An interspeciWc linkage map of SSR and intronic ... - Core

Theor Appl Genet (2010) 121:731–739 DOI 10.1007/s00122-010-1344-3

ORIGINAL PAPER

An interspeciWc linkage map of SSR and intronic polymorphism markers in tomato Kenta Shirasawa · Erika Asamizu · Hiroyuki Fukuoka · Akio Ohyama · Shusei Sato · Yasukazu Nakamura · Satoshi Tabata · Shigemi Sasamoto · Tsuyuko Wada · Yoshie Kishida · Hisano Tsuruoka · Tsunakazu Fujishiro · Manabu Yamada · Sachiko Isobe

Received: 16 December 2009 / Accepted: 11 April 2010 / Published online: 30 April 2010 © The Author(s) 2010. This article is published with open access at Springerlink.com

Abstract Despite the collection and availability of abundant tomato genome sequences, PCR-based markers adapted to large scale analysis have not been developed in tomato species. Therefore, using public genome sequence data in tomato, we developed three types of DNA markers: expressed sequence tag (EST)-derived simple sequence repeat (SSR) markers (TES markers), genome-derived SSR markers (TGS markers) and EST-derived intronic polymorphism markers (TEI markers). A total of 2,047 TES, 3,510 TGS and 674 TEI markers were established and used in the polymorphic analysis of a cultivated tomato (Solanum lycopersicum) ‘LA925’ and its wild relative Solanum pennellii ‘LA716’, parents of the Tomato-EXPEN 2000 mapping population. The polymorphic ratios between parents Communicated by I. Paran. Electronic supplementary material The online version of this article (doi:10.1007/s00122-010-1344-3) contains supplementary material, which is available to authorized users. K. Shirasawa · E. Asamizu · S. Sato · Y. Nakamura · S. Tabata · S. Sasamoto · T. Wada · Y. Kishida · H. Tsuruoka · T. Fujishiro · M. Yamada · S. Isobe (&) Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu, Chiba 292-0818, Japan e-mail: [email protected] E. Asamizu Gene Research Center, University of Tsukuba, Ten-no dai 1-1-1, Tsukuba, Ibaraki 305-8572, Japan H. Fukuoka · A. Ohyama National Institute of Vegetable and Tea Science, 360 Kusawa, Ano, Tsu, Mie 514-2392, Japan Y. Nakamura National Institute of Genetics, 1111 Yata, Mishima, Shizuoka 411-8540, Japan

revealed by the TES, TGS and TEI markers were 37.3, 22.6 and 80.0%, respectively. Those showing polymorphisms were used to genotype the Tomato-EXPEN 2000 mapping population, and a high-density genetic linkage map composed of 1,433 new and 683 existing marker loci was constructed on 12 chromosomes, covering 1,503.1 cM. In the present map, 48% of the mapped TGS loci were located within heterochromatic regions, while 18 and 21% of TES and TEI loci, respectively, were located in heterochromatin. The large number of SSR and SNP markers developed in this study provide easily handling genomic tools for molecular breeding in tomato. Information on the DNA markers developed in this study is available at http:// www.kazusa.or.jp/tomato/.

Introduction The tomato, Solanum lycopersicum, which originated in Latin America, is the second most important vegetable crop and is cultivated throughout the world (Foolad 2007). Tomato belongs to the family Solanaceae, which consists of approximately 100 genera and 2,500 species, including several plants of agronomic importance such as potato, eggplant, pepper and tobacco (Olmstead et al. 2008). Tomato has a relatively compact genome within the Solanaceae species, characterized by its diploidy (2n = 2X = 24). It is approximately 950 Mb in size, and is one of the most intensively characterized Solanaceae genomes (Arumuganathan and Earle 1991). The International Tomato Sequencing Project, established in 2004 with members from 10 countries, promotes structural genome analysis in tomato by sequencing the gene-rich regions of all 12 chromosomes through the generation of high quality sequences from bacterial artiWcial chromosomes (BAC). The data are released

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immediately after completion, principally through the SOL Genomics Network (SGN) website (http://solgenomics.net/ ; Mueller et al. 2005). In addition, ESTs and full-length cDNAs of a miniature tomato cultivar (S. lycopersicum) named Micro-Tom have been published at MiBASE (http:// www.kazusa.or.jp/jsol/microtom/) and KafTom (http:// www.pgb.kazusa.or.jp/kaftom/) (Yamamoto et al. 2005). Currently, more than 320,000 ESTs and 461,000 BAC-end sequences have been registered in the databases. Genetic diversity in the cultivated tomato is generally low, due to the occurrence of population bottlenecks during the domestication and the generation of modern varieties (Rick 1976). Therefore, during the past quarter century, several linkage maps of the tomato genome have been developed with more than 20 mapping populations derived from crosses between cultivated tomato (S. lycopersicum) and its wild relatives, such as S. pennellii, S. pimpinellifolium, S. cheesmaniae, S. neorickii, S. chmielewskii, S. habrochaites and S. peruvianum (reviewed by Foolad 2007). In particular, an F2 mapping population named Tomato-EXPEN 1992, which was derived from a cross between S. lycopersicum cv. VF36 and the inbred accession of S. pennellii LA716, facilitated the development of a high-density restriction fragment length polymorphism (RFLP) linkage map with 1,030 loci (Tanksley et al. 1992). Subsequently, other types of DNA markers, such as random ampliWed polymorphic DNA (RAPD), simple sequence repeats (SSRs), ampliWed fragment length polymorphisms (AFLPs), cleaved ampliWed polymorphic sequence (CAPS) and single nucleotide polymorphisms (SNPs), were developed and mapped onto the Tomato-EXPEN 2000 mapping population derived from the cross between S. lycopersicum LA925 and S. pennellii LA716 (Frary et al. 2005; Fulton et al. 2002). Currently, the Tomato-EXPEN 2000 map comprises a total of 2,604 markers including 1,088 CAPS, 1,342 RFLP, 155 SSR and 19 SNP markers (http://solgenomics.net/cview/map.pl?map_id=9; Fulton et al. 2002). In addition, a total of 349 PCR-based markers are now available at Tomato Mapping Resource Database (http:// www.tomatomap.net), and mapped on interspeciWc crosses between S. pimpinellifolium and S. lycopersicum. Most of the loci mapped on the two Tomato-EXPEN mapping populations consist of RFLP and CAPS markers, which result in a higher cost for the identiWcation of polymorphisms due to their laborious nature and the need for restriction enzymes. The rapid progress in genome analysis during the past several years has enabled large scale segregation analysis in molecular genetics. We therefore considered it essential to develop PCR-based markers designed to adapt large scale genotyping systems, such as SSR and SNP markers, for the promotion of genetics and genomics in tomato species. Recently, Ohyama et al. (2009) developed SSR markers using BAC-end and

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Theor Appl Genet (2010) 121:731–739

cDNA sequences in tomato and mapped a total of 148 SSR loci onto the EXPEN 2000 map. They investigated a total of 89,824 cDNA and 310,583 BAC-end sequences to generate SSR markers. Their results suggested that abundant sequence resources would allow the generation of a larger number of PCR-based markers in tomato. In this study, we developed three types of DNA markers, namely EST-derived SSR markers (TES markers), genomederived SSR markers (TGS markers) and EST-derived intronic polymorphism markers (TEI markers), using the public genome sequence data. As Ohyama et al. (2009) reported that most of the SSR markers derived from BACend sequences mapped to the centromeric regions, we used diVerent polymorphic sources for marker generation. The developed markers were mapped onto the TomatoEXPEN 2000 mapping population to Wll in the gaps in knowledge within and between tomato genomics and genetics.

