International Journal of Plant Genomics

Volume 2012 (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

Erika Asamizu,¹ Kenta Shirasawa,² Hideki Hirakawa,² Shusei Sato,² Satoshi Tabata,² Kentaro Yano,³ Tohru Ariizumi,¹ Daisuke Shibata,² and Hiroshi Ezura¹

¹Faculty of Life and Environmental Sciences, University of Tsukuba, 1-1-1 Tennodai, Tsukuba 305-8572, Japan

²Kazusa DNA Research Institute, 2-6-7 Kazusa-kamatari, Kisarazu 292-0818, Japan

³School of Agriculture, Meiji University, 1-1-1 Higashi-mita, Tama-ku, Kawasaki 214-8571, Japan

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.

Abstract.

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 polymorphisms 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

Tomato (Solanum lycopersicum) is one of the most important vegetable crops cultivated worldwide. Tomato has a diploid (2n = 2x = 24)and relatively compact genome of approximately 950 Mb [1]. Recently, its genome has been completely sequenced by the international genome sequencing consortium [2].

Genetic linkage maps of tomato have been created by crossing cultivated tomato (S. lycopersicum) with several wild relatives, S. pennellii, S. pimpinellifolium, S. cheesmaniae, S. neorickii, S. chmielewskii, S. habrochaites, and S. peruvianum [3]. Introgression lines generated from a cross between S. lycopersicum and S. pennellii have contributed to the isolation of important loci and quantitative trait loci (QTLs) related to fruit size by utilizing DNA markers on the Tomato-EXPEN 2000 genetic map [4–9]. Such interspecies genetic mapping is effective because the divergent genomes provide many polymorphic DNA markers. In contrast, intraspecies mapping is less popular in tomato because of the low genetic diversity within cultivated tomatoes that has resulted from the domestication process and subsequent modern breeding [10]. Recently, we developed SNP, simple sequence repeat (SSR), and intronic polymorphic markers using publicly available EST information and BAC-end sequences (BESs) derived from “Heinz 1706,” a standard line for tomato genomics [11, 12], and applied these markers to create linkage maps between Micro-Tom and either Ailsa Craig, a greenhouse tomato, or M82, a processing tomato, by mapping 1,137 markers [12].

Micro-Tom, a dwarf cultivar, is regarded as a model cultivar for functional genomics of tomato because of several characteristics, including small size (20 cm plant height), short life cycle (3 months), existence of indoor cultivation protocols under normal fluorescent conditions, and high-efficiency transformation methods that have been developed for this line [13–15]. The dwarf phenotype of Micro-Tom is the result of mutations in at least two major recessive loci. dwarf (d) encodes a cytochrome P450 protein, which functions in the brassinosteroid biosynthesis pathway [16]. Another locus, miniature (mnt), is suggested to associate with gibberellin (GA) signaling without affecting GA metabolism, but the causal gene has not been identified to date [17]. In Japan, Micro-Tom genomics resources have been extensively accumulated, mainly in the framework of the National BioResource Project (NBRP) (http://tomato.nbrp.jp/indexEn.html). Large-scale ethyl methanesulfonate (EMS) and gamma-ray-mutagenized populations have been created, and visible phenotype data have been accumulated [18–20]. The availability of Micro-Tom genome sequence data will accelerate the mapping of mutant alleles.

BAC-end sequencing has been performed in the tomato standard line “Heinz 1706” genome project to order BAC clones along the chromosomes [21]. Currently, about 90,000 BESs are available at the Sol Genomics Network (SGN, http://solgenomics.net/). BAC-end sequencing has been conducted for other crop species. In the rice indica cultivar “Kasalath,” 78,427 BESs were generated from 47,194 clones and mapped onto the “Nipponbare” reference genome. As a result, 12,170 paired BESs were mapped that covered 80% of the rice genome [22]. Recently, BAC-end sequencing has been performed in crop plants with higher genome complexity. BESs from a commercial sugarcane variety, an interspecific hybrid with complex ploidy, were generated to analyze microsynteny between sugarcane and sorghum [23]. In wheat, which has a complex hexaploid genome, the short arm of chromosome 3A was flow sorted to make a BAC library, and chromosome arm-specific BESs were generated for DNA marker development [24]. In switchgrass, more than 50,000 SSRs were identified from 330,000 BESs, and this enabled detailed analysis on the evolution of this species [25]. A low level of genetic variation has been observed for cultivated peanuts. Polymorphic SSRs were accumulated from the BESs and successfully used in the construction of a genetic map [26]. BAC-end sequencing can be useful as a resource for performing comparative genomic studies through mapping of the sequences to a reference genome and by facilitating the development of polymorphic DNA markers.

