Double-Stranded RNA in Rice

Oryza sativa ) and wild rice (O. rufipogon) tissues. It is detected at every developmental stage, and is transmitted very efficiently to progeny via seeds (more than 98%). The dsRNA is maintained at a constant level (approximately 100 copies/cell) in almost all tissues. However, the number of copies increases about 10-fold when host cells are grown in suspension culture. Complete nucleotide sequences of cultivated rice (temperate japonica rice, cv. Nipponbare, J-dsRNA) and wild rice (W-1714, W-dsRNA) dsRNAs have been determined. Both wild and cultivated rice dsRNAs have a single long open reading frame (ORF) containing the conserved motifs of RNA-dependent RNA polymerase and RNA helicase. The coding strands of both contain a site-specific discontinuity (nick) at nt 1,211 (J-dsRNA) or at nt 1,197 (W-dsRNA) from the 5′ end of their coding strand. Rice dsRNA has several unique properties and can be regarded as a novel RNA replicon. This paper discusses the origin and evolution of the rice dsRNA. Received 23 October 1998/ Accepted in revised form 15 December 1998     

Inheritance of Oryza sativa endornavirus in F1 and F2 hybrids between japonica and indica rice

We have found a 14 kbp double-stranded RNA (dsRNA) in many cultivars of japonica rice (Oryza sativa L.) but not in any cultivars of indica rice. This dsRNA is an RNA replicon with plasmid-like properties and is proposed to be a novel dsRNA virus, Oryza sativa endornavirus (OSV). Reciprocal crosses between the OSV-carrier japonica variety (Nipponbare) and the OSV-free indica variety (IR 26 or Kasalath) were performed to investigate whether OSV can be transmitted to F1 hybrids. When IR 26 and Nipponbare were used, efficient transmission of OSV from ova (93%) and pollen (89%) was observed. When Kasalath and Nipponbare were used, the OSV transmission efficiency to F1 progeny was 68% from ova and 20% from pollen. The transmission of OSV to F2 progeny plants was also complicated, showing non-Mendelian inheritance. These results suggest that the dsRNA replicon (OSV) is unstable in indica rice plants.     

Characterization of Interspecific Hybrids Between Oryza sativa L. and Three Wild Rice Species of China by Genomic In Situ Hybridization

In the genus Oryza, interspecific hybrids are useful bridges for transferring the desired genes from wild species to cultivated rice （Oryza sativa L.）. In the present study, hybrids between O. sativa （AA genome） and three Chinese wild rices, namely O. rufipogon （AA genome）, O. officinalis （CC genome）, and O. meyeriana （GG genome）, were produced. Agricultural traits of the F1 hybrids surveyed were intermediate between their parents and appreciably resembled wild rice parents. Except for the O. sativa × O. rufipogon hybrid, the other F1 hybrids were completely sterile. Genomic in situ hybridization （GISH） was used for hybrid verification. Wild rice genomic DNAs were used as probes and cultivated rice DNA was used as a block. With the exception of O. rufipogon chromosomes, this method distinguished the other two wild rice and cultivated rice chromosomes at the stage of mitotic metaphase with different blocking ratios. The results suggest that a more distant phylogenetic relationship exists between O. meyeriana and O. sativa and that O. rufipogon and O. sativa share a high degree of sequence homology. The average mitotic chromosome length of O. officinalis and O. meyeriana was 1.25- and 1.51-fold that of O. sativa, respectively. 4＇,6＇-Diamidino- 2-phenylindole staining showed that the chromosomes of O. officinalis and O. meyeriana harbored more heterochromatin, suggesting that the C and G genomes were amplified with repetitive sequences compared with the A genome. Although chromocenters formed by chromatin compaction were detected with wild rice-specific signals corresponding to the C and G genomes in discrete domains of the F1 hybrid interphase nuclei, the size and number of O. meyeriana chromocenters were bigger and greater than those of O. officinalis. The present results provide an important understanding of the genomic relationships and a tool for the transfer of useful genes from three native wild rice species in China to cultivars.     

The extent of linkage disequilibrium in rice (Oryza sativa L.)

