Xinghua Wei

Genome-wide association study of blast resistance in indica rice

BackgroundRice blast disease is one of the most serious and recurrent problems in rice-growing regions worldwide. Most resistance genes were identified by linkage mapping using genetic populations. We extensively examined 16 rice blast strains and a further genome-wide association study based on genotyping 0.8 million single nucleotide polymorphism variants across 366 diverse indica accessions.ResultsTotally, thirty associated loci were identified. The strongest signal (Chr11_6526998, P =1.17 × 10−17) was located within the gene Os11g0225100, one of the rice Pia-blast resistance gene. Another association signal (Chr11_30606558) was detected around the QTL Pif. Our study identified the gene Os11g0704100, a disease resistance protein containing nucleotide binding site-leucine rich repeat domain, as the main candidate gene of Pif. In order to explore the potential mechanism underlying the blast resistance, we further examined a locus in chromosome 12, which was associated with CH149 (P =7.53 × 10−15). The genes, Os12g0424700 and Os12g0427000, both described as kinase-like domain containing protein, were presumed to be required for the full function of this locus. Furthermore, we found some association on chromosome 3, in which it has not been reported any loci associated with rice blast resistance. In addition, we identified novel functional candidate genes, which might participate in the resistance regulation.ConclusionsThis work provides the basis of further study of the potential function of these candidate genes. A subset of true associations would be weakly associated with outcome in any given GWAS; therefore, large-scale replication is necessary to confirm our results. Future research will focus on validating the effects of these candidate genes and their functional variants using genetic transformation and transferred DNA insertion mutant screens, to verify that these genes engender resistance to blast disease in rice.

Temporal changes in SSR allelic diversity of major rice cultivars in China.

Forty simple sequence repeats (SSRs) were used to assess the changes of diversity in 310 major Chinese rice cultivars grown during the 1950s-1990s. Of the 40 SSR loci, 39 were polymorphic. A total of 221 alleles were detected with an average of 5.7 alleles per locus (Na). The Neis genetic diversity index (He) varied drastically among the loci (0.207 to 0.874, mean 0.625). Comparing the temporal changes in Na and He, the cultivars from the 1950s had more alleles and higher He scores than the cultivars from the other four decades. Analysis of molecular variance (AMOVA) indicated that the genetic differentiation among the five decades was not significant in the whole set, but significant within indica and japonica. More changes among the decades were revealed in indica cultivars than in japonica cultivars. Some alleles had been lost in current rice cultivars in the 1990s, occurring more frequently in indica. These results suggest that more elite alien genetic resources should be explored to widen the genetic backgrounds of rice cultivars currently grown in China.

Genome-wide copy number variations in Oryza sativa L.

BackgroundCopy number variation (CNV) can lead to intra-specific genome variations. It is not only part of normal genetic variation, but also is the source of phenotypic differences. Rice (Oryza sativa L.) is a model organism with a well-annotated genome, but investigation of CNVs in rice lags behind its mammalian counterparts.ResultsWe comprehensively assayed CNVs using high-density array comparative genomic hybridization in a panel of 20 Asian cultivated rice comprising six indica, three aus, two rayada, two aromatic, three tropical japonica, and four temperate japonica varieties. We used a stringent criterion to identify a total of 2886 high-confidence copy number variable regions (CNVRs), which span 10.28 Mb (or 2.69%) of the rice genome, overlapping 1321 genes. These genes were significantly enriched for specific biological functions involved in cell death, protein phosphorylation, and defense response. Transposable elements (TEs) and other repetitive sequences were identified in the majority of CNVRs. Chromosome 11 showed the greatest enrichment for CNVs. Of subspecies-specific CNVRs, 55.75% and 61.96% were observed in only one cultivar of ssp. indica and ssp. japonica, respectively. Some CNVs with high frequency differences among groups resided in genes underlying rice adaptation.ConclusionsHigher recombination rates and the presence of homologous gene clusters are probably predispositions for generation of the higher number of CNVs on chromosome 11 by non-allelic homologous recombination events. The subspecies-specific variants are enriched for rare alleles, which suggests that CNVs are relatively recent events that have arisen within breeding populations. A number of the CNVs identified in this study are candidates for generation of group-specific phenotypes.

