甜叶菊微卫星富集文库的构建与多态性标记的筛选  

Construction of Microsatellite-Enriched Library and Isolation of Icrosatellite Markers in Stevia rebaudiana

作　　者：秦海峰[1] 龙宁[1] 吴建国[1,2] 石春海[1] 

机构地区：[1]浙江大学农业与生物技术学院农学系,浙江杭州310058 [2]浙江农林大学农业与食品科学学院/浙江省农业产品品质改良技术研究重点实验室,浙江临安311300

出　　处：《作物学报》2014年第3期447-456,共10页Acta Agronomica Sinica

基　　金：浙江省科技攻关项目(2009C32030)资助

摘　　要：甜叶菊是我国一种重要特种经济作物,其分子标记相关遗传背景研究甚少。本研究基于生物素与链霉亲和素的强亲和性原理,用链霉亲和素顺磁颗粒捕捉人工合成的标记有生物素的寡核苷酸探针(AG)15,间接筛选出含有甜叶菊基因组微卫星序列的DNA酶切片段,将筛选得到的片段连接到pUC-T载体中,构建甜叶菊微卫星序列的富集文库。挑取354个克隆进行菌落PCR检验,从中筛选出158个阳性克隆进行测序。结果表明,134个(84.81%)克隆中含有微卫星序列,其中完美型85个(63.43%)、非完美型15个(11.19%)、复合型34个(25.38%)。根据微卫星序列共设计出71对微卫星引物,其中62对能扩增出稳定的条带。利用24个甜叶菊品系对这62对引物的遗传多样性的分析表明,有16个位点表现出多态性,等位基因数为2～8个,平均每个位点扩增得到4.5个等位基因,多态性信息含量在0.3163～0.7595之间,观测杂合度(Ho)与期望杂合度(He)的范围分别为0.2174～0.9167与0.3555～0.8076。通过聚类分析,将甜叶菊分为大小叶两大类。本研究开发出的微卫星标记可为甜叶菊的分子遗传育种提供有效的遗传标记。The objective of this study was to identify QTLs for seed germination percentage of Brassica napus under the salinity stress and drought stress using the composite interval mapping （CIM） method. The recombinant inbred lines （RIL） population derived from a cross between yellow-seeded female parent GH06 and black-seeded male parent P174 was established by selfing for nine successive generations with single seed propagating from F2. The oilseeds were dealt with NaCl （16 g L–1 solution） for salinity stress, 20% （W/W） PEG-6000 solution for drought stress. The QTLs of germination ratein two different stress conditions were detected using the SNP genetic map constructed in 2013, which contains 2795 SNP markers with the total map length of 1832.9 cM and an average distance of 0.66 cM. A total of 19 QTLs for seed germinationrate under two stresses were located onchromosomes of A01, A03, A06, A07, A09, and C06. Twelve QTLs related to salinity stress were detected, with explained phenotypic variation from 4.9% to 10.9% of, while eight QTLs related to drought stress were detected, with explained phenotypic variation from 3.8% to 6.9% of. Some QTLs located on A03 and A09 under two stresses were detected in a near region. In conclusion, （1） the seed germination percentage is a quantitative trait controlled by many minor-effect genes, and the expression of the QTL is affected by environmental factors greatly; （2） different genes are involved in the oilseed responses to the stresses of different stages.

关 键 词：甜叶菊 富集文库 微卫星 磁珠 遗传多样性 

分 类 号：S566.9[农业科学—作物学]