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Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome Rearrangements and Polymorphisms

Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome... Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2012, Article ID 437026, 8 pages doi:10.1155/2012/437026 Research Article Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome Rearrangements and Polymorphisms 1 2 2 2 2 Erika Asamizu, Kenta Shirasawa, Hideki Hirakawa, Shusei Sato, Satoshi Tabata, 3 1 2 1 Kentaro Yano, Tohru Ariizumi, Daisuke Shibata, and Hiroshi Ezura Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu 292-0818, Japan School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki 214-8571, Japan Correspondence should be addressed to Erika Asamizu, asamizu@gene.tsukuba.ac.jp Received 10 August 2012; Accepted 18 October 2012 Academic Editor: Pierre Sourdille Copyright © 2012 Erika Asamizu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A total of 93,682 BAC-end sequences (BESs) were generated from a dwarf model tomato, cv. Micro-Tom. After removing repetitive sequences, the BESs were similarity searched against the reference tomato genome of a standard cultivar, “Heinz 1706.” By referring to the “Heinz 1706” physical map and by eliminating redundant or nonsignificant hits, 28,804 “unique pair ends” and 8,263 “unique ends” were selected to construct hypothetical BAC contigs. The total physical length of the BAC contigs was 495, 833, 423 bp, covering 65.3% of the entire genome. The average coverage of euchromatin and heterochromatin was 58.9% and 67.3%, respectively. From this analysis, two possible genome rearrangements were identified: one in chromosome 2 (inversion) and the other in chromosome 3 (inversion and translocation). Polymorphisms (SNPs and Indels) between the two cultivars were identified from the BLAST alignments. As a result, 171,792 polymorphisms were mapped on 12 chromosomes. Among these, 30,930 poly- morphisms were found in euchromatin (1 per 3,565 bp) and 140,862 were found in heterochromatin (1 per 2,737 bp). The average polymorphism density in the genome was 1 polymorphism per 2,886 bp. To facilitate the use of these data in Micro-Tom research, the BAC contig and polymorphism information are available in the TOMATOMICS database. 1. Introduction to fruit size by utilizing DNA markers on the Tomato- EXPEN 2000 genetic map [4–9]. Such interspecies genetic Tomato (Solanum lycopersicum) is one of the most important mapping is effective because the divergent genomes provide vegetable crops cultivated worldwide. Tomato has a diploid many polymorphic DNA markers. In contrast, intraspecies (2n = 2x = 24) and relatively compact genome of approxi- mapping is less popular in tomato because of the low genetic mately 950 Mb [1]. Recently, its genome has been completely diversity within cultivated tomatoes that has resulted from sequenced by the international genome sequencing consor- the domestication process and subsequent modern breeding tium [2]. [10]. Recently, we developed SNP, simple sequence repeat Genetic linkage maps of tomato have been created by (SSR), and intronic polymorphic markers using publicly crossing cultivated tomato (S. lycopersicum)withseveral wild available EST information and BAC-end sequences (BESs) relatives, S. pennellii, S. pimpinellifolium, S. cheesmaniae, S. derived from “Heinz 1706,” a standard line for tomato neorickii, S. chmielewskii, S. habrochaites,and S. peruvianum genomics [11, 12], and applied these markers to create [3]. Introgression lines generated from a cross between S. linkage maps between Micro-Tom and either Ailsa Craig, a lycopersicum and S. pennellii have contributed to the isolation greenhouse tomato, or M82, a processing tomato, by map- of important loci and quantitative trait loci (QTLs) related ping 1,137 markers [12]. 2 International Journal of Plant Genomics Micro-Tom, a dwarf cultivar, is regarded as a model (MEXT, Japan) and sent to the Clemson University Genom- cultivar for functional genomics of tomato because of several ics Institute (CUGI) for BAC library construction. The characteristics, including small size (20 cm plant height), genomic DNA was partially digested, and fragments were short life cycle (3 months), existence of indoor cultivation cloned into the Hind III site of pIndigoBAC536. A total of protocols under normal fluorescent conditions, and high- 55,296 clones in Escherichia coli DH10B cells were arrayed in efficiency transformation methods that have been developed 144 384-well plates. for this line [13–15]. The dwarf phenotype of Micro-Tom is the result of mutations in at least two major recessive 2.2. End Sequencing of Micro-Tom BAC Clones. To analyze loci. dwarf (d) encodes a cytochrome P450 protein, which BESs, the BAC DNAs were amplified using a TempliPhi large- functions in the brassinosteroid biosynthesis pathway [16]. construction kit (GE Healthcare, UK), and the end sequences Another locus, miniature (mnt), is suggested to asso- were analyzed according to the Sanger method, using a cycle ciate with gibberellin (GA) signaling without affecting GA sequencing kit (Big Dye-terminator kit, Applied Biosystems, metabolism, but the causal gene has not been identified to USA) with a type 3730xl DNA sequencer (Applied Biosys- date [17]. In Japan, Micro-Tom genomics resources have tems). The resulting sequence reads were quality checked been extensively accumulated, mainly in the framework of with PHRED [27, 28], allowing the identification and the National BioResource Project (NBRP) (http://tomato removal of low-quality (QV < 20) sequences. The 93,682 .nbrp.jp/indexEn.html). Large-scale ethyl methanesulfonate reads clearing the quality criteria were submitted to DDBJ/ (EMS) and gamma-ray-mutagenized populations have been GenBank with accession numbers FT227487-FT321168. created, and visible phenotype data have been accumulated [18–20]. The availability of Micro-Tom genome sequence 2.3. Mapping to the Reference Genome and Analyses. BES data will accelerate the mapping of mutant alleles. reads were subjected to similarity search using the BLASTN BAC-end sequencing has been performed in the tomato program [29, 30]. To isolate unique sequences from repetitive standard line “Heinz 1706” genome project to order BAC ones, 93,682 BESs were searched against the repeat database clones along the chromosomes [21]. Currently, about 90,000 in ITAG2.3 (http://solgenomics.net/) using a cutoff E-value BESs are available at the Sol Genomics Network (SGN, −50 of less than 10 . The remaining sequences were searched http://solgenomics.net/). BAC-end sequencing has been con- against the published version of the “Heinz 1706” genome ducted for other crop species. In the rice indica cultivar (SL2.40), which was accessed from the SGN database “Kasalath,” 78,427 BESs were generated from 47,194 clones (http://solgenomics.net/). From all of the BLAST alignments, and mapped onto the “Nipponbare” reference genome. As BESs were extracted according to the following criteria, a result, 12,170 paired BESs were mapped that covered 80% suggested in a previous report [22]: (1) sequence identity > of the rice genome [22]. Recently, BAC-end sequencing has 90% and alignment coverage > 50%; (2) mapped positions of been performed in crop plants with higher genome com- each pair of ends < 200 kb apart in the same chromosome; plexity. BESs from a commercial sugarcane variety, an (3) direction of each paired end is correct; (4) BLASTN E < interspecific hybrid with complex ploidy, were generated to −100 10 ; (5) a minimum of one hit for one of the paired ends; analyze microsynteny between sugarcane and sorghum [23]. (6) no redundant chromosomal locations. Sequence poly- In wheat, which has a complex hexaploid genome, the short morphisms (SNPs and Indels) between Micro-Tom and arm of chromosome 3A was flow sorted to make a BAC “Heinz 1706” were predicted based on the BLASTN align- library, and chromosome arm-specific BESs were generated ment. Since we did not allow a gap exceeding 27 bases, only for DNA marker development [24]. In switchgrass, more Indels up to 26 basesinlengthwerecounted. than 50,000 SSRs were identified from 330,000 BESs, and this enabled detailed analysis on the evolution of this species [25]. 2.4. Database and Clone Distribution. Mapped data and A low level of genetic variation has been observed for culti- SNP/Indel sites were made accessible through the database vated peanuts. Polymorphic SSRs were accumulated from the TOMATOMICS at http://bioinf.mind.meiji.ac.jp/tomatom- BESs and successfully used in the construction of a genetic ics/. BAC clones are available upon request from NBRP map [26]. BAC-end sequencing can be useful as a resource tomato (http://tomato.nbrp.jp/indexEn.html). for performing comparative genomic studies through map- ping of the sequences to a reference genome and by facilitat- ing the development of polymorphic DNA markers. 3. Results In the present study, we generated 93,682 single-pass end sequences from a Micro-Tom BAC library. To compare 3.1. General Features of the Generated BESs. The BAC insert the structures between the reference tomato “Heinz 1706” size distribution was deduced based on the mapping results. genome, mapping of unique ends was performed, and pos- According to these results, 45.4% (6,396 out of 14,101) of the sible genome rearrangements and polymorphisms were BACs ranged from 100 to 120 kb, with average and median identified. sizes of 101.3 kb and 101.8 kb, respectively (Figure 1). By multiplying by the number of clones (55,296), this BAC library covers 5.9x of the 950 Mb tomato genome. 2. Materials and Methods Micro-Tom BES mapping to the “Heinz 1706” genome 2.1. Micro-Tom BAC Library Construction. Micro-Tom was processed as indicated in Figure 2. By eliminating repet- (TOMJPF00001) seeds were obtained from the NBRP itive, redundant, and unmapped sequences, 28,804 “unique International Journal of Plant Genomics 3 BAC insert size (kb) Figure 1: Distribution of BAC clone insert size. The insert size was deduced by mapping BESs onto the reference “Heinz 1706” genome (SL2.40). 93,682 BES BLASTN versus repeat DB Repetitive sequences (43, 598) 50,084 BES BLASTN versus “Heinz 1706” SL2.40 Multiple hits (10, 943) Low similraity (2, 015) No hit (59) 37,067 BES Both ends mapped One end mapped 28,804 BES 8,263 BES Figure 2: Flow of the BES analysis. To eliminate repetitive sequences, 93,682 BESs were initially searched against the repeat dataset of ITAG −50 2.3 with a BLASTN cutoff value of E <10 . Next, the remaining sequences were mapped onto the “Heinz 1706” pseudomolecule sequences −100 (SL2.40) under the following criteria: identity >90%, coverage >50%; E <10 ; the inclusion of single hits only. Mapped BESs were classified as either unique pair ends, for which both ends were mapped, or unique ends, for which only one end was mapped. pair ends” and 8,263 “unique ends” were selected. Paired-end physical length of the BAC contigs was 495,833,423 bp, cov- sequences were mapped onto the reference tomato genome ering 65.3% of the total chromosomes (Table 1). sequence, and 2,248 hypothetical BAC contigs were con- structed (see details at TOMATOMICS, http://bioinf.mind 3.2. Possible Genome Rearrangements. To assess the occur- .meiji.ac.jp/tomatomics/). The integrity of the hypothetical rence of genome rearrangements, Micro-Tom and the refer- contigs was confirmed by linking to the DNA markers on ence tomato “Heinz 1706” were compared. Possible inver- two genetic maps, AMF and MMF (see Supplementary sions, translocations, and insertions were considered. To 2 2 Table 1 in Supplementary Material available online at eliminate an artificial effect (e.g., chimeric BAC clones), only doi:10.1155/2012/437026). regions covered by more than two BAC clones were selected. The genome coverage of the hypothetical BAC con- After removing regions that had cleared the criteria for tigs was assessed by applying euchromatin/heterochromatin extraction (see Section 2 ) but were either shown to be multi- boundary information from the genetic map EXPEN2000 copy by manual evaluation of the BLAST results or displayed [2]. The results indicated that the euchromatin coverage similarity to transposable elements, we obtained two cases ranged between 45.1% and 71.1% (average, 58.9%) among of a possible rearrangement between Micro-Tom and “Heinz the different chromosomes, while heterochromatin coverage 1706” (Table 2). On chromosome 2, a possible inversion ranged between 57.4% and 75.3% (average, 67.3%). The total was detected. The size of this inversion could be 20–220 kb Number of clones <50 ≤60 ≤70 ≤80 ≤90 ≤100 ≤110 ≤120 ≤130 ≤140 ≤150 >150 4 International Journal of Plant Genomics Table 1: Coverage of chromosomes by hypothetical