Li-yun Chen

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.

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.

Effects of high temperature stress on microscopic and ultrastructural characteristics of mesophyll cells in flag leaves of rice.

Abstract The microscopic and ultrastructural characteristics of mesophyll cells in flag leaves of two rice lines (a thermosensitive line 4628 and a thermo-resistant line 996) under high temperature stress (37°C during 8:00–17:00 and 30°C during 17:00–8:00) were investigated using an optical and a transmission electron microscopy. The membrane permeability and malondialdehyde content increased under the high temperature stress, and the increase of both variables was greater in the line 4628 than in the line 996. Under the high temperature stress, the line 996 showed tightly arranged mesophyll cells in flag leaves, fully developed vascular bundles and some closed stomata, whereas the line 4628 suffered from injury because of undeveloped vascular bundles, loosely arranged mesophyll cells and opened stomata. The mesophyll cells in flag leaves of the line 4628 were severely damaged under the high temperature stress, i.e. the chloroplast envelope became blurred, the grana thylakoid layer was arranged loosely and irregularly, the stroma layer disappeared, many osmiophilic granules appeared within the chloroplast, the outer membrane of mitochondria and the nucleus disintegrated and became blurred, the nucleolus disappeared, and much fibrillar-granular materials appeared within the nucleus. In contrast, the mesophyll cells in flag leaves of the line 996 maintained an intact ultrastructure under the high temperature stress. From these results, it is suggested that the ultrastructural modification of the cell membrane system is the primary plant response to high temperature stress and can be used as an index to evaluate the crop heat tolerance.

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.

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 Diversity of Tropical Hybrid Rice Germplasm Measured by Molecular Markers

Investigation of genetic diversity and relationships among breeding lines is of great importance to facilitate parent selection in hybrid rice breeding programs. In this study, we characterized 168 hybrid rice parents from International Rice Research Institute with 207 simple sequence repeat (SSR) and 353 single nucleotide polymorphism (SNP) markers. A total of 1 267 SSR and 706 SNP alleles were detected with the averages of 6.1 (SSR) and 2.0 (SNP) alleles per locus respectively across all lines. Based on the genetic distances estimated from the SSR and SNP markers separately and combined, the unrooted neighbor-joining cluster and STRUCTURE analyses consistently separated the 168 hybrid rice parents into two major groups: B-line and R-line, which is consistent with known parent pedigree information. The genetic distance matrices derived from the SSR and SNP genotyping were highly correlated (r = 0.81, P < 0.001), indicating that both of the SSR and SNP markers have distinguishable power to detect polymorphism and are appropriate for genetic diversity analysis among tropical hybrid rice parents. A subset of 60 SSR markers were also chosen by the Core Hunter with 368 alleles, and the cluster analysis based on the total and subset of SSR markers highly corresponded at r = 0.91 (P < 0.001), suggesting that fewer SSR markers can be used to classify and evaluate genetic diversity among parental lines.

Diallel analysis of combining ability and heterosis for yield and yield components in rice by using positive loci

Diallel crosses have been widely used for analysis of general combining ability (GCA), specific combining ability (SCA) and heterosis. In the present research, 12 lines were selected from 60 inbred rice bred by International Rice Research Institute with extremely-high or -low yielding records according to the previous three consecutive seasons of yield trial experiments, to construct a half diallel cross. The genetic distances (GDs) revealed by molecular markers for the 12 lines ranged from 0.2288 to 0.7169, averaging at 0.5882; clustering analysis showed the 12 lines were divided into four groups maintaining the original cluster structure of the 60 lines. The positive loci (PLs) including effective-increasing loci (ILs) and effective-decreasing loci (DLs) were screened. The results showed that hybrids derived from those parents with higher GCA effects had better performance for traits of yield and yield components. The SCA effects were strongly correlated to F1 performance as well as heterosis; the GDs based on ILs were significantly positive correlated to SCA effects and heterosis for yield and yield components while the GDs based on DLs showed the significant negative correlations. Based on this research, a new conception, i.e. general sum of combining ability (GSCA) was conceived, which is defined as the sum of GCAs for two parents of a hybrid. The GSCA and SCA showed similar correlations with traits of yield and yield components. The results illustrated that ILs could be used for further study on prediction of heterosis for traits of yield and yield components; and GSCA may be another considerable parameter combined with ILs for breeders in selecting elite hybrid.

Genetic Analysis and Molecular Mapping of Novel White Striped Leaf Mutant Gene in Rice

A new white striped leaf mutant wsl1 was discovered from Nipponbare mutated by ethyl methanesulfonate. The mutant showed white striped leaves at the seedling stage and the leaves gradually turned green after the tillering stage. The chlorophyll content of wsl1 was significantly lower than that of wild-type during the fourth leaf stage, tillering stage and booting stage. The numbers of chloroplast, grana and grana lamella were reduced and the thylakoids were degenerated in wsl1 compared with wild type. Genetic analysis showed that the wsl1 was controlled by a single recessive gene. Molecular mapping of the wsl1 was performed using an F2 population derived from wsl1/Nanjing 11. The wsl1 was finally mapped on the telomere region of chromosome 9 and positioned between simple sequence repeat markers RM23742 and RM23759 which are separated by approximately 486.5 kb. The results may facilitate map-based cloning of wsl1 and understanding of the molecular mechanism of the regulation of leaf-color by WSL1 in rice.

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.