Background
Comparative genomics is a powerful tool for unraveling the evolutionary history and gene functionality of related species. The availability of high resolution genetic and physical maps and genome assemblies has established comparative genomics platforms in organisms ranging from bacteria and fungi to animals and plants 1234567. The majority of sequence-based eukaryotic platforms have focused on comparisons between genera 891011. Although highly informative, the divergence times between genera are often too distant to be useful for the identification of conserved noncoding sequences such as transcription factor binding sites, enhancers and matrix attachment regions (for example, see 1213). Sequence comparisons between species within a single genus have focused primarily on orthologous loci or genomic regions 1415. Whole genome comparative platforms within a genus are still in their infancy, with the most notable exception being the generation of whole genome shotgun assemblies of 12 Drosophila species that span an evolutionary time frame of approximately 60 million years 16.
In plants, rice (Oryza sativa) and thale cress (Arabidopsis thaliana) are important model systems for functional and evolutionary biology. Both genomes have been completely sequenced 1718 and serve as the reference sequences for the major monocot and dicot plant lineages. Rice has added significance by virtue of being the world's most important crop, directly feeding half the human population. The population that depends on rice is expected to double in 25-50 years and there is thus an intense effort by breeders to double current rice yields using less land and water and on poorer soils 19. To achieve this goal, the plant biology community is engaged in a concerted effort to functionally characterize all plant genes in both model plants using approaches including genetics, transgenetics, and comparative genomics. These efforts have significance not only to world food and bio-energy security issues but also to fundamental eukaryotic biology.
Comparative genomics across the cereals and within the genus Oryza will play a major role in these efforts. Oryza is composed of 24 species, including 2 domesticated (O. sativa, and O. glaberrima) and 22 wild species 2021. These species are classified into ten genome types (six diploids and four allotetraploids) based on crossing barriers 2223, chromosome pairing 2425, morphology 2627, and molecular phylogenetics 28. Compared to the domesticates, the wild relatives of rice are phenotypically inferior grass-like plants, but are a virtually untapped reservoir of agriculturally important genes and allelic variants that can be used to improve cultivated rice 2129. Therefore, comparative genomics of wild and domesticated Oryza species may not only provide useful materials for breeding, but will also shed light on the evolution and domestication of cultivated rice.
Our long-term objective is to develop a genome-level comparative experimental system for the genus Oryza (the Oryza Map Alignment Project (OMAP)). Our strategy is to develop bacterial artificial chromosome (BAC)-end sequence (BES) physical frameworks of the ten genome types of Oryza and align them to the reference sequence. These frameworks can then be populated with an array of phenotypic, genetic, biochemical, and physiological data to address fundamental questions in biology and agriculture. Previously, we described the construction and characterization of 12 BAC libraries from representative species that encompass the 10 genome types of Oryza 30. Here we report the construction of 12 BAC/BES framework physical maps derived from these libraries and an analysis of the BESs in terms of transposable element, simple sequence repeat (SSR), microRNA (miRNA), and single nucleotide variation (SNV) content.
Results
Fingerprinting, BAC end sequencing and contig assembly to generate phase I physical maps for the ten genome types of Oryza
BAC-based physical maps for the ten genome types of Oryza were generated using a two-step procedure. First, clones from 12 Oryza BAC libraries 30 were fingerprinted 31 and BAC end sequenced. Fingerprints from each library were then assembled into contigs using FPC 32. Fingerprint, BES and FPC assembly statistics are summarized in Tables 1 and 2. Briefly, we generated a total of 710,536 fingerprints and 1,452,912 BESs (approximately 932 Mb). Fingerprinting and BES success rates ranged from 85-94% and 88-96%, respectively. BESs represented approximately 8-17% of each Oryza genome, corresponding to one sequence tag per every 4-8 kb. From 90-98% of the BESs were paired and ranged in sequence length from 559 bp in O. minuta [BBCC] to 717 bp in O. officinalis [CC]. More importantly, an average of 89% of the fingerprinted clones had paired BESs. An average of 95% of the fingerprints assembled into contigs, with the remainder existing as singletons. The number of assembled contigs for each species ranged from 422 in O. brachyantha [FF] to 3,962 in O. minuta [BBCC], which roughly correlated with the size of each genome. The 12 unedited FPC assemblies are defined as phase I physical maps (Table 2) and are available interactively using WebFPC 33 or CMap, or as downloadable FPC files (see Materials and methods).
<p>Table 1</p>
Summary of BAC end sequences of 12 Oryza species
Species
Genome type
Genome size (Mb)*
No. of GenBank submissions
Average length after trim (in GenBank)
Total sequenced length (in GenBank)
Genome coverage
No. of forward reads
No. of reverse reads
No. of clones with paired reads (% of total BES)
O. nivara
AA
448
106,124
665 bp
~ 71 Mb
16%
53,450
52,674
51,820 (98%)
O. rufipogon
AA
439
70,982
704 bp
~ 50 Mb
11%
35,473
35,509
34,747 (98%)
O. glaberrima
AA
357
66,821
590 bp
~ 39 Mb
11%
33,456
33,365
30,885 (92%)
O. punctata
BB
425
68,384
710 bp
~ 49 Mb
11%
35,051
33,333
32,284 (94%)
O. officinalis
CC
651
101,091
717 bp
~ 72 Mb
11%
49,972
49,052
47,197 (93%)
O. minuta
BBCC
1,124
169,460
559 bp
~ 95 Mb
8%
85,466
83,994
82,248 (97%)
O. alta
CCDD
1,008
128,732
586 bp
~ 75 Mb
7%
64,365
64,367
58,217 (90%)
O. australiensis
EE
965
135,769
625 bp
~ 85 Mb
9%
67,245
68,524
64,081 (94%)
O. brachyantha
FF
362
67,364
672 bp
~ 45 Mb
13%
34,009
33,355
32,258 (96%)
O. granulata
GG
882
138,171
674 bp
~ 93 Mb
11%
69,962
68,209
66,434 (96%)
O. ridleyi
HHJJ
1,283
204,729
632 bp
~ 129 Mb
10%
102,640
102,089
98,160 (96%)
O. coarctata
HHKK
771†
195,285
661 bp
~ 129 Mb
17%
100,341
94,944
91,853 (94%)
Total/Average
1,452,912
650 bp
~ 932 Mb
11%
731,430
719,415
690,184 (95%)
*From [30]. †By K Arumuganathan using flow cytometric methods as previously described [30].
