The sequence of rice chromosomes 11 and 12, rich in disease resistance genes and recent gene duplications

Background Rice is an important staple food and, with the smallest cereal genome, serves as a reference species for studies on the evolution of cereals and other grasses. Therefore, decoding its entire genome will be a prerequisite for applied and basic research on this species and all other cereals. Results We have determined and analyzed the complete sequences of two of its chromosomes, 11 and 12, which total 55.9 Mb (14.3% of the entire genome length), based on a set of overlapping clones. A total of 5,993 non-transposable element related genes are present on these chromosomes. Among them are 289 disease resistance-like and 28 defense-response genes, a higher proportion of these categories than on any other rice chromosome. A three-Mb segment on both chromosomes resulted from a duplication 7.7 million years ago (mya), the most recent large-scale duplication in the rice genome. Paralogous gene copies within this segmental duplication can be aligned with genomic assemblies from sorghum and maize. Although these gene copies are preserved on both chromosomes, their expression patterns have diverged. When the gene order of rice chromosomes 11 and 12 was compared to wheat gene loci, significant synteny between these orthologous regions was detected, illustrating the presence of conserved genes alternating with recently evolved genes. Conclusion Because the resistance and defense response genes, enriched on these chromosomes relative to the whole genome, also occur in clusters, they provide a preferred target for breeding durable disease resistance in rice and the isolation of their allelic variants. The recent duplication of a large chromosomal segment coupled with the high density of disease resistance gene clusters makes this the most recently evolved part of the rice genome. Based on syntenic alignments of these chromosomes, rice chromosome 11 and 12 do not appear to have resulted from a single whole-genome duplication event as previously suggested.


Background
Rice (Oryza sativa) is a major staple food and is consumed by nearly half the world's population. It accounts for more than 21% of global human per capita energy and 15% of per capita protein. In the past few decades, although rice production has doubled due to the introduction of high yielding varieties/hybrids and improved cultivation practices, it is still insufficient to cope with ever-increasing global demand, which is expected to increase at the rate of about 1% per annum [1]. At the same time, inappropriate natural resource use, along with biotic and abiotic stress pressure, is casting a shadow on rice production [2]. Access to the rice genome sequence will enable identification of genes responsible for traits and alleles that will be essential to meet the growing demands of food production in the coming years. Towards this end, wholegenome shotgun-based draft sequences of the indica and japonica subspecies of rice were reported previously [3][4][5] while the International Rice Genome Sequencing Project (IRGSP), using a clone-by-clone approach, focused on generating a high quality, finished sequence of the rice genome [6]. Indeed, access to the rice genome has served as a catalyst for investigations on comparative genomics, functional genomics, map-based gene cloning and molecular breeding in rice [7,8].
Rice is one of the cereals in the Poaceae family, which collectively provides the largest source of calories for human consumption. Within the cereals are larger genome species such as maize, wheat, millet and sorghum [9]. Synteny in the cereals has been reported previously using molecular markers [10] and recently at a higher resolution using sequences available from the rice, sorghum, maize, and wheat genomes [3,11,14]. Thus, access to a complete, high-resolution rice genome sequence will facilitate research on other cereals with larger, partially sequenced genomes.
One of the emerging features of plant genomes appears to be the recent generation of gene copies that have diverged in regulation and function [15]. A common pathway for such a mechanism in plants is whole-genome duplication (WGD), which occurred for instance in maize as little as 5 million years ago [16]. Other mechanisms involve tandem gene amplifications and segmental duplications. In rice, chromosomes 11 and 12 provide such an example. In addition, several genes of agronomic importance, such as blast, bacterial blight, virus and insect resistance, photoperiod-sensitive male-sterility as well as salt tolerance, have been mapped on these two chromosomes [17]. Here, we report the in-depth analysis of chromosomes 11 and 12 of a japonica cultivar of rice with genetic lengths estimated to be 118.6 cM and 110.1 cM, respectively [18]. We have generated high-quality finished sequences for these two chromosomes, annotated the chromosomes for genes and other features, and used these data to examine novel features such as the organization of disease resistance genes. We also asked whether chromosomes 11 and 12 are likely to have resulted from a WGD event by examining duplication events between the two chromosomes a Total number of genes includes the TE-related genes and the non-TE-related genes. b Gene density is reported for all genes and non-TE-related genes (in parentheses). c Average length of the genes is determined using the length of from the start codon to stop codon of the longest isoform.
through alignments of mapped genes of wheat along the rice chromosomes.