Despite its status as one of the world's major crops, linkage disequilibrium (LD) patterns have not been systematically characterized across the genome of Asian rice (Oryza sativa). Such information is critical to fully exploit the genome sequence for mapping complex traits using association techniques. Here we characterize LD in five 500-kb regions of the rice genome in three major cultivated rice varieties (indica, tropical japonica, and temperate japonica) and in the wild ancestor of Asian rice, Oryza rufipogon. Using unlinked SNPs to determine the amount of background linkage disequilibrium in each population, we find that the extent of LD is greatest in temperate japonica (probably >500 kb), followed by tropical japonica (approximately 150 kb) and indica (approximately 75 kb). LD extends over a shorter distance in O. rufipogon (<40 kb) than in any of the O. sativa groups assayed here. The differences in the extent of LD among these groups are consistent with differences in outcrossing and recombination rate estimates. As well as heterogeneity between groups, our results suggest variation in LD patterns among genomic regions. We demonstrate the feasibility of genomewide association mapping in cultivated Asian rice using a modest number of SNPs.     

Simple sequence repeat analyses of interspecific hybrids and MAALs of <Emphasis Type="Italic">Oryza </Emphasis><Emphasis Type="Italic">officinalis</Emphasis> and <Emphasis Type="Italic">Oryza sativa</Emphasis>

Wild rice is a valuable resource for the genetic improvement of cultivated rice (Oryza sativa L., AA genome). Molecular markers are important tools for monitoring gene introgression from wild rice into cultivated rice. In this study, Simple sequence repeat (SSR) markers were used to analyze interspecific hybrids of O. sativa-O. officinalis (CC genome), the backcrossing progenies and the parent plants. Results showed that most of the SSR primers (335 out of 396, 84.6%) developed in cultivated rice successfully amplified products from DNA samples of wild rice O. officinalis. The polymorphism ratio of SSR bands between O. sativa and O. officinalis was as high as 93.9%, indicating differences between the two species with respect to SSRs. When the SSR markers were applied in the interspecific hybrids, only a portion of SSR primers amplified O. officinalis-specific bands in the F(1) hybrid (52.5%), BC(1) (52.5%), and MAALs (37.0%); a number of the bands disappeared. Of the 124 SSR loci that detected officinalis-specific bands in MAAL plants, 96 (77.4%) showed synteny between the A and C-genomes, and 20 (16.1%) showed duplication in the C-genome. Sequencing analysis revealed that indels, substitution and duplication contribute to the diversity of SSR loci between the genomes of O. sativa and O. officinalis.     

Nonindependent domestication of the two rice subspecies, Oryza sativa ssp. indica and ssp. japonica, demonstrated by multilocus microsatellites

The origins of the Asian cultivated rice Oryza sativa from its wild ancestor O. rufipogon have been debated for decades. The question mainly concerns whether it originated monophyletically or polyphyletically. To shed light on the origins and demographic history of rice domestication, we genotyped a total of 92 individual plants from the two O. sativa subspecies and O. rufipogon for 60 microsatellites. An approximate Bayesian method was applied to estimate demographic parameters for O. rufipogon vs. O. sativa ssp. indica and O. rufipogon vs. O. sativa ssp. japonica. We showed that the japonica subspecies suffered a more severe bottleneck than the indica subspecies and thus a greater loss of genetic variation during its domestication. Across microsatellite loci there is a significant positive correlation in the reduction of genetic diversity between the two subspecies. The results suggest that completely independent domestication of indica and japonica subspecies may not explain our data and that there is at least partial sharing of their ancestral populations and/or recent gene flow between them.     

江西东乡野生稻苗期抗旱基因定位

普通野生稻是栽培稻的祖先种,其遗传多样性远远大于栽培稻,蕴涵着栽培品种中难以找到的重要性状.以江西东乡普通野生稻为供体、以桂朝2号为遗传背景的野生稻基因渗入系（BC4F5、BC4F6）为材料,利用30%的PEG人工模拟干旱环境,对渗入系二叶一心苗期进行抗旱鉴定,共定位了12个与抗旱有关的QTL,其中在第2、6和12染色体上发现了4个QTL的加性效应值为正,来自东乡野生稻的等位基因能使渗入系的抗旱性增强,特别是位于第12染色体RM17附近的qSDT12-2在多次重复中均被检测到,在PEG处理后1-8 d能稳定表达.通过对抗旱性QTL的动态分析,发现不同QTL表达时间不同.     

