酿酒葡萄“蛇龙珠”新株系的遗传多样性研究

发布时间：2018-03-01 02:12

  本文关键词： 葡萄 植物学性状 分子标记 ITS2条形码　出处：《鲁东大学》2017年硕士论文　论文类型：学位论文

【摘要】：葡萄(Vitis vinifera L.)隶属于葡萄科(Vitaceae)葡萄属(Vitis),栽培历史悠长、栽培面积广泛,具有重要的经济价值。“蛇龙珠”(Cabernet Gernischt)是中国特色品种,在胶东地区广泛栽培,是中国主要的红色酿酒葡萄品种。引进100多年来,“蛇龙珠”的植物学性状在不同地区表现出明显差异,形成了若干新品系(新株系),但是品种退化问题也逐渐出现。因此,研究酿酒葡萄“蛇龙珠”的遗传多样性,对“蛇龙珠”酿酒新品种的选育、种质资源的保护与管理等都具有重要意义。利用植物学性状及SSR、ISSR等分子标记以及ITS2条形码对酿酒葡萄“蛇龙珠”及其近缘品种进行遗传多样性分析。主要结果如下:(1)根据《葡萄品种学》选择多项植物学性状指标作为形态多样性分析鉴定依据,发现其成龄叶片上刻裂基部形状、叶柄洼形状、果穗重量、果穗紧密度、果粒大小(纵径)、果粉厚度、可溶性固形物等表现出不同程度的多样性;蛇龙珠E04(幸福)最具有经济价值;利用Galet的葡萄叶形结构数值分析方法,得出蛇龙珠E05(莱山)和E06(龙口)亲缘关系最近。(2)利用14对条带清晰、多态性好的SSR引物扩增出139条条带,69个多态性位点,有效等位基因数在1.0216(VMC4F3)到1.5901(VrZAG25),Nei’s基因多样性范围在0.0195(VMC4F3)到0.0320(VrZAG25),香浓信息指数I最低在0.0405(VMC4F3),最高在0.4673(VrZAG25)。聚类分析将蛇龙珠8个株系分为2大组,蛇龙珠E06单独聚为一组,其他7个株系聚为一组。(3)利用11条ISSR引物共扩增出96条条带,11份样品的遗传相似系数在0.7347到0.9388,平均遗传相似系数为0.8256。有效等位基因数Ne在1.0721(UBC817)到1.5848(UBC807)之间。Nei’s基因多样性在0.0628(UBC826)到0.3351(UBC835)之间,平均值0.1858,香浓信息指数I最低在0.0902(UBC826),最高在0.5057(UBC835),平均值0.2754。蛇龙珠E02(栖霞北)和E03(栖霞南)聚为一类,遗传距离与地理距离一致,E05和E08(开发区)聚为一类,E01(蓬莱)、E04、E06、E07(开发区)之间差异显著。(4)基于VvmybA1基因序列片段对11份酿酒葡萄样品进行遗传多样性分析,克隆的片段大小在905bp~914bp,其中,GC含量42.1%~43.5%。葡萄的VvmybA1基因序列片段在编码区仅存在一个基因缺失,在非编码区有5个碱基替换,有较低的遗传多样性水平(Hd=0.673,Pi=0.00221),不能将不同品种的亲缘关系准确表达。3种中性检验,符合中性进化模型,在近期进化史上VvmybA1基因较稳定,在过去一段历史未发生种群扩张。(5)基于ITS2条形码探讨不同产地蛇龙珠新株系的遗传多样性,扩增得到的ITS2序列长度范围在425bp~459 bp,GC含量在66.6%~67.7%,种内的遗传距离K-2-P(Kimura-2-parameter)最大为0.0037,种间遗传距离最大为0.0114。蛇龙珠E01和E02聚为一类,蛇龙珠E04和E07聚为一类,蛇龙珠E05和E08聚为一类。(6)对11份葡萄样品的羧酸酯酶基因片段进行扩增和序列分析,探讨蛇龙珠等酿酒葡萄样品之间的遗传差异。序列对比显示,该基因片段在编码区和非编码区均存在不同程度的碱基替换。11份葡萄材料的基因片段共发现87个多态性位点,平均74bp左右的序列长度上能够检测到一个SNP位点,核苷酸多样度(Pi)0.05076(±0.01149),单倍型多样度(Hd)1.000(±0.039),表现出丰富的遗传多样性。3种中性检验方法比较序列变异模式,结果表明符合中性模型。通过聚类分析可将蛇龙珠E02、E03聚为一类,E07、E08聚为一类,其遗传距离和地理距离一致。该序列片段存在一些突变,这些突变能将蛇龙珠8个新株系以及其他酿酒葡萄品种明显区分,可以作为一种遗传标记用来鉴别葡萄的亲缘关系。
[Abstract]:Grape (Vitis vinifera L.) belongs to Vitaceae vitis (Vitaceae) (Vitis), a long history of cultivation, widely cultivated area, has important economic value. "Shelongzhu" (Cabernet Gernischt) is China characteristics of species, widely cultivated in the area of Jiaodong, China is the main red wine grape varieties introduced in 100. Over the years, "botanical Gernischet" showed significant differences in different regions, the formation of several new lines (new lines), but the variety degeneration problem has also emerged. Therefore, genetic diversity of wine grape "snake dragon", "snake dragon" for breeding new varieties of wine, have the significance of Germplasm resources protection and management. The use of botanical characters and SSR, ISSR molecular markers and ITS2 barcode on the wine grape "snake dragon" and its related species to analyze genetic diversity. The main results are as follows: (1 According to the < ampelography) > choose a number of botanical traits as indicators of morphological diversity analysis basis, found its mature leaves engraved on the base of the crack shape, hollow petiole shape, ear weight, ear tightness, fruit size (vertical diameter), powder thickness, soluble solids exhibit different diversity the dragon pearl; E04 (happiness) has the highest economic value; by using the method of analyzing the grape leaf shape structural numerical Galet, that dragon ball E05 (Laishan) and E06 (Longkou) had the closest relationship. (2) based on 14 clear bands, polymorphic SSR primers amplified 139 bands, 69 polymorphic loci, the effective number of alleles in 1.0216 (VMC4F3) to 1.5901 (VrZAG25), Nei "s gene diversity (VMC4F3) in the range of 0.0195 to 0.0320 (VrZAG25), Shannon's information index I is the lowest in 0.0405 (VMC4F3), the highest in 0.4673 (VrZAG25). The 8 strains divided Gernischet for the clustering analysis 2澶х粍,铔囬緳鐝燛06鍗曠嫭鑱氫负涓，

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