General features of chromosomes 11 and 12
A total of 255 and 269 BAC/PAC clones were sequenced from chromosomes 11 and 12, respectively. Chromo-some 11 is slightly longer than chromosome 12, with 28.4 Mb and 27.5 Mb, respectively, of sequence identified for them (Table 1, Fig. 1). Excluding the telomeres, a few physical gaps remain on the two pseudomolecules; six and one on chromosomes 11 and 12, respectively (Accession Numbers: chromosome 11: DP000010, chromosome 12: DP000011). This was due to a difficulty in Display of features on rice chromosomes 11 and 12 Figure 1 Display of features on rice chromosomes 11 and 12. Using a false-color display, we plotted features present on rice chromosomes 11 and 12. Select genetic markers are plotted with their cM positions noted; physical gaps are plotted with the centromere gap noted in red; gene density (GD) is plotted in two tracks (all genes and non-TE-related genes); expression density is determined by aligment to ESTs; transposable element (TE) density is plotted with a separate track for MITEs; tRNAs are the transfer RNAs, and the CP and MT represent chloroplast and mitochondrial insertions.
obtaining clones that span these regions, especially the centromeres and telomeres, which present technical challenges in sequencing highly repetitive tracts of the genome.
Of the 4,286 and 4,169 genes identified on chromosomes 11 and 12, respectively, 3,148 and 2,845 could be annotated as non-transposable element related (non-TE; Fig.  1). A similar percentage of genes on the two chromosomes could be assigned a putative function or annotated as encoding an expressed protein, leaving a similar percentage annotated as encoding a hypothetical protein (Table 1). With respect to domain composition, there was a striking difference in representation of some Pfam domains between the chromosomes, suggesting the presence of genes coding for different suites of proteins on the two chromosomes. As shown in Tables 2 and 3, there was a large enrichment of proteins on chromosome 11 (166 proteins) containing a leucine-rich repeat (LRR) domain, which is involved in protein-protein recognition and is a hallmark of disease resistance genes (see below). Besides the LRR-domain, there are two other domains common to disease resistance genes, protein kinase and NB-ARC domains, which were also enriched on chromosome 11 relative to chromosome 12; 106 versus 74 proteins containing protein kinase domains and 102 versus 49 proteins with NB-ARC domains on chromosome 11 versus 12, respectively. Based on alignments by a rice transcript, 1,235 (39%) and 1,221 (43%) of non-TE-related genes on chromosomes 11 and 12, respectively, were active (Addi-tional figure 10 [see Additional file 1]). Of these, 952 and 980 non-TE-related genes from chromosomes 11 and 12, respectively, could be aligned with a FL-cDNA. In addition, 152 non-TE-related genes aligned with ESTs from other monocots on each chromosome. For 26 genes from chromosome 11 and 23 from chromosome 12, only nonmonocot evidence was available for expression (Additional figure 10 [see Additional file 1]). A ready set of mutants is available for 800 and 845 genes on chromosomes 11 and 12, respectively, as evidenced by a flanking sequence tag (FST) in or within 500 bp of the transcription unit.
A similar fraction of chromosomes 11 and 12 could be classified as repetitive (29.5% and 31.6%, respectively (Additional tables 5 and 6 [see Additional file 1]), which with the exception of miniature inverted repeat transposable elements (MITEs) were enriched in the centromeric and pericentromeric regions of the two chromosomes ( Fig. 1). As noted previously [19], MITEs, which constitute 18.6 and 16.2% of the repetitive sequences on chromosomes 11 and 12, respectively, were enriched in the euchromatic arms of the two chromosomes ( Fig. 1).
A similar number of non-TE-related proteins with putative homologs in model species were present in chromosomes 11 and 12 (Fig. 2). The highest number of putative homologs was seen in Arabidopsis, with 60.9% and 59.6% of the proteins from chromosomes 11 and 12, respectively, having a potential homolog in Arabidopsis (E value cutoff 10 -5 ). Similar ranges of potential homologs were seen between the two chromosomes and the other model organisms, ranging from 7-30.6% in chromosome 11 and 10-33.6% in chromosome 12.
A high percentage of rice genes could be aligned with genomic assemblies from two other cereals, maize and sorghum. Of the 2,405 maize assemblies with a best hit on rice chromosome 11 or 12, 1,879 overlap with non-TErelated genes while 160 overlap with TE-related genes (Additional table 7 [see Additional file 1]). The remaining 366 assemblies align to intergenic regions, which might include genes or TEs that are degenerated and not recognized by our bioinformatics methods. Interestingly, for sorghum, the occurrence of alignments was similar to maize except that there was a higher incidence of alignment to TE or intergenic regions, which could be due the fact that the sorghum dataset includes only methyl-filtrated and not C o t-enriched sequences [20][21][22]. The 1,469 non-TE-related genes that aligned to both maize and sorghum genomic assemblies suggest that these genes might predate the divergence of the Panicoideae, estimated at 50 mya [23].