Phylogenetic analysis of Oryza rufipogon strains and their relations to Oryza sativa strains by insertion polymorphism of rice SINEs

Oryza rufipogon, the progenitor of the cultivated rice species Oryza sativa, is known by its wide intraspecific variation. In this study, we performed phylogenetic analyses of O. rufipogon strains and their relationships to O. sativa strains by using 26 newly identified p-SINE1 members from O. rufipogon strains, in addition to 23 members previously identified from O. sativa strains. A total of 103 strains of O. rufipogon and O. sativa were examined for the presence and absence of each of the p-SINE1 members at respective loci by PCR with a pair of primers that hybridize to the regions flanking each p-SINE1 member. A phylogenetic tree constructed on the basis of the insertion polymorphism of p-SINE1 members showed that O. rufipogon and O. sativa strains are classified into three groups. The first group consisted of O. rufipogon perennial strains mostly from China and O. sativa ssp. japonica strains, which included javanica strains forming a distinct subgroup. The second group consisted of almost all the O. rufipogon annual strains, a few O. rufipogon perennial strains and O. sativa ssp. indica strains. These groupings, in addition to other results, support the previous notion that annual O. rufipogon originated in the O. rufipogon perennial population, and that O. sativa originated polyphyletically in the O. rufipogon populations. The third group consisted of the other perennial strains and intermediate-type strains of O. rufipogon, in which the intermediate-type strains are most closely related to a hypothetical ancestor with no p-SINE1 members at the respective loci and to those belonging to the other rice species with the AA genome. This suggests that O. rufipogon perennial strains are likely to have originated from the O. rufipogon intermediate-ecotype population.     

A chromosome-level genome assembly of the wild rice Oryza rufipogon facilitates tracing the origins of Asian cultivated rice

Oryza rufipogon Griff. is a wild progenitor of the Asian cultivated rice Oryza sativa. To better understand the genomic diversity of the wild rice, high-quality reference genomes of O. rufipogon populations are needed, which also facilitate utilization of the wild genetic resources in rice breeding. In this study, we generated a chromosome-level genome assembly of O. rufipogon using a combination of short-read sequencing, single-molecule sequencing, BioNano and Hi-C platforms. The genome sequence(399.8 Mb) was assembled into 46 scaffolds on the 12 chromosomes, with contig N50 and scaffold N50 of 13.2 Mb and 20.3 Mb,respectively. The genome contains 36,520 protein-coding genes, and 49.37% of the genome consists of repetitive elements. The genome has strong synteny with those of the O. sativa subspecies indica and japonica, but containing some large structural variations. Evolutionary analysis unveiled the polyphyletic origins of O. sativa, in which the japonica and indica genome formations involved different divergent O. rufipogon(including O. nivara) lineages, accompanied by introgression of genomic regions between japonica and indica. This high-quality reference genome provides insight on the genome evolution of the wild rice and the origins of the O. sativa subspecies, and valuable information for basic research and rice breeding.     

Polyphyletic origin of cultivated rice: based on the interspersion pattern of SINEs

The wild rice species Oryza rufipogon with wide intraspecific variation is thought to be the progenitor of the cultivated rice species Oryza sativa with two ecotypes, japonica and indica. To determine the origin of cultivated rice, subfamily members of the rice retroposon p-SINE1, which show insertion polymorphism in the O. sativa -O. rufipogon population, were identified and used to "bar code" each of 101 cultivated and wild rice strains based on the presence or absence of the p-SINE1 members at the respective loci. A phylogenetic tree constructed based on the bar codes given to the rice strains showed that O. sativa strains were classified into two groups corresponding to japonica and indica, whereas O. rufipogon strains were in four groups, in which annual O. rufipogon strains formed a single group, differing from the perennial O. rufipogon strains of the other three groups. Japonica strains were closely related to the O. rufipogon perennial strains of one group, and the indica strains were closely related to the O. rufipogon annual strains, indicating that O. sativa has been derived polyphyletically from O. rufipogon. The subfamily members of p-SINE1 constitute a powerful tool for studying the classification and relationship of rice strains, even when one has limited knowledge of morphology, taxonomy, physiology, and biochemistry of rice strains.     