Materials and methods Plant material A previously reported F2 mapping population, which was derived from a cross between S. lycopersicum LA925 and the inbred accession S. pennellii LA716 (Fulton et al. 2002), was used for linkage map construction. The mapping parents and 83 F2 individuals were kindly provided by Prof. S. Tanksley from Cornell University. Total DNA was extracted from the leaves of each plant using the DNeasy Plant Mini kit (Qiagen, Germany). Development of TES (tomato EST-SSR) and TGS (tomato Genome-SSR) markers Microsatellite or SSR regions were identiWed in tomato ESTs and BAC-end sequences registered in public databases, namely MiBASE (http://www.kazusa.or.jp/jsol/ microtom/), KafTom (http://www.pgb.kazusa.or.jp/kaftom/) and SGN (http://sgn.cornell.edu/). SSRs longer than 14 bases, which contained all possible combinations of dinucleotide (NN), trinucleotide (NNN) and tetranucleotide (NNNN) repeats, were identiWed using the FINDPATTERNS module in the GCG software package (Accelrys Inc., USA). Oligonucleotides for PCR primers were designed based on the Xanking regions of the identiWed SSRs using the Primer3 program (Rozen and Skaletsky 2000) in such a way that the ampliWed products ranged between 90 and 300 bp in length. Markers corresponding to those previously developed by Fulton et al. (2002) and Ohyama et al. (2009) were identiWed based on the associated nucleotide sequences and excluded from our collection

Theor Appl Genet (2010) 121:731–739

733

of EST-derived SSR (TES) and BAC-end sequencesderived SSR (TGS) markers. PCR was performed using 0.5 ng tomato genomic DNA in a 5 l reaction mix containing 1£ PCR buVer (BIOLINE, UK), 3 mM MgCl2, 0.04 U BIOTAQTM DNA polymerase (BIOLINE, UK), 0.2 mM dNTPs and 0.8 M of each primer. A modiWed ‘touchdown PCR’ protocol was used, as described previously (Sato et al. 2005). PCR products were separated either on a 10% polyacrylamide gel with TBE buVer according to the standard protocol or in a Type 3730 DNA fragment analyzer (Applied Biosystems, USA). In the latter case, the data were analyzed using GeneMapper software (Applied Biosystems, USA).

using the grouping module of JoinMap® and based on an LOD score of 4.0–10.0. Marker order and genetic distance were calculated using a regression mapping algorithm with the following parameters: Kosambi’s mapping function, recombination frequency · 0.35, LOD score ¸ 2.0. Genotype probabilities which show possible genotyping and data entry errors were investigate for all the mapped markers and presented as ¡log10(P). Euchromatic and heterochromatic regions in each linkage group were assumed based on the previously reported positions of markers anchored to heterochromatin (Frary et al. 2005; Ohyama et al. 2009; Tang et al. 2008; Wang et al. 2006).

Development of TEI (tomato EST-derived intronic polymorphism) markers

Results Development of SSR markers

The positions of introns were predicted by comparisons between KTU2 EST unigene sequences in MiBASE and Arabidopsis thaliana genomic sequences (http://www.arabidopsis.org/) using the GAP2 program (Huang 1994). Oligonucleotides for PCR primers were designed based on the Xanking regions of the predicted intron positions in the tomato unigenes using the Primer3 program (Rozen and Skaletsky 2000). PCR was performed as described above. Polymorphic PCR amplicons were identiWed by high-resolution melting (HRM) analysis (Palais et al. 2005) using the LightScanner system (Idaho Technology, USA) with 0.5 l of 10£ LCGreen Plus + Melting Dye (Idaho Technology) as the Xuorescent dye and 0.05 ng of genomic DNA of S. pennellii ‘LA716’ as the control DNA. Linkage analysis Linkage analysis was performed using genotypic data derived from the markers developed in this study along with data derived from a total of 683 published markers. These markers included 449 RFLPs, 61 CAPS markers and 173 SSR markers (Fulton et al. 2002; Ohyama et al. 2009), which were retrieved from the SGN (http://sgn.cornell.edu/) and VegMarks (http://vegmarks.nivot.affrc.go.jp/) databases. The genotypic data of the mapping population generated from the published markers were kindly provided by Prof. S. Tanksley at Cornell University and Dr. A. Ohyama at the National Institute of Vegetable and Tea Science of Japan (NIVTS). Henceforth, we refer to these as Cornell markers and NIVTS markers, respectively. Linkage analysis was performed using the JoinMap® program version 4 (Van Ooijen 2006). The map positions of the Cornell markers reported by Fulton et al. (2002) were used as a frame for the linkage analysis. The genotyped markers were roughly classiWed into 12 linkage groups, which corresponded to the previous EXPEN 2000 map,

A total of 7,599 SSR markers were generated by in silico data mining of 83,785 sequences, as described in the “Materials and methods”, and designated TES (Tomato EST-SSR) markers. Of these generated markers, those corresponding to the Cornell and NIVTS markers were excluded from the TES marker group. Of the SSRs in the TES marker group, 6,043 (80%) were trinucleotide repeats, while 525 (7%) were dinucleotide repeats and 1,031 (13%) were tetranucleotide repeats (Table 1). The poly (AAG)n motif was the most abundant of the trinucleotide repeats (1,712 SSRs, 28%), followed by poly (ATC)n (919 SSRs, 15%), poly (AGC)n (708 SSRs, 12%) and poly (AAC)n (641 SSRs, 11%). While three types of dinucleotide repeats were observed, poly (AT)n and poly (AG)n were the most abundant and comprised 96% of the dinucleotide repeats. Among the tetranucleotide repeats, AT-rich motifs, namely poly (AAAT)n, poly (AAAG)n and poly (AAAC)n, were more frequently observed than other motifs and when combined, comprised up to 59% of the tetranucleotide repeats. In addition to the TES markers, genome-derived SSR markers were generated by extracting SSRs from the 90,763 BAC-end sequences retrieved from the SGN database. A total of 13,501 primer pairs were designed to amplify the SSRs, which were subsequently named TGS (Tomato Genome-SSR) markers. Among the TGS markers, 7,005 of the SSRs (52%) were trinucleotide repeats, while 2,338 (17%) were dinucleotide and 4,158 (31%) were tetranucleotide repeats (Table 1). Poly (AAT)n was the most abundant trinucleotide repeat (2,517 SSRs, 36%), followed by poly (AAG)n (1,771 SSRs, 25%), poly (AAC)n (1,012 SSRs, 14%) and poly (ATC)n (691 SSRs, 10%). Four types of dinucleotide repeats were observed in the SSRs of the BAC-end sequences, and poly (AT)n was the most frequently observed motif (71% of the identiWed dinucleotide repeats). Similar to the TES markers, AT-rich motifs

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734 Table 1 Numbers of SSR motifs in the TES and TGS markers

Theor Appl Genet (2010) 121:731–739

Motif

Dinucleotide

Trinucleotide

Tetranucleotide

TES Designed (%)

Mapped (%)

AC

61 (0.8)

AG

229 (3.0)

AT

235 (3.1)

Designed (%)

Mapped (%)

4 (0.6)

272 (2.0)

27 (4.3)

39 (6.0)

408 (3.0)

35 (5.5)

44 (6.8)

1,653 (12.2)

192 (30.3)

GC

0 (0.0)

0 (0.0)

5 (0.0)

0 (0.0)

AAC

641 (8.4)

56 (8.6)

1,012 (7.5)

38 (6.0)

AAG

1,712 (22.5)

147 (22.7)

1,771 (13.1)

82 (12.9)

AAT

539 (7.1)

84 (13.0)

2,517 (18.6)

117 (18.5)

ACG

121 (1.6)

9 (1.4)

41 (0.3)

1

ACT

188 (2.5)

19 (2.9)

248 (1.8)

11 (1.7)

(0.2)

AGC

708 (9.3)

34 (5.2)

199 (1.5)

5 (0.8)

ATC

919 (12.1)

59 (9.1)

691 (5.1)

17 (2.7)

GGA

491 (6.5)

22 (3.4)

253 (1.9)

5 (0.8)

GGC

230 (3.0)

19 (2.9)

44 (0.3)

1 (0.2)