In the present study, we generated 93,682 single-pass end sequences from a Micro-Tom BAC library. To compare the structures between the reference tomato “Heinz 1706” genome, mapping of unique ends was performed, and possible genome rearrangements and polymorphisms were identified.

2. Materials and Methods

2.1. Micro-Tom BAC Library Construction

Micro-Tom (TOMJPF00001) seeds were obtained from the NBRP (MEXT, Japan) and sent to the Clemson University Genomics Institute (CUGI) for BAC library construction. The genomic DNA was partially digested, and fragments were cloned into the Hind III site of pIndigoBAC536. A total of 55,296 clones in Escherichia coli DH10B cells were arrayed in 144 384-well plates.

2.2. End Sequencing of Micro-Tom BAC Clones

To analyze BESs, the BAC DNAs were amplified using a TempliPhi large-construction kit (GE Healthcare, UK), and the end sequences were analyzed according to the Sanger method, using a cycle sequencing kit (Big Dye-terminator kit, Applied Biosystems, USA) with a type 3730xl DNA sequencer (Applied Biosystems). The resulting sequence reads were quality checked with PHRED [27, 28], allowing the identification and removal of low-quality (QV < 20) sequences. The 93,682 reads clearing the quality criteria were submitted to DDBJ/GenBank with accession numbers FT227487-FT321168.

2.3. Mapping to the Reference Genome and Analyses

BES reads were subjected to similarity search using the BLASTN program [29, 30]. To isolate unique sequences from repetitive ones, 93,682 BESs were searched against the repeat database in ITAG2.3 (http://solgenomics.net/) using a cutoff E-value of less than 10⁻⁵⁰. The remaining sequences were searched against the published version of the “Heinz 1706” genome (SL2.40), which was accessed from the SGN database (http://solgenomics.net/). From all of the BLAST alignments, BESs were extracted according to the following criteria, suggested in a previous report [22]: (1) sequence identity > 90% and alignment coverage > 50%; (2) mapped positions of each pair of ends < 200  kb apart in the same chromosome; (3) direction of each paired end is correct; (4) BLASTN E < 10⁻¹⁰⁰; (5) a minimum of one hit for one of the paired ends; (6) no redundant chromosomal locations. Sequence polymorphisms (SNPs and Indels) between Micro-Tom and “Heinz 1706” were predicted based on the BLASTN alignment. Since we did not allow a gap exceeding 27 bases, only Indels up to 26 bases in length were counted.

2.4. Database and Clone Distribution

Mapped data and SNP/Indel sites were made accessible through the database TOMATOMICS at http://bioinf.mind.meiji.ac.jp/tomatomics/. BAC clones are available upon request from NBRP tomato (http://tomato.nbrp.jp/indexEn.html).

3. Results

3.1. General Features of the Generated BESs

The BAC insert size distribution was deduced based on the mapping results. According to these results, 45.4% (6,396 out of 14,101) of the BACs ranged from 100 to 120 kb, with average and median 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.

Figure 1: Distribution of BAC clone insert size. The insert size was deduced by mapping BESs onto the reference “Heinz 1706” genome (SL2.40).

Micro-Tom BES mapping to the “Heinz 1706” genome was processed as indicated in Figure 2. By eliminating repetitive, redundant, and unmapped sequences, 28,804 “unique pair ends” and 8,263 “unique ends” were selected. Paired-end sequences were mapped onto the reference tomato genome sequence, and 2,248 hypothetical BAC contigs were constructed (see details at TOMATOMICS, http://bioinf.mind.meiji.ac.jp/tomatomics/). The integrity of the hypothetical contigs was confirmed by linking to the DNA markers on two genetic maps, AMF₂ and MMF₂ (see Supplementary Table 1 in Supplementary Material available online at doi:10.1155/2012/437026).

Figure 2: Flow of the BES analysis. To eliminate repetitive sequences, 93,682 BESs were initially searched against the repeat dataset of ITAG 2.3 with a BLASTN cutoff value of E <10⁻⁵⁰. Next, the remaining sequences were mapped onto the “Heinz 1706” pseudomolecule sequences (SL2.40) under the following criteria: identity >90%, coverage >50%; E <10⁻¹⁰⁰; 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.

