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利用多花黑麦草叶斑病抗性和敏感个体杂交构建F 1分离群体,采用分群分析法(bu lked segregan t ana lys is,BSA),通过多花黑麦草表达序列标签(expressed sequence tag,EST)的PCR扩增和扩增产物的限制性内切酶酶切多态性(cleaved am p lified po lym orph ic sequence,CAPS)筛选,获得一个同多花黑麦草草抗叶斑病紧密连锁的EST-CAPS标记p56。对照多花黑麦草细胞质雄性不育(CM S)群体构建的遗传连锁图,该EST-CAPS标记位于多花黑麦草的第5遗传连锁群(LG 5)。对cDNA文库中对应于标记位点p56的EST序列片段分析和基因库搜索结果表明,该EST所编码的氨基酸序列同大麦天冬酰胺合成酶基因H vAS 1和H vAS 2的部分氨基酸序列片段同源性很高,推测位点p56所处的基因为编码多花黑麦草天冬酰胺合成酶基因。该分子标记可用于多花黑麦草分子标记辅助育种。
The F1 segregant population was constructed by crossing the resistant and susceptible individuals of the ryegrass leaf spot with sensitive individuals. The segregation population was analyzed by cluster analysis (BSA) and expressed sequence tag (EST) ), And cleaved amylolipid orphysiological sequence (CAPS) were screened by PCR amplification and amplification products. EST-CAPS marker p56. The genetic linkage map was constructed based on the control of Cytoplasmic Male Sterile (CM S) population of Italian ryegrass, which is located on the 5th genetic linkage group (LG 5) of ryegrass. The fragment analysis and gene bank search of cDNA sequence corresponding to the marker site p56 in the cDNA library showed that the amino acid sequence encoded by the EST was identical to the partial amino acid sequence fragments of the asparagine synthase gene H vAS 1 and H vAS 2 High source, speculated that the locus p56 locus encoding ryegrass asparagine synthase gene. The molecular marker can be used for ryegrass molecular marker assisted breeding.