本文選題：芥菜型油菜　切入點：Illumina測序　出處：《華中農(nóng)業(yè)大學(xué)》2017年碩士論文　論文類型：學(xué)位論文

【摘要】：芥菜型油菜是十字花科蕓薹屬的異源四倍體種,具有抗旱、抗寒、耐貧瘠等特點;主要分布于我國的西部地區(qū),栽培歷史悠久,資源豐富。葉綠體基因組因其分子量小、非重組性、高度保守性、單親遺傳等特點,在揭示物種起源進化、闡述生物多樣性等方面意義重大�；谌~綠體基因組高度保守的特性,在具備有效參考序列的前提下,本研究采用Illumina測序技術(shù),對來源廣泛的芥菜型油菜材料進行高通量測序,用Sanger測序驗證變異頻率的可靠性,并篩選部分基因?qū)ζ涠鄳B(tài)性進行研究,所獲結(jié)果如下:1、對葉綠體的分離方法及條件進行優(yōu)化,以大規(guī)模提取DNA。主要對以下操作進行了優(yōu)化:在葉綠體的分離過程中增加了200 g離心2 min的操作,該操作可以離掉細胞壁碎片和細胞核顆粒,省去了用percoll梯度離心。還用DTT替代了與它作用相似的β-巰基乙醇,刺激性氣味更小、毒性更低,該方法操作簡單,既降低了實驗成本,又提高了純度和產(chǎn)量。2、Illumina測序技術(shù)發(fā)掘葉綠體基因組DNA的多態(tài)性。通過對1549份芥菜型油菜材料的葉綠體基因組進行混樣測序(每50份為一個混樣池,共31個),在2%的變異頻率之上,共找到變異位點573個,其中位于基因間隔區(qū)的有199個,基因上的有374個,且這374個變異位點集中于31個葉綠體基因。3、利用Sanger測序技術(shù)驗證Illumina測序結(jié)果的可靠性。通過對變異頻率為2%-3%、6%-7%、20%、4%-23%、8%、10%、12%-15%等不同變異頻率分別篩選合適的區(qū)段進行Sanger測序驗證,發(fā)現(xiàn)Illumina測序結(jié)果的可靠性在12%以上;淘汰了諸多不可靠的變異頻率對應(yīng)的的變異位點,為后續(xù)的研究奠定了基礎(chǔ),也為后來的研究提供了參考。4、葉綠體基因rps19及一個基因間隔區(qū)的多態(tài)性分析。利用Sanger測序技術(shù)從不同省份篩選的96份材料中,rps19基因只在來自英國的材料1490中有4個變異位點,可能在長期的地理隔離中發(fā)生了進化。在基因間隔區(qū),有52份材料具有兩個相同的變異位點,分別為位點64298,A變?yōu)門,以及位點64767,A變?yōu)镃;另有一個來自瑞典的材料887顯示較多的30個變異位點。由此可見,相對于功能基因,基因間隔區(qū)進化速率相對較快,地域差異可能也是造成DNA多態(tài)性的原因之一。
[Abstract]:Mustard rape is an allotetraploid species of Brassica in the cruciferous family. It has the characteristics of drought resistance, cold resistance and barren tolerance. It is mainly distributed in the western part of China with a long cultivation history and rich resources. The chloroplast genome is small because of its small molecular weight. The characteristics of non-recombination, highly conservative and single parent inheritance are of great significance in revealing the evolution of species origin and biodiversity. Based on the highly conserved nature of chloroplast genome and the availability of effective reference sequences, In this study, high-throughput sequencing of mustard rape was carried out by using Illumina sequencing technique. The reliability of mutation frequency was verified by Sanger sequencing, and some genes were screened to study its polymorphism. The results are as follows: 1. The separation methods and conditions of chloroplast were optimized for large-scale extraction of DNA. the following operations were optimized: 200g centrifugation 2 min was added to the separation of chloroplast. This procedure can remove cell wall fragments and nuclear particles, and obviate the use of percoll gradient centrifugation. DTT is also used to replace 尾 -mercaptoethanol, which has similar effect, with less irritating odor and less toxicity. It not only reduced the cost of experiment, but also improved the purity and yield of Illumina sequencing technique to discover the polymorphism of chloroplast genomic DNA. A total of 31 loci were found above the mutation frequency of 2%, of which 199 were located in the gene spacer and 374 were on the gene. And these 374 mutation sites were concentrated on 31 chloroplast genes. The reliability of Illumina sequencing results was verified by Sanger sequencing technology. Sanger sequencing was conducted by screening the appropriate sections with a mutation frequency of 2% -3% and 6% -7%, 20% -23% and 10% -15%, respectively. It was found that the reliability of Illumina sequencing was more than 12%, and many unreliable mutation sites were eliminated, which laid the foundation for further research. The polymorphism analysis of chloroplast gene rps19 and a gene spacer was also provided for the later study. The rps19 gene was only found in 4 mutation sites of 1490 material from different provinces by Sanger sequencing. It may have evolved during a long period of geographic isolation. In the genetic spacer, 52 materials had two identical mutation sites. The result shows that the locus 64298A becomes Tand the locus 64767A becomes C.The other material 887 from Sweden shows more 30 mutation sites. It can be seen that the rate of evolution of the spacer region is faster than that of the functional gene. Regional differences may also be one of the reasons for DNA polymorphism.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級別】：碩士
【學(xué)位授予年份】：2017
【分類號】：S565.4

