The transfer RNA genes in Oryza sativa L. ssp. indica

The availability of the draft genome sequence of Oryza sativa L. ssp. indica has made it possible to study the rice tRNA genes. A total of 596 tRNA genes, including 3 selenocysteine tRNA genes and one suppressor tRNA gene are identified in 127551 rice contigs. There are 45 species of tRNA genes and the revised wobble hypothesis proposed by Guthrie and Abelson is perfectly obeyed. The relationship between codon usage and the number of corresponding tRNA genes is discussed. Redundancy may exist in the present list of tRNA genes and novel ones may be found in the future. A set of 33 tRNA genes is discovered in the complete chloroplast genome of Oryza sativa L. ssp. indica. These tRNA genes are identical to those in ssp. japonica identified by us independently from the origional annotation.     

Characterization of Imcrop, a Mutator-like MITE family in the rice genome

Sequence comparisons of ammonium transporter 1?C2 genes (OsAMT1-2) in different rice accessions revealed a MITE insertion in the upstream region of the gene. The 391-bp MITE, classified as a Mutator superfamily member and named Imcrop, included terminal inverted repeat (TIR) and 9-bp target site duplication (TSD) sequences. We identified 151 Imcrop elements dispersed on 12 chromosomes of the japonica reference genome. Of these, 12.6% were found in genic regions and 33.1% were located within 1.5 kb of annotated rice genes. We constructed comparative insertion maps with 111 and 102 intact Imcrop elements in the japonica and indica reference genomes, respectively. The Imcrop elements showed relatively even distribution across all chromosomes although their frequency was higher on chromosomes 1, 3, and 4 in both genomes. Seventy seven Imcrop elements were detected in both subspecies, whereas 34 and 25 insertions were found only in the japonica or indica genome, respectively. We compared insertion polymorphisms of 19 Imcrop elements found inside genes in 48 Korean rice cultivars, consisting of 42 japonica and six Tongil-types (indica-japonica cross). Thirteen insertions were common to all cultivars indicating these elements were present before indica-japonica divergence. The six other elements showed insertion polymorphisms among accessions, showing their recent insertion history or no critical positive effect of their insertion on the rice genome.     

Analysis of indica- and japonica-specific markers of Oryza sativa and their applications

Asian rice, Oryza sativa L., is one of the most important crop species. Genetic analysis has established that rice consists of several genetically differentiated variety groups, with two main groups, namely, O. sativa ssp. japonica kata and ssp. indica kata. To determine the genetic diversity of indica and japonica rice, 45 rice varieties, including domesticated rice and Asia common wild rice (O. rufipogon Griff.), were analyzed using sequence-related amplified polymorphism, target region amplified polymorphism, simple sequence repeat, and intersimple sequence repeat marker systems. A total of 90 indica- and japonica-specific bands between typical indica and japonica subspecies were identified, which greatly helped in determining whether domesticated rice is of the indica or japonica type, and in analyzing the consanguinity of hybrid rice with japonica, which were bred from indica and japonica crossed offspring. These specific bands were both located in the coding and non-encoding region, and usually connected with quantitative trait loci. Utilizing the indica-japonica-specific markers, japonica consanguinity was detected in sterile hybrid rice lines. Many indica-japonica-specific bands were found in O. rufipogon. This result supports the multiple-origin model for domesticated rice. Javanica exhibited a greater number of indica-japonica-specific bands, which indicates that it is a subspecies of O. sativa L.     

New insights into <Emphasis Type="Italic">Oryza</Emphasis> genome evolution: high gene colinearity and differential retrotransposon amplification