Micro-Tom BAC contigs. SL2.40ch01 SL2.40ch02 SL2.40ch03 SL2.40ch04 SL2.40ch05 SL2.40ch06 SL2.40ch07 SL2.40ch08 SL2.40ch09 SL2.40ch10 SL2.40ch11 SL2.40ch12 Total (ch01–ch12) Chromosome length 27,903,720 24,734,122 16,423,960 13,871,288 10,836,573 17,576,248 17,480,118 15,552,430 10,522,300 9,129,273 11,175,203 12,034,427 187,239,662 Euchromatin Euchromatin no. of Contigs 100 78 45 34 32 52 53 55 37 25 45 44 600 no. of BACs 533 504 401 231 170 339 224 279 176 197 184 317 3,555 Euchromatin Euchromatin Covered bases 17,310,734 14,644,412 11,678,941 6,261,540 5,377,576 10,621,719 8,336,310 9,541,847 6,047,365 5,473,701 6,855,876 8,119,058 110,269,079 Uncovered bases 10,592,986 10,089,710 4,745,019 7,609,748 5,458,997 6,954,529 9,143,808 6,010,583 4,474,935 3,655,572 4,319,327 3,915,369 76,970,583 Euchromatin Euchromatin % Coverage 62.0% 59.2% 71.1% 45.1% 49.6% 60.4% 47.7% 61.4% 57.5% 60.0% 61.3% 67.5% 58.9% Heterochromatin Chromosome length 62,400,524 25,184,172 48,416,754 50,193,024 54,184,865 28,465,388 47,788,503 47,480,227 57,139,791 55,705,032 42,210,822 53,451,826 572,620,928 no. of Contigs 175 74 147 131 169 76 135 150 149 159 128 155 1,648 Heterochromatin Heterochromatin no. of BACs 1,000 391 903 1,022 752 544 959 856 1,209 1,056 746 992 10,430 Covered bases 39,941,033 15,405,507 32,458,031 34,993,238 31,099,727 19,672,865 35,988,229 33,980,702 40,376,427 37,964,128 27,534,109 36,150,348 385,564,344 Heterochromatin Uncovered bases 22,459,491 9,778,665 15,958,723 15,199,786 23,085,138 8,792,523 11,800,274 13,499,525 16,763,364 17,740,904 14,676,713 17,301,478 187,056,584 Heterochromatin % Coverage 64.0% 61.2% 67.0% 69.7% 57.4% 69.1% 75.3% 71.6% 70.7% 68.2% 65.2% 67.6% 67.3% Heterochromatin Chromosome length 90,304,244 49,918,294 64,840,714 64,064,312 65,021,438 46,041,636 65,268,621 63,032,657 67,662,091 64,834,305 53,386,025 65,486,253 759,860,590 Total no. of Contigs 275 152 192 165 201 128 188 205 186 184 173 199 2,248 Total no. of BACs 1,533 895 1,304 1,253 922 883 1,183 1,135 1,385 1,253 930 1,309 13,985 Total Covered bases 57,251,767 30,049,919 44,136,972 41,254,778 36,477,303 30,294,584 44,324,539 43,522,549 46,423,792 43,437,829 34,389,985 44,269,406 495,833,423 Total Total Uncovered bases 33,052,477 19,868,375 20,703,742 22,809,534 28,544,135 15,747,052 20,944,082 19,510,108 21,238,299 21,396,476 18,996,040 21,216,847 264,027,167 % Coverage 63.4% 60.2% 68.1% 64.4% 56.1% 65.8% 67.9% 69.1% 68.6% 67.0% 64.4% 67.6% 65.3% Total Table 2: Possible genome rearrangement events observed in the Micro-Tom and “Heinz 1706” genome. No. BAC End1 Acc Chr Direction From To End2 Acc Chr Direction From To Possible event 1 MTBAC102D20 T7 FT290741 SL2.40ch02 — 29,374,874 29,375,640 SP6 FT290742 SL2.40ch02 — 29,494,209 29,494,781 Inversion 1 MTBAC084K15 T7 FT278701 SL2.40ch02 — 29,375,421 29,376,188 SP6 FT278702 SL2.40ch02 — 29,462,866 29,463,675 2 MTBAC041L05 T7 FT251747 SL2.40ch03 — 6,601,537 6,602,368 SP6 FT251748 SL2.40ch03 — 55,664,754 55,665,559 Translocation and Inversion 2 MTBAC077O14 SP6 FT274148 SL2.40ch03 — 6,602,568 6,603,163 T7 FT274147 SL2.40ch03 — 55,665,296 55,666,020 International Journal of Plant Genomics 5 depending on which end of the BAC clone is inversed. DNA from all 12 chromosomes. In the Kasalath rice BES Translocation and inversion were observed on chromo- analysis, chromosomal coverage in relation to the reference some 3. For each of two BAC clones (MTBAC041L05 and Nipponbare pseudomolecule was about 80%, despite the MTBAC077O14), one of the ends was mapped to 6,601 kb lower number (78,427) of analyzed BESs [22]. Because we of chromosome 3, while the other end was mapped to used the same criteria for repetitive sequence selection (E < −50 55,665 kb, more than 49 megabases apart. In addition, both 10 ), the discrepancy between the two studies might be ends were mapped on the minus strand. due to the larger genome size of tomato (950 Mb) compared with rice (430 Mb) [34]. Our Micro-Tom BAC coverage is reasonable, taking into account the scale of the BAC library 3.3. Polymorphisms between Micro-Tom and the Reference used. Tomato. SNPs and Indels between Micro-Tom and “Heinz Micro-Tom has been considered as a model cultivar to 1706” were identified. Among the SNPs and Indels found, promote functional genomics studies of tomato by taking 171,792 were mapped on 12 chromosomes, and 2,635 were advantage of its characteristics. Currently, many tools and mapped on pseudomolecules with no chromosomal infor- platforms have been developed, and some of these are already mation (SL2.40ch00 of the tomato whole-genome shotgun available to the research community. The present study char- chromosomes) (Table 3 and Supplementary Table 2, see acterized the overall polymorphisms found between Micro- details at TOMATOMICS). According to these results, Tom BESs and the reference tomato “Heinz 1706” genome. among the mapped SNPs and Indels, a total of 30,930 poly- In addition, two possible genome rearrangement events, on morphisms were found in the euchromatin (1 out of chromosome 2 and chromosome 3, were observed (Table 2). 3,565 bp), and 140,862 were found in the heterochromatin (1 In the case of translocation and inversion on chromosome out of 2,737 bp). The average polymorphism density in the 3, a gene annotated as reverse transcriptase was found in the genome was 1 polymorphism per 2,886 bp. Transversion- flanking region (Solyc03g104840.1). We speculate that this type SNPs were observed in 83,262 cases, while 60,631 were region was translocated by the activity of a retrotransposon, transition-type SNPs. Among the 30,534 Indels, single-base as it was in the case of SUN. Enhanced expression of insertions (on the SL2.40 version of the tomato whole- SUN caused by a gene duplication event mediated by the genome shotgun chromosomes) were observed in 10,740 retrotransposon Rider led to an elongated fruit shape [35]. cases, and single-base deletions were seen in 17,064 cases. In the future, we plan to sequence the entire BAC and expect The remainder were larger Indels, ranging from 2 to 26 bp that this will help us to characterize these events in more (Supplementary Table 2). Classification of polymorphisms detail. In the case of the other rearrangement possibility, on regarding genic or intergenic regions is shown in Table 4. chromosome 2, we could not find any trace of a retrotranspo- son. Since these rearrangements