Yinghui Xiao

Bulked segregant analysis to detect QTL related to heat tolerance in rice (Oryza sativa L.) using SSR markers.

The study was undertaken to assess the genetic effect of quantitative trait loci (QTLs) conferring heat tolerance at flowering stage in rice. A population consisting of 279 F2 individuals from the cross between 996, a heat tolerant cultivar and 4628, a heat-sensitive cultivar, was analyzed for their segregation pattern of the difference of seed set rate under optimal temperature condition and high temperature condition. The difference of seed set rate under optimal temperature condition and high temperature condition showed normal distribution, indicating the polygenic control over the trait. To identify main effect of QTL for heat tolerance, the parents were surveyed with 200 primer pairs of simple sequence repeats (SSR). The parental survey revealed 30% polymorphism between parents. In order to detect the main QTL association with heat tolerance, a strategy of combining the DNA pooling from selected segregants and genotyping was adopted. The association of putative markers identified based on DNA pooling from selected segregants was established by single marker analysis (SMA). The results of SMA revealed that SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The heat tolerance during flowering stage in rice was controlled by multiple gene. The SSR markers, RM3735 on chromosome 4 and RM3586 on chromosome 3 showed significant association with heat tolerance respectively, accounted for 17 and 3% of the total variation respectively. The two genetic loci, especially for RM3735 on chromosome 4, can be used in marker-assistant-selected method in heat tolerance breeding in rice.

Molecular Mapping of the New Blast Resistance Genes Pi47 and Pi48 in the Durably Resistant Local Rice Cultivar Xiangzi 3150

The indica rice cultivar Xiangzi 3150 (XZ3150) confers a high level of resistance to 95% of the isolates of Magnaporthe oryzae (the agent of rice blast disease) collected in Hunan Province, China. To identify the resistance (R) gene(s) controlling the high level of resistance in this cultivar, we developed 286 F(9) recombinant inbred lines (RILs) from a cross between XZ3150 and the highly susceptible cultivar CO39. Inoculation of the RILs and an F(2) population from a cross between the two cultivars with the avirulent isolate 193-1-1 in the growth chamber indicated the presence of two dominant R genes in XZ3150. A linkage map with 134 polymorphic simple sequence repeat and single feature polymorphism markers was constructed with the genotype data of the 286 RILs. Composite interval mapping (CIM) using the results of 193-1-1 inoculation showed that two major R genes, designated Pi47 and Pi48, were located between RM206 and RM224 on chromosome 11, and between RM5364 and RM7102 on chromosome 12, respectively. Interestingly, the CIM analysis of the four resistant components of the RILs to the field blast population revealed that Pi47 and Pi48 were also the major genetic factors responsible for the field resistance in XZ3150. The DNA markers linked to the new R genes identified in this study should be useful for further fine mapping, gene cloning, and marker-aided breeding of blast-resistant rice cultivars.

Quantitative trait loci associated with seed set under high temperature stress at the flowering stage in rice (Oryza sativa L.)

High temperature stress (HTS), an increasingly important problem in rice production, significantly reduces rice yield by reducing seed set percentage (SSP). Breeding rice varieties with tolerance to HTS at the flowering stage is therefore essential for maintaining rice production as the climate continues to warm. In this study, two quantitative trait loci (QTL) underlying tolerance to HTS were identified using the recombinant inbred lines (RILs) derived from a cross between the HTS-tolerant rice cultivar 996 and the sensitive cultivar 4628. SSP was used as the heat-tolerance indicator for the lines, which were subjected to HTS at the flowering stage in both field and growth chamber experiments. Two major QTL that affected SSP in both conditions were detected in the interval between RM5687 and RM471 on chromosome 4, and between RM6132 and RM6100 on chromosome 10. The QTL located on chromosome 4 explained 21.3% in field and 25.8% in growth chamber of the total phenotypic variation in SSP, and increased the SSP of plants subjected to HTS by 9.1% in field and by 9.3% in growth chamber. The second QTL located on chromosome 10 explained 11.5% in field and 11.6% in growth chamber of the total phenotypic variation in SSP, and increased the SSP of plants subjected to HTS by 7.2% in field and 7.0% in growth chamber. The positive additive effects of the two QTL were derived from the 996 alleles. The two major QTL identified in this study could be useful for further fine mapping and cloning of these genes and for molecular marker-aided breeding of heat-tolerant rice cultivars.