<p>Table 2</p>
Summary of phase I FPC physical maps of 12 Oryza species
Species
Average insert size
No. of total attempted
No. of clones FPCed
Genome coverage by all FPCed clones
Success ratio
Total organellar contam + no. inserts containing clones*
No. of FPC clones with paired BES reads
No. of singletons (%)
No. of contigs
Total CB units
Average no. of bands/clone
Deduced size of 1 CB unit† (kb)
Deduced genome size (Mb)‡ (coverage)
O. nivara
161
55,296
51,056
18
92%
4.1%
48,032 (94%)
2,356 (4.6%)
456
384,733
121.6
1.32
509 (114%)
O. rufipogon
134
35,712
33,023
10
92%
3.9%
31,202 (94%)
1,305 (4.0%)
637
406,768
106.2
1.26
513 (117%)
O. glaberrima
130
36,864
33,065
12
90%
3.7%
27,661 (84%)
2,098 (6.3%)
905
309,740
103.9
1.25
388 (109%)
O. punctata
142
36,864
34,224
11
93%
1.8%
30,228 (88%)
1,482 (4.3%)
490
340,240
117.4
1.21
412 (97%)
O. officinalis
141
54,144
45,856
10
85%
6.7%
40,549 (88%)
1,133 (2.5%)
703
462,126
115.5
1.22
564 (87%)
O. minuta
125
92,160
86,861
10
94%
1.2%
77,999 (90%)
9,576 (11.0%)
3,962
1,004,697
96.6
1.29
1,300 (116%)
O. alta
133
73,728
63,860
8
87%
0.4%
50,842 (80%)
3,111 (4.9%)
2,492
870,411
109.3
1.22
1,059 (105%)
O. australiensis
152
73,728
67,416
11
91%
2.7%
58,992 (88%)
3,144 (4.7%)
1,003
691,155
125.1
1.22
840 (87%)
O. brachyantha
131
36,864
33,424
12
91%
1.7%
29,523 (88%)
2,354 (7.0%)
422
215,208
100.9
1.30
279 (77%)
O. granulata
134
73,728
64,836
10
88%
3.4%
58,818 (91%)
3,032 (4.7%)
2,358
1,078,237
120.8
1.11
1,196 (136%)
O. ridleyi
127
110,592
104,393
10
94%
2.1%
93,192 (89%)
5,169 (5.0%)
2,190
1,216,461
111.2
1.14
1,389 (108%)
O. coarctata
123
100,224
92,522
15
92%
2.1%
82,180 (89%)
1,810 (2.0%)
1,250
611,953
100.0
1.23
753 (98%)
Total/Average
136
779,904
710,536
11
91%
2.8%
629,218 (89%)
5.1%
110.7
1.23
*From [30]. †Deduced one CB unit size = average inset size/average number of bands. ‡Deduced genome size = †Deduced one CB unit size × Total CB units.
To determine the approximate genome coverage of each of the 12 phase I physical maps, we estimated the size of the ordered consensus bands for each map in kilobases based on the average insert size of each BAC library and the average number of fingerprint bands observed for each species. Using this estimation, the genome coverage of the phase I physical maps ranged from 136% in O. granulata [GG] to 77% in O. brachyantha [FF]. In total, 7 of the physical maps had coverages greater that 100% (O. nivara, O. rufipogon, O. glaberrima, O. minuta, O. alta, O. granulata, and O. ridleyi), 4 had coverages between 98% and 87% (O. punctata, O. officinalis, O. australiensis, and O. coarctata) and 1 had a coverage of 77% (O. brachyantha). Coverage discrepancies above or below 100% could be due to a number of parameters, including: possible overlaps between the contigs that were not merged by the criteria used in this study; inaccurate estimation of genome size; and over-estimation of average insert sizes of the BAC libraries that would lead to an over-estimation of kilobases per consensus band (CB).
Alignment of phase I physical maps to the O. sativa reference sequence
To evaluate the coverage of the phase I physical maps graphically, we aligned the BESs associated with each FPC contig to the O. sativa reference sequence (RefSeq) 18 using SyMAP 34. SyMAP displays for all 12 physical maps can be viewed at 35 (display details are in Additional data file 1) and are summarized in Table 3 and Additional data file 2. Overall, between 61% and 95% of the contigs from each of the phase I physical maps could be aligned to the O. sativa RefSeq. Contigs not assigned a chromosomal position were generally small (133-289 CB/contig) and had few clones per contig (3-38 clones/contig) (Table 3). The percentage of BESs aligned, under identical parameters, was the least in O. alta [CCDD] (8% alignment) and the greatest in the AA genome type species O. nivara (60% alignment). Interestingly, the FF (O. brachyantha) and HHKK (O. coarctata) genome type species showed substantially more alignments to the O. sativa RefSeq than more closely related species (CC, BBCC, CCDD, EE, GG, and HHJJ).
<p>Table 3</p>
Summary of alignments of 12 OMAP Phase I FPC maps to the O. sativa RefSeq
Not aligned contig
Species
No. of contigs aligned
Total CB aligned
Total size aligned* (Mb)
No. of clones in aligned contig
No. of BESs aligned
Average identity of BES alignments
E-value of BES alignments
CB/contig (average)
No. of clones/contig (average)
O. nivara
368 (81%)
371,561 (97%)
490 (109%)
48,424 (99%)
63,293 (60%)
97%
e-237 to e-33
150
3
O. rufipogon
581 (91%)
399,346 (98%)
503 (115%)
31,398 (99%)
36,725 (52%)
97%
e-224 to e-57
133
6
O. glaberrima
860 (95%)
302,696 (98%)
378 (106%)
30,708 (99%)
33,356 (50%)
97%
e-213 to e-42
157
6
O. punctata
462 (94%)
332,224 (98%)
402 (95%)
31,674 (97%)
25,842 (38%)
93%
e-183 to e-30
286
38
O. officinalis
617 (88%)
448,477 (97%)
547 (84%)
44,194 (99%)
25,699 (25%)
93%
e-204 to e-16
159
6
O. minuta
3,073 (78%)
833,710 (83%)
1,075 (96%)
66,277 (86%)
46,227 (27%)
93%
e-173 to e-07
192
12
O. alta
1,571 (63%)
671,625 (77%)
819 (81%)
50,281 (83%)
10,770 (8%)
93%
e-193 to e-02
216
11
O. australiensis
787 (78%)
630,836 (91%)
770 (80%)
60,538 (94%)
20,429 (15%)
93%
e-182 to e-03
279
17
O. brachyantha
351 (83%)
205,608 (96%)
267 (74%)
30,717 (99%)
25,387 (38%)
91%
e-175 to e-18
135
5
O. granulata
1,444 (61%)
814,383 (76%)
904 (102%)
50,117 (81%)
18,785 (14%)
91%
e-179 to e-05
289
13
O. ridleyi
1,756 (80%)
1,123,902 (92%)
1,281 (100%)
94,565 (95%)
37,300 (18%)
92%
e-170 to e-06
213
11
O. coarctata
1,002 (80%)
579,809 (95%)
713 (92%)
89,775 (99%)
53,920 (28%)
92%
e-184 to e-07
130
4
*Deduced from [1 CB unit size from Table 2 × Total CB unit aligned].