took place in euchromatin, which is rich in genes, these regions could represent an 4. Discussion interesting target to investigate their possible effects on By selecting unique end sequences from 93,682 reads, phenotypic variation between Micro-Tom and the reference 28,804 paired ends (14,402 pairs) and 8,263 unpaired ends tomato. We mapped the polymorphisms and depicted them, were obtained. The majority of the nonselected sequences (43,598) were derived from repetitive regions. For the rest, alongside maps showing covered regions and gaps, in 10,943 had redundant hits to the “Heinz 1706” genome, pos- Figure 3. On chromosomes 2, 5, and 11, polymorphisms seemed to be concentrated in the heterochromatic regions; sibly including repetitive sequences that were not represented in the repeat database in ITAG2.3 (http://solgenomics.net/), however, this tendency was not clearly observed in the other 2,015 showed weak similarity, and 59 showed no similarity chromosomes. For the other regions, the polymorphism dis- (Figure 2). Considering that the genome has been previously covery rate seemed to be somehow correlated with the BAC estimated to be composed of 25% gene-rich euchromatin coverage. Although our analysis indicated little possibility of [31, 32], BES selection in this study (39.6%, (28,804 + large-scale genome rearrangement between Micro-Tom and 8,263)/93,682)) could have eliminated repetitive regions to “Heinz 1706” (Table 2), this uneven polymorphism distribu- a moderate degree. We identified 59 reads showing no sig- tion suggests the existence of highly divergent chromosomal regions. The gaps in the hypothetical Micro-Tom BAC nificant similarity to the “Heinz-1706” genome. Micro-Tom was bred by crossing the home-gardening cultivars, Florida contigs could have resulted from low coverage of the BAC Basket and Ohio 4013-3. The pedigree of Ohio 4013-3 sug- library, but the occurrence of chromosomal segments specific to either Micro-Tom or “Heinz 1706” is also possible. The gested that a wild relative species was used in the breeding history [18, 33]. Such introgressed segments may lead to the ongoing Micro-Tom genome sequencing and de novo assem- introduction of genomic regions not harbored by “Heinz bly of the Micro-Tom genome will clarify the genome struc- 1706.” The Micro-Tom genome is now being sequenced ture in detail, enabling a more solid assessment of the dif- (draft sequence data available at DDBJ with the accession ferences between Micro-Tom and “Heinz 1706.” We had previously developed SNP markers among sev- number DRA000311), and mapping of orphan BESs to the de novo assembly of Micro-Tom genome data will help to clarify eral cultivated tomatoes [12]. By selecting SNPs through in this question. silico analysis using public EST information and previously developed SSR markers, 1,137 markers were obtained and The total physical length of Micro-Tom BAC contigs was 495,833,423 bp, which covers approximately 65.3% of the successfully mapped on linkage groups between Micro-Tom 6 International Journal of Plant Genomics Table 3: Number of polymorphisms found in each chromosome. SL2.40ch01 SL2.40ch02 SL2.40ch03 SL2.40ch04 SL2.40ch05 SL2.40ch06 SL2.40ch07 SL2.40ch08 SL2.40ch09 SL2.40ch10 SL2.40ch11 SL2.40ch12 Total Euchromatin no. of polymorphisms 4,152 4,123 3,700 2,863 969 2,417 3,504 2,113 1,932 1,302 1,694 2,161 30,930 Covered bases 17,310,734 14,644,412 11,678,941 6,261,540 5,377,576 10,621,719 8,336,310 9,541,847 6,047,365 5,473,701 6,855,876 8,119,058 110,269,079 Euchromatin Euchromatin kb/polymorphism 4,169 3,552 3,156 2,187 5,550 4,395 2,379 4,516 3,130 4,204 4,047 3,757 3,565 Heterochromatin no. of polymorphisms 12,319 10,694 10,408 9,995 30,951 5,134 8,347 8,562 10,231 9,209 14,937 10,075 140,862 Covered bases 39,941,033 15,405,507 32,458,031 34,993,238 31,099,727 19,672,865 35,988,229 33,980,702 40,376,427 37,964,128 27,534,109 36,150,348 385,564,344 Heterochromatin Heterochromatin kb/polymorphism 3,242 1,441 3,119 3,501 1,005 3,832 4,312 3,969 3,946 4,123 1,843 3,588 2,737 Total no. of polymorphisms 16,471 14,817 14,108 12,858 31,920 7,551 11,851 10,675 12,163 10,511 16,631 12,236 171,792 Covered bases 57,251,767 30,049,919 44,136,972 41,254,778 36,477,303 30,294,584 44,324,539 43,522,549 46,423,792 43,437,829 34,389,985 44,269,406 495,833,423 Total Total kb/polymorphism 3,476 2,028 3,129 3,208 1,143 4,012 3,740 4,077 3,817 4,133 2,068 3,618 2,886 Table 4: Number of polymorphisms found in genic and intergenic regions in each chromosome. SL2.40ch01 SL2.40ch02 SL2.40ch03 SL2.40ch04 SL2.40ch05 SL2.40ch06 SL2.40ch07 SL2.40ch08 SL2.40ch09 SL2.40ch10 SL2.40ch11 SL2.40ch12 Total 3 UTR 115 100 127 108 62 58 59 87 47 15 0 0 778 Exon 5 UTR 157 58 48 23 26 34 28 14 38 2 0 0 428 Genic CDS 1,152 967 1,031 758 820 603 757 605 669 570 662 615 9,209 Intron 2,035 1,938 2,023 1,971 1,547 1,122 1,778 1,154 1,425 994 1,947 1,172 19,106 Intron Splice 19 15 12 13 14 15 13 8 8 7 12 12 148 junction Intergenic 12,993 11,739 10,867 9,985 29,451 5,719 9,216 8,807 9,976 8,923 14,010 10,437 142,123 Total 16,471 14,817 14,108 12,858 31,920 7,551 11,851 10,675 12,163 10,511 16,631 12,236 171,792 International Journal of Plant Genomics 7 ch01 ch02 ch03 ch04 ch05 ch06 ch07 ch08 ch09 ch10 ch11 ch12 Figure 3: Micro-Tom BAC coverage with respect to the “Heinz 1706” chromosomes and detected polymorphisms. Black boxes indicate covered regions, and white boxes indicate gaps. Bars represent heterochromatic regions. The scale bars for polymorphisms indicate the number of SNPs or Indels per megabase (200 polymorphisms/scale). and either Ailsa Craig or M82. In the present study, we References identified 171,792 SNPs and Indels and mapped them on 12 [1] K. Arumuganathan and E. D. Earle, “Nuclear DNA content chromosomes. The average density was 1 SNP per 3,565 bp of some important plant species,” Plant Molecular Biology in euchromatin and 1 SNP per 2,886 bp in the genome in Reporter, vol. 9, no. 3, pp. 208–218, 1991. general (including both euchromatin and heterochromatin). [2] Tomato Genome Consortium, “The tomato genome sequence Previously, large-scale Micro-Tom full-length cDNA analysis provides insights into fleshy fruit evolution,” Nature, vol. 485, and comparison of exon regions with those on the “Heinz no. 7400, pp. 635–641, 2012. 1706” genome revealed a mean sequence mismatch of [3] M. R. Foolad, “Genome mapping and molecular breeding of 0.061% (1/1,640 bp) [36]. One possible explanation for the tomato,” International Journal of Plant Genomics, vol. 2007, difference is the quality of the reference “Heinz 1706” Article ID 64358, 52 pages, 2007. genome sequence used in the two studies. We used the pub- [4] I. Paran, I. Goldman, S. D. Tanksley, and D. Zamir, “Recombi- lished version of the “Heinz 1706” genome sequence, which nant inbred lines for genetic mapping in tomato,” Theoretical has higher coverage, giving rise to greater accuracy, although and Applied Genetics, vol. 90, no. 3-4, pp. 542–548, 1995. [5] T. M. Fulton, J. C. Nelson, and S. D. 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Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome Rearrangements and Polymorphisms

