【作者】 任启军；

【导师】 郭旺珍；

【作者基本信息】 南京农业大学 ， 作物遗传育种， 2004， 硕士

【摘要】 棉花是世界上重要的纤维和油料作物。棉花的分子生物学研究和系统演化历史历来受人们重视。微卫星属于第二代分子标记，具有基于PCR、共显性、多态性高、信息量大和分布广等特点。目前，微卫星已经用于棉花的分子遗传图谱构建，重要农艺性状QTL的筛选以及品种多样性分析和纯度鉴定等研究。但是至今仍没有对棉花微卫星变异的特点及进化作系统的研究，本研究利用二倍体野生种、陆地棉品种和品系以及四倍体野生种系为材料，对棉花微卫星变异特点及其进化进行了系统研究，并对黄河流域和长江流域的陆地棉推广品种及优质棉品系进行了SNP分析。主要研究结果如下： 1、基因组SSR位点多态性频率是EST-SSR位点的2.24倍，但两者在供试材料中扩增产物电泳带型类型基本相同。EST-SSR位点具有丰富的重复基元类型，扩增产物以三元重复为主，84％的扩增产物含有完整的重复基元；基因组SSR位点重复基元类型较少，扩增产物以二元重复最多，40.4％的扩增产物含有完整的重复基元。基因组微卫星往往比EST微卫星具有更多的重复次数，EST-SSR位点微卫星发生变异的扩增产物有6个，而基因组却有55个，是EST-SSR位点的9倍。不论微卫星重复次数还是重复基元的变异，基因组SSR位点都比EST-SSR位点具有较高的变异倾向。优质棉品系海7124，不论在EST还是在基因组SSR住点，扩增产物的重复序列都比其它品系倾向于缩短。与其它8个品种相比，在基因组SSR位点，中棉所12扩增产物重复序列倾向于缩短。在4个野生种系和野生种中，达尔文氏棉扩增产物的重复序列倾向于缩短。EST-SSR位点比基因组SSR位点更容易发生同源异型，原因是EST微卫星侧翼序列具有较高的碱基替换频率。EST-SSR位点聚类的系统树分布与基因组SSR位点的结果基本相同。EST和基因组SSR位点的单扩增产物位点侧翼序列分子聚类，不能确定扩增产物的可能二倍体来源，而双扩增产物位点都可以确定扩增产物可能二倍体来源，结果证明了双扩增产物位点根据电泳带型确定四倍体棉种扩增产物的可能二倍体来源是可行的。在EST-SSR位点，四倍体棉种的D亚组分化较大，而在基因组SSR位点，四倍体棉种的A亚组分化较大。 2、以TM-1序列为对照，5个长江流域推广品种中，泗棉3号和苏棉12SNP频率为0.0027，渝棉1号和泗棉2号SNP频率为0.0029，岱字棉15的SNP频率为0.0037；4个黄河流域推广品种中，徐州142SNP频率为0.0035，中棉所12SNP中文摘要频率为0.0048，鲁棉1号和中棉所19 SNP频率为0.0037;4个优质棉品系中，Maxxa的SNP频率0，0053，海7124和7235的SNP频率分别为0.0042和0，004。不论长江流域和还是黄河流域推广品种的EST-SSR侧翼序列与基因组SSR侧冀序列SNP比较结果都表明，EST-SSR侧翼序列SNP频率比基因组SSR侧翼序列的SNP频率高。3、26个二倍体棉种在4个EST一SsR位点的多态性频率为48%，单个位点检测到的分子量种类数最多的为15，最少的为8 .39%的扩增产物含有完整的重复基元，59%的扩增产物微卫星区发生了插入缺失和替换.和四倍体棉种相比，二倍体棉种微卫星区具有更大的变异幅度.由于同源异型产生的单一序列数为41个，占序列种类数的460)。4个位点不同染色体组的分子聚类结果与Fryxell的分类结果基本结果吻合，D组内以拟似棉与其它棉种亲缘关系最远，C组内奈尔逊氏棉、和澳洲棉亲缘关系很近.A组的材料在4个位点不能完全聚类。关键词:微卫星;单核普酸多态性;进化更多还原