Fitness estimation through performance comparison of F1 hybrids with their parental species Oryza rufipogon and O. sativa

BACKGROUND AND AIMS: Introgression of crop genes into populations of wild relatives has important implications for germplasm conservation as well as for the persistence of novel transgenes in wild populations. Studies of hybrid fitness can be used to evaluate the potential for introgression to occur following episodes of interspecific hybridization. METHODS: This study estimated relative fitness of interspecific hybrids through performance comparison of F(1) hybrids with their parental species, a cultivated rice (Oryza sativa) Minghui-63 and perennial common wild rice (O. rufipogon) under the cultivation conditions. KEY RESULTS: Compared with their parents, the hybrids had the lowest values of seedling survival ability, pollen viability and seed production; intermediate values of seed germination, spikelet production and flag leaf areas; and the highest values of plant height, number of tillers and panicles. The hybrids performed poorly at the stage of sexual reproduction, although they had a slightly higher hybrid vigour at the vegetative growth stage and better tillering ability than their wild parent. There were no significant differences in composite fitness across the whole life-history between the hybrids and their wild parental species. CONCLUSIONS: Rice genes, including transgenes, might persist in wild rice populations through vegetative and sexual reproduction. Further studies are needed to examine whether the extent of gene flow from rice is sufficiently significant to influence genetic diversity in wild populations of O. rufipogon, a species that has become endangered in some regions of south-east Asia.     

稻属谷氨酰胺合成酶家族的系统分析

比较籼粳栽培稻和野生稻中谷氨酰胺合成酶(GS)基因和蛋白质的结果表明,水稻GS蛋白编码区序列高度保守,而非编码序列变异较大。GS2基因的进化比GS1基因保守。短药野生稻中GS基因进化主要是内含子的变异,但此种内含子的变异在籼粳栽培稻中幅度要小得多。     

Genetic diversity and origin of weedy rice (Oryza sativa f. spontanea) populations found in North-eastern China revealed by simple sequence repeat (SSR) markers

BACKGROUND AND AIMS: Weedy rice (Oryza sativa f. spontanea) is one of the most notorious weeds occurring in rice-planting areas worldwide. The objectives of this study are to determine the genetic diversity and differentiation of weedy rice populations from Liaoning Province in North-eastern China and to explore the possible origin of these weedy populations by comparing their genetic relationships with rice varieties (O. sativa) and wild rice (O. rufipogon) from different sources. METHODS: Simple sequence repeat (SSR) markers were used to estimate the genetic diversity of 30 weedy rice populations from Liaoning, each containing about 30 individuals, selected rice varieties and wild O. rufipogon. Genetic differentiation and the relationships of weedy rice populations were analysed using cluster analysis (UPGMA) and principle component analysis (PCA). KEY RESULTS: The overall genetic diversity of weedy rice populations from Liaoning was relatively high (H(e) = 0.313, I = 0.572), with about 35 % of the genetic variation found among regions. The Liaoning weedy rice populations were closely related to rice varieties from Liaoning and japonica varieties from other regions but distantly related to indica rice varieties and wild O. rufipogon. CONCLUSIONS: Weedy rice populations from Liaoning are considerably variable genetically and most probably originated from Liaoning rice varieties by mutation and intervarietal hybrids. Recent changes in farming practices and cultivation methods along with less weed management may have promoted the re-emergence and divergence of weedy rice in North-eastern China.     

New insights into the history of rice domestication

The history of rice domestication has long been a subject of debate. Recently obtained genetic evidence provides new insights into this complex story. Genome-wide studies of variation demonstrate that the two varietal groups in Oryza sativa (indica and japonica) arose from genetically distinct gene pools within a common wild ancestor, Oryza rufipogon, suggesting multiple domestications of O. sativa. However, the evolutionary history of recently cloned domestication genes adds another layer of complexity to the domestication of rice. Although some alleles exist only within specific subpopulations, as would be expected if the domestications occurred independently, other major domestication alleles are common to all cultivated O. sativa varieties. Our current view of rice domestication supports multiple domestications coupled with limited introgression that transferred key domestication alleles between divergent rice gene pools.     