Disease resistance genes in rice chromosomes 11 and 12
Disease resistance genes (R-like genes) conferring resistance to viral, bacterial, fungal and nematode pathogens have been grouped into five classes on the basis of their encoded protein products [24]. For instance, a genomewide study identified such typical protein domains in the R-like genes of Arabidopsis [25]. Major classes of conserved domains include leucine zipper (LZ), coiled coil (CC), nucleotide binding site (NBS), leucine-rich repeat (LRR), protein kinase, Toll-IL-IR homology region (TIR) and trans-membrane (TM) as well as miscellaneous Rgenes. Apart from the genes containing these typical domains, one maize disease resistance gene, HM1, encodes a reductase that detoxifies HC-toxin [26]. We analyzed the sequences of chromosomes 11 and 12 to identify the type and distribution of the disease resistance as well as downstream defense response genes (Fig. 3). We identified 201 R-like gene models in chromosome 11 (Additional table 8   Presence of potential homologs in rice chromosomes 11 and 12 with other model organisms  [27]. A detailed depiction of duplicated genes in the interval 112-119 cM on chromosome 11 is shown in Fig. 5 and a cladogram of these genes is shown in Additional figure 11 [see Additional file 1], confirming that the genes within a cluster generally belong to the same clade. A tandem array of 12 Xa21-like genes is present on the short arm of chromosome 11 at position 19 cM (between 3,516,492 -3,665,074 bp), while the actual Xa21 gene is present on the long arm of chromosome 11 [28]. A large cluster of 14 defense response genes, 12 of which are chitinases, is present in tandem at 116.2 cM (between 28,056,455 -28,122,601 bp). Several other R-like genes are also arranged in similar but small clusters.
On chromosome 12, 88 gene models showed homology to R-like genes ( Fig. 3 Additional table 8 [see Additional file 1]), which is 2.0% of the total number of gene models predicted for this chromosome. However, 50 of these 88 gene models belonged to the miscellaneous category including viral resistance, Verticillium wilt resistance, BLB resistance genes and those containing LRR motif but without NBS, CC or LZ motifs. On chromosome 12, only 18 gene models showed homology to the NBS-LRR category. Although the R-like genes and defense response genes were distributed throughout the chromosome, they were present in clusters (Fig. 4). A large cluster of 23 miscellaneous resistance genes was found between positions 40-50 cM (between 5,858,249 and 10,009,727 bp) and a cluster of seven defense response genes was found at position 107.4 cM (between 26,803,729 and 26,870,016 bp). The previously described blast resistance gene Pita [29] was present near the centromere of chromosome 12. The total number of R-like genes in chromosome 12 was less than half of the number in chromosome 11, even though the size and total number of genes in these two chromosomes are similar. This can be attributed to the enrichment of NBS-LRR, LZ-NBS-LRR and LRR-TM-like genes in chromosome 11, which was 3-4 times higher than chromosome 12 (Fig. 3). The difference in the number of R-like genes between the two chromosomes also has an impact on the extent of tandem gene arrays. Chromosome 11 has a total of 924 genes (29%) and chromosome 12 has 684 genes (24%) that are duplicated at least once within a short distance (Fig. 6).
A previous report on the analysis of R-like genes in the entire rice genome revealed that most of the R-like genes (24.98%) are present on chromosome 11 [30]. Another report found more than 25% of the physically mapped   NBS-encoding genes on chromosome 11 and claimed that approximately 20% of the NBS-LRR genes in the Nipponbare genome were predicted to be pseudogenes [31].
Comparison of the total number of genes containing the LRR domain (837) in the finished genome sequence [6] with the number of such genes on chromosomes 11 (166 genes) and 12 (60 genes) in the present study (Tables 2  and 3) shows a similar percentage (27%) of R-like genes to be on these two chromosomes. However, the previous total estimate of 536 R-like genes is significantly lower than the one from the finished genome (837), indicating that previous draft sequences did not provide sufficient information for capturing all candidate genes for disease resistance and, in addition, that gene finding methods are not directly comparable. Complete sequence information is also important for accurate sequence alignments of Rlike genes with cloned disease resistance genes from rice (Pita, Xa21, Pib) Arabidopsis (RPM1), tomato, (Cf2, Cf9), barley (Mla1) and wheat (Lr10). The R-like gene and defense response gene hot spots in these two chromosomes will be invaluable for future mapping and cloning of disease resistance genes from naturally occurring disease resistant rice lines. However, manual re-annotation of these R-like genes, as done in case of Arabidopsis [25], and functional validation of the candidate resistance genes, would be important for drawing practical benefits from this information.