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Copyright © 2012 Erika Asamizu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.
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Hindawi Publishing Corporation International Journal of Plant Genomics Volume 2012, Article ID 437026, 8 pages doi:10.1155/2012/437026 Research Article Mapping of Micro-Tom BAC-End Sequences to the Reference Tomato Genome Reveals Possible Genome Rearrangements and Polymorphisms 1 2 2 2 2 Erika Asamizu, Kenta Shirasawa, Hideki Hirakawa, Shusei Sato, Satoshi Tabata, 3 1 2 1 Kentaro Yano, Tohru Ariizumi, Daisuke Shibata, and Hiroshi Ezura Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu 292-0818, Japan School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki 214-8571, Japan Correspondence should be addressed to Erika Asamizu, asamizu@gene.tsukuba.ac.jp Received 10 August 2012; Accepted 18 October 2012 Academic Editor: Pierre Sourdille Copyright © 2012 Erika Asamizu et al. This is an open access article distributed under the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. A total of 93,682 BAC-end sequences (BESs) were generated from a dwarf model tomato, cv. Micro-Tom. After removing repetitive sequences, the BESs were similarity searched against the reference tomato genome of a standard cultivar, “Heinz 1706.” By referring to the “Heinz 1706” physical map and by eliminating redundant or nonsignificant hits, 28,804 “unique pair ends” and 8,263 “unique ends” were selected to construct hypothetical BAC contigs. The total physical length of the BAC contigs was 495, 833, 423 bp, covering 65.3% of the entire genome. The average coverage of euchromatin and heterochromatin was 58.9% and 67.3%, respectively. From this analysis, two possible genome rearrangements were identified: one in chromosome 2 (inversion) and the other in chromosome 3 (inversion and translocation). Polymorphisms (SNPs and Indels) between the two cultivars were identified from the BLAST alignments. As a result, 171,792 polymorphisms were mapped on 12 chromosomes. Among these, 30,930 poly- morphisms were found in euchromatin (1 per 3,565 bp) and 140,862 were found in heterochromatin (1 per 2,737 bp). The average polymorphism density in the genome was 1 polymorphism per 2,886 bp. To facilitate the use of these data in Micro-Tom research, the BAC contig and polymorphism information are available in the TOMATOMICS database. 1. Introduction to fruit size by utilizing DNA markers on the Tomato- EXPEN 2000 genetic map [4–9]. Such interspecies genetic Tomato (Solanum lycopersicum) is one of the most important mapping is effective because the divergent genomes provide vegetable crops cultivated worldwide. Tomato has a diploid many polymorphic DNA markers. In contrast, intraspecies (2n = 2x = 24) and relatively compact genome of approxi- mapping is less popular in tomato because of the low genetic mately 950 Mb [1]. Recently, its genome has been completely diversity within cultivated tomatoes that has resulted from sequenced by the international genome sequencing consor- the domestication process and subsequent modern breeding tium [2]. [10]. Recently, we developed SNP, simple sequence repeat Genetic linkage maps of tomato have been created by (SSR), and intronic polymorphic markers using publicly crossing cultivated tomato (S. lycopersicum)withseveral wild available EST information and BAC-end sequences (BESs) relatives, S. pennellii, S. pimpinellifolium, S. cheesmaniae, S. derived from “Heinz 1706,” a standard line for tomato neorickii, S. chmielewskii, S. habrochaites,and S. peruvianum genomics [11, 12], and applied these markers to create [3]. Introgression lines generated from a cross between S. linkage maps between Micro-Tom and either Ailsa Craig, a lycopersicum and S. pennellii have contributed to the isolation greenhouse tomato, or M82, a processing tomato, by map- of important loci and quantitative trait loci (QTLs) related ping 1,137 markers [12]. 2 International Journal of Plant Genomics Micro-Tom, a dwarf cultivar, is regarded as a model (MEXT, Japan) and sent to the Clemson University Genom- cultivar for functional genomics of tomato because of several ics Institute (CUGI) for BAC library construction. The characteristics, including small size (20 cm plant height), genomic DNA was partially digested, and fragments were short life cycle (3 months), existence of indoor cultivation cloned into the Hind III site of pIndigoBAC536. A total of protocols under normal fluorescent conditions, and high- 55,296 clones in Escherichia coli DH10B cells were arrayed in efficiency transformation methods that have been developed 144 384-well plates. for this line [13–15]. The dwarf phenotype of Micro-Tom is the result of mutations in at least two major recessive 2.2. End Sequencing of Micro-Tom BAC Clones. To analyze loci. dwarf (d) encodes a cytochrome P450 protein, which BESs, the BAC DNAs were amplified using a TempliPhi large- functions in the brassinosteroid biosynthesis pathway [16]. construction kit (GE Healthcare, UK), and the end sequences Another locus, miniature (mnt), is suggested to asso- were analyzed according to the Sanger method, using a cycle ciate with gibberellin (GA) signaling without affecting GA sequencing kit (Big Dye-terminator kit, Applied Biosystems, metabolism, but the causal gene has not been identified to USA) with a type 3730xl DNA sequencer (Applied Biosys- date [17]. In Japan, Micro-Tom genomics resources have tems). The resulting sequence reads were quality checked been extensively accumulated, mainly in the framework of with PHRED [27, 28], allowing the identification