本文關(guān)鍵詞：棉花基于FISH的單染色體圖譜及亞基因組和Oligo序列鑒定　出處：《華中農(nóng)業(yè)大學(xué)》2016年博士論文　論文類型：學(xué)位論文

  更多相關(guān)文章： 棉花 BAC FISH 細(xì)胞遺傳圖譜 LTR-RT Oligo-FISH

【摘要】：棉花是世界上重要的天然纖維作物。異源四倍體棉花是二倍體A、D基因組祖先種經(jīng)過(guò)雜交、加倍形成的雙二倍體,是多倍化物種的典范,是植物多倍化研究的重要模式植物。目前,棉花基因組測(cè)序工作已經(jīng)緊鑼密鼓展開,部分棉種的基因組序列草圖相繼公布,但是受棉屬基因組復(fù)雜度的影響,高質(zhì)量組裝序列的獲得任重道遠(yuǎn),因此,與之相關(guān)的基礎(chǔ)研究工作的開展勢(shì)在必行。細(xì)胞遺傳圖可以將遺傳圖上的分子標(biāo)記位點(diǎn)與其在染色體的具體位置聯(lián)系起來(lái),對(duì)基因組序列組裝具有重要的指導(dǎo)意義。作為重要的細(xì)胞遺傳學(xué)研究手段,熒光原位雜交技術(shù)可以實(shí)現(xiàn)DNA序列直觀準(zhǔn)確的染色體定位,已經(jīng)成為染色體細(xì)胞遺傳圖譜構(gòu)建的有效方法。本研究通過(guò)熒光原位雜交技術(shù),把遺傳錨定的BAC克隆在陸地棉Ah01和Dh01染色體進(jìn)行定位,構(gòu)建相應(yīng)染色體的細(xì)胞遺傳圖譜,與公布的陸地棉基因組序列草圖進(jìn)行了整合分析;初步嘗試了基于基因組序列數(shù)據(jù)的FISH探針的開發(fā)應(yīng)用,設(shè)計(jì)了棉花oligonucleotides(oligos)探針庫(kù),建立了棉花Oligo-FISH技術(shù)體系。主要研究結(jié)果如下:1、陸地棉Ah01和Dh01染色體BAC克隆的獲得。選擇47個(gè)陸地棉Ah01和Dh01染色體共有的SSR分子標(biāo)記,篩選阿非利加棉和海島棉Pima 90-53 BAC文庫(kù),共獲得84個(gè)陽(yáng)性克隆;對(duì)84個(gè)陽(yáng)性克隆以已知的Ah01和Dh01染色體特異BAC克隆為參照進(jìn)行FISH染色體定位,12對(duì)引物篩選所獲得信號(hào)明顯且單一(僅在目標(biāo)染色體上有信號(hào))的BAC克隆15個(gè)。按照對(duì)應(yīng)標(biāo)記在遺傳圖譜的大致位置信息,對(duì)上述15個(gè)BACs兩兩組合進(jìn)行了雙色熒光原位雜交,確定了鄰近兩個(gè)BAC克隆在染色體上的排列次序。2、構(gòu)建陸地棉Ah01和Dh01染色體細(xì)胞遺傳圖譜。將經(jīng)過(guò)FISH定位的BAC克隆通過(guò)染色體分析軟件進(jìn)行相對(duì)位置測(cè)量,BAC克隆在染色體臂的定向,依據(jù)位置已知的染色體特異BAC克隆(A1和D1)來(lái)確定。根據(jù)各BAC的相對(duì)圖距分別構(gòu)建了陸地棉Ah01和Dh01染色體細(xì)胞遺傳圖譜,兩條染色體的細(xì)胞遺傳圖譜分別包括12個(gè)遺傳標(biāo)記錨定的BAC克隆。3、對(duì)不同圖譜進(jìn)行整合分析。將BAC克隆對(duì)應(yīng)的SSR標(biāo)記在全基因組分子標(biāo)記圖譜(Whole-genome marker map,WGMP)的厘摩信息轉(zhuǎn)化為相對(duì)位置(Relative map positon,RMP);SSR標(biāo)記在基因組序列圖譜的堿基位置信息也轉(zhuǎn)化成相對(duì)位置。將構(gòu)建的染色體細(xì)胞遺傳圖譜與對(duì)應(yīng)標(biāo)記所在的遺傳圖、基因組序列草圖進(jìn)行整合分析。結(jié)果表明,(1)ssr標(biāo)記在wgmm中的遺傳位置和它們對(duì)應(yīng)的bac克隆在細(xì)胞遺傳圖譜中位置順序一致性很高;個(gè)別位點(diǎn)相對(duì)位置偏差很大,遺傳距離相距很近的兩個(gè)標(biāo)記nau3433和bnl2921(相對(duì)位置相差11.2%),在細(xì)胞遺傳圖譜中相距卻很遠(yuǎn)(59.4%/dh01和47%/ah01)。(2)細(xì)胞遺傳圖譜與基因組中染色體序列圖譜的整合,初步確定了兩個(gè)scaffolds在染色體上的大致位置:scaffold183_a01(長(zhǎng)度約56kb)的位置在ah01染色體序列3.4%~9.6%(90268610~96488204bp)位置之間,scaffold3710_d01(長(zhǎng)度約191kb)的位置在dh01染色體序列6145600~9387374bp之間。(3)通過(guò)遺傳標(biāo)記錨定的bac克隆在細(xì)胞遺傳圖的位置和順序與e-pcr定位在染色體序列中的位置和順序的比較,發(fā)現(xiàn)在ad1-nbi基因組序列中,ssr標(biāo)記的順序與細(xì)胞遺傳圖譜中ssr標(biāo)記錨定的bac克隆順序與相對(duì)位置基本一致。(4)序列組裝過(guò)程中一些同源序列被作為重復(fù)單元去除,只在部分同源染色體之一保留了序列信息:ah01和dh01染色體細(xì)胞遺傳圖譜中共有的11個(gè)bacs對(duì)應(yīng)的ssr標(biāo)記,在兩條染色體序列圖共有的僅4個(gè),其它7個(gè)分別定位在ah01和dh01這對(duì)部分同源染色體之一。(5)在染色體組裝序列覆蓋度方面稍有缺陷,bac克隆378j07和400l15在細(xì)胞遺傳圖中的相對(duì)位置分別為8.0%和96%,在染色體序列中的相對(duì)位置分別為3.4%和97.8%。4、完善了陸地棉粗線期染色體制片技術(shù)。從材料粗檢入手,明確了顯微觀察過(guò)程中處于粗線期花粉母細(xì)胞的主要細(xì)胞學(xué)特征,方便材料的高效選擇;高濃度混合酶解液(4%纖維素酶+1%果膠酶+1%蝸牛酶)處理,控制酶解時(shí)間在3h左右;60%乙酸涂片之后50℃烤片、酸解,并控制酸解時(shí)間1?2min之間,成功制備陸地棉粗線期染色體制片,相比之前的研究,技術(shù)準(zhǔn)確、可重復(fù)性高。5、對(duì)研究中發(fā)現(xiàn)的富含重復(fù)序列的bac克隆57i23生物信息學(xué)分析。(1)獲得同時(shí)鑒別a、d亞基因組的細(xì)胞遺傳學(xué)標(biāo)記,即bac克隆57i23;(2)bac序列分析,91238bp的bac序列里面,發(fā)現(xiàn)129個(gè)重復(fù)序列元件,占到bac序列的62%以上。結(jié)合fish驗(yàn)證,識(shí)別了其中的gypsy-48_gr-i類ltr-rt,是bac克隆57i23特異性的關(guān)鍵重復(fù)序列元件,推測(cè)其也是影響a、d基因組大小差異的關(guān)鍵元件之一;(3)結(jié)合fish與blastn比對(duì)結(jié)果,驗(yàn)證了基因組特異重復(fù)序列在ad1基因組序列中染色體歸屬的準(zhǔn)確性。6、根據(jù)已經(jīng)測(cè)序的棉花基因組序列設(shè)計(jì)oligos庫(kù),經(jīng)過(guò)乳化pcr擴(kuò)增、體外轉(zhuǎn)錄和反轉(zhuǎn)錄,獲得生物素或地高辛標(biāo)記的oligos探針庫(kù),用該混合探針在相關(guān)棉種進(jìn)行FISH雜交,首次建立了棉花Oligo-FISH技術(shù)體系,為深入解析棉屬基因組提供了一種新的研究方法。