A Comparative Study of SSR Diversity in Chinese Major Rice Varieties Planted in 1950s and in the Recent Ten Years (1995-2004)

Forty pairs of SSR markers were used to compare the genetic diversity changes in 151 Chinese major rice varieties planted in 1950s and in the recent ten years. Of 40 SSR loci, 39 were found to be polymorphic while one locus (RM479) monomorphic. A total of 213 alleles were identified from the 39 polymorphic loci. The average number of alleles per locus (Na) was of 5.5, ranging from 2 to 11. Neis gene diversity index (He) varied drastically among loci from 0.309 at RM174 to 0.869 at RM418, with an average value of 0.649. There existed significant difference in SSR allelic diversity between indica and japonica subspecies, and indica had more variation than japonica both in Na and He. By comparison with the genetic changes in Na and He, it was revealed that the varieties planted in 1950s had more alleles and higher He than those in the recent ten years both for indica and japonica rices. The difference between two subspecies for Na was significant in a tendency over time (indica: z = 2.677, P = 0.007; japonica: z = 3.441, P = 0.001), but not significant for He (indica: z = 1.471, P = 0.141; japonica: z = 1.932, P = 0.053). Analysis of molecular variance (AMOVA) indicated that there existed significant difference (P < 0.05) in genetic variation between the two periods, of which more genetic variation was contributed by indica (Fst = 0.050) and japonica (Fst = 0.082) subsets. Using locus-by-locus AMOVA procedure, significant genetic differentiations were observed in 13 loci (RM21, RM128, RM147, RM169, RM190, RM221, RM231, RM251, RM253, RM317, RM341, RM418, and RM478) for indica varieties and 11 loci (RM101, RM135, RM152, RM159, RM169, RM190, RM251, RM253, RM311, RM418, and RM478) for japonica ones between the two periods. It was found some alleles had been lost in current major rice varieties as comparing with those in 1950s. Therefore, it should be necessary to exploit more alien elite genetic resources for extension of genetic background in current rice breeding program. Key word: rice (Oryza sativa); major varieties; simple sequence repeats; genetic diversity; analysis of molecular variance In general, crop breeding will lead to the increase of food yield and other products to meet various demands of the people, meanwhile will lead to the decline of crop biological diversity resulting in genetic vulnerability (1-7) to adverse environment. This phenomenon has been revealed by the comparative studies on the genetic diversity changes in DNA level in wheat in Europe (8-9) , oat in Canada (10) , and maize in France (11) . However, some scientists did not agree with this idea, thinking that due to the progress of breeding techniques and the increasing germplasm exchange in modern crop breeding, the genetic background of the current varieties was enlarged, which has been indicated by those studies with the molecular biological methods on the modern varieties

Development of novel microsatellite markers for the BBCC Oryza genome (Poaceae) using high-throughput sequencing technology.

Wild species of Oryza are extremely valuable sources of genetic material that can be used to broaden the genetic background of cultivated rice, and to increase its resistance to abiotic and biotic stresses. Until recently, there was no sequence information for the BBCC Oryza genome; therefore, no special markers had been developed for this genome type. The lack of suitable markers made it difficult to search for valuable genes in the BBCC genome. The aim of this study was to develop microsatellite markers for the BBCC genome. We obtained 13,991 SSR-containing sequences and designed 14,508 primer pairs. The most abundant was hexanuclelotide (31.39%), followed by trinucleotide (27.67%) and dinucleotide (19.04%). 600 markers were selected for validation in 23 accessions of Oryza species with the BBCC genome. A set of 495 markers produced clear amplified fragments of the expected sizes. The average number of alleles per locus (Na) was 2.5, ranging from 1 to 9. The genetic diversity per locus (He) ranged from 0 to 0.844 with a mean of 0.333. The mean polymorphism information content (PIC) was 0.290, and ranged from 0 to 0.825. Of the 495 markers, 12 were only found in the BB genome, 173 were unique to the CC genome, and 198 were also present in the AA genome. These microsatellite markers could be used to evaluate the phylogenetic relationships among different Oryza genomes, and to construct a genetic linkage map for locating and identifying valuable genes in the BBCC genome, and would also for marker-assisted breeding programs that included accessions with the AA genome, especially Oryza sativa.

Mapping QTLs for Drought Tolerance at Seedling Stage in Rice Using Doubled Haploid Population

QTLs for drought tolerance at the rice seedling stage were analyzed using a doubled haploid (DH) population consisted of 251 lines from the cross between a japonica parent Maybelle and an indica parent Baiyeqiu. A genetic linkage map with 226 SSR marker loci was constructed. Single-locus analysis following composite interval mapping (CIM) detected a total of five QTLs located on five different chromosomes of rice. Four QTLs were also detected following two-locus analysis, resolving two pairs of epistatic QTLs (E-QTLs) with positive and additive genetic effects. The results indicated that the alleles from the parent Baiyeqiu contributed DH population to improve drought tolerance at the seedling stage.