and the National BioResource Project (NBRP) (http://tomato removal of low-quality (QV < 20) sequences. The 93,682 .nbrp.jp/indexEn.html). Large-scale ethyl methanesulfonate reads clearing the quality criteria were submitted to DDBJ/ (EMS) and gamma-ray-mutagenized populations have been GenBank with accession numbers FT227487-FT321168. created, and visible phenotype data have been accumulated [18–20]. The availability of Micro-Tom genome sequence 2.3. Mapping to the Reference Genome and Analyses. BES data will accelerate the mapping of mutant alleles. reads were subjected to similarity search using the BLASTN BAC-end sequencing has been performed in the tomato program [29, 30]. To isolate unique sequences from repetitive standard line “Heinz 1706” genome project to order BAC ones, 93,682 BESs were searched against the repeat database clones along the chromosomes [21]. Currently, about 90,000 in ITAG2.3 (http://solgenomics.net/) using a cutoff E-value BESs are available at the Sol Genomics Network (SGN, −50 of less than 10 . The remaining sequences were searched http://solgenomics.net/). BAC-end sequencing has been con- against the published version of the “Heinz 1706” genome ducted for other crop species. In the rice indica cultivar (SL2.40), which was accessed from the SGN database “Kasalath,” 78,427 BESs were generated from 47,194 clones (http://solgenomics.net/). From all of the BLAST alignments, and mapped onto the “Nipponbare” reference genome. As BESs were extracted according to the following criteria, a result, 12,170 paired BESs were mapped that covered 80% suggested in a previous report [22]: (1) sequence identity > of the rice genome [22]. Recently, BAC-end sequencing has 90% and alignment coverage > 50%; (2) mapped positions of been performed in crop plants with higher genome com- each pair of ends < 200 kb apart in the same chromosome; plexity. BESs from a commercial sugarcane variety, an (3) direction of each paired end is correct; (4) BLASTN E < interspecific hybrid with complex ploidy, were generated to −100 10 ; (5) a minimum of one hit for one of the paired ends; analyze microsynteny between sugarcane and sorghum [23]. (6) no redundant chromosomal locations. Sequence poly- In wheat, which has a complex hexaploid genome, the short morphisms (SNPs and Indels) between Micro-Tom and arm of chromosome 3A was flow sorted to make a BAC “Heinz 1706” were predicted based on the BLASTN align- library, and chromosome arm-specific BESs were generated ment. Since we did not allow a gap exceeding 27 bases, only for DNA marker development [24]. In switchgrass, more Indels up to 26 basesinlengthwerecounted. than 50,000 SSRs were identified from 330,000 BESs, and this enabled detailed analysis on the evolution of this species [25]. 2.4. Database and Clone Distribution. Mapped data and A low level of genetic variation has been observed for culti- SNP/Indel sites were made accessible through the database vated peanuts. Polymorphic SSRs were accumulated from the TOMATOMICS at http://bioinf.mind.meiji.ac.jp/tomatom- BESs and successfully used in the construction of a genetic ics/. BAC clones are available upon request from NBRP map [26]. BAC-end sequencing can be useful as a resource tomato (http://tomato.nbrp.jp/indexEn.html). for performing comparative genomic studies through map- ping of the sequences to a reference genome and by facilitat- ing the development of polymorphic DNA markers. 3. Results In the present study, we generated 93,682 single-pass end sequences from a Micro-Tom BAC library. To compare 3.1. General Features of the Generated BESs. The BAC insert the structures between the reference tomato “Heinz 1706” size distribution was deduced based on the mapping results. genome, mapping of unique ends was performed, and pos- According to these results, 45.4% (6,396 out of 14,101) of the sible genome rearrangements and polymorphisms were BACs ranged from 100 to 120 kb, with average and median identified. sizes of 101.3 kb and 101.8 kb, respectively (Figure 1). By multiplying by the number of clones (55,296), this BAC library covers 5.9x of the 950 Mb tomato genome. 2. Materials and Methods Micro-Tom BES mapping to the “Heinz 1706” genome 2.1. Micro-Tom BAC Library Construction. Micro-Tom was processed as indicated in Figure 2. By eliminating repet- (TOMJPF00001) seeds were obtained from the NBRP itive, redundant, and unmapped sequences, 28,804 “unique International Journal of Plant Genomics 3 BAC insert size (kb) Figure 1: Distribution of BAC clone insert size. The insert size was deduced by mapping BESs onto the reference “Heinz 1706” genome (SL2.40). 93,682 BES BLASTN versus repeat DB Repetitive sequences (43, 598) 50,084 BES BLASTN versus “Heinz 1706” SL2.40 Multiple hits (10, 943) Low similraity (2, 015) No hit (59) 37,067 BES Both ends mapped One end mapped 28,804 BES 8,263 BES Figure 2: Flow of the BES analysis. To eliminate repetitive sequences, 93,682 BESs were initially searched against the repeat dataset of ITAG −50 2.3 with a BLASTN cutoff value of E <10 . Next, the remaining sequences were mapped onto the “Heinz 1706” pseudomolecule sequences −100 (SL2.40) under the following criteria: identity >90%, coverage >50%; E <10 ; the inclusion of single hits only. Mapped BESs were classified as either unique pair ends, for which both ends were mapped, or unique ends, for which only one end was mapped. pair ends” and 8,263 “unique ends” were selected. Paired-end physical length of the BAC contigs was 495,833,423 bp, cov- sequences were mapped onto the reference tomato genome ering 65.3% of the total chromosomes (Table 1). sequence, and 2,248 hypothetical BAC contigs were con- structed (see details at TOMATOMICS, http://bioinf.mind 3.2. Possible Genome Rearrangements. To assess the occur- .meiji.ac.jp/tomatomics/). The integrity of the hypothetical rence of genome rearrangements, Micro-Tom and the refer- contigs was confirmed by linking to the DNA markers on ence tomato “Heinz 1706” were compared. Possible inver- two genetic maps, AMF and MMF (see Supplementary sions, translocations, and insertions were considered. To 2 2 Table 1 in Supplementary Material available online at eliminate an artificial effect (e.g., chimeric BAC clones), only doi:10.1155/2012/437026). regions covered by more than two BAC clones were selected. The genome coverage of the hypothetical BAC con- After removing regions that