基于ISSR分子标记的叶子花亲缘关系分析和指纹图谱构建

Analysis of genetic relationship and construction of fingerprints in Bougainvillea based on ISSR molecular marker

关键词:  叶子花, ISSR标记, 遗传多样性, 亲缘关系, 指纹图谱

Key words:  Bougainvillea, ISSR marker, genitic diversity, genetic relationship, fingerprints

摘要:

为分析品种遗传多样性和遗传距离并构建品种聚类图和指纹图谱,该研究从DNA模板浓度、引物浓度、退火温度和循环次数等方面优化了叶子花ISSR-PCR反应体系和反应程序,利用11个ISSR引物对131个叶子花品种进行PCR扩增,扩增产物经琼脂糖凝胶电泳检测。结果表明:优化的ISSR-PCR反应体系中DNA模板浓度为0.5 ng·μL^-1,引物浓度为0.5 μmol·L^-1,引物UBC813、UBC814、UBC815、UBC823、UBC824、UBC835、UBC840、UBC841、UBC843、UBC844和UBC876的最佳退火温度分别为52.3、55.9、54.3、54.3、53.6、56.2、56.2、51.9、54.4、54、50 ℃,循环次数为32。用11个ISSR引物对131个叶子花品种扩增出161条带,其中多态性条带156条,多态性比率为96.89%。单个引物的等位基因数、有效等位基因数、Nei's基因多样性指数和Shannon's信息指数分别为1.86～2.00、1.33～1.68、0.21～0.39和0.34～0.57,平均值分别为1.969、1.478、0.294和0.447。引物UBC841的鉴别率最高(80.92%),可有效鉴别106个品种,与引物UBC876结合可将131个叶子花品种完全鉴别开,建立了各品种的指纹图谱。叶子花品种的遗传距离范围为0.00～0.60,平均值为0.365,遗传多样性较低,在遗传距离0.58处,131个品种分为6大类群,聚类分析显示同一个种的品种大多聚在一类,但同一个种仍有品种未聚在一类或亚类、也有多个种的品种聚在一类。该研究较为准确地揭示了叶子花种质资源的遗传多样性,建立的指纹图谱为叶子花品种登记、知识产权保护以及品种鉴定提供了可靠技术和有效手段。

Abstract:

In this study, ISSR-PCR reaction system was optimized from DNA template concentration, primer concentration, annealing temperature and cycle times, a total of 11 ISSR markers were used to amplify DNA samples of the 131 Bougainvillea cultivars, and the ISSR amplicons were detected based on a agarose gel electrophoresis method. Cultivars genetic diversity was analysed, their genetic distances were calculated, and the clustering analysis and fingerprint construction were performed for all the cultivars. The results were as follows: DNA template concentration was 0.5 ng·μL^-1, primer concentration was 0.5 μmol·L^-1, and the optimal annealing temperature of primers UBC813, UBC814, UBC815, UBC823, UBC824, UBC835, UBC840, UBC841, UBC843, UBC844 and UBC876 were 52.3, 55.9, 54.3, 54.3, 53.6, 56.2, 51.9, 54.4, 54, 50 ℃, respectively, the number of rings was 32. A total of 161 bands were generated collectively by the 11 ISSR primers, and the 156 bands were polymorphic, and the polymorphic ratio was 96.89%. Allele number, effective allele number, Nei's gene diversity index and Shannon's information index ranged from 1.86 to 2.00, 1.33 to 1.68, 0.21 to 0.39 and 0.34 to 0.57, with an average of 1.969, 1.478, 0.294 and 0.447 per primer, respectively. Primer UBC841 had the highest identification rate(80.92%), by which 106 cultivars were identified. A total of 131 cultivars were completely identified and their molecular fingerprints were constructed based on combination of UBC841 and UBC876. The genetic distance between 131 cultivars ranged from 0.00 to 0.60, with an average value of 0.365, and genetic diversity of 131 Bougainvillea cultivars was low, which were divided into six groups at 0.58 genetic distance. Clustering analysis indicates that majority of the cultivars within a species tend to fall in the same cluster, but some cultivars of the same species were grouped in different clusters or sub-clusters and certain cultivars from different species grouped in the same cluster. Genetic diversity of Bougainvillea germplasm resources was revealed accurately, the ISSR-based fingerprints of Bougainvillea cultivar provide reliable technique for cultivar registration and intellectual property protection as well as cultivar clarification in production practices.