Figure 1 shows a SyMAP alignment of chromosome 1 of the eight diploid Oryza species to the O. sativa RefSeq as an example. As expected, the most co-linear map alignments were observed with species that were evolutionarily less divergent (for example, AA, BB, CC). Regions of disrupted colinearity were detected mostly in the centromeric regions of the O. sativa genome. Although SyMAP alignments were also performed for the four polyploid species, we cannot conclude anything conclusive about their genome organization until their physical maps can be manually subdivided into each of their subgenomes.
<p>Figure 1</p>
SyMAP view of unedited physical maps of chromosome 1 from eight diploid Oryza species aligned to the O. sativa ssp. japonica chromosome 1 RefSeq
SyMAP view of unedited physical maps of chromosome 1 from eight diploid Oryza species aligned to the O. sativa ssp. japonica chromosome 1 RefSeq. The numbers in the small rectangles on the left are contig numbers of OMAP phase I physical maps. Beige bars on the right represent the O. sativa RefSeq (IRGSP V.4 assembly) and the red crosses on the beige bars represent the CentO position of O. sativa [18]. Purple lines represent BESs aligned to the O. sativa RefSeq. The order of the species from the left to right is; O. nivara [AA], O. rufipogon [AA], O. glaberrima [AA], O. punctata [BB]; O. officinalis [CC], O. australiensis [EE]; O. brachyantha [FF], O. granulata [GG].
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Sequence analysis of the OMAP BES data set: repeats, SSRs, miRNAs, and SNV
The large BES data set produced by this project offered a unique opportunity to explore sequence content and variation across the Oryza. We next investigated repeat and SSR content across all twelve species and identified miRNA content and SNV amongst the three AA and one BB genomes.
Repeats
RepeatMasker 36 in conjunction with a curated rice (O. sativa) repeat database was used to identify and classify previously identified repetitive elements. RECON 37 was used to identify repeats de novo. Results reported here relate to the number of bases covered by each element class, rather than the total number of members of each element class with the exception of small transposable elements (that is, miniature inverted repeat transposable elements (MITEs) and short interspersed elements (SINEs)), as BES reads are too short to span complete elements. The repeat content results ranged from a high of 76% in O. australiensis [EE] to a low of 36% in O. coarctata [HHKK] (Table 4). The repeats extracted by RECON overlapped with those identified by RepeatMasker by 32% in O. coarctata [HHKK] to 74% in O. punctata [BB]. This is expected as the curated repeat library used for RepeatMasker is O. sativa [AA] specific and, therefore, more distantly related species would be expected to share fewer repeats. Interestingly, the total repeat content of two CC genome containing allotetraploid species (O. minuta [BBCC] and O. alta [CCDD]) was less than the CC diploid genome of O. officinalis. It was also observed that the highest amount of unique or species-specific repetitive DNA was present in O. australiensis [EE] (21% corresponding to 203 Mb) and the lowest in O. granulata [GG] (10% corresponding to 92 Mb). These sequences were compiled into species-specific repeat databases.
<p>Table 4</p>
Repeat content analysis of 12 Oryza species using RepeatMasker and RECON
Repeat content by RECON
Species
Repeat content by RepeatMasker
Total
Overlap with RepeatMasker (% of total)
Uniqe (% of total)
Total repeat content
O. sativa (Nipponbare)
29.4%
37.2%
23.8% (64%)
13.4% (36%)
42.8%
O. nivara
37.0%
47.6%
29.5% (62%)
18.1% (38%)
55.1%
O. rufipogon
36.6%
49.7%
34.6% (70%)
15.2% (30%)
51.7%
O. glaberrima
28.5%
30.9%
19.8% (64%)
11.2% (36%)
39.7%
O. punctata
40.8%
41.4%
30.7% (74%)
10.7% (26%)
51.5%
O. officinalis
47.2%
56.5%
37.6% (67%)
18.9% (33%)
66.1%
O. minuta
44.0%
46.3%
32.2% (70%)
14.1% (30%)
58.1%
O. alta
43.2%
47.9%
33.2% (69%)
14.7% (31%)
57.9%
O. australiensis
55.0%
66.4%
45.4% (68%)
21.0% (32%)
76.0%
O. brachyantha
20.9%
27.5%
11.6% (42%)
15.9% (58%)
36.8%
O. granulata
30.1%
30.9%
20.5% (66%)
10.4% (34%)
40.5%
O. ridleyi
35.6%
42.7%
23.0% (54%)
19.7% (46%)
55.2%
O. coarctata
19.7%
24.5%
7.7% (32%)
16.7% (68%)
36.4%
The amount of repeats in each genome was directly proportional to the genome size except for O. granulata [GG] (Additional data file 3), suggesting that repetitive sequences play a major role in genome size expansion in the genus Oryza. O. australiensis [EE] had a high amount of repetitive DNA, consistent with the observation of a rapid burst of retrotransposons in this species 38.