had cleared the criteria for tigs was assessed by applying euchromatin/heterochromatin extraction (see Section 2 ) but were either shown to be multi- boundary information from the genetic map EXPEN2000 copy by manual evaluation of the BLAST results or displayed [2]. The results indicated that the euchromatin coverage similarity to transposable elements, we obtained two cases ranged between 45.1% and 71.1% (average, 58.9%) among of a possible rearrangement between Micro-Tom and “Heinz the different chromosomes, while heterochromatin coverage 1706” (Table 2). On chromosome 2, a possible inversion ranged between 57.4% and 75.3% (average, 67.3%). The total was detected. The size of this inversion could be 20–220 kb Number of clones <50 ≤60 ≤70 ≤80 ≤90 ≤100 ≤110 ≤120 ≤130 ≤140 ≤150 >150 4 International Journal of Plant Genomics Table 1: Coverage of chromosomes by hypothetical Micro-Tom BAC contigs. SL2.40ch01 SL2.40ch02 SL2.40ch03 SL2.40ch04 SL2.40ch05 SL2.40ch06 SL2.40ch07 SL2.40ch08 SL2.40ch09 SL2.40ch10 SL2.40ch11 SL2.40ch12 Total (ch01–ch12) Chromosome length 27,903,720 24,734,122 16,423,960 13,871,288 10,836,573 17,576,248 17,480,118 15,552,430 10,522,300 9,129,273 11,175,203 12,034,427 187,239,662 Euchromatin Euchromatin no. of Contigs 100 78 45 34 32 52 53 55 37 25 45 44 600 no. of BACs 533 504 401 231 170 339 224 279 176 197 184 317 3,555 Euchromatin Euchromatin Covered bases 17,310,734 14,644,412 11,678,941 6,261,540 5,377,576 10,621,719 8,336,310 9,541,847 6,047,365 5,473,701 6,855,876 8,119,058 110,269,079 Uncovered bases 10,592,986 10,089,710 4,745,019 7,609,748 5,458,997 6,954,529 9,143,808 6,010,583 4,474,935 3,655,572 4,319,327 3,915,369 76,970,583 Euchromatin Euchromatin % Coverage 62.0% 59.2% 71.1% 45.1% 49.6% 60.4% 47.7% 61.4% 57.5% 60.0% 61.3% 67.5% 58.9% Heterochromatin Chromosome length 62,400,524 25,184,172 48,416,754 50,193,024 54,184,865 28,465,388 47,788,503 47,480,227 57,139,791 55,705,032 42,210,822 53,451,826 572,620,928 no. of Contigs 175 74 147 131 169 76 135 150 149 159 128 155 1,648 Heterochromatin Heterochromatin no. of BACs 1,000 391 903 1,022 752 544 959 856 1,209 1,056 746 992 10,430 Covered bases 39,941,033 15,405,507 32,458,031 34,993,238 31,099,727 19,672,865 35,988,229 33,980,702 40,376,427 37,964,128 27,534,109 36,150,348 385,564,344 Heterochromatin Uncovered bases 22,459,491 9,778,665 15,958,723 15,199,786 23,085,138 8,792,523 11,800,274 13,499,525 16,763,364 17,740,904 14,676,713 17,301,478 187,056,584 Heterochromatin % Coverage 64.0% 61.2% 67.0% 69.7% 57.4% 69.1% 75.3% 71.6% 70.7% 68.2% 65.2% 67.6% 67.3% Heterochromatin Chromosome length 90,304,244 49,918,294 64,840,714 64,064,312 65,021,438 46,041,636 65,268,621 63,032,657 67,662,091 64,834,305 53,386,025 65,486,253 759,860,590 Total no. of Contigs 275 152 192 165 201 128 188 205 186 184 173 199 2,248 Total no. of BACs 1,533 895 1,304 1,253 922 883 1,183 1,135 1,385 1,253 930 1,309 13,985 Total Covered bases 57,251,767 30,049,919 44,136,972 41,254,778 36,477,303 30,294,584 44,324,539 43,522,549 46,423,792 43,437,829 34,389,985 44,269,406 495,833,423 Total Total Uncovered bases 33,052,477 19,868,375 20,703,742 22,809,534 28,544,135 15,747,052 20,944,082 19,510,108 21,238,299 21,396,476 18,996,040 21,216,847 264,027,167 % Coverage 63.4% 60.2% 68.1% 64.4% 56.1% 65.8% 67.9% 69.1% 68.6% 67.0% 64.4% 67.6% 65.3% Total Table 2: Possible genome rearrangement events observed in the Micro-Tom and “Heinz 1706” genome. No. BAC End1 Acc Chr Direction From To End2 Acc Chr Direction From To Possible event 1 MTBAC102D20 T7 FT290741 SL2.40ch02 — 29,374,874 29,375,640 SP6 FT290742 SL2.40ch02 — 29,494,209 29,494,781 Inversion 1 MTBAC084K15 T7 FT278701 SL2.40ch02 — 29,375,421 29,376,188 SP6 FT278702 SL2.40ch02 — 29,462,866 29,463,675 2 MTBAC041L05 T7 FT251747 SL2.40ch03 — 6,601,537 6,602,368 SP6 FT251748 SL2.40ch03 — 55,664,754 55,665,559 Translocation and Inversion 2 MTBAC077O14 SP6 FT274148 SL2.40ch03 — 6,602,568 6,603,163 T7 FT274147 SL2.40ch03 — 55,665,296 55,666,020 International Journal of Plant Genomics 5 depending on which end of the BAC clone is inversed. DNA from all 12 chromosomes. In the Kasalath rice BES Translocation and inversion were observed on chromo- analysis, chromosomal coverage in relation to the reference some 3. For each of two BAC clones (MTBAC041L05 and Nipponbare pseudomolecule was about 80%, despite the MTBAC077O14), one of the ends was mapped to 6,601 kb lower number (78,427) of analyzed BESs [22]. Because we of chromosome 3, while the other end was mapped to used the same criteria for repetitive sequence selection (E < −50 55,665 kb, more than 49 megabases apart. In addition, both 10 ), the discrepancy between the two studies might be ends were mapped on the minus strand. due to the larger genome size of tomato (950 Mb) compared with rice (430 Mb) [34]. Our Micro-Tom BAC coverage is reasonable, taking into account the scale of the BAC library 3.3. Polymorphisms between Micro-Tom and the Reference used. Tomato. SNPs and Indels between Micro-Tom and “Heinz Micro-Tom has been considered as a model cultivar to 1706” were identified. Among the SNPs and Indels found, promote functional genomics studies of tomato by taking 171,792 were mapped on 12 chromosomes, and 2,635 were advantage of its characteristics. Currently, many tools and mapped on pseudomolecules with no chromosomal infor- platforms have been developed, and some of these are already mation (SL2.40ch00 of the tomato whole-genome shotgun available to the research community. The present study char- chromosomes) (Table 3 and Supplementary Table 2, see acterized the overall polymorphisms found between Micro- details at TOMATOMICS). According to these results, Tom BESs and the reference tomato “Heinz 1706” genome. among the mapped SNPs and Indels, a total of 30,930 poly- In addition, two possible genome rearrangement events, on morphisms were found in the euchromatin (1 out of chromosome 2 and chromosome 3, were observed (Table 2). 