Practices and Prospects of Super Hybrid Rice Breeding

The great progress in super rice breeding both in China and other countries has been made in recent years. However, there were three main problems in super rice breeding: 1) the super rice varieties were still rare; 2) most super rice varieties exhibited narrow adaptability; and 3) current breeding theories emphasized too much on the rice growth model, but they were unpractical in guidance for rice breeding. According to the authors’ experience on the rice breeding, the breeding strategies including three steps (super parent breeding, super hybrid rice breeding and super hybrid rice seed production) were proposed, and the objectives of each step and the key technologies to achieve the goals were elucidated in detail. The super parent of hybrid rice should exhibit excellent performance in all agronomic traits, with the yield or sink capacity reached the level of the hybrid rice control in regional trials. The super hybrid rice combination should meet the following criteria: good rice quality, wide adaptation, lodging resistance, resistance to main insects and diseases, and the yield exceeded above 8% over the control varieties in the national and provincial regional trials. To achieve the goal, the technical strategies, such as selecting optimal combination of the parents, increasing selection pressure, paying more attention to harmony of ideal plant type, excellent physiological traits and all the agronomic traits, should be emphasized. The yield of seed production should reach 3.75 t/ha and 5.25 t/ha for the super hybrid rice combinations derived from early-season and middle-season types of male sterile lines, respectively. The main technologies for raising seed production yield included selecting optimum seed production site, using the male sterile line with large sink capacity and good outcrossing characteristics, and improving the amount of the pollen by intensive cultivation of the male parent. According to the technologies of the three-step breeding on super hybrid rice, two super rice parents, including a male parent 996 and a thermo(photo)-genic male sterile [T(P)GMS] line C815S, were bred. Furthermore, a super early hybrid rice combination, Luliangyou 996, which could be used as a double-season early rice variety in middle and lower reaches of the Yangtze River, China, was bred by using the super rice variety 996 as the male parent, and several hybrid rice combinations with higher yield than control variety in regional trials both of Hunan Province and state were bred with the T(P)GMS line C815S as the female parent.

Quantitative Trait Loci Associated with Pollen Fertility under High Temperature Stress at Flowering Stage in Rice (Oryza sativa)

High temperature stress (HTS), an increasingly important problem in rice production, significantly reduces rice yield by reducing pollen fertility and seed setting rate. Breeding rice varieties with tolerance to HTS at the flowering stage is therefore essential for maintaining rice production as the climate continues to become warm. In this study, two quantitative trait loci (QTLs) underlying tolerance to HTS were identified using recombinant inbred lines derived from a cross between an HTS-tolerant rice cultivar 996 and a sensitive cultivar 4628. Pollen fertility was used as a heat-tolerance indicator for the lines subjected to HTS at the flowering stage in field experiments. Two QTLs that affected pollen fertility, qPF4 and qPF6, were detected between RM5687 and RM471 on chromosome 4, and between RM190 and RM225 on chromosome 6, by using the composite interval mapping (CIM) analysis. The two QTLs explained 15.1% and 9.31% of the total phenotypic variation in pollen fertility, and increased the pollen fertility of the plants subjected to HTS by 7.15% and 5.25%, respectively. The positive additive effects of the two QTLs were derived from the 996 alleles. The two major QTLs identified would be useful for further fine mapping and cloning of these genes and for molecular marker-assistant breeding of heat-tolerant rice varieties.

Dissection of the genetic architecture of rice resistance to the blast fungus Magnaporthe oryzae

Resistance in rice cultivars to the rice blast fungus Magnaporthe oryzae is complex and is controlled by both major genes and quantitative trait loci (QTLs). We undertook a genome-wide association study (GWAS) using the rice diversity panel 1 (RDP1) that was genotyped using a high-density (700 000 single nucleotide polymorphisms) array and inoculated with five diverse M. oryzae isolates. We identified 97 loci associated with blast resistance (LABRs). Among them, 82 were new regions and 15 co-localized with known blast resistance loci. The top 72 LABRs explained up to 98% of the phenotypic variation. The candidate genes in the LABRs encode nucleotide-binding site leucine-rich repeat (NBS-LRR) resistance proteins, receptor-like protein kinases, transcription factors and defence-related proteins. Among them, LABR_64 was strongly associated with resistance to all five isolates. We analysed the function of candidate genes underlying LABR_64 using RNA interference (RNAi) technology and identified two new resistance alleles at the Pi5 locus. We demonstrate an efficient strategy for rapid allele discovery using the power of GWAS, coupled with RNAi technology, for the dissection of complex blast resistance in rice.

Mechanism of Sterility and Breeding Strategies for Photoperiod/Thermo- Sensitive Genic Male Sterile Rice

To understand the male sterility mechanism of photoperiod/thermo-sensitive genic male sterile [P(T)GMS] lines in rice, the research progress on genetics of photoperiod and/or temperature sensitive genic male sterility in rice was reviewed. A new idea was proposed to explain the sterility mechanism of P(T)GMS rice. The fertility transition from sterile to fertile is the result of cooperative regulation of major-effect sterile genes with photoperiod and/or temperature sensitive genes, but not the so-called pgms gene in P(T)GMS rice. The minor-effect genes, which exhibit accumulative effect on sterility, are the important factors for the critical temperature of sterility transition. The more minor-effect genes the sterile line holds, the lower the critical temperature of sterility transition is. The critical temperature of sterility transition will be invariable if all the minor-effect genes are homozygous. The strategies for breeding P(T)GMS rice were also proposed. The selective indices of critical photoperiod and temperature for sterility transition should be set according to varietal type and ecological region. Imposing selection pressure is a key technology for breeding P(T)GMS rice with lower critical temperature for sterility, and improving the comprehensive performance of the whole traits and combining ability is vital for breeding P(T)GMS rice lines.

