中国野生葡萄分类与系统进化的分子研究

中国野生葡萄分类与系统进化的分子研究

MOLECULAR STUDY ON THE CLASSIFICATION AND PHYLOGENY OF CHINESE WILD GRAPES

硕士研究：张永辉　　导师：刘崇怀

中国农业科学院　　果树学2011届硕士

摘　要

我国是葡萄属植物的主要起源地之一，也是世界葡萄属植物种类最多、遗传资源最为丰富的国家之一。迄今为止，已知起源我国的葡萄属植物共有38个种1个亚种和7个变种。如此丰富多彩的葡萄属种质资源不仅对葡萄科学的发展有重大意义，也是阐明世界葡萄起源、演化和生物多样性必不可少的重要证据。本研究对包含16个中国葡萄野生种及其近缘植物的81份材料进行染色体基因组DNA、叶绿体DNA序列和核糖体ITS序列的研究，探讨了我国葡萄属植物的分类与系统进化关系，获得主要结论如下：

（1）利用ISSR标记对包含16个中国葡萄野生种及其近缘植物的81份材料进行分类研究，从100个引物中筛选出13个带型清晰、多态性好、重复性高的引物用于ISSR扩增，共扩增出分子量在300～2000bp之间的185条带，多态性条带为175个，多态百分率为94.6%。根据扩增结果，利用NTSYSpc2.10e软件进行分析，81份真葡萄亚属材料聚成22类，聚类结果与传统的分类结果基本一致。确定该标记技术可以作为传统分类研究的手段。

（2）通过初筛和复筛得到的引物UBC807、UBC810、UBC811、UBC812、UBC818、UBC826、UBC827、UBC835、UBC836、UBC855、UBC856、UBC868和UBC869等，可以作为葡萄属植物分类研究的重要引物。

（3）通过对ISSR-PCR扩增结果的聚类分析，将早期入圃时误定为毛葡萄的“毛葡萄1099”材料确定为桑叶葡萄，进一步证实了“万县野葡萄”、“福建野葡萄”属于华东葡萄，并把“福建野葡萄”和“华东葡萄1058”确定为同物异名材料。

（4）首次利用cpDNA序列和ITS序列构建了16个中国葡萄野生种及其近缘植物的22份材料的分子系统发育树。聚类结果表明，cpDNA序列和ITS序列未能完全阐明22份葡萄属不同种材料之间的亲缘关系，需要结合形态学特征和其他手段对上述材料的分类及系统进化关系作进一步研究。

（5）cpDNA序列聚类结果表明，22份葡萄属不同种材料之间的亲缘进化关系与其大陆性地理分布相一致，其母本为中国野生葡萄的某个种，父本为河岸葡萄；并根据上述实验方法推断桑叶葡萄为一个独立的种。

关键词　中国野生葡萄　ISSR　DNA序列　分类　系统进化关系

Abstract

As one of the major original centers of Vitis species，China has abundant genetic resources in the world。So far，38 species，1 subspecies and 7 varieties of Chinese wild grapes were originated in China. The vitis germplasm resources were not only significant in the development of grapes science,but also provided an indispensable evidence to clarify the origin,evolution and biodiversity of the vitis in the world. In this research,16 Chinese wild grape species and their relatives were used to study their phylogeny and classification by nuclear and chloroplast DNA analyses. The main results were shown as follows:

(1) The classification of 16 Chinese wild grape species and their relatives were studied by ISSR markers。A total of 13 primers selected from 100 primers were used for ISSR amplification。A total of 185 bands were generated，of which 175 bands were polymorphic bands （the percentage of polymorphic band，PPB=94.6%）。The bands size ranged from 300 to 2000bp。According to the results of ISSR amplification，the similarity coefficient were analyzed by NTSYpc2.10e software，the genetic similarity coefficient was 0.57 to 0.99. the clustering dendrogram was constructed by UPGMA method to explore the taxonomic relationship among 81 Vitis materials。The 79 Euvitis materials were classified as 22 groups based on the clustering analysis of ISSR markers。Most of materials were coincided with traditional classification。So we determined ISSR molecular markers could be used as supplementary means to the traditional classification.

(2) The ISSR primers UBC807、UBC810、UBC811、UBC812、UBC818、UBC826、UBC827、UBC835、

UBC836、UBC855、UBC856、UBC868和UBC869 screened from 100 primers could be applied to Vitis molecular classification.

(3) According to the results of ISSR amplification，we determined the “Maoputao 1099” as Vitis ficifolia Bunge. We confirmed “Wanxianyeputao” and “Fujianputao” belonged to V.pseudoreticulata W.T.Wang. we also found “Fujianputao” and “Huadongputao1058” were synonym materials.

(4) A total of 22 different types of vitis materials were used to constructed the Phylogenetic tree by chloroplast DNA and ITS。The clustering results partial illustrated the genetic relationship of 22 Vitis materials,so we need further research of the Phylogenetic relationship on those materials by morphology and other means.

(5) Chloroplast DNA sequences clustering results showed that the relationship of 22 Vitis materials were coincided with the continental geographical distribution，and the impact of the continental geographical isolation to the Vitis evolution was embodied in genetic information from material ancestor.

(6) From the clustering results of cpDNA sequences and ITS sequences，we preliminarily determined “Yanshanputao0947” was a interspecific hybrid，which female and male parent were one species of the Chinese wild grapes and V.riparia Michx,respectively. V.heyneana Roem. &Schult subsp. Ficifolia (Bge.) C.L.Li. could be a separate species from this result.

Key words　Chinese wild grapes　ISSR　DNA sequence　Taxonomy Phylogenetic relationships