水稻PSM标记的发展及抗虫基因的分子定位

Development of Position-specific Microsatellite Markers and Molecular Mapping of Insect Resistant Genes in Rice (Oryza sativa L.)

水稻是世界上最重要的粮食作物之一 ,为世界近一半人口提供食物来源。水稻微卫星图谱的构建有利于遗传基础的研究及分子育种。本研究通过发展位置特异性微卫星标记 ,对微卫星标记进行了图谱的整合 ,并利用微卫星等标记对水稻抗稻瘿蚊基因Gm6和抗褐飞虱基因Bph3进行了分子定位。主要研究结果如下 :1、利用网上公布的序列信息进行位置特异性微卫星引物的设计和微卫星图谱的整合。共发展了 198个PSM标记 ,有 10 5个标记的基序为GA/CT重复 ,占 5 3 3% ,绝大多数标记 (98 0 % )的重复序列长度在 2 0bp以上。将原有微卫星标记和本实验发展的PSM标记整合到RGP遗传图谱上 ,整合后总的SSR标记数为 718个 ,新图谱的遗传距离总长度为 15 2 7 2cM ,平均标记密度为每 2 13cM有一个SSR标记 ,其中第 1染色体的标记最密 (1 73cM /个 ) ,而第 11染色体的标记密度最低 (3 32cM /个 )。将本研究发展的微卫星图谱与IRMI发表的高密度微卫星进行了比较 ,结果显示本研究发展的微卫星图谱标记分布比较均匀 ,只有 3个区域标记间遗传间距在 10cM以上 ,5个区域在 8- 10cM ,而IRMI微卫星图谱分别有 17和 12个。2、采用G2 4 17- 2 - 1×抗蚊青占 (2 39株 )和G30 0 4 - 4×抗蚊青占 (2 4 3株 )两个F2 作图群体对水稻抗稻瘿蚊进行了分?

Rice is the important crop for human consumption, providing staple food for about half the world's population. The construction of rice microsatellite map will contribute greatly to genetic research and molecular breeding. In this study, PSM (position specific microsatellite) markers were developed in rice, and then an integrated microsatellite map was constructed by using these PSM markers and also some other published RM markers. Two insect resistant genes viz. Gm6 for gall midge and Bph3 for brown planthopper were mapped using some SSR markers and one STS marker in rice. The main results were as follows: 1. Based on published sequences on the websites, position specific microsatellite primers were designed and an integrated microsatellite map was constructed in silico. A total of 198 primer pairs were designed, among which 105 primer pairs (53 3%) whose motif types were poly(GA/CT)n. The majority (98%) of developed markers had SSRs20bp in length. Previously mapped SSR markers and newly developed PSM markers were integrated into RGP map and finally a 718 SSR marker genetic map covering 1527 2 cM was constructed. The integrated map was estimated to have one SSR marker at every 2 13 cM on an average. Chromosome 1 has a highest average density of SSR markers i e one at every 1 73 cM, whereas chromosome 11 has a lowest average density of SSR markers i e one at 3 32 cM Compared with newly developed IRMI map with high density of SSR markers, our developed map has more uniform distribution of makers with 3 genetic gaps10 cM and 5 genetic gaps between 8 cM and 10 cM, whereas there are 17 and 12 gaps on IRMI map, respectively. 2 Two segregating populations derived from two crosses between two susceptible varieties, 'G2417-2-1' and 'G3004-4', and the resistant variety 'Kangwenqingzhan' were used for maping and physical mapping of a gall midge resistance gene Gm6 in rice. Based on linkage analysis of these two populations consisting of 239 and 243 F 2 individuals, respectively, Gm6 was mapped on the long arm of chromosome 4 by using two SSR markers (RM348 and RM255) and one STS marker (RG476/ScaⅠ). Position specific microsatellite markers were developed for fine mapping of Gm6 gene on the basis of published sequences on the websites. The results showed that Gm6 was mapped between PSM112 and PSM114 at the genetic interval of 0 4 cM, whereas three additional markers PSM101, PSM106, and PSM115 showed no recombination with Gm6 . Two overlapping BAC clones AL606660 and AL606645 covering Gm6 gene were used for physical mapping. With the known physical information of all the markers in the two overlapping BAC clones, a sequence of ～158kb was defined as the Gm6 containing region. 3 An F 2 mapping population derived from a cross between the resistant variety 'RH' and the susceptible variety 'Qishiruanzhan' was used to map Bph3 gene that confers resistance to brown planthopper of rice. Based on the phenotypic results of F 3 lines, 27 F 2 individuals selected from the population consisting of 237 F 2 individuals were used for molecular mapping of Bph3 gene. Through linkage analysis of polymorphic SSR markers of chromosome 4 with Bph3 gene, two SSR markers (RM261 and RM119) were found to have distances of 8 0 cM and 8 0 cM with Bph3 , respectively. For further analysis, PSM (position specific microsatellite) markers were developed in the region between RM261 and RM119. Two polymorphic PSM markers (PSM323 and PSM194) were mapped with Bph3 gene of 8 0cM and 3 8cM distances, respectively. Therefore, Bph3 gene was mapped between PSM323 and PSM194 on the long arm of chromosome 4.