Large-scale DNA polymorphism study of <Emphasis Type="Italic">Oryza sativa</Emphasis> and <Emphasis Type="Italic">O. rufipogon</Emphasis> reveals the origin and divergence of Asian rice

Polymorphism over ∼26 kb of DNA sequence spanning 22 loci and one region distributed on chromosomes 1, 2, 3 and 4 was studied in 30 accessions of cultivated rice, Oryza sativa, and its wild relatives. Phylogenetic analysis using all the DNA sequences suggested that O. sativa ssp. indica and ssp. japonica were independently domesticated from a wild species O. rufipogon. O. sativa ssp. indica contained substantial genetic diversity (π = 0.0024), whereas ssp. japonica exhibited extremely low nucleotide diversity (π = 0.0001) suggesting the origin of the latter from a small number of founders. O. sativa ssp. japonica contained a larger number of derived and fixed non-synonymous substitutions as compared to ssp. indica. Nucleotide diversity and genealogical history substantially varied across the 22 loci. A locus, RLD15 on chromosome 2, showed a distinct genealogy with ssp. japonica sequences distantly separated from those of O. rufipogon and O. sativa ssp. indica. Linkage disequilibrium (LD) was analyzed in two different regions. LD in O. rufipogon decays within 5 kb, whereas it extends to ∼50 kb in O. sativa ssp. indica. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

The serine carboxypeptidase like gene family of rice (Oryza sativa L. ssp. japonica)

Serine carboxypeptidases (SCPs) comprise a large family of protein hydrolyzing enzymes and have roles ranging from protein turnover and C-terminal processing to wound responses and xenobiotic metabolism. The proteins can be classified into three groups, namely carboxypeptidase I, II and III, based on their coding protein sequences and the fact that each family is characterized by a central catalytic domain of unique topology designated as the “α/β hydrolase fold”. The available SCP protein sequences have been utilized as datasets to build a HMM (hidden Markov model) profile, which is used to search the rice (Oryza sativa L. ssp. japonica) proteome. A total of 71 SCP and serine carboxypeptidase-like (SCPL) protein-coding genes exist in rice. The intron-exon structure, chromosome localization, expression and characteristics of encoded protein sequences of the 71 putative genes are reviewed.     

Identification of Quantitative Trait Loci Controlling Drought Tolerance at Seedling Stage in Chinese Dongxiang Common Wild Rice (Oryza rufipogon Griff.)

Common wild rice (Oryza rufipogon Griff.) is the ancestor of cultivated rice (O. sativa L.), which has a greater genetic diversity and important traits that remain to be employed in cultivated rice. In this study, a set of introgression lines (BC₄F₅ and/or BC₄F₆) carrying various introgressed segments from common wild rice, collected from Dongxiang county, Jiangxi Province, China, in the background of an Indica (O. sativa L. ssp. indica) cultivar, Guichao 2, was used. A total of 12 drought-related quantitative trait loci (QTL) were identified by investigating drought tolerance of introgression lines under 30% PEG treatment at the young seedlings stage. Of these QTLs, the alleles of 4 QTLs on chromosome 2, 6 and 12 from Dongxiang common wild rice were responsible for increased drought tolerance of the introgression lines. In particular, a QTL qSDT12-2, near RM17 on chromosome 12, was consistently detected in different replications, and expressed stably under PEG stress throughout the study. It was also found that the QTLs located on different chromosomes might express at different stages.     

Domestication and geographic origin of Oryza sativa in China: insights from multilocus analysis of nucleotide variation of O. sativa and O. rufipogon

Previous studies have indicated that China is one of the domestication centres of Asian cultivated rice (Oryza sativa), and common wild rice (O. rufipogon) is the progenitor of O. sativa. However, the number of domestication times and the geographic origin of Asian cultivated rice in China are still under debate. In this study, 100 accessions of Asian cultivated rice and 111 accessions of common wild rice in China were selected to examine the relationship between O. sativa and O. rufipogon and thereby infer the domestication and evolution of O. sativa in China through sequence analyses of six gene regions, trnC‐ycf6 in chloroplast genomes, cox3 in mitochondrial genomes and ITS, Ehd1, Waxy, Hd1 in nuclear genomes. The results indicated that the two subspecies of O. sativa (indica and japonica) were domesticated independently from different populations of O. rufipogon with gene flow occurring later from japonica to indica; Southern China was the genetic diversity centre of O. rufipogon, and the Pearl River basin near the Tropic of Cancer was the domestication centre of O. sativa in China.     