GGT

494 (6.5)

45 (6.9)

229 (1.7)

5

(0.8)

AAAC

160 (2.1)

11 (1.7)

403 (3.0)

9 (1.4)

AAAG

274 (3.6)

12 (1.9)

690 (5.1)

10 (1.6)

AAAT

272 (3.6)

20 (3.1)

1,892 (14.0)

45 (7.1)

AACG

3 (0.0)

0 (0.0)

11 (0.1)

0 (0.0)

AAGC

40 (0.5)

1 (0.2)

44 (0.3)

3 (0.5)

AATC

56 (0.7)

5 (0.8)

165 (1.2)

4 (0.6)

AATG

47 (0.6)

1 (0.2)

194 (1.4)

0 (0.0)

AATT

71 (0.9)

8 (1.2)

607 (4.5)

23 (3.6)

AGGC

2 (0.0)

0 (0.0)

1 (0.0)

0 (0.0)

AGGT

2 (0.0)

0 (0.0)

14 (0.1)

0 (0.0) 0 (0.0)

GACG

2 (0.0)

0 (0.0)

4 (0.0)

GACT

5 (0.1)

1 (0.2)

11 (0.1)

0 (0.0)

GAGC

8 (0.1)

0 (0.0)

6 (0.0)

0 (0.0)

GATC

9 (0.1)

2 (0.3)

4 (0.0)

0 (0.0)

GGAC

2 (0.0)

0 (0.0)

4 (0.0)

0 (0.0)

GGAT

27 (0.4)

0 (0.0)

25 (0.2)

1 (0.2)

GGCA

5 (0.1)

0 (0.0)

6 (0.0)

0 (0.0)

GGCC

2 (0.0)

0 (0.0)

1 (0.0)

0 (0.0)

GGCT

6 (0.1)

1 (0.2)

3 (0.0)

0 (0.0)

GGGA

22 (0.3)

2 (0.3)

36 (0.3)

1 (0.2)

GGGC

0 (0.0)

0 (0.0)

3 (0.0)

1 (0.2)

GGGT Total

were more frequently observed in the tetranucleotide repeats of the TGS markers. The combination of poly (AAAT)n, poly (AAAG)n and poly (AATT)n motifs comprised 86% of the tetranucleotide repeats. From all the generated SSR markers, 2,047 TES and 3,510 TGS markers were examined for polymorphisms between the parents of the EXPEN mapping population. A total of 451 TES markers (22.0% of all tested markers) and 229 TGS markers (6.5%) showed co-dominant polymorphisms between the parents. In addition, 313

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TGS

16 (0.2)

3 (0.5)

34 (0.3)

1 (0.2)

7,599 (100)

648 (100)

13,501 (100)

634 (100)

TES markers (15.3% of all tested) and 566 TGS markers (16.1%) showed dominant polymorphisms. Therefore, 41% [313/(451 + 313)] of all TES markers and 71% [566/(229 + 566)] of all TGS markers were dominant. In most of the dominant markers, null alleles were observed in S. pennellii (LA716). The polymorphic ratios for the di-, tri- and tetranucleotide repeats among the TES markers were 13.5, 75.7 and 10.9%, respectively, while those for the TGS markers were 40.8, 43.8 and 15.5%, respectively.

Theor Appl Genet (2010) 121:731–739

Development of TEI (tomato EST-derived Intronic polymorphism) markers A total of 589 unigenes retrieved from a KTU2 unigene set in the MiBASE database were aligned with A. thaliana genome sequences using the GAP2 program (Huang 1994). By taking the GU-AG rule into account, the positions of 1,073 introns were predicted for 206 of the unigene sequences. The mean number of predicted introns per single unigene was 5.2. Primer pairs were designed using sequences of the Xanking regions of the predicted intron positions to generate DNA markers named TEI (tomato EST-derived intronic polymorphism). A total of 674 TEI markers were tested for polymorphisms using HRM analysis, as described in the “Materials and methods”, and 537 of them (80%) showed polymorphisms between the mapping parents. These 537 TEI markers represented 166 independent unigenes. Therefore, 166 non-redundant TEI markers were selected for subsequent linkage analysis. Of these selected TEI markers, 148 and 18 showed co-dominant and dominant polymorphisms between the parents, respectively. Though the SNPs were identiWed by HRM, it is sometimes not possible to identify polymorphisms between heterozygous and one parental speciWc homozygous SNPs, due to the Xanking sequences of the SNPs. These kinds of SNPs were scored as dominant SNPs. Construction of a linkage map Segregation data were generated for a total of 1,725 markers (764 TES, 795 TGS and 166 TEI markers) in the Tomato-EXPEN 2000 mapping population. These data were combined with data derived from 547 Cornell and 136 NIVTS markers and subjected to linkage analysis. The map positions of the 547 Cornell markers reported by Fulton et al. (2002) were used as a frame for the construction of the linkage map. Subsequently, 2,116 loci, including 634 TGS, 648 TES, 151 TEI, 36 NIVTS and 547 Cornell loci, were mapped onto 12 linkage groups corresponding to the 12 chromosomes (Chrs), while 292 loci, including 161 TGS, 116 TES and 15 TEI loci, were excluded from the analysis. The total length of the linkage groups was 1,503.1 cM, as shown in Table 2, Fig. 1 and the Supplementary Table. The total number of newly mapped loci was 1,433, ranging from 105 to 160 in each linkage group. The average distance between two loci was 0.71 cM ranging from 0.59 cM (Chr 9) to 0.88 cM (Chr 5). Of the 2,116 loci mapped in this study, 1,481 and 635 loci were assumed to be located in euchromatic and heterochromatic regions, respectively, according to the position of markers anchored to heterochromatin reported in previous studies (Frary et al. 2005; Ohyama et al. 2009; Tang

735

et al. 2008; Wang et al. 2006). The markers used as anchor markers of euchromatic and heterochromatic regions are listed in the Supplemental Table. In the present map, 48% (305) of the mapped TGS loci were predicted to be present in the heterochromatic regions, while only 18% (119) and 21% (32) of the TES and the TEI loci were in the same regions. Segregation distortion was observed for 38.2% of the mapped marker loci (Fig. 1, Table 2). The distortion ratios varied from chromosome to chromosome; Chr 4, 5, 8, 9 and 12 showed distortions for less than 10% of the mapped loci, while Chr 1, 10 and 11 showed distortions for more than 70% of the loci. Heterochromatin-speciWc segregation distortion was observed for Chr 2 and 7. The ‘LA716’ (S. pennellii) genotypes were more frequently observed in the distorted loci mapped to Chr 1, 2, 3, 6, 10 and 11, while more ‘LA925’ (S. lycopersicum) genotypes were identiWed in the distorted loci mapped to Chr 7.

Discussion In this study, we developed three types of DNA markers designated TES, TGS and TEI. The TES markers that contained trinucleotide repeats exhibited a higher ratio of polymorphisms than those containing di- and tetranucleotide repeats. On the other hand, a signiWcant diVerence was not observed in the frequency of polymorphisms between diand trinucleotide repeats in TGS markers. Dinucleotide repeats in coding regions often causes critical changes, such as frame-shift mutations. The decreased number of polymorphisms present in the dinucleotide repeats in TES markers in comparison to TGS markers suggested that the coding region contained higher sequence conservation than the intergenic regions of S. lycopersicum and S. pennellii. Among the polymorphic markers, the percentage of dominant TES markers was 41% [313/(451 + 313)], while that of dominant TGS markers was 71% [566/(229 + 566)]. In most of the dominant markers, null alleles were observed in S. pennellii. As the primers were designed from the S. lycopersicum genome and EST sequences, sequence divergence between the two species might cause poor annealing of the primers to the S. pennellii genomic DNA. The results also indicate that sequence conservation is higher in the intragenic regions than the intergenic regions. A segregation distortion ratio was observed for more than 70% of loci mapped to Chr 1, 10 and 11, while in less than 10% of loci mapped to Chr 4, 5, 8, 9, and 12. Segregation distortion or transmission ratio distortion (TRD) in a hybrid population can result from various factors, such as hybrid lethality or sterility of gametophytic competition (Harushima et al. 2001). The bias of segregation distortion can be explained by the presence of more TRD factors on

