Analysis of QTLs for yield-related traits in Yuanjiang common wild rice （Oryza rufipogon Griff.）

Using an accession of common wild rice(Oryza rufipogon Griff.)collected from Yunnjiang County,Yunnan Province,China,as the donor and an elite cnltivar 93-11,widely used in two-line indica hybrid rice production in China,as the recurrent parent,an advanced backcross populations were developed.Through genotyping of 187 SSR markers and investigation of six yield-related traits of two generations(BC4F2 and BC4F4),a total of 26 QTLs were detected by employing single point analysis and interval mapping in both generations.Of the 26 QTLs,the alleles of 10(38.5%)QTLs originating from O.rufipogon had shown a beneficial effect for yield-related traits in the 93-11 genetic background.In addition,five QTLs controlling yield and its components were newly identified,indicating that there arc potentially novel alleles in Yuanjiang common wild rice.Three regions underling significant QTLs for several yield-related traits were detected on chromosome 1,7 and 12.The QTL clusters were founded and corresponding agronomic traits of those QTLs showed highly significant correlation,suggesting the pleiotropism or tight linkage.Fine-mapping and cloning of these yield-related QTLs from wild rice would be helpful to elucidating molecular mechanism of rice domestication and rice breeding in the future.     

Novel pleiotropic loci controlling panicle architecture across environments in japonica rice （Oryza sativa L.）

To identify quantitative trait loci (QTLs) controlling panicle architecture in japonica rice, a genetic map was constructed based on simple sequence repeat (SSR) markers and 254 recombinant inbred lines (RILs) derived from a cross between cultivars Xiushui 79 and C Bao. Seven panicle traits were investigated under three environments. Single marker analysis indicated that a total of 27 SSR markers were highly associated with panicle traits in all the three environments. Percentage of phenotypic variation explained by single locus varied from 2% to 35%. Based on the mixed linear model, a total of 40 additive QTLs for seven panicle traits were detected by composite interval mapping, explaining 1.2%-35% of phenotypic variation. Among the 9 QTLs with more than 10% of explained phenotypic variation, two QTLs were for the number of primary branches per panicle (NPB), two for panicle length (PL), two for spikelet density (SD), one for the number of secondary branches per panicle (NSB), one for secondary branch distribution density (SBD), and one for the number of spikelets per panicle (NS), respectively. qPLSD-9-1 and qPLSD-9-2 were novel pleiotropic loci, showing effects on PL and SD simultaneously. qPLSD-9-1 explained 34.7% of the phenotypic variation for PL and 25.4% of the phenotypic variation for SD, respec- tively. qPLSD-9-2 explained 34.9% and 24.4% of the phenotypic variation for PL and SD, respectively. The C Bao alleles at the both QTLs showed positive effects on PL, and the Xiushui 79 alleles at the both QTLs showed positive effects on SD. Genetic variation of panicle traits are mainly attributed to additive effects. QTL × environment interactions were not significant for additive QTLs and additive × additive QTL pairs.     

Molecular evolution and functional divergence of HAK potassium transporter gene family in rice （Oryza sativa L.）

The high-affinity K＋ （HAK） transporter gene family is the largest family in plant that functions as potassium transporter and is important for various aspects of plant life. In the present study, we identified 27 members of this family in rice genome. The phylogenetic tree divided the land plant HAK transporter proteins into 6 distinct groups. Although the main characteristic of this family was established before the origin of seed plants, they also showed some differences between the members of non-seed and seed plants. The HAK genes in rice were found to have expanded in lineage-specific manner after the split of monocots and dicots, and both segmental duplication events and tandem duplication events contributed to the expansion of this family. Functional divergence analysis for this family provided statistical evidence for shifted evolutionary rate after gene duplication. Further analysis indicated that both point mutant with positive selection and gene conversion events contributed to the evolution of this family in rice.     

