小麦耐盐基因的标记和标记的克隆

Molecular Markers and Its Clone for Salt Tolerance Gene in Wheat

普通小麦 (TriticumaesticumL .)耐盐农家品种茶淀红与盐敏感品种农大 85 0 2 1杂交 ,对杂交后代 14 1株F2 分离个体进行耐盐性鉴定。通过遗传分析和卡方检验证实小麦耐盐农家品种茶淀红含有一个主效耐盐基因 ,杂交后代符合 1∶2∶1的分离规律。依据混合群体分组分析法 (BulkedSegregateAnalysis ,BSA)建立耐盐基因池和盐敏感基因池 ,通过RAPD实验 ,从 5 2 0个引物中筛选出了一个在两池间具有多态性的引物OPZ0 9,用双亲、F1、F2 单株DNA进行RAPD实验证明了该引物扩增出的特异性片段OPZ0 9 5 90是一个与茶淀红耐盐基因连锁的RAPD标记 ,用JOINMAPVersion 1 4计算基因与标记间的重组值为 5 6 74 % ,连锁距离为 6 5 5 7cM。从琼脂糖凝胶回收OPZ0 9 5 90与载体pUCm T连接 ,并转入受体菌JM10 9,对克隆片段测序表明其实际大小为 5 91bp ,故此小麦茶淀红耐盐基因的RAPD标记为OPZ0 9 5 91。

Genetic and RAPD analysis were studied among individuals in F 2 segregated population originating from a cross between wheat cultivator Nongda 85021 (female parent, salt sensitive) and Chadian Red (male parent, salt tolerant). For genetic analysis and chi square test gave a good fit of 1∶2∶1 ratio showing that one major gene was controlling the salt tolerant character in Chadian Red. DNA of F 2 population was extracted to develop tolerant and sensitive gene pools, respectively, based the BSA (Bulked Segregate Analysis). By RAPD (Randomly Amplified Polymorphic DNAs), 520 random primers were used to amplify the two types of gene pools. Only primer OPZ09 was found to be polymorphic with a fragment of 590bp in the two parents, F 1 and F 2 populations. So, the specific fragment OPZ09 590 was, a RAPD marker linked to salt tolerance gene in Chadian Red. By the software JOINMAP (Version l.4), the recombination was 5 674% and the linking distance was 6 557 cM. OPZ09 590 was extracted from the agarose and mixed with pUCm T vector, then the vector was transferred into JM109. The clone was determined 591bp after being sequenced. It indicated that the RAPD marker of salt tolerance gene in Chadian Red was OPZ09 591.