【作者】 张宇；

【导师】 李焕秀； 耿三省；

【作者基本信息】 四川农业大学 ， 蔬菜学， 2010， 硕士

【摘要】 根结线虫是辣(甜)椒的主要病害之一,其危害日趋严重。培育抗性品种是防治根结线虫最有效的方法,常规育种周期长、消耗大,利用分子标记辅助育种则会大大提高育种速度。EST-SSR作为一种新型分子标记,来自表达基因,除具备传统基因组来源的SSR标记所有优势外,还可能与基因功能具有直接或间接关系,从而强化了SSR标记在遗传研究中的应用。本研究以辣椒EST数据为基础,通过分析辣椒EST-SSR位点信息,建立了部分辣椒EST-SSR标记,并开发与辣椒抗根结线虫显性单基因Mel紧密连锁的分子标记,为Me1基因的标记辅助选择奠定基础。主要研究结果如下：1.利用SSRIT软件(http://www.gramene.org/gremene/searches/ssrtool)从NCBI(http://www.ncbi.nlm.nih.gov/dbEST)数据库中19173条辣椒EST序列中筛选到612个SSR,检出率为3.19%。共观察到51种重复基元,其中三核苷酸基序是主导类型,占总SSR的60.46%；AG/GA/CT/TC, AAG/AGA/GAA/CTT/TTC/TCT, TAAT, ATATA和CTGCTC分别是二、三、四、五和六核苷酸基序的优势重复基序,在所有SSR中的出现频率各自为13.40%、16.18%、1.80%、3.27%及2.12%。辣椒EST-SSR以5-10次重复为主,基序长度集中在15-20bp范围内。2.利用筛选出含SSR的576条EST序列,设计了357对引物,在合适的PCR条件下,利用非变性聚丙烯酰胺凝胶银染检测这些引物在6个辣(甜)椒品种中的扩增情况和多态性。其中有337对引物显示出清晰条带,引物有效扩增率为94.40%；75对引物表现多态性,占有效扩增引物的21.01%。3.用含抗根结线虫病基因Me1的PM217与典型感线虫品种茄门,及其F1、F2代为试材,采用南方根结线虫(Meloidogyne spp.)人工接种鉴定,每株接种2000卵,两个月后进行抗病性鉴定,根据鉴定结果,利用分离群体分组分析法(bulked segregant analysis, BSA)建立抗感池,共筛选到4对(118、141、142及211)多态性EST-SSR引物在抗感池间存在差异,分别将该4个标记位点暂命名为118、141、142及211。4.利用Joinmap3.0软件,结合抗病性鉴定,对271个单株的F2群体进行SSR分析,研究4个EST-SSR标记位点与根结线虫抗性基因Me1的连锁关系,结果表明,EST-SSR标记141、118及211与Me1连锁程度较高,遗传距离分别为6.984 cM、18.684cM和29.310cM。更多还原

【Abstract】 Root-knot nematode is one of the main disease of pepper, having brought increasingly serious harm. The most effective way to control this disease is breeding resistant pepper. Traditional breeding has the disadvantage of long cycle and high cost, and molecular mark assisted breeding will greatly improve efficiency of breeding. EST-SSR is a new type of molecular marker developed from the databank of sequence of expression sequence tags (EST) and cDNAs. As a new kind of molecular marker, EST-SSR markers have more advantages than the traditional genomic-derived SSRs because they are part of expressed genes. Therefore, EST-SSRs might be involved in gene functioning directly or indirectly. The ability of SSR markers will be greatly enhanced.In this study, we analyzed SSR resource of ESTs data in Capsicum, and preliminary developed soMe EST-SSR markers, and identified molecular markers linked to the dominant resistant gene Me1 laying the foundation for resistance breeding. The main results were following.1. In this report,19173 ESTs of Capsicum annuum were downloaded from NCBI, from which 612 SSRs were screened, by using software SSRIT, and the frequency was 3.19%.51 types of repeat motifs were detected and the dominant type was trinucleotide which occupied 60.46% out of total SSRs. Predominant motifs of di-, tri-, tetra-, penta-and hexa-nucleotide repeats were AG/GA/CT/TC, AAG/AGA/GAA/CTT/TTC/TCT, TAAT, ATATA and CTGCTC, with the frequencies of 13.40%,16.18%,1.80%,3.27% and 2.12% respectively. The repeat number of pepper EST-SSR was mainly 5-10 and the motif length ranged from 15bp to 20bp.2. We designed 357 candidate priMer pairs for 576 EST-SSRs. Under optimal PCR conditions, The amplification and polymorphism displayed by these priMers in 6 varieties of pepper were detected by using silver staining of non-denaturing polyacrylamide gel.337 priMer pairs (94.40%) showed clear amplification, and 75 of them (21.01%) demonstrated polymorphism.3. The materials were F1 and a F2 population from PM217 with the resistant gent Me1, and the typical susceptible variety QieMen. Using artificial inoculation,2000 eggs of southern root-knot nematodes per plant were poured to investigate the number of root knots after inoculated for two months. Base on the results of the investigation, a resistance group and a sensitive group were divided with ’BSA’ Methods to search for the EST-SSR polymorphic priMer combinations priMer 118,141,142 and 211, and tentatively naMed EST-SSR markers 118,141,142 and 211.4. By the software JoinMap V3.0, the relationship of 4 EST-SSR molecular markers and resistant segregation of F2 population were analyzed.The results indicated that the distance between the EST-SSR molecular markers141,118 and 211 and the nematodes resistance Me1 gene in pepper were 6.984cM,18.684cM and 29.310cM respectively.更多还原