Recent duplication on chromosomes 11 and 12
Chromosomes 11 and 12 harbor duplicated regions at the distal ends of their short arms as determined by physical and genetic mapping [32]. Based on previous versions of the rice genome sequence, it became evident that segmental duplications occur throughout the genome [5,7,33].
Here, we focused on segmental duplications between chromosome 11 and 12 only because the finished sequences of these chromosomes permit us to conduct an analysis of the organization of their genes in a detail that was not possible before. At ≥50% coverage and ≥80% identity and excluding TE-related gene models, 546 gene models were identified as duplicated. A subset of these, 350 from chromosome 11 and 352 from chromosome 12, was found to be unique between these chromosomes, excluding repeated gene models. Based on the chromosomal locations of these unique genes, the maximum extent of duplication was confined within the first 3 Mb of both chromosomes (Fig. 7). Possibly, there could be a difference between japonica and indica varieties, as previous comparisons identified a second duplication between chromosomes 11 and 12 in the indica [5] but not the japonica [33] species. Here, most of the duplicated gene models (98%) were found in the same orientation on both the chromosomes (Fig. 8a,b). The 3 Mb region was further analyzed to find the number of duplicated genes at ≥30, ≥40, ≥50, ≥60, ≥70, ≥80 and ≥90% coverage. The

Number of genes in tandems
Number of tandems expression after gene amplification has also been reported in maize [15,34].
To determine the time of the segmental duplication, the full-length coding regions of a subset of homoeologous genes that had been genetically mapped and were distributed over the entire length of the duplication were aligned and pair-wise analyzed for their nucleotide substitution rates (  11 and 12, respectively) were found to be expressed based on homology to an EST or cDNA, in contrast to 56 and 58% in the first 3 Mb region of both the chromosomes. Out of these, nine expressed genes were common to both chromosomes. Also, there were nine and five expressed genes of chromosomes 11 and 12 that Chromosome 12 sequences were used as query against a database of chromosome 11 sequences using MegaBLAST as described under Methods

Figure 7
Chromosome 12 sequences were used as query against a database of chromosome 11 sequences using MegaBLAST as described under Methods. At ≥50% coverage and >80% identity, the frequency distribution of unique duplicate gene models is plotted over the length of chromosomes 11 and 12. Based on the chromosomal locations of these unique genes, duplications were identified throughout the length of both the chromosomes. The maximum extent of duplication, however, was found to be confined within the first 2 Mb region of both chromosomes.  had non-expressed counterparts. This analysis supports the view that the first 3 Mb duplication of chromosome 11 and 12 is of recent origin. However, the loss of order as well as the low percentage of expressed genes, on each chromosome or in duplicate gene pairs, does not support a duplication of the remaining regions of chromosomes 11 and 12, unless it is very ancient in origin and signatures of duplication have largely disappeared.