Genetic Variation and Evolution of the Pi9 Blast Resistance Locus in the AA Genome Oryza Species

The rice nucleotide-binding site–leucine-rich repeat (NBS-LRR)-encoding resistance (R) gene Pi9 confers broad-spectrum resistance to the fungal pathogen Magnaporthe oryzae. The Pi9 locus comprises many NBS-LRR-like genes and is an ancient locus that is highly conserved in cultivated and wild rice species. To understand the genetic variation and molecular evolutionary mechanism of the Pi9 alleles in different rice species, we studied five AA genome Oryza species including two cultivated rice species (Oryza sativa and Oryza glaberrima) and three wild rice species (Oryza nivara, Oryza rufipogon, and Oryza barthii). A 2.9-kb fragment spanning the NBS-LRR core region of the Pi9 gene was amplified and sequenced from 40 accessions. Sequence comparison revealed that the Pi9 alleles had an intermediate-diversified nucleotide polymorphism among the AA genome Oryza species. Sequence variations were more abundant in the LRR region than in the NBS region, indicating that the LRR region has played a more important role for the evolution of the Pi9 alleles. Furthermore, positive selection was found to be the main force promoting the divergence of the Pi9 alleles, especially in the LRR region. Our results reveal the characteristics and evolutionary dynamics of the Pi9 alleles among the two cultivated and three wild rice species.

Effect of High Temperature Stress on Physiological Characteristics of Anther and Pollen Traits of Rice at Flowering Stage

To ascertain the mechanism affecting flower organ of rice by high temperature stress at flowering stage, we treated heat tolerant line 996 and heat-sensitive line 4628 with high temperature (8:00–17:00, 37℃; 17:00–8:00, 30℃) and optimal temperature (8:00–17:00, 30℃; 17:00–8:00, 25℃) in the growth chambers. The antioxidant enzyme activities, membrane permeability and MDA content in anther and pollen characters of rice were studied. The results showed the superoxide dismutase (SOD), catalase (CAT), peroxidase (POD) and ascorbic acid-peroxidase (AsA-POD) activities in anther of rice increased obviously at early stage of high temperature stress, then decreased rapidly, and higher in heat tolerant line 996 than in line 4628, malonyldialdehyde (MDA) content and membrane permeability in heat-sensitive line 4628 were greater than those in heat tolerant line 996. The an- ther dehiscence coefficient, pollen germination rate and pollen grains number on the stigma significantly decreased, pollen diameter increased under high temperature stress. Anther dehiscence coefficient, pollen germination rate and pollen grains number on the stigma in heat tolerant line 996 were significantly higher than those in heat-sensitive line 4628 under high temperature stress. Under high temperature stress, higher antioxidant enzyme activities, better pollen grain pollination and germination character, as well as lower membrane permeability and MDA content of anther in rice could be the physiological basis of high temperature tolerance.

Mapping of the Resistant Gene to Rice Blast in the Dual Purpose Genic Male Sterile Rice, LongS

LongS is a dual purpose genic male sterile rice with broad-spectrum resistance to rice blast. The objective of the present study was to identify the resistance spectrum to rice blast, to analyze the genetic behavior of resistance gene, and to map the major resistance genes in LongS. LongS had a resistance frequency of 100% inoculated with 41 strains of Magnaporthe oryzae. Population genetic analysis showed that the resistance genes in LongS exhibited dominant inheritance, the genetic model of R gene varied depend on the strains of Magnaporthe oryzae. The main-effect resistant gene to rice blast was fine mapped, by using the bulk segregant analysis (BSA) and recessive class analysis (RCA) methods, with the F2 population derived from the resistant parent of LongS and the susceptible parent of Nipponbare. A single resistant gene to the race of 318-2 located on the interval flanked by the SSR markers of M1 and M2 with a genetic distance of 1.3 cM on chromosome 9 were adjacent to the broad-spectrum blast resistance gene, Pi5. Both of the resistance spectrum and resistant frequency of LongS, however, were significantly different to those of resistant gene of Pi5 and Pii. In conclusion, the major-effect resistant gene identified in this study may be a new broad-spectrum blast resistance gene. The DNA markers linked to the new R gene identified in this study should be useful for marker-aided breeding of blast-resistant rice cultivars.