123

123 TES1301

C2_At5g40530 TES0567TES0490 T1096 TES0088 T1202T1554 TM17T1323 C2_At3g26900 TG154

TES1925

TEI0576

cLEX-2-I9

T1306TG27 T283A

TM22 TGS0127TEI0560 TGS2443

C2_At4g37300

T706 TES1753

TES1644

TES0513

C2_At4g37130 ORFX-2 T1744 T1400 TGS3418

TGS2420

TEI0522 cLES3G11 T347TG34 TGS1770 TGS0902 TES1673 cLPT-1-A21

TES1711 TEI0412

TGS2811 TGS3319

TGS2810 TGS1548 TEI0339 T1532 CT38 TEI0960 TES0811 TES0992 TGS1462 cLET3K13 T1395cLEF50C16 cLEX4I9 TES1042 TES1276 SSR580 SSR331 TEI0071 C2_At5g64670 TEI1013TEI0470 T702T86 TM20 TGS2417 SSR598SSR32 TES0614 TES0195 cLET-1-A5 TES0787 TES1579 TES0378 T759T1671 TES1132 TGS3159 TES1270 TES0602 TES1149 TGS2553 TG463 TES0470 P18 cLEC-7-H4 TEI0659 TES1593 TEI0121 TES0373 TES1669 TG645R TES0368 U146494 TG48 T628 TES1387 T147 TG518 TG337 TES1261 TGS1372 TES0533 TGS3413 TEI0392 TGS1448 TES1003 TGS2722 CT9

TES1413

C2_At4g21710 TES0234 TES0912 TES1691

TES0742

TES1976

TGS0336 TES0458

cLEW-17-M24 TEI0633 TEI0246

U242666 T1665 tma0105 TGS0541

cLPT1E8 TG451 T1654 cLEC27M9

TES1676 TGS2649 TES0531 TGS2094 TES0424 T697 T1668 TES0306 TES0074 TGS2022 TES1081 TES0037 T1256 TES0854 T1516 TEI0517

TGS0785 TGS0634 TGS2446 CT255T1768 TES0366

TES0488

TES0203 TM6 C2_At4g34700 TEI0866

#

# #

##

#

# ### ###

tme0140 TES0058 TES1542

TES0782 T1706 TGS0571 T869 TES0872 TEI0387 SSR40

CT140

T1117 TEI0565 T888T1238 TES0055

TES0332

TES0334

T1782TGS2729

C2_At4g34350

tma0143

tme0013 TES1802 TES1971 tme0059 TES1738 TGS2711 TES0915 cLEC-4-E12T1650 TEI0013 cLET-4-N7cTOB8A5 TGS0377 TES0233TES0943 TES0327 TGS0209 TGS3442 TGS3441 TES0821 CD15 TGS1270 TES0976 TES0277 TGS0379 TGS2972 TGS2028 TES0020 TGS0239 tmc0046 TES0939 TES0309 TGS2047 TGS0349 tma0210 TGS1152 TGS0471 TGS1164 TGS0419 tma0096 TES0932 U224105 SSR98 tma0171 TGS2812 CT62T1422 TGS1052TGS0972 tma0211TGS2839 tmc0035TEI0461 TGS2930 tma0268tmb0113 TGS0454TGS2021 TGS2837TGS0358 TGS0289TGS2085 TGS3496TGS3081 TGS2147TGS0888 TGS1686TGS2380 TGS2458 tma0173TES0812 TES1137TES0408 TGS3437 TGS2833 T1619 TES1259 TES0489 TGS1523 TGS2720 TES0217 T1957 TGS2326 TGS2684 tma0180 TES0983 TGS0248 tma0184 cSTA-6-E19 T1704 TGS1030 TGS0983 TGS0981 SSR51 TES0514 TES0085 TES0109 TES1688 TES0609 TES1849 TES0139 TES1262 T1208 cLET3E17CT231 T1627T1662 T1629T1677 SSR270T67 cLEY14C14 cLET2O9 T1958 TES1242 TGS0685 TGS1273 TGS1265TGS0635 TEI0133 TES0163 TES0039 TES1349 TGS1999 TGS1329 TES0175 TES1412 cLEG-27-F13 TES0397 TGS1584 cLET-5-J13 TGS2700 TES0785 TGS3262 SSR135 TES1547 T1284 C2_At4g00090 TES0291 T1162 TES1192 TES1166 TGS2986 TES0464 cLES-5-J1 TEI0104 TGS2362 P51 TES0967 TGS0486 TEI0548 TES1943 TM21 TEI0440 TGS2044 cTOF-17-H17T767 TES1444 cLET-7-E12 TGS2162 SSR29 TES0026 C2_At2g38730 TEI0646T852 TES1414TES0751 TES0687 T1963 TES1752 TES0114 TM40 T664 cLPT-5-M7 T1488 TEI0088 TEI0125 TES0083 TEI0053 TES1886 TGS2878 TES1638 TG142 TGS1141 TES1345 TGS1156 TGS1593 T276 TG161TES0859 TGS3281 TGS0800 TES0134 T141 T1669 TES0238 TGS2231 TG245 TGS2741 TES1036 T1084 TES0219 TES1231 TGS2063 SSR117 TES0239 T890T1109 T1557T1664 TES1683 TEI0586 TG17 T896 SSR156 TES1396 TES0985 TG474 TG573 TGS2899 TGS2980 TGS3285TGS1673 tme0041 T620C2_At2g15890

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C2_At5g37260 TGS0897 TEI0920 TGS0903 TGS3062 TGS3056 TEI0643 TGS2279 TGS1832 TGS2795 TGS0444 cLEC-7-P21 TGS2414TGS0291 TGS2853 tma0077 tma0292 tma0256 tma0121 TG31 TES1791

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TES1187 T677 TGS0831 TES0168 tmc0055 TGS2705 TGS1216 TGS2557 TES1712 TGS0373 TGS0693 TES0392 TES1837 TGS2314 TES1434 TGS0437 TGS0561 TES1297 TES1317TES1084 TES1214TES1049 TGS1681 TES1806 TGS3388 TGS2582 tmb0008 TGS0254 TGS2461 tmc0025 TGS0064 TGS2375 TGS0742 tma0119 TGS1142 tma0069 TGS2538 TGS3005 TGS2159 TGS0705 TGS3007 TEI0278 TGS0749 TGS2927 TEI0599 TEI1066TEI0637 tma0123TES0174 tma0186 TGS0958 TGS2129 TGS0420TGS2301 TES1623TES1907 TGS2270TGS0544 tmc0021 TGS2121TGS2050 TGS1279TGS1684 TGS2440 TES1237 TES0321TES0142 TES0949TES1249 TES1222 TGS0534 TES1504 TES0293 TES1330 TES1253 TGS0530 TEI0539 T1751 TES1772 tme0012 TES0323 TGS0054 TGS2112 TES0876 TGS0500 TES1461 tma0181 TES1063 TGS3492 T1260 T1331 TES1138 TES0883 TGS2668 TES1660 T1278 TEI0030 T1659 TES1272 TGS0479 TEI0146 T772 TES0530 TGS2732 TGS0945 T1511 TES1581 cLET-21-N18TGS1916 T518 TGS3172 TG246 TES0159 TES0050 TGS0583 T196 TEI0390 TES1256 TES0621 TES0877 TES1685 TGS2288 TES0461 T1130 T781T728 TES1255 TEI0057 TES0359 TES1885 TES0056 T577 TES0748 TGS1405 TGS2195 TG284 TES0731 TES1792 TES0132 TGS0964 TGS0207 TGS1323 T1935 TGS1442 TES0546 TEI0002T761 TGS0565 TES1714 T581 TGS3218T482 TES0091 TES0646 TGS1173 CT243 TES0324 TES1874 TES1935 TES0077 TES1026 TGS2504 cLET-5-F17 TES0221 TES0023 TES0835 T1143 TGS0767 TES0498 SSR201 SSR31 SSR601 TES0299 tme0045 TG549 tme0062 SSR300 C2_At5g49830 TGS1371 TES1203 TG244

