98份甘蔗种质资源遗传多样性的AFLP分析

Genetic Diversity Analysis of 98 Collections of Sugarcane Germplasm with AFLP Markers

【目的】甘蔗蔗糖产量约占中国食糖总量的92%。具有遗传多样性的甘蔗种质资源是甘蔗杂交育种的基础,杂交亲本的选择和组合的选配是杂交育种成败的关键,研究98份种质的遗传多样性及亲缘关系,为甘蔗杂交组合选配和种质创新提供参考依据。【方法】采用CTAB法提取甘蔗幼叶基因组DNA,利用扩增片段长度多态性(amplified fragment length polymorphism,AFLP)分子标记技术,对来源于10个国家的98份种质资源的基因组DNA进行酶切、连接、预扩增、选择性扩增,选择性扩增产物在5%变性聚丙烯酰胺凝胶上电泳分离,银染显色。电泳结果得到"0,1"矩阵,使用POPGENE 32软件计算每对引物的多态性条带数、多态性比率、多态信息量、有效等位基因数、遗传多样性指数等指标,同时使用NTSYS pc-V.2.1计算种质间遗传相似系数,根据相似性系数进行UPGMA(unweighted pair group method analysis)聚类分析和PCA(principal component analysis)主效应分析,对甘蔗种质资源进行分类。【结果】采用云南省甘蔗遗传改良重点实验室筛选出的10对引物组合共扩增出1 392条谱带,其中多态性条带为1 344条,多态性条带比率为96.55%,平均每对引物扩增出139.2个位点和134.4个多态性位点。98份种质的相似性系数在0.484—0.929,平均为0.734,多态信息量为0.2495,每个位点的有效等位基因数为1.4092,平均多样性指数为0.3890,遗传相似性系数最高的是KN90-418和KN90-455,达到0.929,最低的是云蔗94-375和IS76-126,为0.484。根据遗传相似性系数进行聚类分析,在遗传相似性系数0.64处切割时,可划分为4个类群,第I类群有5份澳大利亚种质,包括IK76-48、IS76-126、IK76-22、SES309和E.SARPET;第II类群有1份澳大利亚种质是IS76-199;第III类群有3份种质,包括KN93-06、90-110-9和BURMA;第IV类群有89份种质,在遗传相似性系数0.79处切割时,可将第Ⅳ类群划分为9个亚群(A、B、C、D、E、F、G、H和I)。基于Jaccard系数用PCA法对98份甘蔗种质AFLP标记结果进行主效应分析,主效应分析显示了不同种质的分类位置,分子主效应分析结果与分子聚类结果一致,集中在一个区域的种质亲缘关系较为紧密,澳大利亚种质位置较为分散,最分散材料为蔗茅属和细茎野生种。【结论】98份种质资源的遗传基础差异较小,亲缘关系较近,其中,澳大利亚种质遗传多样性相对较丰富。90-110-9、KN93-06和粤糖00-236较为特殊,在杂交组合选配中应给予重点关注。

【Objective】Cane sugar accounts for 92% of sugar production in China, and hybridization is the most widely used and the most effective way for developing new sugarcane cultivars. Sugarcane germplasm is essential for sugarcane breeding.Selecting parental clones and the cross combinations for hybridization contributes directly to the breeding efficiency. Aimed at providing reference for selecting parental clones and cross combinations, the genetic diversity and similarity among 98 sugarcane germplasm were studied.【Method】Good genomic DNA was extracted from young leaves of 98 sugarcane germplasm collected from10 countries following the CTAB method, and then were amplified by sequence-related amplified polymorphism molecular markers to analyze genetic diversity and genetic similarity. Separation of the amplified fragments was performed on 5% denaturing polyacrylamide gels, the gels were stained with Ag NO3, then ＂0,1＂ matrix was obtained according to the electrophoresis result. The number of polymorphic loci, percentage of polymorphic loci, quantity of polymorphic information, effective number of alleles and the indexes of genetic diversity were estimated by POPGENE version 32. The genetic similarity that estimated by NTSYS pc-V. 2.1was used for UPGMA（unweighted pair group method analysis） and PCA（principal component analysis） to group the sugarcane germplasm. 【Result】Among 1 392 bands detected by 10 selective primer pairs proved by Yunnan Key Laboratory of Sugarcane Genetic Improvement, 1 344（96.55%） were polymorphic. On average, each primer combination amplified 139.2 loci and 134.4polymorphic loci. The genetic similarity of 98 sugarcane germplasm ranged from 0.484 to 0.929 with an average of 0.734, the number of polymorphic information was 0.2495, the number of effective alleles for each loci was 1.4092, the average index of genetic diversity was 0.3890. The highest genetic similarity（0.929） was found between KN90-418 and KN90-455, and the lowest（0.484） was found between Yunzhe94-375 and IS76-126. According to the genetic similarity of 0.64, 98 sugarcane germplasm were divided into 4 groups, 5 sugarcane germplasm IK76-48, IS76-126, IK76-22, SES309 and E.SARPET collected from Australia was classified as group I. 1 sugarcane germplasm IS76-199 collected from Australia was classified as group II. KN93-06, 90-110-9 and BURMA were classified as group III; other 89 sugarcane germplasm were classified as group IV which was divided into 9 subgroups（A, B, C, D, E, F, G, H and I） at the genetic similarity of 0.79. The coefficient of Jaccard was used in PCA and indicated a similar result with cluster analysis that the germplasm with the same region shares high similarity, the similarity within Australian sugarcane germplasm was much lower, and the lowest was found within the germplasm belongs to Erianthus fulvus or Saccharum spontaneum.【Conclusion】It was concluded that 98 sugarcane germplasm share high genetic similarity and low genetic diversity, the Australian sugarcane germplasm is relatively high in genetic diversity. 90-110-9, KN93-06 and Yuetang00-236 are 3 unique germplasm and are worth utilizing in hybridization.