Genetic analysis of starch paste viscosity parameters in glutinous rice (Oryza sativa L.).

Starch paste viscosity plays an important role in estimating the cooking, eating, and processing quality of rice. The inheritance of starch paste viscosity in glutinous rice remains undefined. In the present study, 118 glutinous rice accessions were collected, and the genotypes of 17 starch synthesis-related genes (SSRG) were analyzed by using 43 gene-specific molecular markers. Association analysis indicated that 10 of 17 SSRGs were involved in controlling the rapid visco analyzer (RVA) profile parameters. Among these, the PUL gene was identified to play an important role in control of peak viscosity (PKV), hot paste viscosity (HPV), cool paste viscosity (CPV), breakdown viscosity (BDV), peak time (PeT), and paste temperature (PaT) in glutinous rice. Other SSRGs involved only a few RVA profile parameters. Furthermore, interactions between SSRGs were found being responsible for PeT, PaT, and BDV. Some of the RVA parameters, including PKV, HPV, CPV, CSV, and PaT, were mainly governed by single SSRG, whereas other parameters, such as BDV, SBV, and PeT, were controlled by a few SSRGs, functioning cooperatively. Further, three near-isogenic lines (NIL) of a japonica glutinous cv. Suyunuo as genetic background, with PUL, SSIII-1, and SSIII-2 alleles replaced with those of indica cv. Guichao 2, were employed to verify the genetic effects of the various genes, and the results were consistent with those obtained from the association analysis. These findings indicated that starch paste viscosity in glutinous rice had a complex genetic system, and the PUL gene played an important role in determining the RVA profile parameters in glutinous rice. These results provide important information for potentially improving the quality of glutinous rice.

[Isolation and characterization of the centromeric BAC clones from different genomes in genus Oryza].

Centromeres play an important role in ensuring the correct segregation and transmission of chromosome during mitosis and meiosis in eukaryotes. In this research, we constructed five BAC libraries for diploid wild rice with different genomes. Together with the technique of colony blot hybridization and fluorescence in situ hybridization (FISH), centromere-related BAC clones were screened and characterized from different genomes. Meanwhile, co-hybridization was detected between these clones and the five genomes. The results from this study demonstrated that: (1) there were centromere-specific satellite repeat in Oryza officinalis (CC genome) and O. brachyantha (FF genome), respectively, and centromere-specific CRR-related sequence was found in O. brachyantha; (2) homology sequences of CentO and CRR of O. sativa (AA genome) were detected on all centromeres of O. glaberrima (AA genome), O. punctata (BB genome) and O. australiensis (EE genome); And (3) the two somatic chromosomes of O. officinalis comprised of homology sequences of CentO satellites as revealed FISH analysis probed with RCS2. Homology sequences of CRR of O. sativa were also detected on all centromeres of O. officinalis. The results provided a foundation toward cloning the centromeric sequences from different genomes of genus Oryza, studying centromere organization and evolution of different genome, analyzing the relationship between centromeric structure and function among different genome.

[Agrobacterium-mediated transformation of rice mature embryos and regeneration of transgenic plants with Metr gene].

The mature embryos of a Japonica rice, Guanglingxiangnuo, were used for the study on optimization of Agrobacterium-mediated transformation. Several factors affecting the transformation were investigated and a suitable transformation system was set up. For this transformation system, the HRM medium, based on the MS medium, was suitable for inducing callus from rice mature embryos. The suitable time span of initial culture in this medium was 7-8 days before co-culturing with Agrobacterium and suitable medium for selection was CC medium. Using this transformation system, the Metr gene was introduced into Guanglingxiangnuo, and many transgenic plants were obtained. Most of these transgenic rice plants were confirmed by PCR technique and basta resistance, indicating the T-DNA had been integrated into the genome of transgenic rice plants.

[Mapping quantitative trait loci associated with rice grain shape based on an indica/japonica backcross population].

Appearance of rice grain represents a major character of rice quality in many rice-producing areas of the world, especially in hybrid rice production in China. In this study, we conducted a molecular marker-based genetic analysis of the traits that are determinants of the appearance quality of rice grains, including grain length, grain width and grain shape (measured as grain length to grain width ratio). Two typical indica/japonica varieties Balilla and Nantehao(NTH) were selected to construct Balilla/NTH//Balilla backcross population containing 142 individuals. In the population, grain length, grain width and grain shape all conform to the normal distribution with certain transgressive segregation. It can be deduced that all of three traits were controlled by some quantitative trait loci (QTLs). In order to explore the QTLs effect, number and location, a linkage map consisting of 108 SSR markers based on the backcross population was constructed, and QTLs mapping was carried out for grain length, grain width and grain shape. A QTL, qGL-12, was detected for grain length at the interval RM101-RM270 on chromosome 12, its additive effect was 0.26 mm, and can explain 16.7% genetic variation. As for grain width trait, two QTLs were found, qGW-2 located at RM154-RM211 interval on chromosome 2, and qGW-3 at interval RM257-RM175 on chromosome 3, accounting for 11.5% and 16.6% genetic variation, respectively. The alleles at qGW-2 and qGW-3 from parent Balilla can increase grain width by 0.10 mm and 0.12 mm. For grain shape, 3 QTLs, qLW-2, qLW-6 and qLW-7 were found, located on chromosome 2, 6, and 7, respectively. qLW-2 and qLW-7 had positive effect, and they can explain 12.7% and 18.3% genetic variation, while qLW-6 had negative effect and contributed 11.5% genetic variation to the backcross population. The prospects of application of linkage relationship between SSR marker and QTLs in marker based selection (MAS) in rice breeding, and the improvement of grain shape and rice appearance quality were discussed.