An ∼247-kb genomic region from FF genome of wild rice Oryza brachyantha, possessing the smallest Oryza genome, was compared to the orthologous ∼450-kb region from AA genome, O. sativa L. ssp. japonica. 37 of 38 genes in the orthologous regions are shared between japonica and O. brachyantha. Analyses of nucleotide substitution in coding regions suggest the two genomes diverged ∼10 million years ago. Comparisons of transposable elements (TEs) reveal that the density of DNA TEs in O. brachyantha is comparable to O. sativa; however, the density of RNA TEs is dramatically lower. The genomic fraction of RNA TEs in japonica is two times greater than in O. brachyantha. Differences, particularly in RNA TEs, in this region and in BAC end sequences from five wild and two cultivated Oryza species explain major genome size differences between sativa and brachyantha. Gene expression analyses of three ObDREB1 genes in the sequenced region indicate orthologous genes retain similar expression patterns following cold stress. Our results demonstrate that size and number of RNA TEs play a major role in genomic differentiation and evolution in Oryza. Additionally, distantly related O. brachyantha shares colinearity with O. sativa, offering opportunities to use comparative genomics to explore the genetic diversity of wild species to improve cultivated rice. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users. Data deposition: Sequence data from this article were deposited with GenBank Library under accession number DQ810282. Shibo Zhang and Yong Qiang Gu contributed equally to the work     

Comparative proteomic analysis of indica and japonica rice varieties

Indica and japonica are two main subspecies of Asian cultivated rice (Oryza sativa L.) that differ clearly in morphological and agronomic traits, in physiological and biochemical characteristics and in their genomic structure. However, the proteins and genes responsible for these differences remain poorly characterized. In this study, proteomic tools, including two-dimensional electrophoresis and mass spectrometry, were used to globally identify proteins that differed between two sequenced rice varieties (93–11 and Nipponbare). In all, 47 proteins that differed significantly between 93–11 and Nipponbare were identified using mass spectrometry and database searches. Interestingly, seven proteins were expressed only in Nipponbare and one protein was expressed specifically in 93–11; these differences were confirmed by quantitative real-time PCR and proteomic analysis of other indica and japonica rice varieties. This is the first report to successfully demonstrate differences in the protein composition of indica and japonica rice varieties and to identify candidate proteins and genes for future investigation of their roles in the differentiation of indica and japonica rice.     

Phylogenetic Genomewide Comparisons of the Pentatricopeptide Repeat Gene Family in indica and japonica Rice

More than 400 pentatricopeptide repeat (PPR) genes have been found in higher plants, but most of them have not been functionally analyzed and their origins are still obscure. In this study, we performed phylogenetic genomewide comparisons of the PPR gene family in indica and japonica rice to explore the expansion mechanisms of these genes in higher plants. The functions of PPR genes in plant CMS/Rf systems are also discussed. The results indicate that (1) unequal crossing over participated in the expansion of the newly evolved PPR genes in indica and japonica rice genomes, (2) CMS/Rf systems are different in monocots and dicots, (3) the BT-type CMS/Rf system exists in both indica and japonica rice, and (4) both the PPR gene family and the BT-type CMS/Rf system may have existed before the divergence of indica and japonica rice.     

Population genomics identifies the origin and signatures of selection of Korean weedy rice

Weedy rice is the same biological species as cultivated rice (Oryza sativa); it is also a noxious weed infesting rice fields worldwide. Its formation and population‐selective or ‐adaptive signatures are poorly understood. In this study, we investigated the phylogenetics, population structure and signatures of selection of Korean weedy rice by determining the whole genomes of 30 weedy rice, 30 landrace rice and ten wild rice samples. The phylogenetic tree and results of ancestry inference study clearly showed that the genetic distance of Korean weedy rice was far from the wild rice and near with cultivated rice. Furthermore, 537 genes showed evidence of recent positive or divergent selection, consistent with some adaptive traits. This study indicates that Korean weedy rice originated from hybridization of modern indica/indica or japonica/japonica rather than wild rice. Moreover, weedy rice is not only a notorious weed in rice fields, but also contains many untapped valuable traits or haplotypes that may be a useful genetic resource for improving cultivated rice.     

Genome re-sequencing suggested a weedy rice origin from domesticated indica-japonica hybridization: a case study from southern China

Main conclusion

Whole-genome re-sequencing of weedy rice from southern China reveals that weedy rice can originate from hybridization of domesticated indica and japonica rice.