TES0480

TES2046 TGS0740

TES1197

TGS1138

T1286

TES0894

TEI0712

TES1207TES0133 CAB3

TGS0827 TEI0528 TES1139

TG479

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TM31

SSRB60800

TES1225 TGS2080 TG464 TG163cLPT2K6 cLPT1K12 T1203 TEI0211

C2_At1g47830 T360 T1122

TEI0380 TES1505TES0995 T861 CT173 T739

TES1652

TES1100 TEI0491 TM13 TGS1126 TES1513 cLED-19-B12 TGS2756 TES0051

T974

TG443

TGS2379 TGS1027 TGS0407 TES0491 TGS0283 TGS1244 TGS2910 TGS2238 tmb0140 T635 TES0710 tma0271 TGS2528 tma0228 tmb0019 TGS2814 tmc0041 TGS1008 tme0078tma0220 TGS0646TGS2131 TGS1791 TES0484 TGS1160 TGS1518TES0734 tma0303TGS0777 TGS3333TGS2243 TGS2944TGS0442 TGS2495TGS2258 TGS0580TGS2134 TGS0554 TES0574 TGS0215 TGS2016 TGS1366 T891 tma0290 TES1657 TES1379 T725 TGS2652 TES1500 TGS2803 C2_At1g05055 TGS3254 TES0060TES1040 CT258 TES0853 TES0476 TGS2760 TEI0348 TG287 CLV2 T1107 T877 TGS0602 TGS3058 T883 T819 TES0272 TES0523 TES1709 CT178 T1050 T1929 TES2033 TES0310 TES1210 T737 TES0829 TES1180 TES1809 TGS0292 TEI0139 T1232 T1975 TG574 TES0180 TES0287 TES0722 TM19 P56 TES0032 TES0582 TES1921 TES1927 TES0087 tma0100 TG555 TEI0050 TGS2285 T1955 TES1364 TG155T769 TES0098 TGS1998 T1794T708 cLEC-7-M20 CT50 TGS1157 TGS2730

TES1141

TES0177

TES1023 TGS0778 TEI0645 TGS2046 TGS2802 TES0294 T703 TES0968 TES1955 T1068

TGS0411

T506 TEI0670 cLET5G20T208 TM41 T741

SSR43 TES1741

TES0619 CT229 TES0826

TG15

TGS0899

TES0161 TES0030 TEI0494

CD59

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TGS0242 T633

TES1319 TES1626 TES1546 TES0671

TG69

T1584 TES2039 TES0099

cLET-7-N9 TES1230 TES0382 TGS3224 TES0062 TGS0639 TGS0232 TGS0161 TGS0131 TGS3246 TGS1557 tma0146 T40 tma0252 TGS0914 TGS0645 TGS2113 tmb0058 TGS0070 TGS2682 TGS0669 TGS3461 TGS1544 TGS2370 tma0098 T328TG504 C2_At1g27980 T1319T181 TG379 tma0053tma0094 TGS1901TGS0429 TGS2421tmb0030 tma0107TGS0364 TGS1714tma0296 TGS0504TGS0874 TGS3302TGS3494 TGS3190TGS2645 TGS0567 tma0298 TEI0063TGS0595 TEI0326 TGS0155 TES0188 TGS2277 TGS0661 TGS3107 TES0269 TES1771TGS0341 TGS0633 TES1687 TES0804 TGS0576 TGS0443 TGS2572 TGS2459 TGS2519 TEI0186 TG318 TES0145 TES2006 TG96 TES1318 TES0089 TES0045 TES0103 TES0358 TES0353 T730 TES0454 TES0069 U221402 T1746 TES1044 CT172 TES0674TES1750 TEI0061 TES1594 C2_At5g49510 TGS0986 TES1523 TES1265 TES1838 TGS1606 TES0818 TES1307 tma0305 TES0599

TGS0038

TEI0467

TGS1499

CT93

TES0151 CD64

TES0510 T1335

TES1325 TES0534

TGS1360

SSR115

TES0012 TES0155 CT167 TES0941

TGS1457

TES1224

TES0240

TES0053

TG441 TGS2385

TES0261

TEI0145

T1592

TES0363 U227536

T876

T1181 T1252 TES0729 TES0430TES0898 TEI0331 T1440 TES0690 TEI0772 TES1770 TG623 TES0204 TGS1036 TGS3258 TES0110 TES0460 TEI0177 tme0134

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TG581 SSR350 TES0213TGS1802 C2_At4g28530 TG482cLEY-13-J2 TG115

TGS0588 TES1563 TEI0447

TES1406 TEI0304TGS2097 TEI0629

CT206

cLES-1-K3

TGS2911 TES1386

cLEX-2-F13

TES0945

TG548 TES0495

Fer T693 TGS0862 C2_At4g27700 TGS2907 T1079 TEI0949 TEI0978 TES0502 cLET-4-G23 TES0157 TES1094 TEI0367 TGS3054 TES0449 CT83 TES1179 TEI1058 TEI0147 TES1190 TGS0467 TES0870 TES0752 TGS0228 TES0292 C2_At1g52200 TES0211 TES0298 TG472 TES0305 TES0702 TES0335 TES0606TEI0869 TGS2372TGS2253 TGS1922SSR578 TES0154 TES0355TES1041 TES1469TES0689 TES1805 TM38cLEC15N20 CKIIbeta TGS1094 TG177 cLET5C8 TM43 TES0550 TGS3469 TGS0892 TES0697 TGS1145 TEI0140 TG365 TES0094 TGS0760 T570 CT204 TGS0993 TGS2894 TES0494 TG253 TEI0235 TGS0812 TGS2940 T1556 TEI0094 TES1103 TES0816 TG292 TES1899 TES0422 TES0014 TES1344 TES0660TES0771 cLET-19-J2 C2_At5g22620 TGS1056TGS3488 T405TG579 T1049TEI0400 T585 CT146 TEI0205 TGS1113 U144275 T1169 T1399 TGS1973 TGS0864

TGS2216 TES1872 TES1743 TG590TG325 TGS3083

TGS1863

TGS0266

T1928 T687 CD14 T1188T1082 T270 tme0122 TES0428 T1198 T1636 TGS0330 tmc0068 TGS0149 TES0628 TES0924 TGS2480 TES0111 TES1873 TGS2204 TGS2102TGS1057 TGS0871 TGS0917TGS0919 TEI0374 TGS2108TGS0045 TGS3509TEI0947 T1456T1500 cLET5A4T244 TG178 TES0922 TEI0962 TGS3051 TGS0230 TGS0447 TEI0618 TGS2236 TES0823 TES0312 tma0261

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T463A

TGS2259

TES0549 TES1718 TES0509 TG499 TGS2303

T848

TGS0939

TES0079 TGS2352 TES0264 TGS2361 TGS0514 T1428 TGS3249 tma0046 TGS2585 TGS2355 T1962 TES0678TGS1430 TES1075 tma0139 TES1351 T1518 CT135T656 T1974 TGS3112TGS0492 TGS2522tma0245 TES0130 TGS2005TGS2841 TGS2276TGS3401 tme0050TGS1396 TGS0044 tma0203TGS1411 TGS0540 TGS3387 TGS2163TEI1069 TGS3116 tme0020 TGS0397 tma0054 TGS0844 TGS3487 TGS2307 tma0124 TES1146 TGS0787 TGS0176 cLED-22-K8 TES0357 TG329 TGS1181 TGS1487 TES0573 T643 TES0682 TGS0192 tmc0067 T676 TGS2505 TGS2182 TGS3323 TES0903 TES1567 TGS0925 tma0137 T1510 TGS3483 TES1755 TG183cTOB-9-O3 cTOS18H13 TEI0768 TEI0407TEI0009 TEI0563TEI0085 TGS2373TGS3163 TES0680 TES0966TES1288 TES0863 TES1597 TES1675 TES0980 TES0018 TGS0953 TES1057 TES0115 TES0986 TG572 TES1543 TGS2715 TGS0412 TEI0655 TEI0193 TES0263 T1624 SSR45 TG216 TES1464 TES1074 TES1708 cLET-14-A10 TGS1187 TES0805 TGS0821 T1366 T1257 TEI0305 TG438 TEI0510 T966 TES1521 TES1284 TES1865 TGS2586 cLEX-13-I15