[Abstract]:Cotton is an important natural fiber crop in the world. Allotetraploid cotton is a diploid A and D genome that has been hybridized and doubled. It is a model of polyploidy and an important model plant for polyploidy research. At present, the work of cotton genome sequencing has expanded genome draft sequence wildly beating gongs and drums, cotton seed parts have been released, but the effect of cotton genomic complexity, high quality of assembly sequence obtained has a long way to go, therefore, the basic research work related to the development of imperative. Cytogenetics can link the molecular marker loci in genetic map with its specific location in chromosomes, which has important guiding significance for assembly of genome sequences. As an important method of cytogenetics, fluorescence in situ hybridization (DNA) can realize the direct and accurate chromosomal location of DNA sequences, and has become an effective way to construct chromosome genetic map. This study by fluorescence in situ hybridization, BAC cloning genetic anchoring positioning in Upland Cotton Ah01 and Dh01 cell chromosome, genetic map construction corresponding chromosomes, analyzes and published upland cotton genome draft sequence; preliminary development and application of FISH probe genome sequence based on the data, the design of cotton oligonucleotides (oligos) probe library, established the technical system of cotton Oligo-FISH. The main results are as follows: 1. The BAC cloning of Ah01 and Dh01 chromosomes of upland cotton. Selection of 47 upland cotton Ah01 and Dh01 chromosome shared SSR molecular marker, screening Africa cotton and island cotton Pima 90-53 BAC library, 84 positive clones were obtained; 84 positive clones with known Ah01 and Dh01 chromosome specific BAC clones as reference for FISH chromosome mapping, 12 pairs of primers screened the signal obviously and (single signal only in the target chromosome) BAC 15 clones. According to the location information of corresponding markers in the genetic map, two color fluorescence in situ hybridization of the 15 BACs 22 combinations was carried out, and the order of two BAC clones on chromosome was determined. 2. The genetic map of Ah01 and Dh01 chromosomes of upland cotton was constructed. The BAC location of FISH was cloned through chromosome analysis software, and the relative position was measured by chromosome analysis software. BAC cloned on the chromosome arm was determined according to the known chromosome specific BAC clones (A1 and D1). According to the relative distance of BAC, the genetic linkage map of Ah01 and Dh01 chromosomes of upland cotton was constructed. The cytogenetic map of two chromosomes includes 12 BAC clones anchored by genetic markers. 