Abstract

Weedy rice (Oryza sativa f. spontanea Rosh.), which harbors phenotypes of both wild and domesticated rice, has become one of the most notorious weeds in rice fields worldwide. While its formation is poorly understood, massive amounts of rice genomic data may provide new insights into this issue. In this study, we determined genomes of three weedy rice samples from the lower Yangtze region, China, and investigated their phylogenetics, population structure and chromosomal admixture patterns. The phylogenetic tree and principle component analysis based on 46,005 SNPs with 126 other Oryza accessions suggested that the three weedy rice accessions were intermediate between japonica and indica rice. An ancestry inference study further demonstrated that weedy rice had two dominant genomic components (temperate japonica and indica). This strongly suggests that weedy rice originated from indica-japonica hybridization. Furthermore, 22,443 novel fixed single nucleotide polymorphisms were detected in the weedy genomes and could have been generated after indica-japonica hybridization for environmental adaptation.     

Subspecies — Specific Microsatellite Markers for Rice (Oryza sativa L)

Subspecific classification of Asian rice (Oryza sativa L) into indica and japonica has always been a subject of interest althrough for rice breeders and geneticists. The present study aims at identifying subspecies specific microsatellite markers in six genotypes, each of indica and japonica using 372 microsatellite primers covering the entire genome. Only 36 primers gave clear polymorphism on 3% agarose gel and these can be used as diagnostic markers for routine and easy identification of the subspecies.     

Tandem duplications of receptor-like kinase genes reshaped the homologous chromosome 1 regions in <Emphasis Type="Italic">Oryza sativa</Emphasis> ssp. <Emphasis Type="Italic">indica</Emphasis> and <Emphasis Type="Italic">japonica</Emphasis> and their possible ancestral genomes

Plant receptor-like kinase (Rlk) genes form a large family, each encoding a protein with a signal motif, a single transmembrane region, and a cytoplasmic kinase domain. Various gene duplications have contributed to the establishment and expansion of the family. Here, we characterized the formation and evolution of the Rlk gene family in cultivated rice and their possible progenitors. Using wheat Rlk gene sequences, we identified orthologs from the genomes of domesticated rice subspecies Oryza sativa ssp. japonica and ssp. indica and their putative progenitors O. glaberrima and O. rufipogon. The four chromosome 1 orthologous regions ranged from 103 to 281 kb comprising 181 syntenic blocks with 75 to 100% sequence identity. These regions contained 11–19 Triticum aestivum kinases (Taks) and 10–15 Lr10 receptor-like kinases (Lrks) organized in clusters and 3–12 transposable elements (TEs). Dot plot analyses showed that the 4 regions had 21–37 conserved catalytic domains, mainly in protein kinases (PKs) and tyrosine kinases (TyrKs) in coupling state. Over 50% of the sequences of glaberrima/rufipogon and japonica/indica pairs were colinear, while japonica/indica displayed a marked sequence expansion with duplicated genes and TEs. A total of 2312 single nucleotide polymorphisms (SNPs) and insertion-deletions (INDELs) were identified between japonica and indica. Duplication of the Rlk genes in O. glaberrima and O. rufipogon occurred after the grass species radiation and before the divergence of O. rufipogon from O. glaberrima; the orthologous Rlk genes from O. japonica and O. indica duplicated after O. sativa separated from O. rufipogon; paralogs, obtained through extensive duplication, happened after the separation of rice from maize. Tandem duplication was the major factor contributing to the gene copy number variation and genome size expansion.     

The DROOPING LEAF and OsETTIN2 genes promote awn development in rice

The awn is a long needle‐like appendage that, in some grass species, is formed on the lemma that encloses floral organs together with the palea. In rice, most wild species and most strains of Oryza sativa ssp. indica generate an awn, whereas most strains of O. sativa ssp. japonica do not. In japonica, the long‐awn characteristic appears to have been lost during domestication and breeding programs. Here, we found that the genes DROOPING LEAF (DL) and OsETTIN2 (OsETT2) are involved in awn development in the awned indica strain Kasalath. Genetic analyses and RNA‐silencing experiments indicate that DL and OsETT2 act independently in awn formation, and that either gene alone is not sufficient for awn development. Scanning electron microscopy revealed that the top region of the lemma (a putative awn primordium) is larger in an awned floret than in an awnless floret. OsETT2 is expressed in the awn primordium in the awned indica floret, but not in the awnless japonica floret except in the provascular bundle. DL is expressed underneath the primordium at similar levels in both indica and japonica florets, suggesting non‐cell‐autonomous action. We hypothesize that loss of expression of OsETT2 in the awn primordium is probably associated with the failure of awn formation in japonica strains.     

