCTACT CCTGGTGATT ATGTAGATCG TGTTGATTCA CATGGATACC CGCAATTGTT CTTGTCGAGA


 


AATTCATCTG TAGTGTTTAG CTCAGGGCCA TGGACTGGTG CTGCATTTTC TAGTAGTCCT AGTAATAAAC


 


CATCTTTGTA TTATACGTTC GAGTTTGTTA TCAATCAGAA GGAAATTTAC TTCAAATATG AGCTTAAGAG


 


TGACTCTTTG CCCACCAGGG TGGTGCTCAA CCCGGATGGA GTGATACAAC ACCTAATATG GATTGAGCAT


 


ACTCAGAGCT GGTTTCTCTA CTTGACAGCA CAACTTGATA ATTGTGATCG TTTTGCTTTA TGCGGACCTT


 


ATTCAAGTTG CAACATCAAT AACTCCCCTC CATGTGACTG TTTGAAAGGT TTTGAGCCTA GGTATCCTCA


 


AGAATCTGCA GCAGACTGGT CTAGTGGTTG CGTAAGGAGA ACTTCTTTAA ATTGTACCCA TGATGGTTTT


 


CTTAAATTTA CGCGTATCAA GATGCCTGAT TCTAGAAACT CCTGGTATAA TGAGAGAATG AACCTTGAAG


 


ATTGCGAGAA AATGTGTTTA GCTGATTGCA ATTGTACAGC CTACTCAGAT CTTGATGTTA GAAATGGCGG


 


AAGTGGATGC TTACTATGGT TTGGAGAACT CATAGATATA CGAGAATTCA GCCAAAATGA GCAAAATCTA


 


TATGTGAGAG TAGCTGCTTC AGAATTAGGC GAATGTATAT TGACAGGTTC AAAAGTTGAA AATGAGGACA


 


TGGAGCTTCC ATTGTTTGAT TTAGTTACTG TTACAAGTTC CACTGGAAAC TTCTCTTCTG CTAATGTGAT


 


TGGGGAAGGC GGATTTGGAC CGGTCTACAG GGGTATCCTA CCAAGTGGAC AAGAGATAGC AGTAAAGAGG


 


CTATCGAAGT ATTCTGGACA AGGCATTCAA GAGTTAAAAA ATGAAATCGT TCTCATTTCC AAGCTGCAAC


 


ATAGGAACCT TGTCAAGTTA TTGGGTTGCT GTCTTGAAGG AGAAGAACGG ATGCTAATAT ATGAGTTCAT


 


GCCCAACGCT AGCTTGGACT ATTTCATTTT TGATCCAAGC AGAAAAGCTT CACTTGGATG GAAGAATCGT


 


TTTGAAATTG CTATGGGAAT ATCTCGTGGT CTTCTTTACC TTCACCAGGA TTCAAGATTG AGAATTATTC


 


ACCGAGATCT CAAGACCAGC AACATTTTAT TAGATACTGA CATGAATGCC AAAATTTCTG ACTTTGGCCT


 


TGCCAAAATT TTTGGTGGTG ACCAAGAGGA AGGAAAAACT AAGAGAGTAA TAGGGACATA TGGATATATG


 


TCCCCGGAGT ATGCTGTTGA TGGGAAATAC TCAGTAAAAT CAGATGTATT CAGCATCGGT GTAATCATTC


 


TTGAAATAGT TAGTGGCAGA AAGAACAGAA AATTTCGTCA TTTGGAACAT CATCACAATC TCTTGGGACA


 


TGCATGGTTA CTTTGGATTG AAGGCAACGC GTTGGAACTG ATAGACGAAT GTATCAAAGA ATCCTTTTCA


 


GAATCTCAAG TGCTGAGATG CATCCAGGTT GGTTTGTTAT GCGTCCAAAA ACTCCCCGAG GATAGGCCTA


 


CAATGGCATC AGTAGTTTTC TGGTTAGGCA ATGAAGGTCT GGTTCTTCCT CAACCAAAGC AACCTGGTTT


 


TTTCATAGAG AGGAATTCAA TGGAATCAAC AGAATCATCA ACTGATGAAG TATATGTAAG TAGCAGCGTG


 


