棉花基于FISH的单染色体图谱及亚基因组和Oligo序列鉴定

发布时间：2017-12-27 12:06

本文关键词：棉花基于FISH的单染色体图谱及亚基因组和Oligo序列鉴定　出处：《华中农业大学》2016年博士论文　论文类型：学位论文

【摘要】：棉花是世界上重要的天然纤维作物。异源四倍体棉花是二倍体A、D基因组祖先种经过杂交、加倍形成的双二倍体,是多倍化物种的典范,是植物多倍化研究的重要模式植物。目前,棉花基因组测序工作已经紧锣密鼓展开,部分棉种的基因组序列草图相继公布,但是受棉属基因组复杂度的影响,高质量组装序列的获得任重道远,因此,与之相关的基础研究工作的开展势在必行。细胞遗传图可以将遗传图上的分子标记位点与其在染色体的具体位置联系起来,对基因组序列组装具有重要的指导意义。作为重要的细胞遗传学研究手段,荧光原位杂交技术可以实现DNA序列直观准确的染色体定位,已经成为染色体细胞遗传图谱构建的有效方法。本研究通过荧光原位杂交技术,把遗传锚定的BAC克隆在陆地棉Ah01和Dh01染色体进行定位,构建相应染色体的细胞遗传图谱,与公布的陆地棉基因组序列草图进行了整合分析;初步尝试了基于基因组序列数据的FISH探针的开发应用,设计了棉花oligonucleotides(oligos)探针库,建立了棉花Oligo-FISH技术体系。主要研究结果如下:1、陆地棉Ah01和Dh01染色体BAC克隆的获得。选择47个陆地棉Ah01和Dh01染色体共有的SSR分子标记,筛选阿非利加棉和海岛棉Pima 90-53 BAC文库,共获得84个阳性克隆;对84个阳性克隆以已知的Ah01和Dh01染色体特异BAC克隆为参照进行FISH染色体定位,12对引物筛选所获得信号明显且单一(仅在目标染色体上有信号)的BAC克隆15个。按照对应标记在遗传图谱的大致位置信息,对上述15个BACs两两组合进行了双色荧光原位杂交,确定了邻近两个BAC克隆在染色体上的排列次序。2、构建陆地棉Ah01和Dh01染色体细胞遗传图谱。将经过FISH定位的BAC克隆通过染色体分析软件进行相对位置测量,BAC克隆在染色体臂的定向,依据位置已知的染色体特异BAC克隆(A1和D1)来确定。根据各BAC的相对图距分别构建了陆地棉Ah01和Dh01染色体细胞遗传图谱,两条染色体的细胞遗传图谱分别包括12个遗传标记锚定的BAC克隆。3、对不同图谱进行整合分析。将BAC克隆对应的SSR标记在全基因组分子标记图谱(Whole-genome marker map,WGMP)的厘摩信息转化为相对位置(Relative map positon,RMP);SSR标记在基因组序列图谱的碱基位置信息也转化成相对位置。将构建的染色体细胞遗传图谱与对应标记所在的遗传图、基因组序列草图进行整合分析。结果表明,(1)ssr标记在wgmm中的遗传位置和它们对应的bac克隆在细胞遗传图谱中位置顺序一致性很高;个别位点相对位置偏差很大,遗传距离相距很近的两个标记nau3433和bnl2921(相对位置相差11.2%),在细胞遗传图谱中相距却很远(59.4%/dh01和47%/ah01)。(2)细胞遗传图谱与基因组中染色体序列图谱的整合,初步确定了两个scaffolds在染色体上的大致位置:scaffold183_a01(长度约56kb)的位置在ah01染色体序列3.4%~9.6%(90268610~96488204bp)位置之间,scaffold3710_d01(长度约191kb)的位置在dh01染色体序列6145600~9387374bp之间。(3)通过遗传标记锚定的bac克隆在细胞遗传图的位置和顺序与e-pcr定位在染色体序列中的位置和顺序的比较,发现在ad1-nbi基因组序列中,ssr标记的顺序与细胞遗传图谱中ssr标记锚定的bac克隆顺序与相对位置基本一致。(4)序列组装过程中一些同源序列被作为重复单元去除,只在部分同源染色体之一保留了序列信息:ah01和dh01染色体细胞遗传图谱中共有的11个bacs对应的ssr标记,在两条染色体序列图共有的仅4个,其它7个分别定位在ah01和dh01这对部分同源染色体之一。(5)在染色体组装序列覆盖度方面稍有缺陷,bac克隆378j07和400l15在细胞遗传图中的相对位置分别为8.0%和96%,在染色体序列中的相对位置分别为3.4%和97.8%。4、完善了陆地棉粗线期染色体制片技术。从材料粗检入手,明确了显微观察过程中处于粗线期花粉母细胞的主要细胞学特征,方便材料的高效选择;高浓度混合酶解液(4%纤维素酶+1%果胶酶+1%蜗牛酶)处理,控制酶解时间在3h左右;60%乙酸涂片之后50℃烤片、酸解,并控制酸解时间1?2min之间,成功制备陆地棉粗线期染色体制片,相比之前的研究,技术准确、可重复性高。5、对研究中发现的富含重复序列的bac克隆57i23生物信息学分析。(1)获得同时鉴别a、d亚基因组的细胞遗传学标记,即bac克隆57i23;(2)bac序列分析,91238bp的bac序列里面,发现129个重复序列元件,占到bac序列的62%以上。结合fish验证,识别了其中的gypsy-48_gr-i类ltr-rt,是bac克隆57i23特异性的关键重复序列元件,推测其也是影响a、d基因组大小差异的关键元件之一;(3)结合fish与blastn比对结果,验证了基因组特异重复序列在ad1基因组序列中染色体归属的准确性。6、根据已经测序的棉花基因组序列设计oligos库,经过乳化pcr扩增、体外转录和反转录,获得生物素或地高辛标记的oligos探针库,用该混合探针在相关棉种进行FISH杂交,首次建立了棉花Oligo-FISH技术体系,为深入解析棉属基因组提供了一种新的研究方法。
[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.
【学位授予单位】：华中农业大学
【学位级别】：博士
【学位授予年份】：2016
【分类号】：S562

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