本文關(guān)鍵詞： 葡萄 植物學(xué)性狀 分子標(biāo)記 ITS2條形碼　出處：《魯東大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：葡萄(Vitis vinifera L.)隸屬于葡萄科(Vitaceae)葡萄屬(Vitis),栽培歷史悠長、栽培面積廣泛,具有重要的經(jīng)濟(jì)價(jià)值�！吧啐堉椤�(Cabernet Gernischt)是中國特色品種,在膠東地區(qū)廣泛栽培,是中國主要的紅色釀酒葡萄品種。引進(jìn)100多年來,“蛇龍珠”的植物學(xué)性狀在不同地區(qū)表現(xiàn)出明顯差異,形成了若干新品系(新株系),但是品種退化問題也逐漸出現(xiàn)。因此,研究釀酒葡萄“蛇龍珠”的遺傳多樣性,對“蛇龍珠”釀酒新品種的選育、種質(zhì)資源的保護(hù)與管理等都具有重要意義。利用植物學(xué)性狀及SSR、ISSR等分子標(biāo)記以及ITS2條形碼對釀酒葡萄“蛇龍珠”及其近緣品種進(jìn)行遺傳多樣性分析。主要結(jié)果如下:(1)根據(jù)《葡萄品種學(xué)》選擇多項(xiàng)植物學(xué)性狀指標(biāo)作為形態(tài)多樣性分析鑒定依據(jù),發(fā)現(xiàn)其成齡葉片上刻裂基部形狀、葉柄洼形狀、果穗重量、果穗緊密度、果粒大小(縱徑)、果粉厚度、可溶性固形物等表現(xiàn)出不同程度的多樣性;蛇龍珠E04(幸福)最具有經(jīng)濟(jì)價(jià)值;利用Galet的葡萄葉形結(jié)構(gòu)數(shù)值分析方法,得出蛇龍珠E05(萊山)和E06(龍口)親緣關(guān)系最近。(2)利用14對條帶清晰、多態(tài)性好的SSR引物擴(kuò)增出139條條帶,69個多態(tài)性位點(diǎn),有效等位基因數(shù)在1.0216(VMC4F3)到1.5901(VrZAG25),Nei’s基因多樣性范圍在0.0195(VMC4F3)到0.0320(VrZAG25),香濃信息指數(shù)I最低在0.0405(VMC4F3),最高在0.4673(VrZAG25)。聚類分析將蛇龍珠8個株系分為2大組,蛇龍珠E06單獨(dú)聚為一組,其他7個株系聚為一組。(3)利用11條ISSR引物共擴(kuò)增出96條條帶,11份樣品的遺傳相似系數(shù)在0.7347到0.9388,平均遺傳相似系數(shù)為0.8256。有效等位基因數(shù)Ne在1.0721(UBC817)到1.5848(UBC807)之間。Nei’s基因多樣性在0.0628(UBC826)到0.3351(UBC835)之間,平均值0.1858,香濃信息指數(shù)I最低在0.0902(UBC826),最高在0.5057(UBC835),平均值0.2754。蛇龍珠E02(棲霞北)和E03(棲霞南)聚為一類,遺傳距離與地理距離一致,E05和E08(開發(fā)區(qū))聚為一類,E01(蓬萊)、E04、E06、E07(開發(fā)區(qū))之間差異顯著。(4)基于VvmybA1基因序列片段對11份釀酒葡萄樣品進(jìn)行遺傳多樣性分析,克隆的片段大小在905bp~914bp,其中,GC含量42.1%~43.5%。葡萄的VvmybA1基因序列片段在編碼區(qū)僅存在一個基因缺失,在非編碼區(qū)有5個堿基替換,有較低的遺傳多樣性水平(Hd=0.673,Pi=0.00221),不能將不同品種的親緣關(guān)系準(zhǔn)確表達(dá)。3種中性檢驗(yàn),符合中性進(jìn)化模型,在近期進(jìn)化史上VvmybA1基因較穩(wěn)定,在過去一段歷史未發(fā)生種群擴(kuò)張。(5)基于ITS2條形碼探討不同產(chǎn)地蛇龍珠新株系的遺傳多樣性,擴(kuò)增得到的ITS2序列長度范圍在425bp~459 bp,GC含量在66.6%~67.7%,種內(nèi)的遺傳距離K-2-P(Kimura-2-parameter)最大為0.0037,種間遺傳距離最大為0.0114。蛇龍珠E01和E02聚為一類,蛇龍珠E04和E07聚為一類,蛇龍珠E05和E08聚為一類。(6)對11份葡萄樣品的羧酸酯酶基因片段進(jìn)行擴(kuò)增和序列分析,探討蛇龍珠等釀酒葡萄樣品之間的遺傳差異。序列對比顯示,該基因片段在編碼區(qū)和非編碼區(qū)均存在不同程度的堿基替換。11份葡萄材料的基因片段共發(fā)現(xiàn)87個多態(tài)性位點(diǎn),平均74bp左右的序列長度上能夠檢測到一個SNP位點(diǎn),核苷酸多樣度(Pi)0.05076(±0.01149),單倍型多樣度(Hd)1.000(±0.039),表現(xiàn)出豐富的遺傳多樣性。3種中性檢驗(yàn)方法比較序列變異模式,結(jié)果表明符合中性模型。通過聚類分析可將蛇龍珠E02、E03聚為一類,E07、E08聚為一類,其遺傳距離和地理距離一致。該序列片段存在一些突變,這些突變能將蛇龍珠8個新株系以及其他釀酒葡萄品種明顯區(qū)分,可以作為一種遺傳標(biāo)記用來鑒別葡萄的親緣關(guān)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鍗曠嫭鑱氫負(fù)涓，

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