基于SSR和SRAP标记的红花玉兰品种遗传关系分析及分子鉴定

张妹; 何正权; 马江; 桑子阳; 朱仲龙; 张德春; 马履一; 陈发菊

doi:10.13332/j.1000-1522.20190204

基于SSR和SRAP标记的红花玉兰品种遗传关系分析及分子鉴定

Genetic relationship analysis and molecular identification of Magnolia wufengensis cultivars based on SSR and SRAP markers

摘要

摘要:
目的为了解析红花玉兰各品种间的遗传多样性和遗传差异等问题，本研究采用SSR和SRAP分子标记方法对红花玉兰不同品种的遗传多样性水平和遗传分化程度进行评估，以建立其分子标记体系。
方法以35个红花玉兰品种为实验材料，分别进行SSR和SRAP标记扩增，计算出SSR和SRAP分子标记的各遗传参数。利用NTSYS-pc2.1软件计算各品种间的遗传相似系数并用非加权法（UPGMA）进行聚类分析。通过R语言分析筛选出能够完全区分红花玉兰品种的引物对组合。
结果经筛选共获得18对具有多态性且条带清晰的SSR引物，分别以35个红花玉兰品种基因组DNA为模板，共扩增出DNA条带128条，每对引物扩增条带在2 ~ 15之间，平均每对引物扩增7.1条，平均带型数为10.6，平均有效带型数为5.4，平均分辨能力（D）为0.72；不同引物的多态性位点百分比变化范围很大，在0.142 9 ~ 1.000 0之间，均值为0.730 2；Shannon’s指数平均为0.304 2，范围是0.086 4 ~ 0.433 7；平均期望杂合度为0.248 8，范围是0.059 2 ~ 0.282 3。利用筛选获得的11对SRAP引物对35个红花玉兰品种进行鉴定，共扩增156条DNA条带，引物扩增条带数在7 ~ 18之间，平均每对引物扩增14.2条，平均带型数为22.7，平均有效带型数为13.2，平均分辨能力（D）为0.93；11对引物的多态性位点百分比平均数为0.815 6，变异范围是0.657 1 ~ 1.000 0；平均Shannon’s指数为0.375 9，变异区间在0.287 2 ~ 0.455 3；平均期望杂合度为0.240 4，范围在0.180 2 ~ 0.311 6之间。
结论红花玉兰具有较高的遗传多样性，经筛选和优化后的SSR和SRAP引物组合均能将现有红花玉兰品种完全区分开，实现了对红花玉兰品种的简便、快速、准确地鉴定，并为红花玉兰的保护和繁育，以及新品种的选育提供了重要的参考。

Abstract:
ObjectiveGenetic diversity and genetic variation of Magnolia wufengensis cultivars were examined by simple sequence repeats (SSR) and sequence-related amplified polymorphism (SRAP) molecular markers for establishing its molecular marker system.
MethodThirty-five simples of M.wufengensis cultivars were applied to PCR amplification for SSR and SRAP markers. The genetic similarity coefficient of each cultivar was calculated by NTSYS-pc2.1 and cluster analysis was carried out using UPGMA.The locus data of combination of primer pairs that can discriminate all cultivars were screened by R language.
ResultA total of 18 pairs of SSR primers with polymorphism and clear bands were obtained. With the genome DNA of 35 M. wufengensis cultivars as the PCR template, a total of 128 polymorphic bands were generated. The number of polymorphic bands at each polymorphic primer ranged from 2 to 15, with mean band number of 7.1, mean pattern number of 10.6, mean effective pattern number of 5.4 and mean D of 0.72.The mean value of the percentage of polymorphic loci was 0.730 2, ranging from 0.142 9 ~ 1.000 0. The mean Shannon's index was 0.304 2, ranging from 0.086 4 to 0.433 7. The mean expected heterozygosity among cultivars was 0.248 8, ranging from 0.059 2 to 0.282 3.Eleven pairs of effective SRAP primers were acquired through screening. A total of 156 polymorphic bands were produced with the genome DNA of 35 M.wufengensis cultivars as the template. The number of polymorphic bands at each polymorphic primer ranged from 7 to 18, with mean band number of 14.2, mean pattern number of 22.7, mean effective pattern number of 13.2 and mean D of 0.93. The mean value of the percentage of polymorphic loci was 0.815 6, ranging from 0.657 1 ~ 1.000 0. The mean Shannon’s index was 0.375 9, ranging from 0.287 2 to 0.455 3. The mean expected heterozygosity among cultivars was 0.240 4, ranging from 0.180 2 to 0.311 6.
ConclusionM. wufengensis has relatively high genetic diversity. The analysis of molecular variation of both SSR and SRAP marker systems indicates that most genetic diversity is within M. wufengensis cultivars. The locus data of combination of primer pairs can discriminate all cultivars for identifying M. wufengensis cultivars efficiently and accurately.These results provide important references for the protection and breeding of M. wufengensis.

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