TCGATAACAG TTCTAGAGCC AAGATAG


 


SL1.0sc07408_289.1.1 (SEQ ID NO: 57)


ATGAAAGGGA AACATTTTTT ATTTTCTTGC TCAATTTTTC TTCCAGTCTT ACTAATTTCC ACTGCATTAG


 


ACACAATCAC AACAGAGAAA CCAATTAGAG ATGGTGACAC AATTATTTCA GCTGGAGGGG TTTTTGAGCT


 


TGGATTTTTC AGCCCTGGAA ATTCGAAGAA TCGTTACGTT GGCATATGGT TTAAGAAGAT AGCAACTAGA


 


ACTGTTGTCT GGGTTGCCAA CAGAAACTTC CCACTGAATG ACAATTCAGG AGTGTTATCA CTCAATCCCA


 


ATGGAATTCT TGTACTTCTT CGTAATTCCA ATGCCTCAAT TTGGTCTTCA AACTCATCAA GATTGTTGAC


 


GAATCCAAAA GCATGGCTCC TGGATTCTGG TAACCTTGTT GTGACTGATG GAAATGATAG TGATCCAGAA


 


GTTAATTTCG CGTGGCAGAG TTTTGATTAT CCAGGAGATA CTTTACTACC TGGGATGAAG CTTGGACGTA


 


ATCTGGTCAC GGGCATGGAT TGGTACATAG AGTCATGGAA GAGCAGTGAT GATCCTGCGC CTGGTGAATA


 


TATAGAACGT CTTGATTCTC ATGGATACCC ACAATTTTTC GTGTGGCAAA ATTCATCTAT AGTATATAGC


 


ACAGGGCCAT GGAATGGTAT CACATTTAGC AGTAGTCCAA AAAATCAACC AGCTATATAT TATGCTTTCG


 


AGTTTGTTAT TAAACAGAAG GAGATTTACT TTAAATACGA GCTAAACGAG TCCCTGCCCA CCAGGGTAGT


 


GATCAATCAG GCTGGAATGG TAGAACACCT AACATGGATT GAGCGAAATC AGAGATGGAT AGTCTATGTA


 


TCAACACAAT CTGATAATTG TGATCGTTTT GCTTTATGTG GTCCTTATGC AAGTTGCAAC ATCAATAACT


 


CTCCTCCATG TGACTGCTTG CAAGGTTTCG AGCCTAGGTA TCCTGAACAA TGGTATGCAG TGGACTGGTC


 


TAATGGTTGT ATAAGGAAAA CTTCTTTGTC TTGTAACCAA GATGGTTTTC TTAAATTTAC GAATATCAAG


 


ATGCCGGATT CTAGACACTC CTGGTATAAT GTAAGCATGA ATCTTGAAGA ATGCAAGAAA ATGTGCTTGG


 


CTGATTGCAA TTGTACAGCC TACTCAAATC TTGATATAAG AAATGGCGGA AGTGGATGTT TACTATGGTT


 


CGGTGAGCTC ATTGATATTA GAGAGTACAA CAAAAATGAG CAACGCCTGT TTGTGAGAGT TGCTGCTTCA


 


GAATTAGATC CAGTCAGGAC TTGGAGGGGA AAGTGGCCAG CTCTGATTGC GGTCATTTCA GCACTAGCAG


 


CAACTTTTAT CCTCATCTTT GTAGCTTGGT TTACCTTCCA AAGAAGGAAC AAAAAAACAG ACAAACATAC


 


TGGAGGTTCA GAAGTTGGAA AGAATGACCT TGAGTTGCCA TTGTTTGATT TAGTTACTGT TACTACTTCC


 


ACTGAAAGTT TCTCTTCTGC GAATGTGATT GGTGAGGGTG GCTTTGGACA GGTTTACAAG GGTATTCTAC


 


CAGATGGACA AGAGATAGCA GTAAAGAAGC TATCGAAGTA TTCTGGACAA GGCGTTCAAG AGTTAAAAAA


 


TGAAATTGTT TTCATTTCCA AGCTGCAACA TAGAAACCTT GTCAAGCTTT TGGGTTGCTG CCTTGAAGGA


 