We attempted to sub-classify the repetitive elements identified by their class. Only the results from the RepeatMasker search were used for classification (Figure 2) as the de novo repeats identified by RECON were mostly truncated. Retrotransposons (both long terminal repeats (LTRs) and non-LTRs) comprised the major fraction of the total repeats in all species and ranged from 46% of the genome in O. australiensis [EE] to 9% in O. brachyantha [FF]. Among all types of retroelements, the Ty-3/gypsy elements were the most predominant, followed by Ty-1/copia, LINEs and SINEs, respectively. The second most abundant class was DNA (non-MITE) transposons consisting mainly of the cacta-En/Spm, mutator, mariner, ping/pong/snoopy, hAT, and Ac/Ds elements, with En/Spm being the most predominant. These non-MITE DNA transposons ranged from 7% of the genome in O. ridleyi [HHJJ] to 2% in O. coarctata [HHKK]. An exception to this trend was O. brachyantha [FF], which had more MITEs (7%) than DNA (non-MITE) transposons (2%). MITEs were the third most abundant elements in the genus Oryza with the exception of O. brachyantha; MITEs ranged from 0.9% of the genome in O. australiensis to 7% in O. brachyantha. The three AA genome species, as well as O. sativa, have approximately equal amounts of both DNA (non-MITE) transposons as well as MITEs, although with slight differences. Since MITEs represent a large class of repeats with many different types, we calculated the percentage of different MITEs from the overall total as shown in Additional data file 4. Some types of MITEs were more abundant than others and also some were specific to a particular species - for instance, the three AA genome species illustrated a similar pattern for each element. Similarly, the distribution pattern of the MITE elements to the classification of MITE types and their proportion was more comparable among O. punctata [BB], O. officinalis [CC], O. minuta [BBCC] and O. alta [CCDD] genome species rather than those of the other genomes. A similar pattern was also detected between O. ridleyi [HHJJ] and O. coarctata [HHKK] species. This result indicates that the abundance and composition of the MITE elements are closely related to genome type evolution in the genus Oryza.
<p>Figure 2</p>
Comparison of classified repeat composition in the genomes of 12 Oryza species and the O. sativa RefSeq
Comparison of classified repeat composition in the genomes of 12 Oryza species and the O. sativa RefSeq.
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Analysis of O. alta [CCDD] revealed that approximately 9% of the BESs were categorized as unclassified repeats, high relative to other 12 Oryza species (2% in O. minuta [BBCC] to 0.2% in O. coarctata [HHKK]), implying the existence of one or more unique classes of repeats in O. alta or the presence of highly diverged repeats that were recognized by our current stringency parameters. On closer inspection, we identified a copia type LTR retrotransposon in a fully sequenced BAC (OA_BBa0237I11) representing a large family of retrotransposons that accounts for the majority of these unclassified repeats. This element, considered in detail elsewhere 39, accounted for two-thirds (2,939,525 bases of 4,392,322 bases) of these unclassified repeat sequences when assayed using cross_match alone.
Simple sequence repeats
SSRs are extremely useful for detecting variation between species and generating molecular genetic maps. A total of 16,980 non-redundant SSRs were detected from the 1.45 million BESs analyzed, which equates to approximately 18 SSRs/Mb of Oryza sequence. Overall, the AA and FF genome species (O. nivara [AA], O. rufipogon [AA], O. glaberrima [AA] and O. brachyantha [FF]) showed the highest SSR content, with an average of 27 SSRs/Mb, whereas the tetraploid (O. minuta [BBCC], O. alta [CCDD], O. ridleyi [HHJJ] and O. coarctata [HHKK]) and non-AA genome diploid species (O. punctata [BB], O. officinalis [CC], O. australiensis [EE], and O. granulata [GG]) showed 17.6 and 17.2 SSRs/Mb, respectively (Table 5). Among the 16,980 non-redundant SSRs, di- and tri-nucleotide SSRs were the most abundant, representing 48.6% and 26.7% of the total dataset, respectively. The relative frequency of di-nucleotide SSRs in all the Oryza species was 6-7% higher than in the O. sativa reference genomes (Table 5; Additional data file 5). In contrast, the relative frequency of tri-nucleotide SSRs was 4-5% lower than in the O. sativa reference genomes and was inversely related to that of the di-nucleotide repeats in each species (Additional data file 5).
<p>Table 5</p>
Distribution of OMAP non-redundant SSRs by motif type
Non-redundant
Di
Tri
Tetra
Penta
Hexa
Total
Species
Ploidy
Total no. of SSRs containing BESs (%)
No.
%
No.
%
No.
%
No.
%
No.
%
No.
SSR density (no. of SSRs/Mb)
O. sativa spp japonica
2
-
8,636
41.81
6,259
30.31
2,439
11.81
2,628
12.72
691
3.35
20,653
55.8
O. sativa spp indica
2
-
8,317
42.94
6,059
31.28
2,285
11.80
2,142
11.06
567
2.93
19,370
47.2
O. nivara
2
2,129 (2.01)
739
46.33
451
28.28
189
11.85
170
10.66
46
2.88
1,595
22.5
O. rufipogon
2
1,599 (2.25)
696
49.40
375
26.61
169
11.99
137
9.72
32
2.27
1,409
28.2
O. glaberrima
2
1,289 (1.93)
518
45.00
319
27.72
124
10.77
153
13.29
37
3.21
1,151
29.5
O. punctata
2
1,043 (1.53)
408
44.54
268
29.26
105
11.46
100
10.92
35
3.82
916
18.7
O. officinalis
2
1,397 (1.35)
545
47.43
308
26.81
111
9.66
146
12.71
39
3.39
1,149
16.0
O. australiensis
2
1,306 (0.96)
597
54.17
230
20.87
94
8.53
128
11.62
53
4.81
1,102
13.0
O. brachyantha
2
1,630 (2.42)
590
48.68
287
23.68
135
11.14
166
13.70
34
2.81
1,212
26.9
O. granulata
2
1,174 (0.85)
603
55.99
313
29.06
64
5.94
73
6.78
24
2.23
1,077
11.6
O. minuta
4
1,939 (1.14)
921
54.27
386
22.75
176
10.37
168
9.90
46
2.71
1,697
17.9
O. alta
4
1,582 (1.23)
812
52.83
363
23.62
182
11.84
141
9.17
39
2.54
1,537
20.5
O. ridleyi
4
2,305 (1.13)
709
35.36
741
36.96
238
11.87
257
12.82
60
2.99
2,005
15.5
O. coarctata
4
2,671 (1.37)
1,108
52.02
500
23.47
257
12.07
198
9.30
67
3.15
2,130
16.5
OMAP total
20,064 (1.38)
8,246
48.56
4,541
26.74
1,844
10.86
1,837
10.82
512
3.02
16,980
18.2
The top ten predominant motifs from the five most frequent SSRs in each species comprised between 68% (O. sativa ssp. japonica) and 80% (O. granulata) of all the SSRs identified (Figure 3). The TA, GA and CGG motifs were the most abundant and accounted for a total of 51% (30%, 15%, and 6%, respectively) of all the Oryza SSRs. Interestingly, two SSR motifs showed biased compositions in a species-specific manner. The TAA and CAA motifs were significantly higher in O. ridleyi (9.6% for TAA, 13% for CAA) and O. granulata (9% for CAA) compared to the average (4.2% and 3%, respectively) for all Oryza species (Figure 3). Further investigation using the rice repeats revealed that 137 of 235 BESs (58%) containing the TAA motif in O. ridleyi were associated with an uncharacterized LTR retrotransposon (Additional data file 6a). We failed to detect the known interspersed rice repeats in BESs containing the CAA repeat, but a BLAST search against O. granulata and O. ridleyi BESs revealed that more than 76% of total CAA-BESs in both species hit other BESs more than 50 times. This suggests the amplification of certain repeat elements contributed to increase the TAA and CAA repeats in both species (Additional data file 6b).