Genetic analysis and molecular mapping of a novel gene for zebra mutation in rice （Oryza sativa L.）

A novel zebra mutant, zebra-15, derived from the restorer line JinhuilO （Oryza sativa L. ssp. indica） treated by EMS, displayed a distinctive zebra leaf from seedling stage to jointing stage. Its chlorophyll content decreased （55.4%） and the ratio of Chla/Chlb increased （90.2%） significantly in the yellow part of the zebra-15, compared with the wild type. Net photosynthetic rate and fluorescence kinetic parameters showed that the decrease of chlorophyll content significantly influenced the photosynthetic efficiency of the mutant. Genetic analysis of F2 segregation populations derived from the cross of XinonglA and zebra-15 indicated that the zebra leaf trait is controlled by a single recessive nuclear gene. Ninety-eight out of four hundred and eighty pairs of SSR markers showed the diversity between the XinonglA and the zebra-15, their F2 population was then used for gene mapping. Zebra-15 （Z-15） gene was primarily restricted on the short arm of chromosome 5 by 150 F2 recessive individuals, 19.6 cM from marker RM3322 and 6.0 cM from marker RM6082. Thirty-six SSR markers were newly designed in the restricted location, and the Z-15 was finally located between markers nSSR516 and nSSR502 with the physical region 258 kb by using 1,054 F2 recessive individuals.     

Development of microsatellite markers and their utilization in genetic diversity analysis of cultivated and wild populations of the mud carp （ Cirrhina molitorella）

Microsatellite markers have been increasingly used in genetic studies on fishery species because of their high applicability in selective breeding programs.Here we reported the development of microsatellite markers and their utilization in mud carp(Cirrhina molitorella).An (CA)15 enriched library has been constructed for mud carp,using the magnetic beads enrichment procedure.Sequence analysis of 60randomly picked positive colonies indicate that 56 (93.3%) of the colonies contain microsatellites.Microsatellite polymorphism was as-sessed using 10 mud carp individuals,and 12 microsatellite loci turned out to be polymorphic.We utilized these loci to study the genetic diversity of a wild population (WM) and a cultured population (CM) of the mud carp.A total of 109 alleles were detected with an average of 9.08 alleles per locus.The mean value of the observed heterozygosity of WM and CM was 0.6361 and 0.6417,respectively,and sig-nificant decrease of genetic diversity in CM was not observed.The genetic distance between the two populations was 0.1546 and the value of Gsr was 0.0473.This showed that there existed a slight genetic differentiation between WM and CM.     

Oxalate Accumulation as Regulated by Nitrogen Forms and Its Relationship to Photosynthesis in Rice （Oryza sativa L.）

Four-leaf rice seedlings (Oryza sativa L.), which had been cultivated in Kimura B complete nutrient solution, were treated with two nitrogen forms by replacing the nitrogen element in the complete solution with sole nitrate or ammonium (2.86 mmol/L). Nitrate-N nutrition tended to increase oxalate content in all parts of the plant, including the leaves, stems, roots, and root exudates, whereas ammonium had the opposite effect. Consequently, marked differences in oxalate content were observed between the two treatments throughout the time tested (0-12 d), with maximal differences of approximately 12-fold at 6d after treatment. Photosynthetic/respiratory parameters were examined over time simultaneously with changes in oxalate content. Net photosynthetic rate, chlorophyll fluorescence parameters (i.e. maximal photochemical efficiency (Fv/Fm) and photochemical quantum yields of photosystem (PS)II (ΦPSⅡ)), and respiratory rate were not significantly different between plants treated with the two nitrogen forms, although ammonium-fed plants had apparently higher leaf chlorophyll content than nitrate-fed plants. Leaf glucose content was altered little, but the content of fructose, sucrose, and total soluble sugar was significantly higher in the leaves of ammonium-fed plants than nitrate-fed plants. The results indicate that nitrate/ammonium may serve as efficient regulators of oxalate accumulation owing to regulation of metabolism in rice leaves rather than oxalate downward transfer and root excretion, and that photosynthetic metabolism is not directly correlated with the regulation of oxalate accumulation in rice plants.     