Does the Upstream Region Possessing MULE-Like Sequence in Rice Upregulate PsbS1 Gene Expression?

The genomic nucleotide sequences of japonica rice (Sasanishiki and Nipponbare) contained about 2.7-kb unique region at the point of 0.4-kb upstream of the OsPsbS1 gene. In this study, we found that japonica rice with a few exceptions possessing such DNA sequences [denoted to OsMULE-japonica specific sequence (JSS)] is distinct by the presence of Mutator-like-element (MULE). Such sequence was absent in most of indica cultivars and Oryza glaberrima. In OsMULE-JSS1, we noted the presence of possible target site duplication (TSD; CTTTTCCAG) and about 80-bp terminal inverted repeat (TIR) near TSD. We also found the enhancement ofOsPsbS1 mRNA accumulation by intensified light, which was not associated with the DNA methylation status in OsMULE/JSS. In addition, O. rufipogon, possible ancestor of modern rice cultivars was found to compose PsbS gene of either japonica (minor) or indica (major) type. Transient gene expression assay showed that the japonica type promoter elevated a reporter gene activity than indica type.     

Whole-Genome Analysis Revealed the Positively Selected Genes during the Differentiation of indica and Temperate japonica Rice

To investigate the selective pressures acting on the protein-coding genes during the differentiation of indica and japonica, all of the possible orthologous genes between the Nipponbare and 93–11 genomes were identified and compared with each other. Among these genes, 8,530 pairs had identical sequences, and 27,384 pairs shared more than 90% sequence identity. Only 2,678 pairs of genes displaying a Ka/Ks ratio significantly greater than one were revealed, and most of these genes contained only nonsynonymous sites. The genes without synonymous site were further analyzed with the SNP data of 1529 O. sativa and O. rufipogon accessions, and 1068 genes were identified to be under positive selection during the differentiation of indica and temperate japonica. The positively selected genes (PSGs) are unevenly distributed on 12 chromosomes, and the proteins encoded by the PSGs are dominant with binding, transferase and hydrolase activities, and especially enriched in the plant responses to stimuli, biological regulations, and transport processes. Meanwhile, the most PSGs of the known function and/or expression were involved in the regulation of biotic/abiotic stresses. The evidence of pervasive positive selection suggested that many factors drove the differentiation of indica and japonica, which has already started in wild rice but is much lower than in cultivated rice. Lower differentiation and less PSGs revealed between the Or-It and Or-IIIt wild rice groups implied that artificial selection provides greater contribution on the differentiation than natural selection. In addition, the phylogenetic tree constructed with positively selected sites showed that the japonica varieties exhibited more diversity than indica on differentiation, and Or-III of O. rufipogon exhibited more than Or-I.     

Gene Expression Profiles Deciphering Rice Phenotypic Variation between Nipponbare (Japonica) and 93-11 (Indica) during Oxidative Stress

Rice is a very important food staple that feeds more than half the world''s population. Two major Asian cultivated rice (Oryza sativa L.) subspecies, japonica and indica, show significant phenotypic variation in their stress responses. However, the molecular mechanisms underlying this phenotypic variation are still largely unknown. A common link among different stresses is that they produce an oxidative burst and result in an increase of reactive oxygen species (ROS). In this study, methyl viologen (MV) as a ROS agent was applied to investigate the rice oxidative stress response. We observed that 93-11 (indica) seedlings exhibited leaf senescence with severe lesions under MV treatment compared to Nipponbare (japonica). Whole-genome microarray experiments were conducted, and 1,062 probe sets were identified with gene expression level polymorphisms between the two rice cultivars in addition to differential expression under MV treatment, which were assigned as Core Intersectional Probesets (CIPs). These CIPs were analyzed by gene ontology (GO) and highlighted with enrichment GO terms related to toxin and oxidative stress responses as well as other responses. These GO term-enriched genes of the CIPs include glutathine S-transferases (GSTs), P450, plant defense genes, and secondary metabolism related genes such as chalcone synthase (CHS). Further insertion/deletion (InDel) and regulatory element analyses for these identified CIPs suggested that there may be some eQTL hotspots related to oxidative stress in the rice genome, such as GST genes encoded on chromosome 10. In addition, we identified a group of marker genes individuating the japonica and indica subspecies. In summary, we developed a new strategy combining biological experiments and data mining to study the possible molecular mechanism of phenotypic variation during oxidative stress between Nipponbare and 93-11. This study will aid in the analysis of the molecular basis of quantitative traits.     