<p>Figure 3</p>
Comparison of the ten most frequent SSR motif compositions in 14 Oryza species (japonica, O. sativa ssp. japonica; indica, O. sativa ssp. indica; OMAP, average of 12 OMAP species)
Comparison of the ten most frequent SSR motif compositions in 14 Oryza species (japonica, O. sativa ssp. japonica; indica, O. sativa ssp. indica; OMAP, average of 12 OMAP species).
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The di-nucleotide SSRs showed the longest tracks of repeats, with an average of 44.5 ± 45.2 bp in the OMAP SSRs (Table 6). The TA repeat class showed the most variable repeat length among SSRs analyzed, with an average of 53.3 ± 55 bp (Additional data file 7). O. coarctata showed the least variable TA repeat length (31 ± 22.8 bp) and O. rufipogon showed the most variable length (73.8 ± 92.1 bp) in the Oryza species. We observed that the length of tri-nucleotide SSRs was inversely related to the G/C composition of their motifs (Additional data file 7). G/C rich motifs (CGA, CAG, CGG, TGG and GGA) revealed a shorter repeat length, with an average of 24.6 bp ± 4.5 bp in OMAP SSRs. The same trend was detected in japonica (24.9 ± 5.6 bp) and indica (24.8 ± 5.6 bp). However, A/T rich tri-nucleotide repeats (CAA, CAT, TAG, GAA and TAA) showed relatively longer tracks of repeats, with a length of 33.6 ± 18.2 bp in OMAP SSRs, 42.3 ± 30.1 bp in japonica, and 40.2 ± 25.9 bp in indica.
<p>Table 6</p>
Distribution of SSR length by the repeat types
OMAP SSR
japonica SSR
indica SSR
Repeat
No.
Average length
Standard deviation
No.
Average length
Standard deviation
No.
Average length
Standard deviation
Di
8,246
44.51
45.15
8,636
43.32
24.94
8,317
40.78
22.2
Tri
4,541
28.75
13.61
6,259
29.26
17.59
6,059
28.89
15.71
Tetra
1,844
27.69
12.69
2,439
34.25
28.81
2,285
29.9
16.51
Penta
1,837
25.11
7.73
2,628
25.24
6.39
2,142
25.52
9
Hexa
512
27.46
10.5
691
29.96
41.33
567
28.04
20.58
[C/G]A DNR*
3,151
30.29
11.08
3,617
32.31
14.76
3,722
31.55
12.45
TA DNR*
5,086
53.32
55
4,996
51.38
27.62
4,573
48.38
25.32
G/C rich TNR†
2,442
24.56
4.51
4,673
24.85
5.59
4,448
24.79
5.56
A/T rich TNR†
2,099
33.64
18.24
1,586
42.25
30.07
1,611
40.19
25.86
*Di-nucleotide repeat. †Tri-nucleotide repeat.
Identification of miRNAs in four Oryza species based on OMAP alignment data
miRNAs play an important role in plant developmental and physiological processes by negatively controlling gene expression 40414243. To detect evolutionarily conserved miRNAs between wild rice and O. sativa genomes, we anlayzed BES datasets from one domesticated and three wild Oryza genomes using an alignment-based approach.
In total, 64 paralogous miRNA precursors of O. sativa belonging to 22 miRNA families were identified among the four Oryza species (Table 7). O. rufipogon showed the highest number of conserved O. sativa miRNAs while O. punctata showed the lowest (22 and 7, respectively). This is consistent with our expectations since, as the evolutionary distance among species increases, the divergence within the miRNA precursor structure also increases 44. The miRNA families of miR166 and miR171, known to play a key role in apical meristem development, were detected in all four species; the highest number of copies of each was 9 and 7, respectively. Both of these miRNAs are known to be widely shared among plant lineages. In addition, four unknown locus miRNAs (miR420, miRNA441, miR442, and miR446) appeared to be AA genome specific.
<p>Table 7</p>
miRNAs conservation between O. sativa and four wild rice species detected from their BES datasets
O. sativa miRNA ID hits by wild rice species*
O. sativa miRNA family
Target mRNA class
O. nivara
O. rufipogon
O. glaberrima
O. punctata
Total no. of loci
miR156
ND
d
1
miR159
MYB and TCP TFs
e
b
2
miR160
ND
d
1
miR162
DICER-LIKE 1
b
1
miR164
ND
a
1
miR166
HD-Zip TFs
f, h, k
h, k, n
h, n
c
9
miR167
Auxin response factors TFs
a, d, f
j
d
a
6
miR168
ARGONAUTE
b
1
miR169
CCAAT binding factor and HAP2-like TFs
n, p
c
3
miR171
SCARECROW-like TFs
c, e, f
c
c, d
a
7
miR319
ND
a
1
miR395
ATP sulphurylases
i, j, k
3
miR396
GRF TFs, rhodenase-like, and kinesin-like protein B
b
1
miR397
Laccases and beta-6 tubulin
a
a
2
miR399
Phosphatase TFs
e
b, e
3
miR418
ND
x
x
2
miR420
ND
x
x
x
3
miR441
ND
b
a, b, c
c
5
miR442
ND
x
x
x
3
miR446
ND
x
x
x
3
miR531
ND
x
x
2
miR535
ND
x
x
x
x
4
Total no. of loci
20
22
15
7
64
*Named miRNA loci ID [69] are shown by species (for example, a putative miR156d locus ID is noted using a 'd' in the O. rufipogon column). For miRNA without a locus ID (for example, miR408), an 'x' is shown. ND, not determined; TF, transcription factor.