Genetic Analysis and Preliminary Mapping of Two Recessive Resistance Genes to Brown Planthopper, Nilaparvata lugens Stål in Rice

An F2 population derived from the cross of WB01, an introgression line resistant to brown planthopper (BPH) originated from Oryza rufipogon Griff. and a susceptible indica variety 9311, was developed for genetic analysis and gene mapping. The population with 303 F2:3 families was genotyped by 141 simple sequence repeat (SSR) markers and used for gene mapping. Two softwares, Mapmaker/Exp 3.0 and Windows QTL Cartographer V2.0 were applied to detect QTLs. Totally, two QTLs resistant to BPH, named temporarily as bph22(t) and bph23(t), were identified to locate on chromosomes 4 and 8, individually had LOD values of 2.92 and 3.15, and explained 11.3% and 14 .9% of the phenotypic variation, respectively.

Association Mapping Reveals Novel Genetic Loci Contributing to Flooding Tolerance during Germination in Indica Rice

Rice (Oryza sativa L.) is the only cereal crop that possesses the ability to germinate under flooded or other oxygen-deficient conditions. Rapid elongation of the coleoptile is a perfect response to flooding during germination, with coleoptile length differing among various rice varieties. Despite multiple studies have uncovered valuable information concerning this trait by focusing on the physiological metabolism of oxygen stress, the underlying genetic mechanism still remains unknown. In the present study, we screened coleoptile lengths of 432 indica varieties germinated in two environments (normal and flooded) and found more variation existing in flooded coleoptile length (FCL) rather than in normal coleoptile length (NCL). With the phenotypic data of NCL, FCL and FTI (flooding tolerance index), a genome-wide association study was performed by using 5291 single nucleotide polymorphism (SNP) markers. We detected 2, 11, and 9 significant SNPs under a mixed linear mode for NCL, FCL, and FTI, respectively. Of these SNPs, five were shared by FCL and FTI. Haplotype and phenotype effect analysis on the highest ranking locus indicated one of the two haplotypes contributed to coleoptile elongation remarkably. To better understand the controlling gene of this locus, reported expression profile data was applied. We focused on LOC_Os06g03520, a candidate gene which was highly induced by anoxia (∼507 fold). Sequence analysis in 51 varieties demonstrated Hap.2 associated perfectly with flooding tolerance. Further studies on this gene may help explore the molecular mechanism of rice flooding tolerance during germination. We believe our discoveries may conduce to isolating major genes and aid the improvement of flooding tolerance in modern breeding programs.

Genetic Diversity and Structure of New Inbred Rice Cultivars in China

Abstract A total of 408 inbred rice cultivars bred in the last decade were analyzed for 24 SSR markers. The results showed the genetic diversity of indica cultivars was higher than that of japonica cultivars, and the genetic diversity of new cultivars raised in recent years was lower. Among the six rice cropping regions (RCRs) in China, genetic diversity was the highest in the central rice region (RCR-II) and the southwest rice region (RCR-III). Genetic differences among subpopulations of japonica were more complex than those in indica. Differentiation among seasonal ecotypes and RCRs in indica populations was unclear, but differentiation between RCR-II and northeast rice region (RCR-V) was more distinct for japonica cultivars. Considering the North rice region (RCR-IV) has very low genetic diversity among the tested cultivars, it is important to broaden the genetic background for future cultivars in rice breeding programs.

SSR Analysis on Diversity of AA Genome Oryza Species in the Southeast and South Asia

To investigate genetic diversities among the AA genome Oryza species in the Southeast and South Asia, a total of 428 accessions of the AA genome Oryza species were genotyped using 36 simple sequence repeats (SSR) markers distributed throughout the rice genome. All of the 36 SSR markers generated polymorphic bands, revealing 100% polymorphism. The number of alleles per locus ranged from 3 to 17 with the mean of 8.6. The Nei’s genetic diversity index (He) ranged from 0.337 at RM455 to 0.865 at RM169 with an average value of 0.650. The genetic diversity of the AA genome Oryza species in the Southeast Asia was obviously higher than that in the South Asia. Among the detected Oryza species in the South and Southeast Asia, O. rufipogon showed the highest genetic diversity. Meanwhile, a higher genetic differentiation (Fst) was found among the detected Oryza species in the Southeast Asia than in the South Asia. The Fst value between O. nivara and O. sativa was the highest. The results from the number of specific alleles, specific loci, and allele frequency confirmed the greater genetic variation among the detected species. In addition, the specific allele in RM161 displayed higher frequency (0.193), suggesting its important function in identifying Oryza species of AA genome.