Molecular mapping of a novel gene, Grh5, conferring resistance to green rice leafhopper (Nephotettix cincticeps Uhler) in rice, Oryza sativa L.

The green rice leafhopper (GRH), Nephotettix cincticeps Uhler, is one of the most serious insect pests affecting cultivated rice (Oryza sativa L.) in temperate regions of East Asia. An accession of the wild rice species, Oryza rufipogon Griff. (W1962), was found to be highly resistant to GRH by an antibiosis test. To understand the genetic basis of the GRH resistance, a BC₁F₁ population derived from a cross between a susceptible Japonica variety, Taichung 65 (T65), and a highly resistant accession W1962 was analyzed by quantitative trait loci (QTL) mapping. A single major QTL for GRH resistance was detected on rice chromosome 8. A nearly isogenic population containing segments of the targeted QTL region derived from W1962 was then developed through advanced backcrossing with marker-assisted selection. Further molecular mapping using a BC₄F₂ population revealed that a new resistance gene, designated as Green rice leafhopper resistance 5 (Grh5), was located on the distal region of the long arm of chromosome 8 and tightly linked to the simple sequence repeat markers RM3754 and RM3761. A nearly isogenic line (NIL) carrying Grh5 was subsequently developed in the progeny of the mapping population. The resistance level of Grh5-NIL was compared with those of developed NILs for GRH resistance and was found to have the highest resistance. The DNA markers found to be closely linked to Grh5 would be useful for marker-assisted selection for the improvement of resistance to GRH in rice.     

Enigmatic double-stranded RNA in Japonica rice

We have found a linear, 16 kb, double-stranded RNA (dsRNA) in symptomless Japonica rice (Oryza sativa L.) that is not found in Indica rice (Oryza sativa L.). The dsRNA was detected in every tissue and at every developmental stage, and its copy number was approximately constant (about 20 copies/cell). Double-stranded RNA was also detected in two strains of Oryza rufipogon (an ancestor of O. sativa). Hybridization experiments indicated that the dsRNA of O. rufipogon was homologous but not identical to that of O. sativa. The sequence of about 13.2 kb of the dsRNA was determined and two open reading frames (ORFs) were found. The larger ORF (ORF B) was more than 12 351 nucleotides long and encoded more than 4 117 amino acid residues.     

Identification and linkage mapping of complementary recessive genes causing hybrid breakdown in an intraspecific rice cross

One outcome of hybrid breakdown is poor growth, which we observed as a reduction in the number of panicles per plant and in culm length in an F₂ population derived from a cross between the genetically divergent rice (Oryza sativa L.) cultivars ‘Sasanishiki’ (japonica) and ‘Habataki’ (indica). Quantitative trait locus (QTL) analysis of the two traits and two-way ANOVA of the detected QTLs suggested that the poor growth was due mainly to an epistatic interaction between genes at QTLs located on chromosomes 2 and 11. The poor growth was likely to result when a plant was homozygous for the ‘Habataki’ allele at the QTL on chromosome 2 and homozygous for the ‘Sasanishiki’ allele at the QTL on chromosome 11. The results suggest that the poor growth found in the F₂ population was due to hybrid breakdown of a set of complementary genes. To test this hypothesis and determine the precise chromosomal location of the genes causing the hybrid breakdown, we performed genetic analyses using a chromosome segment substitution line, in which a part of chromosome 2 from ‘Habataki’ was substituted into the genetic background of ‘Sasanishiki’. The segregation patterns of poor growth in plants suggested that both of the genes underlying the hybrid breakdown were recessive. The gene on chromosome 2, designated hybrid breakdown 2 (hbd2), was mapped between simple sequence repeat markers RM3515 and RM3730. The gene on chromosome 11, hbd3, was mapped between RM5824 and RM1341. Electronic supplementary material The online version of this article (doi:) contains supplementary material, which is available to authorized users.     

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.     