【參考文獻】

相關(guān)期刊論文 前10條

1 蔡夢鮮;喬江偉;閆貴欣;吳金鋒;李再云;伍曉明;;EcoTILLING高通量分析甘藍型油菜種質(zhì)葉綠體rps16基因的多態(tài)性[J];分子植物育種;2014年06期

2 劉果;謝耀堅;張黨權(quán);羅建中;曾艷飛;;基于20種桉樹葉綠體matK基因序列的系統(tǒng)發(fā)育分析[J];桉樹科技;2014年03期

3 盧萍;高利霞;金鳳;恩和巴雅爾;;基于psbA-trnH序列的十種棘豆屬植物分子系統(tǒng)學(xué)研究[J];植物分類與資源學(xué)報;2014年03期

4 唐云濤;粟桂蓉;謝毓;易浪波;;基于PsbA-trnH,RbcL和trnL-F序列的野生葡萄系統(tǒng)發(fā)育[J];吉首大學(xué)學(xué)報(自然科學(xué)版);2014年02期

5 楊金宏;孔衛(wèi)青;;基于rbcL和matK序列探討馬鞭草科部分植物的系統(tǒng)學(xué)位置[J];熱帶亞熱帶植物學(xué)報;2013年02期

6 李西文;高歡歡;王一濤;宋經(jīng)元;Henry Robert;吳和珍;胡志剛;姚輝;羅紅梅;羅q;潘宏林;陳士林;;荷花玉蘭葉綠體全基因組高通量測序及結(jié)構(gòu)解析[J];中國科學(xué):生命科學(xué);2012年12期

7 王玲;董文攀;周世良;;被子植物葉綠體基因組的結(jié)構(gòu)變異研究進展[J];西北植物學(xué)報;2012年06期

8 王博;高磊;蘇應(yīng)娟;王艇;;基于葉綠體基因組全序列分析真葉植物葉綠體基因的適應(yīng)性進化[J];中山大學(xué)學(xué)報(自然科學(xué)版);2012年03期

9 李西文;胡志剛;林小涵;李卿;高歡歡;羅國安;陳士林;;基于454FLX高通量技術(shù)的厚樸葉綠體全基因組測序及應(yīng)用研究[J];藥學(xué)學(xué)報;2012年01期

10 張韻潔;李德銖;;葉綠體系統(tǒng)發(fā)育基因組學(xué)的研究進展[J];植物分類與資源學(xué)報;2011年04期

相關(guān)博士學(xué)位論文 前3條

1 陳勛;基于簡化基因組測序的油菜高通量SNP分析及白菜基因組DNA甲基化解析[D];華中農(nóng)業(yè)大學(xué);2014年

2 張同武;植物細胞器基因組測序，組裝及比較基因組學(xué)研究[D];浙江大學(xué);2012年

3 孔芳;蕓薹屬種間關(guān)系及其人工合成甘藍型油菜早期世代蛋白質(zhì)組學(xué)的研究[D];揚州大學(xué);2010年

相關(guān)碩士學(xué)位論文 前2條

1 陳春梅;基于cpDNA序列的茶樹及其近緣植物的親緣關(guān)系研究[D];中國農(nóng)業(yè)科學(xué)院;2014年

2 白雪菲;基于混合樣品高通量測序數(shù)據(jù)的植物葉綠體基因組拼接和分析[D];浙江大學(xué);2013年

，

本文編號：1631142