基于ISSR标记的猕猴桃品种遗传多样性分析及指纹图谱构建

Genetic diversity analysis and fingerprinting construction of cultivars of Actinidia spp. based on ISSR marker

采用ISSR标记对中华猕猴桃(Actinidia chinensis Planch.)、美味猕猴桃[A.chinensis var.deliciosa(A.Chev.)A.Chev.]、软枣猕猴桃[A.arguta(Sieb.et Zucc.)Planch.ex Miq.]和毛花猕猴桃(A.eriantha Benth.)的32个样本进行了遗传多样性分析,并以ISSR标记为基础构建了DNA指纹图谱。结果表明:筛选的10个多态性高且条带清晰的引物共扩增出200个条带(位点),其中,多态性位点195个,多态性位点百分率(PPL)达97.50%;各引物的多态性信息含量(PIC)以及供试样本的观测等位基因数(Na)、有效等位基因数(Ne)、Nei’s基因多样性指数(H)和Shannon’s多样性指数(I)的总均值分别为0.908 0、1.980 0、1.356 9、0.225 5和0.361 3,4个猕猴桃种间的遗传分化系数(G_(st))为0.414 6,基因流(N_m)为0.705 9,且32个样本间的Na、Ne、H和I值差异极显著(P<0.001)。供试32个样本间的遗传相似系数(GS)为0.565 0~0.965 0,平均值为0.716 4;基于GS值进行UPGMA聚类分析,在GS值为0.76处将32个样本分为4组,基本对应供试的4个猕猴桃种类,其中,第Ⅰ组的大多数样本属于美味猕猴桃品种,第Ⅱ组的样本均属于中华猕猴桃品种,第Ⅲ组的样本属于毛花猕猴桃品种,第Ⅳ组的样本均属于软枣猕猴桃品种。分子方差分析结果表明:4个猕猴桃的种间变异占总变异的40.84%,种内变异占总变异的59.16%。研究结果表明:供试的猕猴桃品种间遗传分化程度较高,基因交流频率较低,且总遗传变异的近60%存在于种内,说明供试的猕猴桃品种具有较丰富的遗传多样性。另外,根据10个ISSR引物的扩增结果,筛选出引物UBC818、UBC824、UBC854和UBC895扩增的15个多态性位点构建的DNA指纹图谱可用于供试32个猕猴桃样本的鉴定。

Genetic diversity of 32 samples of Actinidia cAinensis Planch., A. cAinensis var. deliciosa （ A. Chev.） A. Chev., A. arguta （Sieb. et Zucc.） Planch. ex Miq. and A. eriantAa Benth. were analyzed by using ISSR marker,and DNA fingerprinting was constructed based on ISSR marker. The results show that 200 bands （loci） are amplified by 10 primers screened with high polymorphic and clear band,in which, there are 195 polymorphic loci with percentage of polymorphic loci （PPL） of 97. 50%. The overall averages of polymorphism information content （PIC） of each primer,observed number of alleles （ Aa）, effective number of alleles （Ne） , Nei’ s gene diversity index （H）and Shannon’ s diversity index （ I ） of samples tested are 0.908 0, 1.980 0, 1.356 9, 0.225 5 and 0.361 3, respectively. The genetic differentiation coefficient （ Gst ） and gene flow （ IVin） of four species in Lindl. are 0.0. 705 9,respectively. The differences in Na, Ne, H and I values among 32 samples are extremely significant （ P 〈0.001）. The genetic similarity coefficient （GS） among 32 samples tested is 0？ 565 0 - 0. 965 0 with average of 0. 716 4. UPGMA cluster analysis is carried out according to GS value,and 32 samples are divided into four groups at GS value of 0. 76, which correspond to the four species in Actinidia tested. Among them, most of samples in group I belong to cultivars of A. chinensis var. deliciosa,samples in group II belong to cultivars of A. chinensis, sample in group W belongs to cultivar of A. eriantha,and samples in group ~N belong to cultivars of A. arguta. The results of molecular variance analysis show that interspecies variation of four species in Actinidia accounts for 40. 84% of the total variation,while intraspecies variation accounts for 59. 16% of the total variation. It is suggested that cultivars of Actinidia spp. tested have high degree of genetic differentiation and low gene exchange frequency,and nearly 60% of the total genetic variation is existed in species, indicating that there is abundant genetic diversity in cultivars of Actinidia spp. tested. In addition,15 polymorphic loci amplified by primers UBC818, UBC824, UBC854 and UBC895 are screened based on amplification result of ten ISSR primers, and DNA fingerprinting is constructed, which can be used to identify 32 samples of Actinidia spp. tested.