3,565 bp), and 140,862 were found in the heterochromatin (1 In the case of translocation and inversion on chromosome out of 2,737 bp). The average polymorphism density in the 3, a gene annotated as reverse transcriptase was found in the genome was 1 polymorphism per 2,886 bp. Transversion- flanking region (Solyc03g104840.1). We speculate that this type SNPs were observed in 83,262 cases, while 60,631 were region was translocated by the activity of a retrotransposon, transition-type SNPs. Among the 30,534 Indels, single-base as it was in the case of SUN. Enhanced expression of insertions (on the SL2.40 version of the tomato whole- SUN caused by a gene duplication event mediated by the genome shotgun chromosomes) were observed in 10,740 retrotransposon Rider led to an elongated fruit shape [35]. cases, and single-base deletions were seen in 17,064 cases. In the future, we plan to sequence the entire BAC and expect The remainder were larger Indels, ranging from 2 to 26 bp that this will help us to characterize these events in more (Supplementary Table 2). Classification of polymorphisms detail. In the case of the other rearrangement possibility, on regarding genic or intergenic regions is shown in Table 4. chromosome 2, we could not find any trace of a retrotranspo- son. Since these rearrangements took place in euchromatin, which is rich in genes, these regions could represent an 4. Discussion interesting target to investigate their possible effects on By selecting unique end sequences from 93,682 reads, phenotypic variation between Micro-Tom and the reference 28,804 paired ends (14,402 pairs) and 8,263 unpaired ends tomato. We mapped the polymorphisms and depicted them, were obtained. The majority of the nonselected sequences (43,598) were derived from repetitive regions. For the rest, alongside maps showing covered regions and gaps, in 10,943 had redundant hits to the “Heinz 1706” genome, pos- Figure 3. On chromosomes 2, 5, and 11, polymorphisms seemed to be concentrated in the heterochromatic regions; sibly including repetitive sequences that were not represented in the repeat database in ITAG2.3 (http://solgenomics.net/), however, this tendency was not clearly observed in the other 2,015 showed weak similarity, and 59 showed no similarity chromosomes. For the other regions, the polymorphism dis- (Figure 2). Considering that the genome has been previously covery rate seemed to be somehow correlated with the BAC estimated to be composed of 25% gene-rich euchromatin coverage. Although our analysis indicated little possibility of [31, 32], BES selection in this study (39.6%, (28,804 + large-scale genome rearrangement between Micro-Tom and 8,263)/93,682)) could have eliminated repetitive regions to “Heinz 1706” (Table 2), this uneven polymorphism distribu- a moderate degree. We identified 59 reads showing no sig- tion suggests the existence of highly divergent chromosomal regions. The gaps in the hypothetical Micro-Tom BAC nificant similarity to the “Heinz-1706” genome. Micro-Tom was bred by crossing the home-gardening cultivars, Florida contigs could have resulted from low coverage of the BAC Basket and Ohio 4013-3. The pedigree of Ohio 4013-3 sug- library, but the occurrence of chromosomal segments specific to either Micro-Tom or “Heinz 1706” is also possible. The gested that a wild relative species was used in the breeding history [18, 33]. Such introgressed segments may lead to the ongoing Micro-Tom genome sequencing and de novo assem- introduction of genomic regions not harbored by “Heinz bly of the Micro-Tom genome will clarify the genome struc- 1706.” The Micro-Tom genome is now being sequenced ture in detail, enabling a more solid assessment of the dif- (draft sequence data available at DDBJ with the accession ferences between Micro-Tom and “Heinz 1706.” We had previously developed SNP markers among sev- number DRA000311), and mapping of orphan BESs to the de novo assembly of Micro-Tom genome data will help to clarify eral cultivated tomatoes [12]. By selecting SNPs through in this question. silico analysis using public EST information and previously developed SSR markers, 1,137 markers were obtained and The total physical length of Micro-Tom BAC contigs was 495,833,423 bp, which covers approximately 65.3% of the successfully mapped on linkage groups between Micro-Tom 6 International Journal of Plant Genomics Table 3: Number of polymorphisms found in each chromosome. SL2.40ch01 SL2.40ch02 SL2.40ch03 SL2.40ch04 SL2.40ch05 SL2.40ch06 SL2.40ch07 SL2.40ch08 SL2.40ch09 SL2.40ch10 SL2.40ch11 SL2.40ch12 Total Euchromatin no. of polymorphisms 4,152 4,123 3,700 2,863 969 2,417 3,504 2,113 1,932 1,302 1,694 2,161 30,930 Covered bases 17,310,734 14,644,412 11,678,941 6,261,540 5,377,576 10,621,719 8,336,310 9,541,847 6,047,365 5,473,701 6,855,876 8,119,058 110,269,079 Euchromatin Euchromatin kb/polymorphism 4,169 3,552 3,156 2,187 5,550 4,395 2,379 4,516 3,130 4,204 4,047 3,757 3,565 Heterochromatin no. of polymorphisms 12,319 10,694 10,408 9,995 30,951 5,134 8,347 8,562 10,231 9,209 14,937 10,075 140,862 Covered bases 39,941,033 15,405,507 32,458,031 34,993,238 31,099,727 19,672,865 35,988,229 33,980,702 40,376,427 37,964,128 27,534,109 36,150,348 385,564,344 Heterochromatin Heterochromatin kb/polymorphism 3,242 1,441 3,119 3,501 1,005 3,832 4,312 3,969 3,946 4,123 1,843 3,588 2,737 Total no. of polymorphisms 16,471 14,817 14,108 12,858 31,920 7,551 11,851 10,675 12,163 10,511 16,631 12,236 171,792 Covered bases 57,251,767 30,049,919 44,136,972 41,254,778 36,477,303 30,294,584 44,324,539 43,522,549 46,423,792 43,437,829 34,389,985 44,269,406 495,833,423 Total Total kb/polymorphism 3,476 2,028 3,129 3,208 1,143 4,012 3,740 4,077 3,817 4,133 2,068 3,618 2,886 Table 4: Number of polymorphisms found in genic and intergenic regions in each chromosome. SL2.40ch01 SL2.40ch02 SL2.40ch03 SL2.40ch04 SL2.40ch05 SL2.40ch06 SL2.40ch07 SL2.40ch08 SL2.40ch09 SL2.40ch10 SL2.40ch11 SL2.40ch12 Total 3 UTR 115 100 127 108 62 58 59 87 47 15 0 0 778 Exon 5 UTR 157 58 48 23 26 34 28 14 38 2 0 0 428 Genic CDS 1,152 967 1,031 758 820 603 757 605 669 570 662 615 9,209 Intron 2,035 1,938 2,023 1,971 1,547 1,122 1,778 1,154 1,425 994 1,947 1,172 19,106 Intron Splice 19 15 12 13 14 15 13 8 8 7 12 12 148 junction Intergenic 12,993 11,739 10,867 9,985 29,451 5,719 9,216 8,807 9,976 8,923 14,010 10,437 142,123 Total 16,471 14,817 14,108 12,858 31,920 7,551 11,851 10,675 12,163 10,511 16,631 12,236 171,792 International Journal of Plant Genomics 7 ch01 ch02 ch03 ch04 ch05 ch06 ch07 ch08 ch09 ch10 ch11 ch12 Figure 3: Micro-Tom BAC coverage with respect to the “Heinz 1706” chromosomes and detected polymorphisms. Black boxes indicate covered regions, and white boxes indicate gaps. Bars represent heterochromatic regions. 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