Biased distribution of microsatellite motifs in the rice genome

Microsatellites are useful tools to study the extent of divergence between two taxonomic groups that show high sequence similarity. We have compared microsatellite distribution to illustrate genetic variation between the two rice genomes, Oryza sativa L. ssp. indica and Oryza sativa L. ssp. japonica. Microsatellite distribution proved to be non random as certain regions of very high microsatellite density have been identified. Microsatellite density in the subspecies japonica was computed marginally higher than in the subspecies indica in the genomic regions compared between the two subspecies. Unexpectedly high microsatellite densities were observed in 5′-untranslated regions of genes. These regions also displayed a clear motif bias. Some of the longest microsatellite repeats were found in intron sequences. Frequency, as well as motif bias was also noted with respect to the association of microsatellites with transposable elements. Microsatellite mutability values were exemplarily estimated for 90 loci by aligning the microsatellite containing regions between the two genomes. Poor rates of finding an orthologue corresponded with high microsatellite mutability in rice. These insights are likely to play a significant role in selecting microsatellite loci to be used in molecular breeding and studying evolutionary dynamics of the two subspecies.     

Mapping 49 quantitative trait loci at high resolution through sequencing-based genotyping of rice recombinant inbred lines

Mapping chromosome regions responsible for quantitative phenotypic variation in recombinant populations provides an effective means to characterize the genetic basis of complex traits. We conducted a quantitative trait loci (QTL) analysis of 150 rice recombinant inbred lines (RILs) derived from a cross between two cultivars, Oryza sativa ssp. indica cv. 93-11 and Oryza sativa ssp. japonica cv. Nipponbare. The RILs were genotyped through next-generation sequencing, which accurately determined the recombination breakpoints and provided a new type of genetic markers, recombination bins, for QTL analysis. We detected 49 QTL with phenotypic effect ranging from 3.2 to 46.0% for 14 agronomics traits. Five QTL of relatively large effect (14.6–46.0%) were located on small genomic regions, where strong candidate genes were found. The analysis using sequencing-based genotyping thus offers a powerful solution to map QTL with high resolution. Moreover, the RILs developed in this study serve as an excellent system for mapping and studying genetic basis of agricultural and biological traits of rice.     

Genome-wide selection and introgression of Chinese rice varieties during breeding

China is the largest rice-producing country, but the genomic landscape of rice diversity has not yet been clarified. In this study, we re-sequence 1070 rice varieties collected from China(400) and other regions in Asia(670). Among the six major rice groups(aus, indica-I, indica-II, aromatic, temperate japonica, and tropical japonica), almost all Chinese varieties belong to the indica-II or temperate japonica group. Most Chinese indica varieties belong to indica-II, which consists of two subgroup...     

The Oryza Map Alignment Project: The Golden Path to Unlocking the Genetic Potential of Wild Rice Species

The wild species of the genus Oryza offer enormous potential to make a significant impact on agricultural productivity of the cultivated rice species Oryza sativa and Oryza glaberrima. To unlock the genetic potential of wild rice we have initiated a project entitled the ‘Oryza Map Alignment Project’ (OMAP) with the ultimate goal of constructing and aligning BAC/STC based physical maps of 11 wild and one cultivated rice species to the International Rice Genome Sequencing Project’s finished reference genome – O. sativa ssp. japonica c. v. Nipponbare. The 11 wild rice species comprise nine different genome types and include six diploid genomes (AA, BB, CC, EE, FF and GG) and four tetrapliod genomes (BBCC, CCDD, HHKK and HHJJ) with broad geographical distribution and ecological adaptation. In this paper we describe our strategy to construct robust physical maps of all 12 rice species with an emphasis on the AA diploid O. nivara – thought to be the progenitor of modern cultivated rice.