Variation discovery in four wild rice species and O. sativa
Candidate SNVs between wild rice BESs and the O. sativa genome were detected with a multi-tiered alignment strategy 45 using BLASTZ 46 and cross_match followed by a base-by-base analysis of the resulting high quality alignment (see Materials and methods). Putative nucleotide variations were further filtered using sequence quality scores and quality scores of neighboring regions to exclude variations that were due to possible sequencing error.
Our initial data set contained 330,993 high quality BESs (73,716 from O. rufipogon, 110,589 from O. nivara, 73,344 from O. glaberrima and 73,344 from O. punctata). Since two assemblies of the O. sativa ssp. japonica RefSeq (International Rice Genome Sequencing Project (IRGSP) V.4 and The Institute for Genomic Research (TIGR) V.4) were available, we detected variations on both assemblies using a quality score cutoff of 23. We were able to accurately map 217,202 BESs (53,426 from O. rufipogon, 76,886 from O. nivara, 50,736 from O. glaberrima, and 36,154 from O. punctata) to the IRGSP V.4 assembly and 217,258 BESs (53,273 from O. rufipogon, 76,833 from O. nivara, 50,833 from O. glaberrima, and 36,319 from O. punctata) to the TIGR V.4 assembly. A total of 1,619,446 non-redundant SNVs (Additional data file 8a) and 319,974 insertions and deletions (INDELs; Additional data file 8b) were detected between the BESs from the four Oryza species (O. rufipogon, O. nivara, O. glaberrima, and O. punctata) and the O. sativa genome assembly from IRGSP V.4. Likewise 1,619,557 SNVs (Additional data file 8c) and 320,592 INDELs (Additional data file 8d) were detected on the TIGR V.4 assembly. The overall divergence rate was found to be 1.22%, 1.32%, 1.59% and 7.15% for O. rufipogon, O. nivara, O. glaberrima and O. punctata, respectively. The variation rate was found to be 12.2, 13.2, 15.9 and 71.5 SNVs/kb and 1.5, 1.6, 2.0 and 11.4 INDELs/kb for O. rufipogon, O. nivara, O. glaberrima and O. punctata, respectively. In comparison, the polymorphism rate reported by the Beijing Genomics Institute 47 between the indica and japonica cultivars of O. sativa was 1.70 SNPs/kb and 0.11 INDELs/kb 48. Therefore, the rate of variation between Oryza species is roughly an order of magnitude greater than the polymorphism rate within species, as we would expect. The variation data are available through dbSNP and Gramene 49. An example view of the variation data in Gramene is shown in Additional data file 9.
Discussion
We describe here the generation and analysis of a comparative genomics resource for the genus Oryza that can be used as a research platform to address grand challenge questions in basic and applied biology. The primary resources developed were 1,452,912 BESs, 710,536 fingerprints, 12 phase I physical maps aligned to the rice RefSeq, and repeat, SSR, miRNA and SNV analyses. All sequences and variations have been deposited in GenBank and dbSNP, and all physical maps can be visualized using SyMAP 34 and CMAP at the OMAP 50 and Gramene 49 websites, respectively. The BAC libraries, BESs, SNaPshot fingerprints, and phase I physical maps have been publicly available since January 2006 and have been extensively accessed by the international community 3138515253545556.
Estimates of genome coverage of the physical maps relative to flow cytometry data were quite close except for O. granulate. To make the Oryza physical maps more useful for future research, they must now be manually edited to merge contigs and resolve any conflicts and mis-assemblies, as was done for O. punctata [BB] 31. Manual editing will be particularly challenging for the four allotetraploid species, where contigs need to be assigned to one of two subgenomes for each species. Fortunately for two tetraploids, O. minuta [BBCC] and O. alta [CCDD], we can use BES information from their diploid progenitors to assist in this process.
The BES data set represents an unprecedented sampling of genomic sequence from the Oryza. This resource is enhanced by the fact that the majority of BESs are binned in FPC contigs where 6-60% of BESs (depending on species) can be mapped to the rice RefSeq, and approximately 95% are paired. Here, we mined the BES data set for repeat, SSR, miRNA and SNV content. Repetitive sequences, especially transposable elements, are major components of plant genomes and contribute significantly to genome size variation. Detailed analysis of the BES resource for O. australiensis [EE] and O. granulata [GG] BESs have already demonstrated that retrotranspositional bursts of RIRE2, Wallaby and Kangaroo in the EE genome 38 and Gran3 in the GG genome 51 account for 50% and 25% of the genome sizes of these species, respectively. The RECON data described here for O. alta [CCDD] may likely represent yet a third retrotransposon burst accounting for genome size variation, subsequent to speciation 39.
Further analysis of the Oryza BES data identified 16,980 and 1,619,446 non-redundant SSRs and SNVs, respectively. The SSRs and SNVs can be immediately translated into polymorphic markers for breeding crosses between the two sequenced O. sativa genomes with the AA genome OMAP accessions to accelerate crop improvement. Further, the SSRs will serve as candidates to identify polymorphic genetic makers for intraspecific Oryza mapping populations currently under development (for example, BB × BB; CC × CC, and so on). The availability of molecular genetic maps tied to our physical maps for all 12 accessions will make these resources even stronger for functional and map-based cloning applications.
Paired-end data are also extremely important for studying structural variation, such as expansions, contractions, inversions and translocations 5758. Using our paired BES database, we plan to catalogue all such variation across the Oryza in order to address fundamental questions on the roles of such variation on the speciation process.
To our knowledge, the combined resources described herein comprise the most comprehensive within-genus comparative genomics framework available for any higher eukaryote, exceeded only by Drosophila, for which 12 draft genome sequences of wild flies are available 16. We envisage that the OMAP system will be a model for other within-genus comparative frameworks, including large genome plant genera like Gossypium (cotton), Solanum (tomato), Zea (corn) and Phaseolus (soybean). Having physical map frameworks for all genome types of a particular genus aligned to a reference genome provides immediate access to any orthologous or unique region of a genome(s) for functional and evolutionary analysis. Such frameworks are also highly compatible with next generation sequencing technologies whereby whole genomes can be sequenced in 4-8 Mb chunks, thereby reducing assemblies to the size of a bacterial genome 59.
Conclusion
The development and analysis of genus-wide research platforms is the next frontier in comparative systems biology. The genus Oryza, which includes the world's most important cereal, rice, is composed of 24 species (2 domesticated and 22 wild) with wide geographical distribution and contains 10 distinct genome types (6 diploid (2N = 24) and 4 polyploid (2N = 48)) and a 3.6-fold genome size variation (357-1,283 Mb).