Evidences of introgression from cultivated rice to Oryza rufipogon (Poaceae) populations based on SSR fingerprinting: implications for wild rice differentiation and conservation

Crop-to-wild introgression may play an important role in evolution of wild species. Asian cultivated rice (Oryza sativa L.) is of a particular concern because of its cross-compatibility with the wild ancestor, O. rufipogon Griff. The distribution of cultivated rice and O. rufipogon populations is extensively sympatric, particularly in Asia where many wild populations are surrounded by rice fields. Consequently, gene flow from cultivated rice may have a potential to alter genetic composition of wild rice populations in close proximity. In this study, we estimated introgression of cultivated rice with O. rufipogon based on analyses of 139 rice varieties (86 indica and 53 japonica ecotypes) and 336 wild individuals from 11 O. rufipogon populations in China. DNA fingerprinting based on 17 selected rice simple sequence repeat (SSR) primer pairs was adopted to measure allelic frequencies in rice varieties and O. rufipogon samples, and to estimate genetic associations between wild and cultivated rice through cluster analysis. We detected consanguinity of cultivated rice in O. rufipogon populations according to the admixture model of the STRUCTURE program. The analyses showedz that four wild rice populations, DX-P1, DX-P2, GZ-P2, and HL-P, contained some rare alleles that were commonly found in the rice varieties examined. In addition, the four wild rice populations that scattered among the rice varieties in the cluster analysis showed a closer affinity to the cultivars than the other wild populations. This finding supports the contention of substantial gene flow from crop to wild species when these species occur close to each other. The introgressive populations had slightly higher genetic diversity than those that were isolated from rice. Crop-to-wild introgression may have accumulative impacts on genetic variations in wild populations, leading to significant differentiation in wild species. Therefore, effective measure should be taken to avoid considerable introgression from cultivated rice, which may influence the effective in-situ conservation of wild rice species.     

Identification of a Drought Tolerant Introgression Line Derived from Dongxiang Common Wild Rice (O. rufipogon Griff.)

Construction of introgression lines using cultivated rice as recipient and wild rice is a novel approach to explore primitive and broad genetic resources in rice breeding. We recently generated a set of 159 introgression lines via a backcrossing program using an elite Indica cultivar rice Guichao 2 (O. sativa L. ssp. indica) as recipient and a common wild rice Dongxiang accession (O. rufipogon Griff.) as donor. In this study, we have evaluated the previously constructed 159 introgression lines for drought-tolerance. A total of 12 quantitative trait loci (QTLs) related to drought tolerance were mapped. Furthermore, a drought tolerant introgression line, IL23, was identified and characterized. Genotype analysis of IL23 demonstrated that IL23 contained two QTLs associated with drought tolerance, qSDT2-1 and qSDT12-2, which were located on chromosome 2 and 12 within the two introgressed segments derived from the common wild rice, respectively. Physiological characterization, including measurement of water loss, osmotic potential, electrolytical leakage, MDA content, soluble sugars content and the leaf temperature, revealed that IL23 showed the characteristics associated with drought tolerance. Identification and characterization of IL23 would provide a useful basis for isolation of novel genes associated with drought tolerance and for molecular breeding of drought tolerant rice. Furthermore, the results in this study indicated that construction of introgression lines from common wild rice should be an appropriate approach to obtain favorable genetic materials.     

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     

Introgression of genes from Oryza officinalis Well ex Watt to cultivated rice,O. sativa L.

Summary Sterile AC hybrids between cultivated Oryza sativa (AA) and a distant wild species, O. officinalis (CC), were backcross to O. sativa. Most of the BC₁ progenies were allotriploid (AAC), a few were hypotriploid. AAC progenies were again backcrossed to O. sativa. BC₂ progenies consisting of disomic or aneuploid individuals were examined for the presence of O. officinalis traits. Eleven different traits from O. officinalis were identified in these progenies. Segregation data in the subsequent generations suggest that these traits are monogenic in nature. Two of these genes — for resistance to BPH and WBPH — are of value in rice improvement. The extremely low recovery of recombinant progenies is in agreement with the very low amount of pairing between A and C genomes. Because of this restricted recombination, the genotype of the recurrent parent was reconstituted after two backcrosses only. Thus, the BC₂ progenies look remarkably similar to O. sativa. Most of them are stable and fertile and also interfertile with other O. sativa breeding lines. Some of the BPH-and WBPH-resistant progenies are comparable in yield to the best O. sativa parents and are being evaluated as varietal possibilities.     

Use of asymmetric somatic hybridization for transfer of the bacterial blight resistance trait from Oryza meyeriana L. to O. sativa L. ssp. japonica

Bacterial blight is one of the major diseases affecting rice productivity. To improve the resistance of cultivated rice to bacterial blight, we introduced a bacterial blight resistance trait from Oryza meyeriana, a wild rice species, into an elite japonica rice cultivar (Dalixiang) using asymmetric somatic hybridization. One hundred and thirty-two independent lines were regenerated. The hybrid plants possessed several morphological features of the donor species, O. meyeriana. Random amplified polymorphic DNA analysis revealed that hybrid plants exhibited banding patterns derived from their parental genotypes. For the majority of the hybrids, resistance to bacterial blight pathogens was intermediate to that observed for O. meyeriana and O. sativa (cv. Dalixiang). Four of the hybrid lines exhibited a high bacterial blight resistance, but it was less than that observed for O. meyeriana. These results demonstrate that O. meyeriana can be used as a good genetic source for improving bacterial blight resistance in commercial rice cultivars through asymmetric somatic hybridization.Abbreviations 2,4-D: 2,4-Dichlorophenoxyacetic acid - IOA: Iodoacetamide - NAA: -Naphthaleneacetic acid - PEG: Polyethylene glycol Communicated by P. Lakshmanan     

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.     