TES0407 TGS2685 T1328 TES0525

SSR241 TGS0501 TES0178 TES1816 TGS2602 TES0852 TES0520 TES0584 TES1008 TES0881 TGS0943 tme0069cLED7F15 TG418CT52 U231219SSR285 T1355TM29 CP52C2_At4g30580 cTOF11I4TG355 TEI0872 TES0181TES0683 TES0764 SSR52 cLED-21-J7 TES0054 TES1614 TG61

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CT68 T760A T763

TES0481

CT252

TES2003 TES1918TES0125 T1433 TES0537 TGS0810

T1558 TGS3110 TES0786

TES0297

T1522 TES1784

cLPT-1-J10 TES1352 T1581 tme0030 TES1757 TGS2002 TES1903 TES1883 tme0100 CT148 TES1535 TES0248 TEI0697tme0075 TES0052 T487 T608 TES0265 TGS2230 TES1969 T337 TGS2727 C2_At4g11560

TGS2376 SSR594 SSR38 TEI0839 TEI0346 TES0741 TGS2194 TGS0947 TES1833 U230185 TEI0254 TGS2819 CT111 TGS1335

TES0232 TES1884 TES1665 TES0954 T1341

TES0747

TES1088 TES0349 TES1719 cLET-5-O24 TES1077

TES1496

TES0611 TEI0580

TES1024 TES1761 TES0172 TES0241 CD40 TES1982 T721 TES1427 TES0254 TEI0153 TES0113TGS0398 TGS3192 TGS3361 TGS0868 TGS2946 TES0286 cLEG-16-L5 TGS0559 TES0065 tma0099 TES0736 TES0990TES0610 TES0285 TGS1172 TGS3003tma0304 TES0515 TG45 cLET3O9 T1179T720 T718T1358 TGS0493tma0193 tma0260 TEI0115 TGS0547 TEI0796TEI0541 TGS1894 TGS0202TGS0224 TGS2132TGS0469 tma0075tmb0063 TGS0368 TGS1143TGS2900 TGS0697 TGS0610 CT92 TEI0607 TGS0689TGS2256 TES0410 TGS0834 TGS0354 T1435 CT228 TES1281 TES1350 T1352 TEI0649 TGS3314 TGS3508 TGS3011 TGS2706 TGS0769 TGS0607 TES0289 TGS0842 TES0923 TG349 TES1502 C2_At4g32770

CT156B TES0999 TES0948 tme0052 tme0016 C2_At4g17380 cLEN9M1T1123 T1737 cLEF51A13TG176 C2_At3g52860

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TES1393TG9 TGS2174 T1673 TGS0426 T532 cLET-7-O3 SSR73 TES1059 TES0559 TGS0240 Brix9-2-5 TGS1315 TES1985 TES1183 T1617TGS1354 TGS0170 TES1157 tme0014 TGS2744 TES0176 cTOB1K3 tma0288 TES1143 TGS0365 TES0551 TES0184 TES0707 TGS1193 TGS0160 TES1418 TES0042 TGS2552 TES1099 T1212 tma0144 TGS2762 TGS1359 TES0148 TES1246 TES1815 TGS1629 TES0719 TES1728TGS1032 TGS1380 TGS0880TGS0235 TGS1722TES1473 TGS2919TGS0935 TGS3078 TES1378 TES0035TES0340 TES0059 TES0033TES0635 TES1123 TES0793 TGS0828TGS0766 TGS1417 TGS2669TGS3346 TGS0321TGS1378 TGS2149tmb0128 tma0224 TGS0906TGS1441 TEI0291TEI0642 tmb0082tma0227 tmb0033tma0067 tma0208 TGS2331TGS3498 TGS2575TEI0267 T1267TGS1713 cLEX-14-M4T1582 SSR19 TGS1180 CD3C2_At3g63200 T565 tme0123 cLET-2-D4 C2_At3g63190 TGS0213tma0127 T556 TGS3468 TGS1336 TGS2114 TEI0680 TGS0781 T182T431 TGS3371 TGS0274 TGS0470 TES0840 tmb0006 TGS1268 TEI0742 TGS3324 TGS0296 TGS1929 TGS1365 TGS0801 T1421 TGS2037 T1407 cLET-4-D15 TEI0184 C2_At2g47580 TES0472 TG348 TEI0174 TES1690 CT74 T732 TEI0318 cLET-7-D17 TES0226 TEI0382 TES0667 TEI0233 TGS0034 TEI0546 tme0035 TES0325 TES0101 TES1674 cLES-17-G6 TES0252 TGS2463 TES0319 TES0467 TGS0560 TG421 TES0871 tme0004 TES0623 TG424TES1028 GP101 C2_At3g24050 TES1891TES0562 TES0842 TEI0161 TES1037 TES1576 TES0503 tme0072 TES0914 SSR599 TGS1305TES0544 TES0889 TES1592 TEI0573 TEI0466 TG328 TES0322 T1065 tma0229

TEI0336

TES1696

T1641 TGS2555 TES1477 TGS0435

TES0036

cLPT-4-C24

SSR479

TES0171 TEI0190 TGS3422 T1391 TES1432 TES1740 TEI0220 T787 TGS0656 TES0440 CT234 TES0048 TGS1326 TGS2789 TGS0617 TGS1921 TES0720 U228331 TG560 TES0487 TEI0683 TG280 tma0044 T55 TES0790 TES1534 cLED-11-F6SSR248 TGS1123TES1409 TGS0643 TGS1918 TGS2189 tma0223 TGS2294TGS2358 TGS2431 TES0393TES1554 TGS0833TGS1331 TES0444 TEI0123TEI0482 TEI0570tma0244 T1577tma0235 T590cLET3F16 T1681 TES0950TES1169 TES1290TGS1585 TGS1263TG386 T1164TGS0139 TGS2247TGS1621 TGS0520tma0147 tmb0116tma0089 TEI0952tma0070 TGS0204 TGS1539TGS3269 TGS0165TES1762 TGS0551 TGS0385 tma0108 TGS0403 TGS0938 TGS2524 TGS3450 TGS3460 TES1266 TGS2261 TGS0725 TGS0934 TGS0472 TGS0073 TGS1572 TGS2913 TGS3353 TGS0898 TGS2646 TEI0268 tma0118 TGS1349 TG408 TGS2982 TGS0166 TES0477 tmc0040 TES0642 T571 TES1407 TGS2241 T1482TGS0579 T246 TGS0133 TEI0222 TES1859 TES0555 TES1756 K28 cLEX-11-C19 TES1384 TES0140 TGS0841 TGS1217 TGS3117 TGS2224 TES1177 cTOB-8-M7 TES0169 TEI0351 TGS2758 T615 TES0040 TES0075 TEI0272 TES0485 U241351 CT124 TES1842TES0433 TGS1951 TEI0396 C2_At3g09740 TES1540 TES0250 TES0545 TGS1282 T1521 TEI0282 TG63 TES0718 TG233CD32B T724 TES1966