The genome coverage of the hypothetical BAC contigs was assessed by applying euchromatin/heterochromatin boundary information from the genetic map EXPEN2000 [2]. The results indicated that the euchromatin coverage ranged between 45.1% and 71.1% (average, 58.9%) among the different chromosomes, while heterochromatin coverage ranged between 57.4% and 75.3% (average, 67.3%). The total physical length of the BAC contigs was 495,833,423 bp, covering 65.3% of the total chromosomes (Table 1).

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)

Euchromatin
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
no. of Contigs
100
78
45
34
32
52
53
55
37
25
45
44
600

Euchromatin
no. of BACs
533
504
401
231
170
339
224
279
176
197
184
317
3,555

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

Euchromatin
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
% 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

Heterochromatin
no. of Contigs
175
74
147
131
169
76
135
150
149
159
128
155
1,648

Heterochromatin
no. of BACs
1,000
391
903
1,022
752
544
959
856
1,209
1,056
746
992
10,430

Heterochromatin
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%

Total
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
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

Total
% 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%

3.2. Possible Genome Rearrangements

To assess the occurrence of genome rearrangements, Micro-Tom and the reference tomato “Heinz 1706” were compared. Possible inversions, translocations, and insertions were considered. To eliminate an artificial effect (e.g., chimeric BAC clones), only regions covered by more than two BAC clones were selected. After removing regions that had cleared the criteria for extraction (see Section 2) but were either shown to be multicopy by manual evaluation of the BLAST results or displayed similarity to transposable elements, we obtained two cases of a possible rearrangement between Micro-Tom and “Heinz 1706” (Table 2). On chromosome 2, a possible inversion was detected. The size of this inversion could be 20–220 kb depending on which end of the BAC clone is inversed. Translocation and inversion were observed on chromosome 3. For each of two BAC clones (MTBAC041L05 and MTBAC077O14), one of the ends was mapped to 6,601 kb of chromosome 3, while the other end was mapped to 55,665 kb, more than 49 megabases apart. In addition, both ends were mapped on the minus strand.

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

3.3. Polymorphisms between Micro-Tom and the Reference Tomato

SNPs and Indels between Micro-Tom and “Heinz 1706” were identified. Among the SNPs and Indels found, 171,792 were mapped on 12 chromosomes, and 2,635 were mapped on pseudomolecules with no chromosomal information (SL2.40ch00 of the tomato whole-genome shotgun chromosomes) (Table 3 and Supplementary Table 2, see details at TOMATOMICS). According to these results, among the mapped SNPs and Indels, a total of 30,930 polymorphisms were found in the euchromatin (1 out of 3,565 bp), and 140,862 were found in the heterochromatin (1 out of 2,737 bp). The average polymorphism density in the genome was 1 polymorphism per 2,886 bp. Transversion-type SNPs were observed in 83,262 cases, while 60,631 were transition-type SNPs. Among the 30,534 Indels, single-base insertions (on the SL2.40 version of the tomato whole-genome shotgun chromosomes) were observed in 10,740 cases, and single-base deletions were seen in 17,064 cases. The remainder were larger Indels, ranging from 2 to 26 bp (Supplementary Table 2). Classification of polymorphisms regarding genic or intergenic regions is shown in Table 4.

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

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

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

Heterochromatin
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
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

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
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

Genic
Exon
3′ UTR
115
100
127
108
62
58
59
87
47
15
0
0
778

5′ UTR
157
58
48
23
26
34
28
14
38
2
0
0
428

CDS
1,152
967
1,031
758
820
603
757
605
669
570
662
615
9,209

Intron
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

Splice junction
19
15
12
13
14
15
13
8
8
7
12
12
148

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

4. Discussion

By selecting unique end sequences from 93,682 reads, 28,804 paired ends (14,402 pairs) and 8,263 unpaired ends were obtained. The majority of the nonselected sequences (43,598) were derived from repetitive regions. For the rest, 10,943 had redundant hits to the “Heinz 1706” genome, possibly including repetitive sequences that were not represented in the repeat database in ITAG2.3 (http://solgenomics.net/), 2,015 showed weak similarity, and 59 showed no similarity (Figure 2). Considering that the genome has been previously estimated to be composed of 25% gene-rich euchromatin [31, 32], BES selection in this study (39.6%, (28,804 + 8,263)/93,682)) could have eliminated repetitive regions to a moderate degree. We identified 59 reads showing no significant similarity to the “Heinz-1706” genome. Micro-Tom was bred by crossing the home-gardening cultivars, Florida Basket and Ohio 4013-3. The pedigree of Ohio 4013-3 suggested that a wild relative species was used in the breeding history [18, 33]. Such introgressed segments may lead to the introduction of genomic regions not harbored by “Heinz 1706.” The Micro-Tom genome is now being sequenced (draft sequence data available at DDBJ with the accession number DRA000311), and mapping of orphan BESs to the de novo assembly of Micro-Tom genome data will help to clarify this question.