Rice-wheat synteny
To investigate further whether rice chromosomes 11 and 12 were related to each other and arose by a WGD event, we compared each gene model predicted from these two chromosomes against 586,577 wheat ESTs. Comparison of bin-mapped wheat ESTs with earlier versions of rice genome sequence provided a glimpse of the structural similarities between these two important cereal genomes [11,13]. However, the present study involves comparative analysis of the finished version of rice chromosomes 11 and 12, which allowed a higher resolution analysis with wheat and a comparative analysis between rice chromosomes 11 and 12. A gene-by-gene comparison of sequence homology of all the predicted rice gene models with the wheat ESTs revealed that 1,588 (35.8%) and 2,220 (51%) of the gene models in chromosomes 11 and 12, respectively, have significant homology with the wheat ESTs at a bit cut-off score of 100. Of these, 416 gene models (26.2%) from chromosome 11 and 552 gene models (24.86%) from chromosome 12 showed significant similarity with bin-mapped wheat EST contigs, a number that is much larger than the levels reported earlier for chromosomes 11 and 12 [13]. A complete list of the matching genes along with their annotated functions is provided in Additional tables 11 and 12 [see Additional file 1].
Gene duplication between rice chromosomes 11 and 12 over the whole length (a) and in the first 3 Mb region (b) Figure 8 Gene duplication between rice chromosomes 11 and 12 over the whole length (a) and in the first 3 Mb region (b). Blue and red lines connect duplicate gene models in the same and opposite orientation, respectively.
Out of the 416 gene models from rice chromosome 11, 338 (81.3%) mapped to single wheat homoeologous groups, with the maximum number (35.2%) located on group 4 chromosomes of wheat. The distribution of rice genes that mapped to the wheat chromosome groups 1, 2, 3, 5, 6 and 7 was 9.2%, 9.5%, 12.4%, 15.4%, 8.9% and 9.5%, respectively ( Fig. 9 Additional figures 12 and 13 [see Additional file 1]). The majority of rice chromosome 11 gene models mapped to group 4 chromosomes of wheat indicating their common origin. Many of these genes were clustered in the distal region of the long arm of wheat chromosome 4A, but the same gene models mapped to the short arms of wheat chromosomes 4B and 4D, reflecting significant rearrangements and a dynamic state of gene organization in homoeologous chromosomes of wheat and rice chromosome 11. Similar to chromosome 11, a high percentage of chromosome 12 gene models (441, 79.9%) mapped to single wheat homoeologous groups. Of these, the maximum percent-age (30.6%) mapped to group 5 chromosomes of wheat whereas the remainder were distributed almost uniformly on the other six wheat homoeologous groups (Fig. 9 Additional figures 12 and 13 [see Additional file 1]).
A comparative distribution of rice gene homologs from chromosomes 11 and 12 to the seven wheat homoeologous groups clearly indicates that the two rice chromosomes have different origins and, apart from the recent 3 Mb duplication, there is not much in common between the two chromosomes. This contradicts earlier observations that the whole of the rice chromosomes 11 and 12 may have evolved as a result of chromosome duplication via polyploidy about 70 mya, i.e. before the divergence of cereals [7]. Furthermore, these results are also consistent with the alignment of the genetic maps of cereal genomes [10], where rice aligns with two homoeologous regions of maize.

Conclusion
With the completion of the sequences of rice chromosomes 11 and 12, we were able to identify 289 R-like and 28 defense response-like genes, accurately date a 3 Mb recent duplication between the two chromosomes, and show significant synteny between these two chromosomes and wheat chromosome groups 4 and 5, respectively. Rice chromosome 11 has several large clusters of fast evolving disease resistance and defense response genes that have originated by the process of tandem duplication and subsequent divergence under the selective pressure of rice pathogens. This sequence and annotation will be an essential resource for the engineering of rice for tolerance to biotic and abiotic stresses to accommodate growing production needs.

