摘要
四一是课题组在我国现有玉米种质资源的基础上,筛选出的一份综合农艺性状优良的自交系,其含有两个显性互补的抗玉米矮花叶病基因,即RSCMV1和RSCMV2。其中RSCMV1位于第6染色体的短臂,RSCMV2位于第3染色体的长臂。国内外多个课题组利用不同的抗源均在这两个区域发现了抗病基因或抗病QTL。对四一中的这两个抗病基因的效应进行了分析时发现,位于第6染色体短臂上的抗病基因RSCMV1控制植株的早期抗病性,在我国的很多抗病材料中均发现了该位点的存在。而位于第3染色体上的抗病基因RSCMV2位点特异在成株期表现出抗病性,是我国玉米育种材料中特异的抗病基因资源。分离这两个区域的抗病基因不仅对玉米矮花叶病抗病育种具有重要的实践作用,而且为解析其抗病的分子机制有重要的意义。结合玉米基因组测序公布的BAC信息,利用课题组构建的近等基因系,利用各种方法在抗病基因区域内开发分子标记,并对抗病基因进行定位,为其图位克隆奠定基础。主要工作包括以下方面:
1.07年利用BC4F3、BC4F4群体(共676株)对抗病基因RSCMV1进行初步定位,将其定位于微卫星标记umc1018和umc2311之间,两者之间的物理距离为6.83Mb。针对两个标记之间的BAC信息,在不同的位置上随机选取14个BAC,设计了47对BAC-SSR引物,17对ILP引物,经凝胶电泳检测,有13对引物在亲本间为共显性,4对引物在亲本间表型为显性。利用开发的标记对umc1018和umc2311之间的18个交换单株检测,并对其衍生后代进行表型鉴定,将抗病基因RSCMV1定位在A16-18与a1-1之间,两者相距2.6Mb。08年利用BC4F3、BC4F4、BC4F5群体(1279株)对玉米矮花叶病基因RSCMV1进行定位,将其定位在BAC-SSR标记A5-1和ILP标记a1-1之间,分别相距0.3cM和0.3cM,其物理距离为0.8Mb,其中A4-3、A4-2A、a2-1与抗病基因共分离。其中在A5-1和a1-1之间发生交换的抗病单株有4株。
2.07年利用BC4F3、BC4F4群体(共549株)对抗病基因RSCMV2进行初步定位,将其定在于分子标记bnlg1456与umc2020之间,两者之间的物理距离为16.61Mb,大约113个BAC。在不同位置上选取41个BAC,共设计了129对BAC-SSR引物,共有26对引物在亲本间有多态性;有69对引物在亲本中无多态性;另有34对引物没有扩增出条带。利用开发的标记对bnlg1456和umc2020之间的交换单株检测,将抗病基因定位在bnlg1456与3-AC16-20之间,并对其衍生后代进行接种鉴定。08年利用为将近7000株的BC4F5群体和5922粒的F2代种子及其部分单株对抗病基因进行定位,最终将抗病基因RSCMV2定位在3-ac36-9与3-AC16-20之间。两者之间的物理距离为5.54Mb。在BC4F5群体中共检测到154株在3-ac36-9与3-AC16-20之间交换的单株,其中抗病单株40株,感病单株114株。08年冬在海南对部分交换单株进行了接种鉴定,其结果与室内分子标记检测结果一致。
Maize dwarf mosaic virus is one of the most important virus diseases of maize and causes serious yield losses in the world. The most effective way to control the disease is to breed and popularize resistant hybrids based on screenings of resistant materials and understanding on the inheridity of resistance. In our previous study, one inbred line Siyi with elite agronomy traits was singled out and its inheritance of resistance to SCMV was studied. Two dominant complementary genes were detected to determine the resistance to sugarcane mosaic virus in Siyi.They were mapped on chromosome 3 and chromosome 6, respectively using microsatellite markers. They were named RSCMV2 and RSCMV1. Many people were found resistance genes or disease resistance QTL in these regions. Action mode analysis of two dominant complementary genes using NILs. These results suggest that RSCMV1 is likely a predominant gene and shows basal resistance to SCMV, while RSCMV2 shows specific resistance at adult stage. Separation of the two resistance gene is important for the resistance breeding practice and the analysis of its molecular mechanism of disease resistance. Combination of maize genome sequencing information, development of molecular markers and mapping of resistance genes by using near isogenic lines in Maize is base of map-based cloning. The results were obtained as following:
1. 674 F2 individuals derived from Mo17AaBB were used to mapping of the RSCMV1 in 2007. The resistant gene RSCMV1 was flanked by SSR markers umc1018 and umc2311. The physical distance between the two SSR markers is 6.83 MB, which contained about 50 BAC. Based on 41 BAC’s sequences of different regions by randomly selected , 47 pairs of BAC-SSR primers and 17 pairs of ILP primers were developed, 13 pairs of primers exhibited polymorphisms between Siyi and Mo17 and 4 pairs of exhibited dominant phenotyp . Consequently, the resistant gene RSCMV1 was mapped between A16-18 and a1-1, The physical distance between the two markers is 2.6 MB. This result is proved by the offsprings of recombinat plants phenotype. 1279 F3 individuals derived from Mo17AaBB were used to conduct fine mapping of the RSCMV1 in 2008.finally, the resistant gene RSCMV1 was located in a 0.8×106 bp long region on chromosome 6 and co-segregation markers A4-3, A4-2 and a2-1were detected.
2. In 2007, 549 F2 individuals derived from Mo17AaBB were used to mapping of the RSCMV2. The resistant gene RSCMV2 was located in SSR markers bnlg1456and umc2020. The physical distance between the two SSR markers is 16.61MB, which contained 113 BAC. Based on 14 BAC’s sequences of different regions by randomly selected , 129 pairs of BAC-SSR primers were developed and 26 pairs of primers exhibited polymorphisms between Siyi and Mo17 .Consequently, the resistant gene RSCMV2 was located between bnlg1456 and 3-AC16-20. This result is proved by the offsprings of recombinat plant’s phenotype. in 2008, 7000 F3 individuals derived were used to conduct fine mapping of the RSCMV2.finally, the resistant gene RSCMV1 was located in BAC-SSR markers 3-ac36-9 and 3-AC16-20, the physical distance is 5.54MB. Totally 154 individual plants showed recombination between RSCMV1 and SSR markers.
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