【Abstract】 Cotton is an important fiber and oil crop in the world. Molecular biology and systematic evolution of cotton have attached great importance all the time. Microsatellite or SSR belongs to molecular markers of the second generation, and it has many advantages such as PCR-based, codominent, hypervariable, infomative and extensive in genome. At present, SSR had been used for the construction of cotton molecular linkage map, the screening of QTL of important agronomic traits, the evaluation of diversity of variaties and characterization of seeds purity. To date, there was not any research on SSR variation and its evolution in cotton. In this study, variaties and accessions of upland cotton, wild races of diploid and allotetraploid were used to characterize of SSR variation and evaluate SNP frequency of cotton variaty in Yangtse and Yellow Rivers and accessions with high fiber quality. The main results were listed as follows:1. The results reveal that the frequency of polymorphism at GENOME-SSR loci is as 2.24 times many as that at EST-SSR loci, electrophoretic patterns of PCR products are identical. There are a variety of motifs at EST-SSR locus, three nucleotide mtoif is predominent among them, 84% PCR products contain perfect motifs. However, there are fewer motifs at GENOME-SSR loci, two nucleotide motif is abundant among them, 40.4% PCR products contain perfect motifs. Microsatellites from Genome library often have more motifs numbers than those from EST. There are six products with micosatellite motif variation at EST-SSR loci, while there are 55 at GENOME-SSR loci, which is 9 times as many as those at EST-SSR loci. There is higher variation frequency of either microsatellite motif variation or motif numbers at GENOME-SSR locus than EST-SSR locus. Hai7124 is one of cotton accessions with high fiber quality, its microsatellites sequence is prone to contract at either EST-SSR or GENOME-SSR locus. With comparison to 8 cultivars, microsatellite sequence of Zhongmiansuol2 is inclined to shorten. Among 4 wild races, microsatellite sequence of G.Darwinii shows high inclination to contract. Size homoplasy is prone to occur at EST-SSR locus because there was higher base substitution at flanking sequence ofEST microsatellite .The distribution of phylogenetic tree is similar at EST-SSR and Genome-SSR locus. The putative diploid ancestor of alloteraploid amplicon was hard to determine at EST-SSR and Genome-SSR loucus with single product, but it was easy to identify at EST-SSR and Genome-SSR locus with double products. The results demonstrated that it was feasible to determine the putative diploid ancestors of alloteraploid amplicon accoring to the pattern of electrophoresis band. D subgenome of alloteraploid was more divergent at EST-SSR locus, A subgenome of alloteraploid was more divergent at Genome-SSR locus instead.2. SNP frequency of Simian3 and Sumianl2 was 0.0027 and SNP frequency of Yumianl and Simian2 was 0.0029 among 4 upland cotton cultivars from Yangtse River. SNP frequency of Xuzhou 142 was 0.0035. The frequency of Zhongmiansuol2 was 0.0048, and the value of Lumianl and Zhongmiansuo 19 was 0.0037. Among 4 accessions with high fiber quality, SNP frequency of Maxxa was 0.0053,and that of Hai7124 and 7235 was 0.0042 and 0.004 respectively. The results demonstrate SNP frequency of flanking sequence at EST-SSR locus was higher than that at GENOME-SSR locus according to flanking sequence from EST-SSR products with comparison to that from GENOME-SSR products.3. The frequency of polymorphism of 26 diploid cottons was 48% at 4 EST-SSR loci. The maximum of polymorphic MWs at single locus was 15, the minimum was 8. Thirty-nine percent of amplicons contain perfect motif, and 59% of amplicons contain indel and substitution at microsatellite motifs. With comparision to alloteraploid cotton, microsatellite motif of diploid has more variation. Size homoplasy causes 41 unique sequences, occupying 46% of the sum of the unique sequences. The phylogenetic results of different chromsome groups were consistent with Fry更多还原