Evolutionary relationships among rice species with AA genome based on SINE insertion analysis

Previous studies based on morphological and molecular markers indicated that there are two cultivated and five wild rice species within the Oryza genus with the AA genome. In the cultivated rice species, Oryza sativa, a retroposon named p-SINE1 has been identified. Some of the p-SINE1 members characterized previously showed interspecific insertion polymorphisms in the species with the AA genome. In this study, we identified new p-SINE1 members showing interspecific insertion polymorphisms from representative strains of four wild rice species with the AA genome: O. barthii, O. glumaepatula, O. longistaminata, and O. meridionalis. Some of these members were present only in strains of one species, whereas the others were present in strains of two or more species. The p-SINE1 insertion patterns in the strains of the Asian and African cultivated rice species O. sativa and O. glaberrima were very similar to those of the Asian and African wild rice species O. rufipogon and O. barthii, respectively. This is consistent with the previous hypothesis that O. sativa and O. glaberrima are derived from specific wild rice species. Phylogenetic analysis based on the p-SINE1 insertion patterns showed that the strains of each of the five wild rice species formed a cluster. The strains of O. longistaminata appear to be distantly related to those of O. meridionalis. The strains of these two species appear to be distantly related to those of three other species, O. rufipogon, O. barthii and O. glumaepatula. The latter three species are closely related to one another with O. barthii and O. glumaepatula being most closely related. A phylogenetic tree including a hypothetical ancestor with all loci empty for p-SINE1 insertion showed that the strains of O. longistaminata are related most closely to the hypothetical ancestor. This indicates that O. longistaminata and O. meridionalis diverged early on, whereas the other species diverged relatively recently, and suggests that the Oryza genus with AA genome might have originated in Africa, rather than in Asia.     

Genetics of Hybrid Sterility and Hybrid Breakdown in an Intersubspecific Rice (Oryza Sativa L.) Population

F(1) hybrid sterility and ``hybrid breakdown' of F(2) and later generations in rice (Oryza sativa L.) are common and genetically complicated. We used a restriction fragment length polymorphism linkage map and F(4) progeny testing to investigate hybrid sterility and hybrid breakdown in a cross between ``widely compatible' O. sativa ssp. japonica cultivar Lemont from the Southern U.S. and ssp. indica cultivar Teqing from China. Our results implicate different genetic mechanisms in hybrid sterility and hybrid breakdown, respectively. Hybrid sterility appeared to be due to recombination within a number of putative differentiated ``supergenes' in the rice genome, which may reflect cryptic structural rearrangements. The cytoplasmic genome had a large effect on fertility of both male and female gametes in the F(1) hybrids. There appeared to be a pair of complementary genes that behaved like ``wide compatibility' genes. This pair of genes and the ``gamete eliminator' (S(1)) or ``egg killer' (S-5) may influence the phenotypic effects of presumed supergenes in hybrids. Hybrid breakdown appeared to be largely due to incompatibilities between indica and japonica alleles at many unlinked epistatic loci in the genome. These proposed mechanisms may partly account for the complicated nature of postreproductive barriers in rice.     

Subspecies-specific intron length polymorphism markers reveal clear genetic differentiation in common wild rice （Oryza rufipogon L.） in relation to the domestication of cultivated rice （O. sativa L.）

It is generally accepted that Oryza rufipogon is the progenitor of Asian cultivated rice （O. sativa）. However, how the two subspecies of O. sativa （indica and japonica） were domesticated has long been debated. To investigate the genetic differentiation in O. rufipogon in relation to the domestication of O. sativa, we developed 57 subspecies-specific intron length polymorphism （SSILP） markers by comparison between 10 indica cultivars and 10 japonica cultivars and defined a standard indica rice and a standard japonica rice based on these SSILP markers. Using these SSILP markers to genotype 73 O. rufipogon accessions, we found that the indica alleles and japonica alleles of the SSILP markers were predominant in the O. rufipogon accessions, suggesting that SSILPs were highly conserved during the evolution of O. sativa. Cluster analysis based on these markers yielded a dendrogram consisting of two distinct groups： one group （Group I） comprises all the O. rufipogon accesions from tropical （South and Southeast） Asia as well as the standard indica rice; the other group （Group II） comprises all the O. rufipogon accessions from Southern China as well as the standard japonica rice. Further analysis showed that the two groups have significantly higher frequencies of indica alleles and japonica alleles, respectively. These results support the hypothesis that indica rice and japonica rice were domesticated from the O. rufipogon of tropical Asia and from that of Southern China, respectively, and suggest that the indica-japonica differentiation should have formed in O. rufipogon long before the beginning of domestication. Furthermore, with an O. glaberrima accession as an outgroup, it is suggested that the indica-japonica differentiation in O. ruffpogon might occur after its speciation from other AA-genome species.     