GAAGAAAAGA TGCTAATCTA TGAGTTTATG CCCAACTCTA GCTTGGACTG TTTCATTTTT GATCCAAGCA


 


GAAAAGCTTC ACTTACATGG AAGAATCGTT TTGAAATTGC TGTGGGAATA TCTCGAGGTC TTCTATACCT


 


TCATCAGGAC TCAAGATTTA GAATTATTCA TAGAGACCTC AAGACCAGCA ACATTTTACT AGATGGCAAC


 


ATGAATGCCA AAATTGCTGA CTTTGGCCTT GCCAAAATTT TTGGCGGAGA ACAAGTGGAA GGAAATACTA


 


AGAGAGTGAT AGGGACATAT GGATATATGT CACCTGAGTA TGCTGTTGAT GGGAAATATT CAATAAAATC


 


CGATGTATTC AGTATTGGCG TCATCATTCT AGAACTAGTT AGTGGCAGAA GGAACAGGAA ATTTCGTCAT


 


TTGGAACATC ATCACAATCT TTTGGGACAT GCATGGTTAC TTTGGACTGA AGACAAAGCG TTGGAACTGA


 


TGGACGAATG TTTGAAAGAA TCATTTGCGG AATCTCAAGT GTTGAGATGC ATCCAGGTTG GTTTGTTGTG


 


CGCCCAAAAA CACCCTGAGG ATAGGCCTAC AATGGCATCA GTAGTTTTCT GGTTGGGAAA TGAAGGCCTG


 


GTTCTTCCTC AACCAAAGCA GCCTGGATTT TTTATCGAAA GGAATTCAAT GGAATCAACA GAATCAGCTC


 


AGTTTATAAA CACGATCACA ACAGATAGAT CCATTAGAGA CGGTGACACA ATTGTTTCAG CTGGTGGGAT


 


TTATGAGCTT GGATTTTTCA GTTCTGGAAA TGCGAAGAAT CGTTACGTAG GCATATGGTA CAAGAAGATA


 


TCAACTCAAA CTGTTGTCTG GGTAGCAAAC AGAGATATTC CACTTATTGA CACATCAGGA GTGTTAATAC


 


TCAAACCCAA TGGAATTCTT GTACTTGTTG ATAATTCCAA TACATCAATT TGGTCATCAA ACTCATCAAG


 


ACCGTTAAAG GATCCAAAAG CACGGATCCT GGATTCCGGG AACCTTGTTG TCAATGATGG AAATGAAAGA


 


GACCTGGAAA TTAACTTCGC ATGGCAGAGT TTTGATTATC CAGGAAATAC TTTTATACCT GGAATGAAAC


 


TTGGACGTAA TTTGGTCACG GGCATGGATT GGTATATGTC GTCTTGGAAG AGCATTGATG ATCCTTCTCC


 


TGGTGAATAT ATAAACCGTC TTGATTCTCA TGGATACCCG CAATTGTTTG TGTGGAAAAA TTCAACTATA


 


GTGTCTAGCT CAGGGATATG GAAAGGTAAT GCATTTACTG TTAGTGCTAA CAGTAGACCA AATACACATT


 


ACACTTCCGA GTTTATAATT AATCAGCAGG AAATTTACTA CCAATTCAAG CTTAAGAACG AGTCACTGCC


 


CAGCAGGATG GTGCTCAACC CGGAAGGGCT GATAGAACAC CTAACATGGA TTGAGAGCAG TCAAAGCTGG


 


TTTCTGTACT CAACAGTACA GTTTGATAGC TGTGGTCGTT TTGCTTTATG CGGTCCTTAT TCAAGTTGCA


 


ACATCAATAA CTCCCCTCCA TGTGACTGTT TGCAAGGTTT CAATCCTAGG GTTCCTCAAC AGTCTGCAGC


 


AGATTGGTCT TCTGGTTGTG TTAGGAGCAC TTCTTTGGAT TGTAACAAAG ACGGTTTTCT TAAATTTACA


 


GGCATCAAGA TGCCTGATTC TAGAAACTCC TGGTTTAATA AGAGCATTAA CCTTGAAGAA TGTGAGAAAT


 


