核桃优良品种及无性系DNA指纹图谱构建

Construction of DNA Fingerprints of Good Walnut Varieties and Clones

核桃作为我国重要的经济和油料树种,随着其育种工作的不断深入发展,核桃优良品种和无性系的数量持续增加,然而其种间种内频繁杂交及无性繁殖接穗的广泛流通,导致难以从表型性状上对不同的品种和无性系进行有效地分类辨别。本研究利用SSR分子标记技术对100份核桃优良品种和无性系进行DNA指纹图谱的构建,为核桃种质资源的准确、高效评价鉴定和保护利用提供理论与技术支撑。采集100份核桃优良品种及无性系的幼嫩叶片,利用CTAB法提取基因组DNA;对87对已发表的核桃SSR引物进行筛选;利用筛选出的多态性良好的SSR引物对100份核桃优良品种及无性系进行遗传多样性分析并构建DNA指纹图谱。筛选出11对稳定且多态性良好的SSR引物。各引物扩增的总条带数范围为45~84个,平均每个SSR位点扩增出62个片段。多态性条带比率的范围为91%~100%,均值为98%。有效等位基因数的范围是1.119~1.415个,平均值为1.297个。各引物的Nei's遗传多样性指数(H)和Shannon's信息指数(I)的变化趋势相一致,均值分别为0.191和0.313。多态信息含量(PIC)变化为0.275~0.704,平均值为0.519,其中8个位点(JH89978、JR3773、JR1165、JM78331、JM68820、JR6439、WGA-89和WGA-321)具有高多态性(PIC>0.5)。邻接法聚类结果显示,参试的100个核桃样品被聚为3大类。各引物对供试核桃样本的区分率从68%(JM78331)~100%(JR4964和WGA-321),选择区分率大且PIC值高(0.704)的引物WGA-321成功构建了100份核桃优良品种及无性系的DNA分子指纹图谱。研究结果为核桃品种鉴别、种苗纯度检测、品种追溯等提供了理论依据。

As an important economic and oilseed tree species in China,the number of good varieties and clones of walnut continues to increase with the deepening development of its breeding.However,the frequent interspecific and intraspecific crosses and the wide circulation of asexually propagated scions make it difficult to effectively classify and discriminate different varieties and clones in terms of phenotypic traits.In this study,the SSR molecular marker technology was used to construct DNA fingerprints of 100 excellent cultivars and clones of walnut,providing theoretical and technical supports for the accurate and efficient evaluation,identification,protection and utilization of walnut germplasm resources.The young leaves of 100 fine varieties and clones of walnut were collected and the total genomic DNA was extracted by CTAB method.Eighty-seven pairs of published SSR primers for walnut were screened,and the screened SSR primers with good polymorphisms were used to analyze the genetic diversity of the 100 good walnut varieties and clones and to construct DNA fingerprints.Eleven pairs of stable and well polymorphic SSR primers were screened.The total number of bands amplified by per pair of primers ranged from 45 to 84,with an average of 62 fragments amplified from each SSR locus.The polymorphic band ratio ranged from 91% to 100%,with an average of 98%.The number of effective alleles ranged from 1.119 to 1.415,with a mean value of 1.297.The variation trends of Nei's genetic diversity index(H) and Shannon's information index(I) of all primers were consistent,and the mean values were 0.191 and 0.313,respectively.The polymorphism information content(PIC) ranged from 0.275 to 0.704,with a mean value of 0.519,in which eight loci(JH89978,JR3773,JR1165,JM78331,JM68820,JR6439,WGA-89 and WGA-321) showed high polymorphism(PIC > 0.5).The results of adjacency clustering showed that 100 walnut samples were grouped into 3 categories.The differentiation rate of each primer on the test walnut samples ranged from 68%(JM78331) to 100%(JR4964 and WGA-321),and the primer WGA-321,with a high differentiation rate and PIC value(0.704),was selected to successfully construct DNA molecular fingerprints of 100 good walnut varieties and asexual lines.The results provided a theoretical basis for walnut variety identification,seedling purity testing,and variety tracing.