Additive and additive x additive interaction make important contributions to spikelets per panicle in rice near isogenic （Oryza sativa L.） lines

Epistasis plays an important role in the genetic basis of rice yield traits. Taking interactions into account in breeding programs will help the development of high-yielding rice varieties. In this study, three sets of near isogenic lines (NILs) targeting three QTLs for spikelets per panicle (SPP), namely qSPP1, qSPP2 and qSPP7, which share the same Zhenshan 97 genetic background, were used to produce an F₂ population in which the three QTLs segregated simultaneously. The genotypes of the individual F₂ plants at the three QTLs were replaced with three markers that are closely linked to the corresponding QTLs. These QTLs were validated in the F₂ and F₃ populations at the single marker level. qSPP7 exhibited major pleiotropic effects on SPP, plant height and heading date. Multifactor analysis of variance was performed for the F₂ population and its progeny. Additive (additive interaction between qSPP2 and qSPP7 had significant effects on SPP in both the F₂ population and its progeny. Both additive and additive (additive interactions could explain about 73% of the total SPP phenotypic variance. The SPP performance of 27 three-locus combinations was ranked and favorable combinations were recommended for rice breeding in different ecosystems.     

Introgression of qPE9-1 allele, conferring the panicle erectness, leads to the decrease of grain yield per plant in japonica rice （Oryza sativa L.）

Panicle architecture is closely related to yield formation. The qPE9-1 gene has been proved to be widely used in high-yield rice cultivar developments, conferring erect panicle character in japonica rice. Recently, qPE9-1 has been successfully cloned; however, the genetic effect on grain yield per plant of the erect panicle allele qPE9-1 is controversial yet. In the present study, a drooping panicle parent Nongken 57, carrying qpe9-1 allele, was used as recurrent parent to successively backcross to a typical erect panicle line from the double haploid (DH) population (Wuyunjing 8/Nongken 57), which was previously shown to carry qPE9-1 allele. Thus a pair of near-isogenic lines (NILs) was developed. The comparison of agronomic traits between the NILs showed that, when qpe9-1 was replaced by qPE9-1, the panicle architecture was changed from drooping to erect; moreover, the panicle length, plant height, 1000-grain weight and the tillers were significantly decreased, consequently resulting in the dramatic decrease of grain yield per plant by 30%. Therefore, we concluded that the qPE9-1 was a major factor controlling panicle architecture, and qPE9-1 had pleiotropic nature, with negative effects on grain yield per plant. This result strongly suggests that the erect panicle allele qPE9-1 should be used together with other favorable genes in the high-yield breeding practice. In addition, the effect of qPE9-1 on eating and cooking quality was also discussed in the present study.     

Activation of gibberellin 2-oxidase 6 decreases active gibberellin levels and creates a dominant semi-dwarf phenotype in rice （Oryza sativa L.）

Gibberellin (GA) 2-oxidase plays a key role in the GA catabolic pathway through 2β-hydroxylation.In the present study,we isolated a CaMV 35S-enhancer activation tagged mutant,H032.This mutant exhibited a dominant dwarf and GA-deficient phenotype,with a final stature that was less than half of its wild-type counterpart.The endogenous bioactive GAs are markedly decreased in the H032 mutant,and application of bioactive GAs (GA3 or GA4) can reverse the dwarf phenotype.The integrated T-DNA was detected 12.8 kb upstream of the OsGA2ox6 in the H032 genome by TAIL-PCR.An increased level of OsGA2ox6 mRNA was detected at a high level in the H032 mutant,which might be due to the enhancer role of the CaMV 35S promoter.RNAi and ectopic expression analysis of OsGA2ox6 indicated that the dwarf trait and the decreased levels of bioactive GAs in the H032 mutant were a result of the up-regulation of the OsGA2ox6 gene.BLASTP analysis revealed that OsGA2ox6 belongs to the class III of GA 2-oxidases,which is a novel type of GA2ox that uses C20-GAs (GA12 and/or GA53) as the substrates.Interestingly,we found that a GA biosynthesis inhibitor,paclobutrazol,positively regulated the OsGA2ox6 gene.Unlike the over-expression of OsGA2ox1,which led to a high rate of seed abortion,the H032 mutant retained normal flowering and seed production.These results indicate that OsGA2ox6 mainly affects plant stature,and the dominant dwarf trait of the H032 mutant can be used as an efficient dwarf resource in rice breeding.     