3. Integrated analysis of different atlas. The cloning information of BAC corresponding to SSR markers is transformed into Whole-genome marker map (WGMP) and the relative position (Relative map positon, RMP) is converted to the relative position (Relative map positon, RMP), and the base position information of SSR marker in genome sequence map is also transformed into relative position. The genetic map of the chromosomes was constructed and analyzed with the genetic map and the genome sequence of the corresponding markers. The results show that (1) BAC SSR clonal genetic marker in the wgmm and their corresponding position in the cell genetic map sequential consistency is very high; the relative position deviation of individual loci, the genetic distance is close to the two markers nau3433 and bnl2921 (relative position is 11.2%), the cell genetic map distance far away (59.4%/dh01 and 47%/ah01). (2) the integration of the genetic map and cell chromosome sequences in the genome, two scaffolds on chromosome location was determined: scaffold183_a01 (length 56KB) position in the chromosome ah01 sequence 3.4%~9.6% (90268610~96488204bp) position between scaffold3710_d01 (length 191kb) position between the DH01 chromosome sequence 6145600~9387374bp. (3) was cloned by BAC genetic markers anchored comparative position and order in the cell genetic map and location of the sequence and location of e-PCR in the chromosome sequence, found in the ad1-nbi genome sequence, sequence and cell genetic map of SSR markers in the BAC clone SSR anchor marker sequence is consistent with the relative position. (4) the sequence assembly process some homologous sequences were as repeat units removed, only in part one of the homologous chromosomes retains the sequence information: SSR markers ah01 and DH01 chromosome genetic map common 11 BACs corresponding to the two chromosomes in the sequence shared only 4, the other 7 were located in ah01 and this part of one of the homologous chromosomes dh01. (5) there is a slight defect in the coverage degree of chromosome assembly sequence. The relative positions of BAC clone 378j07 and 400l15 in cell genetic map are 8% and 96%, respectively, and the relative positions in chromosome sequences are 3.4% and 97.8%, respectively. 4. The technique of chromosome making in the coarse line period of land cotton was perfected. Starting from the material in the coarse detection, clear the main cytological features of pachytene pollen mother cells were observed in the process, convenient efficient selection of materials; high concentration mixed enzyme solution (4% +1% cellulase pectinase +1% snail enzyme) treatment, the control of enzymolysis time was about 3H; after 60% acetic acid smear toast, 50 c acid hydrolysis, acid hydrolysis and control time of 1? 2min, the successful preparation of Upland Cotton pachytene chromosome, compared to the previous researches, the technology is accurate and reproducible. 5. Bioinformatics analysis of BAC clone 57i23 rich in repetitive sequences found in the study. (1) to get the cytogenetic marker identifying a and D subgenomes simultaneously, that is, BAC clone 57i23; (2) BAC sequence analysis; 129 BAC repeat sequences were found in 91238bp BAC sequence, accounting for more than 62% of BAC sequences. Combined with fish verification, we identified gypsy-48_gr-i class LTR-RT, which is a key repetitive sequence element of BAC clone 57i23, and speculated that it is also one of the key components that affect the size difference between a and D genome. (3) combining fish and BLASTN alignment.
【學(xué)位授予單位】：華中農(nóng)業(yè)大學(xué)
【學(xué)位級(jí)別】：博士
【學(xué)位授予年份】：2016
【分類號(hào)】：S562

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