TATGCTTAGC TAATTGCAAC TGTACAGCCT ACTCAAATCT TGATGTCAGA AATGGCGGAA GTGGATGCTT


 


ACTATGGTTC GGAGATCTCA TTGATATTCG AGAGTTGAGC CAAAATGAGC AAAACCTGTT TGTGAGAGTT


 


GCTGCTTCAG AAATAGACAG GAAGCAAAGG AGAAAGATGT CAGTCCTGAT TGGTGTCATT TCAGCAGTGG


 


TAGCAACATT TATCCTCAGC TTTTTAGCTT GGTTTTACTT CCAAAGAAGG AAAAGAAGAA TAGGTCCAGA


 


AGTTGAAAAT GAGGACATGG AGCTTCCATT GTTTGATTTA GTTACTGTTA CTACTGCCAC TGGGGACTTC


 


TCTGCTATGA ATGTGATCGG GAAGGGTGGA TTTGGACCGG TTTACAAGGG TATCCTACCA AATGGACAAG


 


AGATAGCAGT GAAGAGGTTA TCAAAGCATT CTGGACAAGG CTTACGAGAG TTAAAAAATG AATTCGTTCT


 


CATTTCCAAG CTGCAACACA GGCCCCTTGT CAAGCTTTTG GGTTGCTGCC TTGAAAGAGA AGAACGGATG


 


CTCATCTATG AGTTTATGCC CAATGCTAGC TTGGACTATT TCATTTTTGA TCCAAGCAGA AAAACTTCAC


 


TTTCATGGAA GAACCGCTTT GAAATTGCTA TAGGAATATC TAGAGGTCTT CTTTACCTTC ACCAGGACTC


 


AAGATTAAGA ATTATTCACA GAGATCTCAA GACCAGCAAC ATCTTATTAG ATACTGACAT GAATGCCAAA


 


ATTTCTGACT TCGGCCTTGC CAAGATATTT GGTGGAGATC AAGTGGAAGG AGAAACTAAG AGTATAGTAG


 


GGACATATGG ATATATGTCC CCGGAGTATG TTGTTGATGG GAAATATTCA GTAAAATCCG ATGTATTCAG


 


CATTGGTGTA ATTATTCTTG AAATAGTTAG TGGCAGAAAG AACAGGAATT TTCGTCATTT GGAACATCAT


 


CACAATCTCT TGGGACATGC ATGGTTACTT TGGACTGAAG GCAACGCGTT GGAATTCATG GATGAACGTT


 


TGAAAGAATC ATTTTCAGAA TCTCAAGTGT TGAGATGCAT CCAGGTCGGT TTGTTATGCG TCCAGAAACT


 


CCCAGAGGAC AGGCCTATAA TGGCATCAGT GGTTTTTTGG TTGGGAAATG AAGGTCTGGT TCTTCCTCAA


 


CCAAGGCATC CAGGTTTCTT CACAGAGAGG AATCCAATGG AATCTACTGA TGAAGAATGT CTAAGTAACA


 


ACGCGACGTT AACTGTTCTT GAGCCAAGAT AG


 


SL1.00sc07408_288.1.1 (SEQ ID NO: 58)


ATGGCAATGC TTCAATGGTT TCCTCTTCTT TGCATTTTCT TCATCTCAGC TTCTGCTGCT AAAGTTCAAA


 


CCAAGGTAAC TGATAATCCT GCTGATGAGC TGGTATCTGC CCTTAATAGT AACAGAACTG CGAATAAATT


 


ATCCTCCTTA TACAGTAACC CTGGCTTGGC ATGCTTGGCT CTGCAATATA TAAAAGCATA CGGAGGTGAT


 


TGTAAAGTAG TTGGAGGGCC AGATGGAAAG AAACCTGCTG AATCTGAATT CGCCCAAGAA TTTGCCCCCA


 


ACTGTGGTGT GCAGGCATCA TCGCTTGCTC AAATAACTGG AAGATTTCTC GCATGTCAAT CTAAGTATGC


 


GGAACCTTCT GAAGCATTCA ATGATATTCT TATAAGAAAT ACCAAGAGTT TGGATATTCT TGTGTACTCT


 