Identification of quantitative trait loci for four morphologic traits under water stress in rice （Oryza sativa L.）

Late season drought coinciding with the rice booting to heading stage affects the development of plant height,panicle exsertion,and flag leaf size,and causes significant yield loss.In this study,a recombinant inbred line population derived from a cross between paddy and upland cultivars was used for data collection of the morphologic traits under well water and drought stress conditions.bought stress was applied at the stage of panicle initiation in the field in 2002 and at the booting stage in PVC pipes in 2003.The data from stress con ditions and their ratios(tait measured under stress condition/trait measured under well water condition)or differences(trait measured under stress condition minus trait measured under well water condition)were used for OTL analysis.Totally,17 and 36 QTLs for these traits were identified in 2002 and 2003,respectively,which explained a range of 2.58%-29.82%Of the phenotypic variation.Among them,six QTLs were commonly identified in the two years,suggesting that the drought stress in the two years was different.The genetic basis of these traits will provide useful information for improving rice late season drought resistance,and their application as indirect indices in rice late season drought resistance screening was also discussed.     

A large-scale field study of transgene flow from cultivated rice (Oryza sativa) to common wild rice (O. rufipogon) and barnyard grass (Echinochloa crusgalli)

The introgression of transgenes into wild relatives or weeds through pollen-mediated gene flow is a major concern in environmental risk assessment of transgenic crops. A large-scale (1.3–1.8 ha) rice gene flow study was conducted using transgenic rice containing the bar gene as a pollen donor and Oryza rufipogon as a recipient. There was a high frequency of transgene flow (11%−18%) at 0–1 m, with a steep decline with increasing distance to a detection limit of 0.01% by 250 m. To our knowledge, this is the highest frequency and longest distance of gene flow from transgenic rice to O. rufipogon reported so far. On the basis of these data, an adequate isolation distance from both conventional and transgenic rice should be taken for in situ conservation of common wild rice. Meanwhile, there is no evidence of transgene introgression into barnyard grass, even when it has coexisted with transgenic rice containing the bar gene for five successive years. Thus, the environmental risk of gene flow to this weedy species is of little concern.     

Comparison of the genetic diversity of common wild rice (Oryza rufipogon Griff.) and cultivated rice (O. sativa L.) using RFLP markers

Forty fourth single-copy RFLP markers were used to evaluate the genetic diversity of 122 accessions of common wild rice (CWR, Oryza rufipogon Griff.) and 75 entries of cultivated rice (Oryza sativa L. ) from more than ten Asian countries. A comparison of the parameters showing genetic diversity, including the percentage of polymorphic loci (P), the average number of alleles per locus (A), the number of genotypes (Ng), the average heterozygosity (Ho) and the average genetic multiplicity (Hs) of CWR and indica and japonica subspecies of cultivated rice from different countries and regions, indicated that CWR from China possesses the highest genetic diversity, followed by CWR from South Asia and Southeast Asia. The genetic diversity of CWR from India is the second highest. Although the average gene diversity (Hs)of the South Asian CWR is higher than that of the Southeast Asian CWR, its percentage of polymorphic loci (P), number of alleles (Na) and number of genotypes (Ng) are all smaller. It was also found that the genetic diversity of cultivated rice is obviously lower than that of CWR. At the 44 loci investigated, the number of polymorphic loci of cultivated rice is only 3/4 that of CWR, while the number of alleles, 60%, and the number of genotypes is about 1/2 that of CWR. Of the two subspecies studied, the genetic diversity of indica is higher than that of japonica. The average heterozygosity of the Chinese CWR is the highest among all the entries studied. The average heterozygosity of CWR is about two-times that of cultivated rice. It is suggested that during the course of evolution from wild rice to cultivated rice, many alleles were lost through natural and human selection, leading to the lower heterozygosity and genetic diversity of the cultivated rice. Received: 19 May 1999 / Accepted: 26 April 2000     

PCR-based molecular markers for the fragrance gene in rice (Oryza sativa. L.)

The genomic DNA clone RG28, linked to the major fragrance gene of rice (fgr), was assessed for polymorphism in order to produce a PCR-based marker for fragrance. A small mono-nucleotide repeat, that was polymorphic between a pair of fragrant and non-fragrant cultivars, was identified and developed into a co-dominant PCR-based marker. The polymorphism-information-content determinations for three microsatellite markers, that have been genetically mapped near RG28, are also presented. These PCR-based markers will be highly useful in distinguishing fragrance-producing alleles from non-fragrance-producing alleles at the fgr locus. Received: 19 October 1999 / Accepted: 16 December 1999     

Search for and analysis of single nucleotide polymorphisms (SNPs) in rice (Oryza sativa, Oryza rufipogon) and establishment of SNP markers.