We generated BAC-based physical maps of 12 Oryza species representing all the 10 genome types and aligned them to the O. sativa RefSeq. The 12 physical maps covered between 77% and 136% of each Oryza genome and revealed extensive colinearity to the O. sativa RefSeq. We analyzed the BES for repeats, SSRs, miRNA and SNVs. The repeat analysis not only cataloged the repeat content of each species, it suggested the possible roll they have played on genome size variation during the course of evolution. The SSR analysis identified 16,908 non-redundant SSRs from OMAP BESs, providing 18 SSRs/Mb of SSR density in the Oryza. We also found that the amplification of certain repeat elements contributed to an increase of the TAA and CAA motif repeats in the O. granulata [GG] and O. ridleyi [HHJJ] genomes. A total of 1,619,446 non-redundant SNVs and 319,974 insertions and deletions (INDELs) were detected between the BESs from four Oryza species (O. rufipogon, O. nivara, O. glaberrima, and O. punctata) and the O. sativa RefSeq. The SSR, SNV and INDEL markers can be immediately used for marker assistant selection as well as evolutionary studies. The miRNA analysis identified 64 paralogous miRNA precursors of O. sativa with more conservation in evolutionarily closer species. In addition, we observed species-wide miRNA families (miR166 and miR171) and AA genome specific miRNAs (miR420, miRNA441, miR442, and miR446).
All biological reagents are available at the AGI BAC/EST Resource Center 60 and all fingerprints and BESs are available at 4950 and GenBank. We envision that the resources and analysis presented here will serve as a model for the establishment of similar genus-wide frameworks for plants and animals. The genus-wide physical framework also fits in well with next generation sequencing technologies where an entire genome or targeted regions across a genus can be reduced to bacterial genome size chunks that can be easily sequenced and assembled as opposed to a whole genome sequencing approach.
Materials and methods
BAC libraries, end sequencing, fingerprinting and FPC assembly
All BAC libraries and methods have been described previously 3031.
FPC map alignment to the O. sativa RefSeq
SyMAP 34 was used for alignment and display of FPC contigs from each of the 12 phase I physical maps to the O. sativa RefSeq (IRGSP V.4). Briefly, the 12 BES data sets were repeat-masked 36 and then searched for sequence similarity to the rice genome using BLAT 61. To capture weaker sequence similarities from the more divergent species, BLAT parameters were adjusted as follows (-minIdentity = 70 -tileSize = 10 -minScore = 30 -qMask = lower -maxIntron = 10,000). BLAT results were filtered, to reduce false-positives, by retaining the top two hits for each query, as measured by match length, and subject to the additional criteria that no retained hit had a match length within 25% of a discarded hit. Approximate linear chains were computed from the retained hits using dynamic programming and then merged to form synteny blocks that were displayed by SyMAP. After the Oryza BESs were positioned on the O. sativa RefSeq, contigs containing the aligned BESs were anchored to the chromosomes and then renumbered in FPC. Contigs that aligned to less than 200 bp of O. sativa RefSeq were ignored in the alignment analysis (Table 3). All 12 SyMAP alignment results are available at 35.
Synteny analysis pipeline and incorporation of data in the CMap, Genome Browser and SyntenyView displays in Gramene
We constructed a pipeline to align clones and contigs from the OMAP species to O. sativa by combining data from the BES alignments to O. sativa and the phase I physical maps. The pipeline consisted of 6 steps: 1) upload phase I physical maps and BES data, 2) align BES to the RefSeq, 3) determine the best alignments for each clone, 4) assemble the clone positions to determine the region where the contig is found to align, 5) create blocks of synteny between selected OMAP species and O. sativa, 6) utilize the data to create visualizations in CMap, the Genome Browser, and SyntenyView in Gramene. Details on each step can be found at 62 Screen shots of the data in Gramene are shown in Additional data file 9.
Repeat analysis
RepeatMasker (V3.1.5), loaded with a custom Oryza repeat database, was used to identify repeats from the Oryza BES data set and the O. sativa RefSeq. The custom database was composed of annotated repeats from: TIGR rice repeat database 63; Dr Susan Wessler (University of Georgia, USA); and Dr Tom Bureau (McGill University, Canada).
RECON 37 was used to identify de novo repeats from the Oryza BES data set. To increase the speed and efficiency of the program, the BLAST output was parsed to discard self hits as well as hits with an e-value greater than 1e-5. The RECON output, which identified repetitive elements and classified them into distinct families, was parsed for sequences greater than 40 bp in length that were found at least 5 times/family. Overlap between the de novo and the custom library was determined using RepeatMasker. Sequences left unmasked by this process and, thus, were not a part of our custom repeat database, were extracted, assembled using PHRAP 64 and annotated using BLASTN at an e-value = 1e-4 against the NCBI non-redundant nucleotide database 65 and a dataset of 2,050 full length LTR-retrotransposons identified from the whole genome sequence of Nipponbare through LTR_STRUC 66. Finally, these sequences were compiled into repeat databases specific to each species. The procedure is outlined in Additional data file 10.
Simple sequence repeat analysis
A total of 20,064 SSRs were identified from 1,452,912 BESs using RepeatMasker (V3.1.5) with a cutoff of longer than 20 bp and less than 5% sequence divergence. BESs containing SSRs (SSR-BES) were assembled for each species using CAP3 67 to collect a total of 16,980 non-redundant SSRs. An SSR was considered redundant when the BAC clones came from the same FPC contig, and an assembled CAP contig contained the same SSR motif, repeat length and sequence divergence. Abundance and relative frequency of SSRs from the Oryza BESs were compared to SSRs identified from two rice genomic sequences: 20,653 SSRs from the japonica RefSeq 18 and 19,370 from the indica draft 6869 sequences using identical criteria. Other interspersed repeats in SSR-BESs were detected using RepeatMasker with a custom repeat database (courtesy of Dr Ning Jiang, Michigan State University).