Molecular marker analysis and genetic basis for sterility of typical indica/japonica hybrids.

To explore the genes differentiated between typical indica and japonica varieties, two typical indica/japonica varieties, Balilla (japonica) and Nantehao (NTH, indica), were selected to construct genetic populations based on the widely surveying for spikelet and pollen fertility of 90 indica/japonica F1 hybrids, which also were used as the wide compatability testers. In order to analyze the genes (QTLs) related to spikelet and pollen fertility, two reciprocal backcross populations Balilla/NTH//Balilla and Balilla/NTH//NTH were constructed and the spikelet and pollen fertility of each individuals were assessed. In both populations, two traits all appeared distorted normal distribution, but in the first population, they forwarded to low-level fertility type, the later population, forwarded to high-level fertility type relatively. The results indicated that both of male and female gametophytes of Balilla/NTH hybrids were partial sterile. Then we analyzed the SSR marker genotype of each individuals of Balilla/NTH//Balilla population containing 142 individuals, and constructed a SSR linkage map, in which, there were 108 information markers distributing on all 12 chromosomes equably, average marker distance was about 11.9 cM. Therefore the linkage map was qualified for QTL analysis. Two methods were employed to conduct QTLs analysis, i.e., single marker analysis and interval mapping. According to single marker analysis, 17 and 12 markers were found significantly responsible for spikelet and pollen fertility, respectively. And further study by means of MAPMAKER/QTL software, for spikelet fertility trait, two QTLs were detected, qSPTF1 on chromosome 1 and qSPTF6 on chromosome 6, and their additive effect were 13.501 and -16.414, respectively. According to previous studies, qSPTF6 was deduced to be the same locus as S-5. For pollen fertility, qPLLN7 on chromosome 7 and qPLLN9 on chromosome 9 were detected, and their additive effects were -12.003 and -11.012, respectively. Because the QTLs detected cannot explain completely the total variance of mapping population, other genetic factors must be existed to be responsible for spikelet and pollen partial sterility. Hence we let two random markers as putative covariates, and divide the 142 individuals into four groups according to the two marker genotypes, then the average values of spikelet and pollen fertility of each groups were calculated for two-way ANOVA (analysis of variance). The results indicated that there existed strong interaction for both spikelet fertility and pollen fertility. At a significance level of 0.005, there over 61 and 51 pairs loci interactions detected playing an important role in spikelet and pollen sterility expression, respectively. These results indicated that epistasis also was one of major genetic components controlling indica/japonica hybrid sterility.

Development and high-throughput genotyping of substitution lines carring the chromosome segments of indica 9311 in the background of japonica Nipponbare.

Chromosome segment substitution lines (CSSLs) are useful for the precise mapping of quantitative trait loci (QTLs) and dissection of the genetic basis of complex traits. In this study, two whole-genome sequenced rice cultivars, the japonica Nipponbare and indica 9311 were used as recipient and donor, respectively. A population with 57 CSSLs was developed after crossing and back-crossing assisted by molecular markers, and genotypes were identified using a high-throughput resequencing strategy. Detailed graphical genotypes of 38 lines were constructed based on resequencing data. These CSSLs had a total of 95 substituted segments derived from indica 9311, with an average of about 2.5 segments per CSSL and eight segments per chromosome, and covered about 87.4% of the rice whole genome. A multiple linear regression QTL analysis mapped four QTLs for 1000-grain weight. The largest-effect QTL was located in a region on chromosome 5 that contained a cloned major QTL GW5/qSW5 for grain size in rice. These CSSLs with a background of Nipponbare may provide powerful tools for future whole-genome discovery and functional study of essential genes/QTLs in rice, and offer ideal materials and foundations for japonica breeding.

Identification and characterization of a major QTL responsible for erect panicle trait in japonica rice (Oryza sativa L.).

Panicle erectness (PE) is one of the most important traits for high-yielding japonica cultivars. Although several cultivars with PE trait have been developed and released for commercial production in China, there is little information on the inheritance of PE traits in rice. In the present study, 69 widely cultivated japonica cultivars and a double haploid (DH) population derived from a cross between a PE cultivar (Wuyunjing 8) and a drooping panicle cultivar (Nongken 57) were utilized to elucidate the mechanisms of PE formation and to map PE associated genes. Our data suggested that panicle length (PL) and plant height (PH) significantly affected panicle curvature (PC), with shorter PL and PH resulting in smaller PC and consequently more erect. A putative major gene was identified on chromosome 9 by molecular markers and bulk segregant analysis in DH population. In order to finely map the major gene, all simple sequence repeats (SSR) markers on chromosome 9 as well as 100 newly developed sequence-tagged site (STS) markers were used to construct a linkage group for quantitative trait locus (QTL) mapping. A major QTL, qPE9-1, between STS marker H90 and SSR marker RM5652, was detected, and accounted for 41.72% of PC variation with pleiotropic effect on PH and PL. another QTL, qPE9-2, was also found to be adjacent to qPE9-1. In addition, we found that H90, the nearest marker to qPE9-1, used for genotyping 38 cultivars with extremely erect and drooping panicles, segregated in agreement with PC, suggesting the H90 product was possibly part of the qPE9-1 gene or closely related to it. These data demonstrated that H90 could be used for marker-aided selection for the PE trait in breeding and in the cloning of qPE9-1.