The total physical length of Micro-Tom BAC contigs was 495,833,423 bp, which covers approximately 65.3% of the DNA from all 12 chromosomes. In the Kasalath rice BES analysis, chromosomal coverage in relation to the reference Nipponbare pseudomolecule was about 80%, despite the lower number (78,427) of analyzed BESs [22]. Because we used the same criteria for repetitive sequence selection (E < 10⁻⁵⁰), the discrepancy between the two studies might be 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 used.

Micro-Tom has been considered as a model cultivar to promote functional genomics studies of tomato by taking advantage of its characteristics. Currently, many tools and platforms have been developed, and some of these are already available to the research community. The present study characterized the overall polymorphisms found between Micro-Tom BESs and the reference tomato “Heinz 1706” genome. In addition, two possible genome rearrangement events, on chromosome 2 and chromosome 3, were observed (Table 2). In the case of translocation and inversion on chromosome 3, a gene annotated as reverse transcriptase was found in the flanking region (Solyc03g104840.1). We speculate that this region was translocated by the activity of a retrotransposon, as it was in the case of SUN. Enhanced expression of SUN caused by a gene duplication event mediated by the retrotransposon Rider led to an elongated fruit shape [35]. In the future, we plan to sequence the entire BAC and expect that this will help us to characterize these events in more detail. In the case of the other rearrangement possibility, on chromosome 2, we could not find any trace of a retrotransposon. Since these rearrangements took place in euchromatin, which is rich in genes, these regions could represent an interesting target to investigate their possible effects on phenotypic variation between Micro-Tom and the reference tomato.

We mapped the polymorphisms and depicted them, alongside maps showing covered regions and gaps, in Figure 3. On chromosomes 2, 5, and 11, polymorphisms seemed to be concentrated in the heterochromatic regions; however, this tendency was not clearly observed in the other chromosomes. For the other regions, the polymorphism discovery rate seemed to be somehow correlated with the BAC coverage. Although our analysis indicated little possibility of large-scale genome rearrangement between Micro-Tom and “Heinz 1706” (Table 2), this uneven polymorphism distribution suggests the existence of highly divergent chromosomal regions. The gaps in the hypothetical Micro-Tom BAC contigs could have resulted from low coverage of the BAC library, but the occurrence of chromosomal segments specific to either Micro-Tom or “Heinz 1706” is also possible. The ongoing Micro-Tom genome sequencing and de novo assembly of the Micro-Tom genome will clarify the genome structure in detail, enabling a more solid assessment of the differences between Micro-Tom and “Heinz 1706.”

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).

We had previously developed SNP markers among several cultivated tomatoes [12]. By selecting SNPs through in silico analysis using public EST information and previously developed SSR markers, 1,137 markers were obtained and successfully mapped on linkage groups between Micro-Tom and either Ailsa Craig or M82. In the present study, we identified 171,792 SNPs and Indels and mapped them on 12 chromosomes. The average density was 1 SNP per 3,565 bp in euchromatin and 1 SNP per 2,886 bp in the genome in general (including both euchromatin and heterochromatin). Previously, large-scale Micro-Tom full-length cDNA analysis and comparison of exon regions with those on the “Heinz 1706” genome revealed a mean sequence mismatch of 0.061% (1/1,640 bp) [36]. One possible explanation for the difference is the quality of the reference “Heinz 1706” genome sequence used in the two studies. We used the published version of the “Heinz 1706” genome sequence, which has higher coverage, giving rise to greater accuracy, although our selection may still contain sequence errors because BESs are single-pass sequences.

The information provided in this study will be useful in the development of DNA markers between Micro-Tom and cultivated tomatoes, which will facilitate a better understanding of the physiological and metabolic differences between them. It would also be useful in the genetic mapping of Micro-Tom mutants through the generation of F₂ segregating populations.

Authors’ Contribution

E. Asamizu and K. Shirasawa equally contributed to this work.

Acknowledgments

The authors are grateful to Shinobu Nakayama and Akiko Watanabe for technical assistances. Construction of the Micro-Tom BAC library was supported by Japan Solanaceae Consortium (JSOL). This work was supported by the Genome Information Upgrading Program “Micro-Tom BAC-end sequencing” of the National BioResource Project in 2009.

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