Identification of Oryza miRNA genes
O.sativa japonica miRNA precursors from miRBase (Version 8.2) 70, including 250 nucleotides upstream and downstream, were extracted from the O. sativa RefSeq 18 and aligned, using BLASTN 71, against BES of four closely related wild rice species (AA and BB genomes). Alignments were filtered and multiple hits to the same region of a BES were discarded, keeping the hit with the lowest e-value. The best orthologous match typically had more conservation in the region surrounding the precursor and, therefore, had a much lower e-value. If two miRNAs aligned to different regions of the same BES, they were not discarded, since they were likely to be tandemly duplicated miRNA genes. To obtain the exact coordinates and secondary structure of the miRNA precursor within the remaining BES, a pattern matching approach was used to align the mature miRNA precursor to the BES. Once the mature miRNA was mapped to the BES, the extended sequence was then submitted to MFold to determine if it could form a stable hairpin structure 72. MFold alignments were checked for the following criteria: had conserved orientation; contained fewer than eight mismatches; was not overlapping the loop region; and lacked some types of asymmetric bulges. A precursor was discarded if the bulge had four or more nucleotides that failed to match with any nucleotide from the opposite strand. A precursor was retained if a bulge of four or fewer nucleotides on one stem corresponded, although not necessarily matching, with one or more nucleotides on the opposite strand. If the sequence that aligned to the mature miRNA met these criteria, it was categorized as the opposing stem (miRNA*). Both stems and the intervening loop region were extracted and resubmitted as input to MFold. If this refined sequence formed a stable hairpin structure under the four conditions, it was categorized as a paralogous miRNA precursor.
Variation discovery
BES trace files from O. rufipogon, O. nivara, O. glaberrima and O. punctata were compared to the O. sativa genome with BLASTZ 46 using the S1-S2 scoring method to select only the best alignment for each read. Each chromosome was then divided into 5 Mb segments and BESs aligned to each region from the previous step were re-aligned to the segment using cross_match 64 (-bandwidth 100 -alignments -discrep_lists) with variation filtering using the neighborhood quality standard (NQS) 73. Settings for NQS were such that candidate variation in the read had a Phred quality value (Q) of at least 23, its neighboring 7 bases on either side of the candidate variation all had Phred quality values of ≥ 15 and at least 11 of the 14 neighbors matched. If a read aligned to more than one place in the genome, then only the longest alignment with the fewest SNVs was reported. To decrease the number of false positive insertions or deletions, variations were filtered out if they had a single base variation and the quality score was less than 40 or if they were multiple base variations and the quality score was less than 23. Since two assemblies of the O. sativa RefSeq were available (IRGSP V.4 and TIGR V.4), variations were detected using both assemblies (Additional data file 8a-d). All data have been deposited into dbSNP and can be viewed at Gramene 49.
Abbreviations
BAC, bacterial artificial chromosome; BES, BAC end sequence; CB, consensus band; INDELs, insertions and deletions; IRGSP, International Rice Genome Sequencing Project; LTR, long terminal repeat; miRNA, microRNA; MITE, miniature inverted repeat transposable element; OMAP, Oryza map alignment project; RefSeq, reference sequence; SNV, single nucleotide variation; SSR, simple sequence repeat; TIGR, The Institute for Genomic Research.
Authors' contributions
HK, LS, DW, SAJ, and RAW designed the research; HK, KC, PS, MW, DK, and JLG performed the research; WN, MB, and CS contributed analytical tools; HK, BH, YY, NG, PS, JCM, and CM analyzed the data; HK, BH, YY, NG, PS, CM, WN, LS, DW, SAJ, CS, and RAW wrote the paper.
Additional data files
The following additional data are available with the online version of this paper. Additional data file 1 is a figure describing the SyMAP display details. Additional data file 2 is a table listing distribution of CB units from the O. sativa reference genome aligned contigs to each chromosome of 12 OMAP phase I physical maps. Additional data file 3 is a figure showing correlation of genome size and repeat content in the genus Oryza. Additional data file 4 is a figure presenting a comparison of MITE compositions in 12 OMAP genomes and the O. sativa genome. Additional data file 5 is a figure showing distribution of OMAP non-redundant SSR by motif types. Additional data file 6 is a table listing repeat association analysis for TAA and CAA motifs of O. ridleyi and O. granulata and BLAST analysis of CAA-BESs. Additional data file 7 is a figure comparing the SSR length and sequence compositions of OMAP SSR motifs. Additional data file 8 is a table listing SNVs and INDELs found between the BESs for O. rufipogon, O. nivara, O. glaberrima, O. punctata and the IRGSP V.4 pseudomolecules (or the TIGR V.4 pseudomolecules). Additional data file 9 is a figure describing wild rice BESs and variation data at Gramene. Additional data file 10 is a figure showing the strategy of repeat analysis and classification using the OMAP BES resources.
<p>Additional data file 1</p>
SyMAP display details
SyMAP display details.
Click here for file
<p>Additional data file 2</p>
Distribution of CB units from the O. sativa reference genome aligned contigs to each chromosome of 12 OMAP phase I physical maps
Distribution of CB units from the O. sativa reference genome aligned contigs to each chromosome of 12 OMAP phase I physical maps.
Click here for file
<p>Additional data file 3</p>
Correlation of genome size and repeat content in the genus Oryza
Correlation of genome size and repeat content in the genus Oryza.
Click here for file
<p>Additional data file 4</p>
Comparison of MITE compositions in 12 OMAP genomes and the O. sativa genome
Comparison of MITE compositions in 12 OMAP genomes and the O. sativa genome.
Click here for file
<p>Additional data file 5</p>
Distribution of OMAP non-redundant SSR by motif types
Distribution of OMAP non-redundant SSR by motif types.
Click here for file
<p>Additional data file 6</p>
Repeat association analysis for TAA and CAA motifs of O. ridleyi and O. granulata and BLAST analysis of CAA-BESs
Repeat association analysis for TAA and CAA motifs of O. ridleyi and O. granulata and BLAST analysis of CAA-BESs.
Click here for file
<p>Additional data file 7</p>
Comparison of the SSR length and sequence compositions of OMAP SSR motifs
Comparison of the SSR length and sequence compositions of OMAP SSR motifs.
Click here for file
<p>Additional data file 8</p>
SNVs and INDELs found between the BESs for O. rufipogon, O. nivara, O. glaberrima, O. punctata and the IRGSP V.4 pseudomolecules (or the TIGR V.4 pseudomolecules)
SNVs and INDELs found between the BESs for O. rufipogon, O. nivara, O. glaberrima, O. punctata and the IRGSP V.4 pseudomolecules (or the TIGR V.4 pseudomolecules).
Click here for file
<p>Additional data file 9</p>
Wild rice BESs and variation data at Gramene
Wild rice BESs and variation data at Gramene.
Click here for file
<p>Additional data file 10</p>
The strategy of repeat analysis and classification using the OMAP BES resources
The strategy of repeat analysis and classification using the OMAP BES resources.
Click here for file