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.     

Retracted: Screening of candidate genes and fine mapping of drought tolerance quantitative trait loci on chromosome 4 in rice (Oryza sativa L.) under drought stress

Due to severe water resource shortage, genetics of and breeding for DT (drought tolerance) in rice (Oryza sativa L.) have become one of the hot research topics. Identification of grain yield QTLs (quantitative trait loci) directly related to the DT trait of rice can provide useful information for breeding new drought‐resistant and water‐saving rice varieties via marker‐assisted selection. A population of 105 advanced BILs (backcross introgression lines) derived from a cross between Zhenshan97B and IRAT109 in Zhenshan97B background were grown under drought stress in a field experiment and phenotypic traits were investigated. The results showed that in the target interval of RM273‐RM255 on chromosome 4, three main‐effect QTLs related to panicle length, panicle number, and spikelet number per panicle were identified (LOD [logarithm of the odds] > 2.0). The panicle length‐related QTL had two loci located in the neighboring intervals of RM17308‐RM17305 and RM17349‐RM17190, which explained 18.80% and 20.42%, respectively, of the phenotypic variation, while the panicle number‐related QTL was identified in the interval of RM1354‐RM17308, explaining 11.47% of the phenotypic variation. As far as the spikelet number per panicle‐related QTL was concerned, it was found to be located in the interval of RM17308‐RM17305, which explained 28.08% of the phenotypic variation. Using the online Plant‐GE query system, a total of 13 matched ESTs (expressed sequence tags) were found in the target region, and of the 13 ESTs, 12 had corresponding predicted genes. For instance, the two ESTs CB096766 and CA765747 were corresponded to the same predicted gene LOC_Os04g46370, while the other four ESTs, CA754286, CB000011, CX056247, and CX056240, were corresponded to the same predicted gene LOC_Os04g46390.     

Construction of introgression lines carrying wild rice (Oryza rufipogon Griff.) segments in cultivated rice (Oryza sativa L.) background and characterization of introgressed segments associated with yield-related traits

Introgression lines (ILs) are useful tools for precise mapping of quantitative trait loci (QTLs) and the evaluation of gene action or interaction in theoretical studies. A set of 159 ILs carrying variant introgressed segments from Chinese common wild rice (Oryza rufipogon Griff.), collected from Dongxiang county, Jiangxi Province, in the background of Indica cultivar (Oryza sativa L.), Guichao 2, was developed using 126 polymorphic simple sequence repeats (SSR) loci. The 159 ILs represented 67.5% of the genome of O. rufipogon. All the ILs have the proportions of the recurrent parent ranging from 92.4 to 99.9%, with an average of 97.4%. The average proportion of the donor genome for the BC₄F₄ population was about 2.2%. The mean numbers of homozygous and heterozygous donor segments were 2 (ranging 0–8) and 1 (ranging 0–7), respectively, and the majority of these segments had sizes less than 10 cM. QTL analysis was conducted based on evaluation of yield-related traits of the 159 ILs at two sites, in Beijing and Hainan. For 6 out of 17 QTLs identified at two sites corresponding to three traits (panicles per plant, grains per panicle and filled grains per plant, respectively), the QTLs derived from O. rufipogon were usually associated with an improvement of the target trait, although the overall phenotypic characters of O. rufipogon were inferior to that of the recurrent parent. Of the 17 QTLs, 5 specific QTLs strongly associated with more than one trait were observed. Further analysis of the high-yielding and low-yielding ILs revealed that the high-yielding ILs contained relatively less introgressed segments than the low-yielding ILs, and that the yield increase or decrease was mainly due to the number of grain. On the other hand, low-yielding ILs contained more negative QTLs or disharmonious interactions between QTLs which masked trait-enchancing QTLs. These ILs will be useful in identifying the traits of yield, tolerance to low temperature and drought stress, and detecting favorable genes of common wild rice.