TGS0738 TES1174 T1551

TES1154

TES1454 TG395

cLEC-5-E20 TGS3021

TG230

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TG393

TGS0346

TES1695

TES0344

TGS0830

TES0008 TGS2854 TGS2393 TES1697 TGS2218 TES1850 TGS2478 TGS0340 TGS0383 TGS3272 SSR46 TES0598 C2_At4g08230 TGS2103 T675 TES0617 TES0246 TGS0956 TGS2333 TGS1186 TGS2081 tma0279 TES1807 TGS2775 TES0281 TES1556 TES1832 TGS0719TGS3071 TEI0099 TGS2425TGS0414 TGS0104 TGS0410TGS2565 TGS1978 TES1810tma0035 tma0095tma0170 tma0285TES0608 TGS0384tma0258 TES0434tma0182 tma0248tma0187 tme0119TGS0959 TES1722 TGS0949TES0078 TEI0690 TGS2757TGS2818 TGS3026 tme0007 TES1039cLEX-4-G10 cSTA-37-E18 TEI0001 TES1060 TGS0606tmb0046 TGS0285 TGS0187 TGS1887 TG47 TEI0209 TGS0973 TGS2769 TGS0522 TGS3464 TES1449 TES0124 TES0095 cTOE-14-L16 TGS0991 TES0899TEI0699 TES1108 T1787T1071 C2_At4g01560 T1648 C2_At3g44880 TGS1476 TES1000 TES0006 T1014 TES1912 TES0727 TES1667 cLET-10-O11TG46 TES0813 TEI0069 TEI0771 TES0713 TEI0795 TEI0360 TG36 TGS1262 TES0701 TES0478

CT182 TEI0504 TGS0188

TGS3378 TES0011

TGS1281

TES0152 TGS0293 TG523

TGS2433 TES0808 TES0333 TES1442 TEI0130 TGS2887 T408

T1125 TEI0429

TES1001

TG651

TGS0794

SSR136

TGS2073

TES1229 TES1021

T1657

TES1970 TES0426

CT269

T1968

TES1975

TES0402 TG629 cTOA-5-G7

TGS2060

P47 TES1394 TES1102 TES1622

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CD2

TGS0591

TES1724

TGS1543

cTOA-22-C23

TES1889 TES1420 TGS2885 TM26 TEI0474 T28 TEI0737 TGS1078 TEI0311 TES1125 T989 CT211 TGS0244 TEI0111 TES0538 TES0716 T1263 TEI0256 TES0316 TES1501 TES1978 TGS0144 TES1564 TGS1200 TGS0456 TES2042 TGS1083 TGS0724 TGS3266 TG360 TES0406 TGS1502 TES1571 TGS1093 TGS1698 TGS1988 TGS0752 TES0500 TES0471 C2_At3g13180 TGS1162 TES1779 T1266 TGS0698 TES0123 TEI0815 TG111 TGS3485TGS2098 T1684 tma0076 TES0367 TEI0206 TGS0512TES1677 TES0249 TGS0253 TES1016 tmb0012 TGS0879TGS3097 TGS0363TES1800 cLPT-1-F6 tmb0050tmb0101 TGS1966TGS2938 TGS0793TGS2093 TGS1694TGS1202 cLET8G15TGS2450 SSR124tma0052 TGS1738 TGS1031 cLEG-24-B23TGS0199 TGS0339 tma0270 TGS2328 TGS2058tmb0075 TGS2087tma0197 tmb0076TGS0684 TGS2342 TGS3490 TGS1082 tmb0025TGS3362 TGS0153 TGS3032 TGS2978 TES1243 tma0022 TES0064 TES1421 T1499 tma0036 TGS0362 TES0317 TGS0145 TES0585 TGS0428 TGS0107 TES1980 TES1788 TG394 TG367 TGS0318 TGS0376 P68 TGS2479 TGS0329 TGS0658 TGS3343 TGS1493 TES0743 TES1686 P64 tma0294 TGS0927 T1483 tme0089 C2_At4g16580 TES1152TES0338 TES0112 TES0717 TES0839 TES2045 TGS0838 TEI0015 TEI0119 T1784cTOF31A1 TG296 TES1481 T882 TES0991 T770 TES1122 C2_At1g48300 TES0620

TES0158 tme0054 TES1515

U216669

TES0027

TES1919

TES0618

cLPT-6-E9 TEI0315

TES1635

TM14B

Fig. 1 Genetic linkage map of the tomato genome. Twelve linkage groups were constructed using 634 TGS markers (red lines), 648 TES markers (blue lines), 151 TEI markers (green lines) and 683 reported markers (black lines). The gray box in each linkage group indicates the estimated heterochromatic region. The distorted loci are indicated by hatch symbols in green (increases in S. pennellii alleles) and black (increases in S. lycopersicum alleles), as determined using the Chi-square test (#P < 0.05, ##P < 0.01, ###P < 0.005, ####P < 0.001, #####P < 0.0005 and ######P < 0.0001). Scale bar 10 cM

#

# # #

#

#### #

# # ##

# # #

### #

# ### #### # # # # # # # # ### ### #### ##### ##### ###### ### ###### ## #### ###### ###### ##### ###### ### #### ### ##### ###### ###### ##### ### ###### ##### #### ### ###### #### ### ##### ### #### ### ##### ###### ###### ###### ##### ###### ###### #### ###### ###### ###### ###### ###### ##### ###### ###### ###### ###### ##### ###### ###### ##### ##### #### ###### ###### ###### ###### ###### ###### ###### ###### ###### ###### ##### ###### ##### #### ### ### ##### ######

TES0566 CT197 TES1991

TGS2126 TES0856

TES1974

SSR92

CT233 SSR478 tma0313

TES1019

736 Theor Appl Genet (2010) 121:731–739

Theor Appl Genet (2010) 121:731–739

737

Table 2 Markers in the euchromatic, heterochromatic and entire chromosomal regions of the tomato EXPEN 2000 map Chromosome

Length (cM)

Number of loci TGS

TES

TEI

Cornell

NIVTS

Total

Average distance between two loci (cM)

Segregation distortion ratio (%)a

Entire region chr01

153.7

75

72

13

77

16

253

0. 61

chr02

162.6

36

56

18

71

6

187

0.87

18.7

chr03

137.5

65

73

12

34

12

196

0.70

43.4

chr04

132.3

47

49

9

56

10

171

0.77

8.8

chr05

137.9

50

54

9

31

12

156

0.88

5.8

chr06

108.5

46

49

18

56

3

172

0.63

63.4

chr07

107.3

50

50

11

43

11

165

0.65

40.0

chr08

110.1

41

53

11

43

11

159

0.69

1.3

chr09

109.0

58

56

15

39

17

185

0.59

8.6

chr10

101.5

57

41

13

36

11

158

0.64

94.9

chr11

122.3

49

48

12

29

12

150

0.82

84.7

chr12

120.4

60

47

10

32

15

164

0.73

9.8

1,503.1

634

648

151

547

136

2,116

0.71

38.2

Total

70.8

Euchromatic regionb chr01

144.4 (94)

44 (59)

62 (86)

12 (92)

71 (77)

5 (31)

194 (77)

0.74

61.9

chr02

159.9 (98)

33 (92)

56 (100)

18 (100)

70 (96)

2 (33)

179 (96)

0.89

16.2

chr03

120.5 (88)

25 (38)

46 (63)

7 (58)

33 (58)

2 (17)

113 (58)

1.07

55.8

chr04

118.7 (90)

22 (47)

38 (78)

8 (89)

50 (70)

1 (10)

119 (70)

1.00

10.1

chr05

128.5 (93)

17 (34)

47 (87)

6 (67)

22 (61)

3 (25)

95 (61)

1.35

9.5

chr06

90.1 (83)

30 (65)

42 (86)

14 (78)

49 (79)

1 (33)

136 (79)

0.66

79.4

chr07

100.1 (94)

28 (56)

46 (92)

10 (91)

37 (75)

3 (27)

124 (75)

0.81

24.2

chr08

103.8 (94)

