The Study Of Photoperiod And Thermo-sensitive Genic Male Sterile Heredity And Gene Mapping In Rice (Oryza Sativa L.)

Male sterility is universal phenomenon in advanced plants in the nature. While following two types are the most usable ones: cytoplasmic male sterility (CMS), and photoperiod and thermo-sensitive genic male sterile (PTGMS).In 1970s, twentieth century, the breeders successfully developed the "three-line" hybrid rice since the wild abortive CMS was first discovered by Bihu Li. This hybrid rice has been spread widely in large areas, which is very important for the continuous development of food both in China and all over the world. Almost at the same period, the natural mutant of photoperiod sensitive genic male sterile (PSGMS) in rice was discovered by Mingsong Shi in 1973, which inaugurated a new method to realize the heterosis in rice. Thanks to the pollen fertility of PTGMS line can be altered by change of day length and temperature, it can be used to produce hybrid seeds as male sterile line under long day and high temperature environment, and also can be self pollinated to obtain sterile seeds under short day and low temperature conditions. This system simplified the procedure of hybrid seeds production because the maintain line, which used to produce the sterile seeds in the "three-line" system, is not necessary in the "two-line" system. Therefore, the "two-line" system is used widely in rice production.The genetic proportion of male fertility was consistently subjected to typical single locus segregation in crosses between Nongken 58S and Nongken 58. The sterile lines derived from Nongken 58S have diversity of photoperiod and thermo sensitive characteristics, the sterile traits are usually controlled by two loci, and the genes are not always allelic under different genetic background. The Pei'ai64S, derived from Nongken58S, is a PTGMS line that has significant thermo sensitive characteristics. The F₁ combination named Liangyoupeijiu (Pei'ai64S/93-11) was widely spread as a super hybrid rice variety in south China. Although many researches of gene mapping for PTGMS have been conducted, the significant elite, Pei'ai64S, has not been involved in the previous studies. So, it is necessary to further investigating the genetic principle of PTGMS genes in Pei'ai64S, to localize and predict the candidate genes, and to estimate the genetic effects of them. Firstly, in this study, in order to improve the issues of DNA extraction of large scale samples, high cost and the shortage of SSR (Simple Sequence Repeats) markers, the application procedures of SSR marker analysis system were optimized for gene mapping. Secondly, the problem of male sterile stability of Jiazhe A was analyzed by investigating the pollen fertility and seed setting rate with the control of Xieqingzao A and Pei'ai64S. Finally, a unique large F₂ population was used to localize and predict the candidate genes of PTGMS, which will greatly facilitate the final isolation of PTGMS genes by a map-based cloning approach and be useful for molecular marker-assisted selection for new PTGMS lines in hybrid rice breeding. The main results of this study are showed as follow:A set of integrated technology system for rice SSR marker assaying was presented. This improved approach includes three parts: (1) high efficient DNA extraction for large population; (2) development of new SSR markers for specific genomic region and predict the polymorphism between indica and japonica variety, 93-11 and Nipponbare, by bioinformatic analysis based on the genomic sequence; (3) PAGE (Poly-Acrylamid Gel Electrophoresis) and silver staining. This method will greatly decrease the expense of experiment and save much time.The percentage of normal fertile pollen is varied from 0 to 30.00 % in the population of Jiazhe A, while that is 0 to 11.67% for Xieqingzao A. The fertility of different branches in these two sterile lines were also different, the highest percentage of normal fertile pollen was on the fifth branch for Jiazhe A and on the third branch for Xieqingzao A. By comparison, the peak value of NF percentage for Pei'ai64S was observed at 22nd, April (95.50%) and at 18th, October (87.67%). However, there has no difference for the fertility in different branches of Pei'ai64S during the long-day and high temperature condition from early-month of July to later-month of August, which showed stable sterile with more than 90% TS pollens.The correlation analysis between pollen fertility and temperature indicated that the pollen fertility restoration was correlated with the maximum, mean and minimum temperature of 9 days to 11 days before the heading date. The percentage of NF pollens would reach the highest value (SS+NF 46.84%) when the mean temperature is 25.2±1.4℃; while the value would be gradient reduced and showed stable sterile when the mean temperature reached at 29.8±2.1℃. As to Pei'ai64S, the highest absolute value of minus correlation coefficient between the pollen fertility restoration and the maximum, mean and minimum temperature was occurred at the period of 12 days to 16 days before the heading date. The critical mean temperature of fertility transformation was 24.8±1.7℃under natural long-day condition. Therefore, the security period for hybrid seed production is from the last ten days of July to the first ten days of August in Hangzhou.Genetic analysis by using two populations, F₂ (Pei'ai64S/93-11) and BC₁F₁ (Pei'ai64S//Pei'ai64S/93-11) , indicated that the sterile traits of Pei'ai64S were controlled by two recessive duplicated genes. By using an F₂ (Pei'ai64S/93-11) mapping population, the PTGMS genes were mapped by a strategy of bulked-extreme and recessive-class approach with SSR and In-Del markers. The mapping population was constructed by composed of 320 extremely male sterile individuals in total. Seventy-two pairs of primers on chromosome 7 were synthesized for polymorphism screen by BSA (Bulked Segregation Analysis) method, and 32 polymorphic markers were linked with pms1 locus. The pms1 was co-segregated with RM6776, and delimitated to a region between closest marker RM21242 (0.2cM) and YF11 (0.2cM) on the short arm of chromosome 7. According to the genomic sequence of Nipponbare, the interval between the both sides of tightly linked markers is 101.1 kb. The TIGR genomic annotation shows that there exist 14 predicted loci in this region. The locus of LOC_Os07g12130, which encodes a myb-like DNA-binding domain containing protein and has correlation with thermo-sensory response, is most likely the candidate gene of the allelic pms1 in light of GO (Gene Ontology ) analysis. The other PTGMS gene, pms3, on chromosome 12 was linked with eight SSR markers, and was located between RM7018 and RM28390 with the genetic distance of 1.0 cM and 4.1 cM, respectively. The pollen fertility and seed setting rate data was classified by marker genotypes, and the ANOVA analysis results indicated that both pms1 and pms3 displayed major effects on fertility. While the effect value of pms1 is 1.3 to 1.4 times larger than pms3.