AATCACACTG AGGTTGGTGC TGCTGTGAGT GGCTCCGGTG GTGGTGGCCC CTATTTCTGG TGTGTACTCT


 


TCAGTAATGG CAAACCAAAG AGTAGCTTTT CCAGAGGTGG AGTTGAGCCT AAGGTAAGTA GACCTGGATG


 


CTTCAGTGGT TCTAATGACC AATGCAGTGG TGCTAATACT TTGTCTCAAA CCATATATCT CTGGACAATC


 


ACTGTAGGAG CTTCCGTTGC ACTGCTTTAT GCCTTAGGAG TATGA


 


SL1.00sc07408_287.1.1 (SEQ ID NO: 59)


ATGGGAATAT CAAGAGGGAT TCTTTATCTT CACCAGGATT CCAGATTAAG GATCATACAT AGAGATCTAA


 


AGACGAGCAA CATTTTACTG GACAGTGAAT GGAATCCCAA GATTTCAGAC TTTGGACTGG CTAGGATTAT


 


TGGCTGTGAC CAAAACGAAG CAAGAACAAA AAGAGTAATA GGGACATATG GAGATATGTC TCCAGAATAC


 


GCAGTTGATG GGAAATTTTC AGTGAAATCA GATGTTTTCA GTCTTGGTGT TCTTCTGCTA GAAATAGTAA


 


GTGGAAGAAA GAACAGAACA TTTCGTCATC CAGATCACCA CCATAGTCTT ATAGGACATG CTTGGTTATT


 


ATGGAACGAA GGAAAAGCCT TGGAACTAAT CGATGATTGT TTAAAAGAAT CGTTTGTGGA ATCCCAAGTG


 


CTAAGATGTG TTCATGTGGC ACTGTTATGT GTTCAACGAC TAACAGATGA AAGACCAACA ATGTCATCAG


 


TGGTATTCAT GTTAAGCCAT GAAGAAGTGG CATTACCTCA ACCAAAGGAG CCTGGTTTCT TCATAGAGAG


 


AAGTATAGCT GAAACAGATG ATTCAAATGA GAAAAGGTGT ATCAGTGACA ATGTTTTGAC ATTAACAATT


 


CTTCAACCAA GATAG

## Claims

1. A method for producing a tomato plant that is tolerant to exposure to continuous light, the method comprising the steps of:
(a) providing a donor tomato plant which is tolerant to exposure to continuous light, selected from the group consisting of S. neorickii, S. chilense, S. pennellii, S. peruvianum, S. habrochaites and S. chmielewskii; 
b) crossing said donor tomato plant with at least one recipient S. lycopersicum plant which is not tolerant to exposure to continuous light, to produce offspring tomato plants,
wherein said crossing results in the introduction of genomic material from said donor tomato plant into the corresponding region of the genome of said at least one recipient tomato plant; and

c) detecting in chromosome 7 of at least one offspring plant the presence of a genomic region from the donor tomato plant linked to tolerance to continuous light, wherein the genomic region comprises a Light-Harvesting Chlorophyll B-Binding Protein 3 (LHCB3) gene;
wherein said genomic region is located on chromosome 7 in a region bordered by markers “7-20-1” and “7-20-2” between 72 and 75 cM based on the EXPEN2000 linkage map; and wherein said genomic region in DNA isolated from the offspring plant is detected by use of a genetic marker, which marker is linked to said genomic region comprising the LHCB3 gene,

thereby producing at least one offspring tomato plant which is tolerant to exposure to continuous light, said offspring plant comprising said LHCB3 gene.

2. A method for increasing the yield of a tomato plant line, said method comprising:
(a) obtaining a tomato plant produced by the method according to claim 1;
(b) crossing the selected plant with a plant of said tomato plant line the yield of which is to be increased to produce offspring plants;
(c) optionally backcrossing or selfing said offspring plant to produce further offspring plants, and
(d) selecting from said offspring plants of step (b) or (c) a plant having said genomic region linked to continuous light-tolerance and exhibiting tolerance to exposure to continuous light and having increased yield as compared to said plant of said tomato plant line used in step (b);
wherein tolerance to exposure to continuous light is determined by exposing the tomato plant for a period of 3 weeks to 90 days of continuous light having an intensity of about 131 μmol m−2 s−1 from fluorescent tubes, wherein the tomato plant is grown at 21° C. and a relative humidity between 60-80%.

