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采用从其他作物转移与利用自身EST序列设计两种方法为高羊茅(Festuca arundinacea L.)开发EST-SSR分子标记,并利用所开发标记对高羊茅品种(系)进行遗传多样性分析。利用来自小麦、大麦、玉米、高粱和水稻的732对EST-SSR在高羊茅上进行通用性分析,得到406个有效的扩增引物对(55.46%)。利用高羊茅EST数据库(Gen Bank/db EST)中63 853条EST序列进行微卫星序列的查找,共发现包含微卫星的EST序列420条,占整个EST总数的0.658%。其中三核苷酸基序出现频率最高(43.33%),次之为二核苷酸基序(33.57%)。二核苷酸基序以CT/GA出现频率最高(16.90%),三核苷酸基序以CAG/GTC出现的频率最高(10.63%)。进一步运用Primer 5.0软件设计了108个引物对,并交上海桑尼公司合成。PCR检测表明,92个引物对(85.19%)在高羊茅中均可以扩增出稳定清晰的带型。从498对(其中5种作物的406对,高羊茅的92对)有效扩增的EST-SSR引物中随机选取81对引物,对12个高羊茅品种(系)进行扩增。79个引物对显示出多态性(97.53%)。共检测到133个等位变异,平均每对引物有1.68个等位变异;多态性信息含量(PIC)值最高为0.66,最低为0.14,平均为0.48。聚类分析结果表明,12份材料在相似系数为0.61处可分为5大类:第Ⅰ类为‘爆发力’和‘法思’;第Ⅱ类为‘强劲’、‘家园’和‘翠碧A’;第Ⅲ类为‘可其思3号’和‘凌志’;第Ⅳ类为‘雅典娜2号’、‘美洲虎3号’、‘火凤凰’和‘TF160’;‘球道’单独为第Ⅴ类。
EST-SSR markers were developed for the transfer of Festuca arundinacea L. using two methods of transferring and using their own EST sequences. The genetic diversity of Festuca arundinacea cultivars (lines) was analyzed by using the developed markers. A total of 406 valid amplification primer pairs (55.46%) were obtained from 732 pairs of EST-SSRs from tall fescue using wheat, barley, maize, sorghum, and rice. A total of 420 ESTs including microsatellites were found in 63 853 EST sequences of EST database (GenBank / db EST), accounting for 0.658% of the total ESTs. Among them, the trinucleotide motif appeared the highest frequency (43.33%), followed by the dinucleotide motif (33.57%). The dinucleotide motifs showed the highest frequency of CT / GA (16.90%) and the trinucleotide motifs showed the highest frequency of CAG / GTC (10.63%). Primer 5.0 software was further used to design 108 primer pairs and submitted to the synthesis of the company. PCR detection showed that 92 primer pairs (85.19%) were able to amplify stable bands in tall fescue. 81 pairs of primers were randomly selected from 498 pairs of EST-SSR primers (406 pairs of 5 pairs of plants and 92 pairs of tall fescue), and 12 varieties of tall fescue were amplified. 79 primer pairs showed polymorphism (97.53%). A total of 133 alleles were detected with an average of 1.68 alleles per primer pair. The highest PIC value was 0.66 and the lowest was 0.14 with an average of 0.48. The results of cluster analysis showed that the 12 materials could be divided into five groups at the similarity coefficient of 0.61: the first class was ’explosive force’ and ’Fasi’; the second class was ’strong’, ’homeland’ A ’; Class III’ Keke 3 ’and’ Lexus’; Class IV ’Athena 2’, ’Jaguar 3’, ’Phoenix’ and ’TF160’; ’Fairway’ alone For the fifth category.