| Mutant is a basic tool for the exploitation of gene function and a useful resource for the genetic improvement of crops. It is of great significance to strengthen the work on gene mining, identification and utilization, which would be not only beneficial for revealing the molecular mechanism underlying the formation of the important agronomic traits, but also for establishing the marker-assisted-selection (MAS) technology of directional genetically improvement, creating specific germplasm and breeding new varieties. In this study, we obtained a series of mutants from an indica maintainer line T98B treated by 60Co-y radiation, and focused on the identification, heredity and utilization of the thermo-sensitive male sterile line T98S and the newly leaf color mutant grc2.The main results are stated as following:1.We established an unsaturated mutant library based on the genetic background of T98B, and proved that the mutants were identical to T98B by molecular fingerprint verification.With 60Co-y irradiation on mature embryos of T98B, we got 168 mutants with a mutation efficiency of 2.97%. The phenotypes of mutants were related to fertility, leaf color, grain size, plant type and other traits. Moreover, the selected four mutants, T98S (a thermo-sensitive male sterile line), grc2 (a newly leaf color mutant), tsm (a thermo-sensitive monoculm mutant) and dsep (a drought-sensitive enclosed panicle mutant), were all proven completely identical to their wild type T98B by detecting the genetic diversity using 48 SSR markers.2. We have discovered the thermo-sensitive male sterile line T98S, analyzed its fertility response to temperature, agronomic traits, flowering habits, gene heredity pattern and its utilization in breeding, and further discussed the strategy and approaches how to effectively breed two-line sterile line and combination with strong-heterosis through transforming a maintainer into a thermo-sensitive male sterile line.The thermo-sensitive male sterile line T98S showed a phenotype with sterility under high temperature and fertility under low temperature. The line of T98S has its flowering blossom time from 9:00 to 11:00 on the morning, and it had a total stigma exsertion rate of 64.49% and a heavy neck-enclosed rate of 19.72%.The T98S keep complete consistency with T98A in the growth period, plant morphology, tiller number and panicle-grain structure. Moreover, we found that, T98S showed a promising utilization of heterosis, by investigating the agronomic traits on the combination of T98S crossed with some early indica restorer line without carrying the wild abortive type male sterile restorer gene. As for wild-abortive type restorer line, the adaptability of its combination with T98S as female parent was better than that of T98A, although the two type of combinations seemed alike in agronomic traits. From above, we proposed a breeding strategy of transforming a maintainer to be a two-line sterile line, so as to breed combination with strong-heterosis. The basic idea of this strategy was to select an elite maintainer as material with its superiority on comprehensive characters, combining ability and habits of flowering and sterility, to be effectively modified to a photo-thermo sensitive sterile line by a treatment with irradiation or knock-out of TMS5gene.3. We identified the newly leaf color mutant grc2, analyzed its morphological, cytological, spectroscopic and genetic features, fine mapped the grc2 gene, and preliminary understood the effects of grc2 on the expression of genes involved in chlorophyll biosynthesis.The mutant of grc2 (green-revertible chlorina 2,grc2) is unique in phenotype with every leaf greening independently. Each leaf of grc2 is initially chlorotic, and then turns green after growing about 7-15 days. The mutant grc2 showed a new pattern of virescence which refreshed green regardless of its plant growth stage. Compared with the wild type T98B, the plant height, the effective panicle, grain number per panicle and 1000 grain weight decreased in grc2, but the growth period and the seed setting rate were almost not affected. On the other hand, the total chlorophyll and chlorophyll b content reduced significantly in the yellowish leaves of grc2 and chloroplast remained in the etioplast stage for a long time, suggesting that grc2 would probably be an essential gene functioning in the development of young leaves.Genetic analysis revealed that, grc2 was controlled by a single recessive nuclear gene. The gene of grc2 was fine mapped between STS markers S254 and S258 with a physical interval of 31 kb on the short arm of chromosome 6, by using 960 F2 plants with mutant phenotype from a cross between grc2 and Nipponbare. This region contained five annotated genes that had not published. Moreover, the results of RT-qPCR revealed that the expression of PORA was down-regulated in yellowish leaves of grc2, and the related genes encoding PS complex proteins such as Rbcs, PsaA, Cab1R were also significantly inhibited, which will provide important information for the investigation on gene function of grc2.In addition, in order to improve the positioning efficiency of the gene controlling qualitative traits, the SLAF-seq method based on simplified sequenced genome was employed to map grc2.It is found that, the position with SLAF-seq was just deviate about 39 kb from the one with the traditional method, indicating that Super BSA would be an effective method in gene mapping. |
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