3. The method according to claim 1, further comprising:
e) backcrossing said at least one offspring tomato plant which is tolerant to exposure to continuous light to said at least one recipient S. lycopersicum plant which is not tolerant to exposure to continuous light, to produce a S. lycopersicum plant which is tolerant to exposure to continuous light,
whereby said S. lycopersicum plant which is tolerant to exposure to continuous light is characterized by the presence of a genomic region from the donor tomato plant linked to tolerance to continuous light, wherein the genomic region comprises a Light-Harvesting Chlorophyll B-Binding Protein 3 (LHCB3) gene on chromosome 7 in a genomic region bordered by markers “7-20-1” and “7-20-2” between 72 and 75 cM based on the EXPEN2000 linkage map.

4. A method of producing a continuous light-tolerant inbred tomato plant, the method comprising the steps of:
a) obtaining a continuous light-tolerant Solanum lycopersicum tomato plant produced by the method according to claim 3;
b) crossing said continuous light-tolerant tomato plant with itself or another tomato plant to yield progeny tomato seed;
c) growing said progeny tomato seed of step b) to yield an additional continuous light-tolerant tomato plant and;
d) repeating the crossing and growing steps from 0 to 7 times to generate a continuous light-tolerant inbred tomato plant;
wherein said tolerance is determined by the presence of a genomic region linked to tolerance to continuous light, wherein the genomic region comprises a Light-Harvesting Chlorophyll B-Binding Protein 3 (LHCB3) gene of S. neorickii, S. chilense, S. peruvianum, S. habrochaites, S. chmielewskii, or S. pennellii, wherein the genomic region is bordered by markers “7-20-1” and “7-20-2” between 72 and 75 cM based on the EXPEN2000 linkage map; wherein tolerance to exposure to continuous light is determined by exposing the tomato plant for a period of at least 3 weeks to continuous light having an intensity of 131 μmol m−2 s−1 from fluorescent tubes, wherein the tomato plant is grown at 21° C. and a relative humidity between 60-80%.

5. The method according to claim 4, wherein step d) further comprises the step of identifying plants that exhibit continuous light tolerance and possess commercially desirable characteristics.

6. The method according to claim 4, wherein said method further comprises the step of selecting inbred tomato plants homozygous for a genomic region comprising the LHCB3 gene linked to continuous light-tolerance.

7. The method of claim 1, wherein the genomic region comprising the Light-Harvesting Chlorophyll B-Binding Protein 3 (LHCB3) gene is detected by using a molecular marker selected from the group consisting of SEQ ID NO:19 and SEQ ID NO:23.

8. A method for producing a S. lycopersicum tomato plant that is tolerant to exposure to continuous light, the method comprising the steps of:
a) providing a donor S. lycopersicum tomato plant obtained by the method according to claim 3;
b) crossing said donor tomato plant with at least one recipient S. lycopersicum plant which is not tolerant to exposure to continuous light, to produce offspring S. lycopersicum tomato plants, wherein said crossing results in the introduction of genomic material from said donor tomato plant into the corresponding region of the genome of said at least one recipient tomato plant; and
c) detecting in chromosome 7 of at least one offspring plant the presence of a genomic region comprising a Light-Harvesting Chlorophyll B-Binding Protein 3 (LHCB3) gene linked to tolerance to continuous light;
wherein said genomic region is bordered by markers “7-20-1” and “7-20-2” between 72 and 75 cM based on the EXPEN2000 linkage map; said genomic region is detected by use of a genetic marker in DNA isolated from said offspring plant, which marker is linked to said genomic region comprising the LHCB3 gene,
thereby producing at least one offspring S. lycopersicum tomato plant which is tolerant to exposure to continuous light, said offspring plant comprising said genomic region comprising said LHCB3 gene.

9. The method according to claim 1, wherein said donor tomato plant which is tolerant to exposure to continuous light is selected from the group consisting of S. neorickii, S. chilense, and S. pennellii.