We searched for SNPs in 417 regions distributed throughout the genome of three Oryza sativa ssp. japonica cultivars, two indica cultivars, and a wild rice (O. rufipogon). We found 2800 SNPs in approximately 250,000 aligned bases for an average of one SNP every 89 bp, or one SNP every 232 bp between two randomly selected strains. Graphic representation of the frequency of SNPs along each chromosome showed uneven distribution of polymorphism-rich and -poor regions, but little obvious association with the centromere or telomere. The 94 SNPs that we found between the closely related cultivars 'Nipponbare' and 'Koshihikari' can be converted into molecular markers. Our establishment of 213 co-dominant SNP markers distributed throughout the genome illustrates the immense potential of SNPs as molecular markers not only for genome research, but also for molecular breeding of rice.     

Water-use efficiency in rice (Oryza sativa L.) in relation to resistance to salinity

Abstract The effects of sodium chloride on water-use efficiency of a number of varieties of rice have been investigated. Sensitivity to salinity in rice arises in large part from excessive rates of sodium ion transport to the shoot consequent upon high rates of transpirational water loss. Varietal differences in water-use efficiency were found: these were greater when measured for whole shoots over a period of one week, than when made as instantaneous measurements on individual leaves. Salinity had rather little effect on water use efficiency but overall resistance of seedlings to salt was, in general, greater the greater the water-use efficiency. The reasons for the differences between varieties are discussed in terms of differences in growth pattern: among the seven varieties investigated, water-use efficiency and salt-resistance were lower in dwarfed as opposed to non-dwarfed varieties.     

Allozyme variation and population genetic structure of common wild rice Oryza rufipogon Griff. in China

In order to determine the genetic diversity and genetic structure of populations in common wild rice Oryza rufipogon, an endangered species, allozyme diversity was analyzed using 22 loci in 607 individuals of 21 natural populations from the Guangxi, Guangdong, Hainan, Yunnan, Hunan, Jiangxi and Fujian provinces in China. The populations studied showed a moderate allozyme variability (A=1.33, P=22.7%, Ho=0.033 and He=0.068), which was relatively high for the genus Oryza. The levels of genetic diversity for Guangxi and Guangdong were significantly higher than those for the other regions, and thus South China appeared to be the center of genetic diversity of O. rufipogon in China. A moderate genetic differentiation (F_ST=0.310, I=0.964) was found among the populations studied. Interestingly, the pattern of population differentiation does not correspond to geographic distance. An estimate of the outcrossing rate (t=0.324) suggests that the species has a typical mixed-mating system. The deficit of heterozygotes (F=0.511) indicates that some inbreeding may have taken place in outcrossing asexual populations because of intra-clone outcrossing events and ”isolation by distance” as a result of human disturbance. In order to predict the long-term genetic survival of fragmented populations, further studies on gene flow among the remaining populations and the genetic effects of fragmentation are proposed. Finally, some implications for the conservation of endangered species are suggested. Received: 22 June 1999 / Accepted: 20 December 1999     

普通野生稻小种群的交配系统与遗传多样性

小种群的遗传动态是保育遗传学关注的核心问题之一,而种群遗传动态又与交配系统密切相关.普通野生稻(Oryza rufipogon Griff.)是具有重要经济价值的濒危物种,目前其种群规模都较小,研究其小种群交配系统与遗传变异性对普通野生稻的保护具有重要意义.运用7对SSR引物,对采自江西东乡普通野生稻小种群的36份种茎和其中20个家系共计601份子代进行了分析.结果显示:该种群的表观异交率为0.318,多位点法估计(MLTR)的多位点异交率为0.481;50%以上的子代共享亲本,非随机交配明显;东乡普通野生稻种群交配系统属于混合交配类型.比较亲本和子代种群的遗传变异性显示:子代种群比亲本种群遗传变异性更丰富;子代种群的杂合子不足与种群变小自交比例上升有关;而亲本种群杂合子过剩可能与杂合基因型的选择优势有关.这些结果说明创造条件扩大种群规模对普通野生稻的原生境保护显得尤为重要.