25 (61)

50 (94)

7 (64)

36 (79)

7 (64)

125 (79)

0.83

1.6

chr09

94.8 (87)

19 (33)

33 (59)

11 (73)

25 (50)

4 (24)

92 (50)

1.03

15.2

chr10

90.1 (89)

21 (37)

29 (71)

7 (54)

25 (53)

1 (9)

83 (53)

1.09

92.8

chr11

116.7 (95)

30 (61)

37 (77)

9 (75)

26 (68)

0 (0)

102 (68)

1.14

79.4

chr12

116.4 (97)

35 (58)

43 (91)

10 (100)

27 (73)

4 (27)

119 (73)

0.98

12.6

1,384.8 (92)

329 (52)

529 (82)

119 (79)

471 (70)

33 (24)

1,481 (70)

0.94

37.8

Total

Heterochromatic regionb chr01

9.3 (6)

31 (41)

10 (14)

1 (8)

6 (23)

11 (69)

59 (23)

0.16

100.0

chr02

2.7 (2)

3 (8)

0 (0)

0 (0)

1 (4)

4 (67)

8 (4)

0.34

75.0

chr03

17 (12)

40 (62)

27 (37)

5 (42)

1 (42)

10 (83)

83 (42)

0.20

26.5

chr04

13.6 (10)

25 (53)

11 (22)

1 (11)

6 (30)

9 (90)

52 (30)

0.26

5.8

chr05

9.4 (7)

33 (66)

7 (13)

3 (33)

9 (39)

9 (75)

61 (39)

0.15

0.0

chr06

18.5 (17)

16 (35)

7 (14)

4 (22)

7 (21)

2 (67)

36 (21)

0.51

2.8

chr07

6.3 (6)

22 (44)

4 (8)

1 (9)

6 (25)

8 (73)

41 (25)

0.15

87.8

chr08

6.3 (6)

16 (39)

3 (6)

4 (36)

7 (21)

4 (36)

34 (21)

0.19

0.0

chr09

14.2 (13)

39 (67)

23 (41)

4 (27)

14 (50)

13 (76)

93 (50)

0.15

2.2

chr10

11.4 (11)

36 (63)

12 (29)

6 (46)

11 (47)

10 (91)

75 (47)

0.15

97.3

chr11

5.6 (5)

19 (39)

11 (23)

3 (25)

3 (32)

12 (100)

48 (32)

0.12

95.8

chr12

4 (3)

25 (42)

4 (9)

0 (0)

5 (27)

11 (73)

45 (27)

0.09

2.2

118.3 (8)

305 (48)

119 (18)

32 (21)

76 (30)

103 (76)

635 (30)

0.19

39.2

Total a b

SigniWcant at P < 0.05 Numbers in parentheses indicate percentage of the entire region

123

738

some chromosomes than on others, and the number of markers that were linked to those TRD. In addition, genotyping errors may have resulted in segregation distortion on some markers. The segregation distortion of loci is often reported in interspeciWc crosses between cultivated tomato and its wild relatives. In a cross between S. lycopersicum and its close relative S. pimpinellifolium, 8% of loci indicated segregation distortion (Grandillo and Tanksley 1996), while in wider crosses such as S. lycopersicum £ S. cheesmaniae and S. lycopersicum £ S. neorickii, 51 and 69% of loci showed segregation distortion, respectively (Paterson et al. 1988, 1991). Rieseberg et al. (1995) reported that TRD in hybrid populations potentially represents some level of reproductive isolation due to chromosome rearrangements or genetic interactions. The results of our study can therefore be considered indicative of genetic diversity between S. lycopersicum and S. pennellii that is wider in Chr 1, 10 and 11, and narrower in Chr 4, 5, 8, 9 and 12. The total length of the linkage map developed in this study was 1,503 cM, which was slightly longer than the 1,460 cM of the Tomato-EXPEN 2000 map reported by Fulton et al. (2002). However, the diVerence in the total length was considered to be within the error range of the linkage analysis, and the map was well saturated because most of the end markers mapped on each linkage were Cornell markers. A total of 292 markers were excluded from the analysis and not mapped. We speculated that this exclusion might be due to genotyping error, for example, if markers producing non-speciWc bands were screened as polymorphic markers. The average physical distance per cM was calculated to be 632 kb, deduced from the entire genome size of 950 Mb (Arumuganathan and Earle 1991). The length of the euchromatic and heterochromatic regions were 1,384 and 118 cM, respectively, and the physical distance per cM in both regions was estimated to be 172 and 6,042 kb, respectively, assuming a proportion of euchromatin and heterochromatin of 25% (238 Mb) and 75% (713 Mb), respectively, of the entire genome (Peterson et al. 1998). These results suggest that the physical distance per cM in the heterochromatin region is approximately 35 times longer than in the euchromatin region. The average distance between two loci in euchromatin was 0.94 cM, which was Wve times longer than in heterochromatin, which measured 0.19 cM. In the present map, 48% of the mapped TGS loci were located within heterochromatic regions, while 18 and 21% of TES and TEI loci, respectively, in heterochromatin regions. These results agree with previous reports (Frary et al. 2005; Ohyama et al. 2009) showing that genome-derived SSR markers are clustered within heterochromatin, while EST-derived SSR markers tend to be randomly distributed across the chromosomes in tomato. The heterochromatin region comprises 75% of the tomato genome with the gene density 10–100 times lower

123

Theor Appl Genet (2010) 121:731–739

than that of euchromatin (Wang et al. 2006). Moreover, recombination events are suppressed in the heterochromatic regions in tomato (Sherman and Stack 1995; Peters et al. 2009). These characteristics of the tomato genome could explain the clustering of TGS markers into such short genetic distances. We therefore conclude that EST-derived markers are superior in their ability to cover the entire genome compared to markers derived from randomly selected genomic regions, as described by Ohyama et al. (2009). In this study, we performed a large scale PCR-based marker development and newly mapped a total of 1,433 SSR and intronic polymorphism loci on the TomatoEXPEN 2000 mapping population. Information on these DNA markers is available at http://www.kazusa.or.jp/ tomato/. The DNA markers developed in this study are scheduled to be applied to anchoring scaVold sequences in an ongoing whole genome shotgun sequencing project (Mueller et al. 2005). The large number of SSR and intronic polymorphic markers developed in this study provide easily handled and abundant seed points for tomato genome sequencing. The EXPEN 2000 mapping population was generated from cross between a cultivated tomato (S. lycopersicum) and its wild relative, S. pennellii (Fulton et al. 2002). As genetic diversity in S. lycopersicum is low (Miller and Tanksley, 1990), saturated linkage maps have not been constructed in intraspeciWc mapping populations of S. lycopersicum (reviewed by Foolad 2007) and required more DNA markers. The large number of PCR-based markers developed in this study should be a useful genomic resource for interspeciWc genetic analysis in S. lycopersicum, although there is a possibility that some markers might not contain polymorphisms in intraspeciWc mapping. In addition, the existence of the dense linkage map allows map-based cloning of genes from wild relatives, such as abiotic stress tolerance and disease resistance genes, with the utilization of whole genome sequence that will be available soon. Once the utilizing genes are identiWed, the large amount of available markers should facilitate selection in introgressive breeding of S. lycopersicum and its wild relatives. The DNA markers and linkage map developed in this study are expected to enhance breeding by allowing a better understanding of whole genome sequences in tomato. Acknowledgments We thank Prof. S. Tanksley from Cornell University for kindly providing DNAs of the mapping population and the genotype data of Cornell markers. This work is supported by the Kazusa DNA Research Institute Foundation and the Ministry of Agriculture, Forestry, and Fisheries of Japan with the cooperation of the Genomics for Agricultural Innovation (DD-4010). Open Access This article is distributed under the terms of the Creative Commons Attribution Noncommercial License which permits any noncommercial use, distribution, and reproduction in any medium, provided the original author(s) and source are credited.

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