Sequencing and bioinformatic methods
Bacterial artificial chromosome and P1-derived artificial chromosome (PAC) clones from chromosomes 11 and 12 were sequenced to 8-10× coverage using shotgun DNA sequencing [35,36] and standard high-throughput methods [6]. Gaps and ambiguities were resolved using resequencing, alternative chemistries, PCR and primer walk-ing [6]. Pseudomolecules or virtual contigs for chromosomes 11 and 12 were constructed as described previously [27]. The pseudomolecules and annotation are available in GenBank under the accession numbers DP000010 and DP000011. Genes were identified using the ab initio gene finder FGENESH [37] and gene model structure was improved with EST and full-length cDNA (FL-cDNA) evidence using the Program to Assemble Spliced Alignments [38]. Genes were annotated for function as described [27]. Transposable element-related (TE) genes were identified using either TBLASTN similarity to known repetitive elements in the TIGR Oryza Repeat Database [39] or the presence of repetitive element-related Pfam domains [40]. Flanking sequence tags (FSTs) from various insertion mutagenesis projects were aligned with the two pseudomolecules using flast [41] with a cutoff of 95% identity over 80% of the FST length. Transfer RNAs were identified using tRNA-ScanSE [42]. Organellar insertions were determined using BLASTN with the rice mitochondrion and chloroplast genome sequences using a cutoff of 95% identity (Fig. 1). Sequenced genetic markers (4,619 sequences [18,43]) were searched against the pseudomolecules with flast; a cutoff criterion of ≥95% identity over ≥90% length of the marker was used to align the markers to the Syntenic mapping of rice genes from rice chromosomes 11 and 12 to wheat Figure 9 Syntenic mapping of rice genes from rice chromosomes 11 and 12 to wheat. Distribution of the rice gene homologues from rice chromosomes 11 (blue bars) and 12 (yellow bars) on to the wheat chromosomes of seven homoeologous groups. Non-TE-related genes with rice transcript evidence were identified by searching chromosomes 11 and 12 against the TIGR rice gene index (Release 16 [45]). To identify genes with FL-cDNA support, we searched 33,678 FL-cDNAs available from the KOME database [46]. Genes with an alignment of ≥95% identity and a minimum of 50% length of the alignment covered by a gene index sequence were considered supported by a rice EST or FL-cDNA. Seven monocot gene indices [47] were also used to demonstrate support of expressed genes (wheat release 9.0, maize release 15.0, barley release 9.0, sorghum release 8.0, rye release 3.0, onion release  [20,49]. Sorghum genomic reads (593,969) were downloaded from NCBI and processed using the Lucy program [50] to remove vector and low quality sequences. The remaining "good" reads (504,458) were assembled into 163,908 clusters as described [20]. The assemblies from sorghum and maize were searched against the TIGR rice pseudomolecules [Release 3.0, January 2005] using the BLASTZ program [51]. Homologs were identified in other model organisms by searching the non-TE-related proteins from chromosomes 11 and 12 against the E. coli, Synechocystis, yeast, Drosophila, C. elegans, human and Arabidopsis predicted proteomes using BLASTP.
Chromosome 11-12 duplication analysis was performed using the predicted gene models. Chromosome 12 sequences were used as query against a database of chromosome 11 sequences using MegaBLAST [52]. Only non-TE gene models at ≥50% coverage were used for further analysis. To compare the expression pattern of duplicated genes, a homology search was performed against the NCBI EST and KOME cDNA databases using the unique duplicated non-TE gene models at ≥90% coverage and identity. Wheat-rice synteny was determined using BLASTN of the chromosome 11 and 12 gene model sequences against Triticum aestivum sequences obtained from dbEST. Search parameters were as described previously [12]. Matching gene models were compared with wheat contigs containing bin-mapped ESTs [53] and plotted on the 21 wheat chromosomes.

List of abbreviations used
bacterial artificial chromosome (BAC), flanking sequence tag (FST), leucine-rich repeat (LRR), million years ago (mya), nucleotide binding site (NBS), resistance gene (Rgene), transposable element (TE), whole-genome duplication (WGD). At AGI, WW, DK and CM contributed to shotgun library construction and clone management. YY, TR, JC, KC, HRK, DS and RAW contributed to shotgun sequencing, sequence assembly and finishing aspects of this project.

Authors' contributions
At CSHL, MK and LS provided sequence assembly and finishing, LN, RP and TZ production sequencing, LP, AO and SD contributed to data analysis and annotation, and WRM directed the research.
At PGIR, EL contributed to clone management, EL and BT to shotgun library construction and sequence/assembly, CD, GF and JM to data analysis, and JM to writing the manuscript; JM is the corresponding author.
At RGP, JW, HM and TM contributed to physical map construction, Fiber-FISH analysis, and physical map extension, NM carried out data analysis and TS directed the research.
At UW, WJ and JJ contributed to gap analysis using fiber-FISH.