Extraordinarily polymorphic ribosomal DNA in wild and cultivated rice.

A collection of 481 rice accessions was surveyed for ribosomal DNA (rDNA) intergenic spacer length polymorphism to assess the extent of genetic diversity in Chinese and Asian rice germplasm. The materials included 83 accessions of common wild rice, Oryza rufipogon, 75 of which were from China; 348 entries of cultivated rice (Oryza sativa), representing almost all the rice growing areas in China; and 50 cultivars from South and East Asia. A total of 42 spacer length variants (SLVs) were detected. The size differences between adjacent SLVs in the series were very heterogeneous, ranging from ca. 21 to 311 bp. The 42 SLVs formed 80 different rDNA phenotypic combinations. Wild rice displayed a much greater number of rDNA SLVs than cultivated rice, while cultivated rice showed a larger number of rDNA phenotypes. Indica and japonica groups of O. sativa contained about equal numbers of SLVs, but the SLV distribution was significantly differentiated: indica rice was preferentially associated with longer SLVs and japonica rice with shorter ones. The results may have significant implications regarding the origin and evolution of cultivated rice, as well as the inheritance and molecular evolution of rDNA intergenic spacers in rice. Key words : rDNA, Oryza rufipogon, Oryza sativa, germplasm diversity, evolution.     

基于线粒体基因片段核苷酸多态性的亚洲栽培稻起源进化研究

亚洲栽培稻的祖先是普通野生稻,已成为世界公认的观点,然而亚洲栽培稻的2个亚种:粳稻和籼稻是一次起源还是二次起源仍存在很大争议,其起源地是国内还是国外依然是国际学者间争论的焦点。本文通过对184份亚洲栽培稻和203份普通野生稻3段基因序列cox3、cox1、orf 224和2段基因间序列ssv-39/178、rps2-trnfM的多样性研究,验证了以下观点:1)粳稻起源于中国,籼稻起源于中国和国外;2)亚洲栽培稻的起源为二次起源,即普通野生稻存在偏籼和偏粳2种类型,亚洲栽培稻的2个亚种籼稻和粳稻在进化过程中分别由偏籼型的普通野生稻和偏粳型的普通野生稻进化而来。     

Molecular characterization of two endogenous double-stranded RNAs in rice and their inheritance by interspecific hybrids

We completely sequenced 13,936 nucleotides (nt) of a double-stranded RNA (dsRNA) of wild rice (W-dsRNA). A single long open reading frame (13,719 nt) containing the conserved motifs of RNA-dependent RNA polymerase and RNA helicase was located in the coding strand. The identity between entire nucleotide sequence of W-dsRNA and that of the dsRNA of temperate japonica rice (J-dsRNA, 13,952 nt) was 75.5%. A site-specific discontinuity (nick) was identified at nt 1,197 from the 5' end of the coding strand of W-dsRNA. This nick is also located at nt 1,211 from the 5' end in the coding strand of J-dsRNA. The dsRNA copy number was increased more than 10-fold in pollen grains of both rice plants. This remarkable increase may be responsible for the highly efficient transmission of J-dsRNA via pollen that we already reported. J-dsRNA and W-dsRNA were also efficiently transmitted to interspecific F1 hybrids. Seed-mediated dsRNA transmission to F2 plants was also highly efficient when the maternal parent was wild rice. The efficiency of dsRNA transmission to F2 plants was reduced when the maternal parent was temperate japonica rice; however, the reduced rates in F2 plants were returned to high levels in F3 plants.