【作者】 张广平；

【导师】 邹学校；

【作者基本信息】 中南大学 ， 植物学， 2012， 博士

【摘要】 公共序列数据库中辣椒的EST序列急剧增长,为EST-SSR标记开发提供了丰富的序列资源。但目前,对辣椒EST序列信息的利用还十分有限,本研究利用公共序列数据库(NCBI)中来自辣椒的近12万条EST序列信息对辣椒的EST-SSR标记进行鉴定和开发。首先,研究利用生物信息学手段和数据挖掘技术,分析了辣椒EST-SSR的发生和分布特征。对118060条辣椒EST序列进行处理和拼接,获得了30759个Unigene,覆盖基因组的长度为23.12Mbp。在Unigene上鉴定出3758个SSR位点,其发生频率为1/6.15kb。二核苷酸和三核苷酸重复基序的SSR位点分别占总数的70.7%和26.8%。83.5%的SSR位点的重复次数在10次以内。出现最多的基序类型是AG/TC,占EST-SSR总数的43.8%；AAG/TTC是出现最多的三核苷酸重复基序,占总数的6.8%。二核苷酸基序的EST-SSR在5’UTR的分布密度为20/10kb,高于CDS区的8/20kb。而三核苷酸基序的EST-SSR在5’UTR的分布密度为4/10kb,与CDS区的5/10kb相当。这些结果为辣椒SSR标记的开发提供了有用的信息。其次,利用公共序列数据库中EST序列来源的异质性和冗余性,通过序列比对的方法鉴定了一批多态性EST-SSR位点,并高效地开发出了一批辣椒EST-SSR标记。对含有冗余EST序列的contig进行搜索,鉴定出68个多态性的SSR位点,其中有65个位点可以设计引物。利用31个辣椒材料进行试验验证,获得了33个多态性EST-SSR标记,其基序重复次数分布在2-10次之间,重复次数在5次以下的多态性EST-SSR位点有18个,占总数的55%。有27个EST-SSR位点与已知功能基因高度同源,这些功能基因涉及到种子成熟、逆境响应等众多生理过程。33个多态性EST-SSR位点中,分别有27个、18个和15个EST-SSR位点在茄子、番茄和马铃薯等作物上具有可转移性。在茄子、番茄和马铃薯上同时具有可转移性的标记有14个,在3种作物上都没有可转移性的标记有6个。研究结果不仅为辣椒遗传育种研究提供了一批可利用的转录区的SSR标记,同时还为在辣椒和其它作物上进一步挖掘公共序列数据库中的序列信息、开发低重复次数的SSR标记提供了实验依据。最后,利用鉴定出的33个EST-SSR标记,对31份来自不同地区的常规种或杂交种进行遗传多样性分析。33个多态性EST-SSR标记共扩增出91个等位基因,平均每个标记扩增出2.76个等位基因,最多检测出6个等位基因；EST-SSR位点的平均观测杂合度和平均期望杂合度分别为0.28和0.39。EST-SSR位点的多态性信息含量(PIC)分布范围为0.03～0.74,平均为0.38。聚类分析在相似系数为0.48处,将31份种质分为2大组。第一组5份材料,多为非常规栽培种Capsicum Chinense Jacquin,第二组26份材料,又可以被细分为6个亚组。主坐标分析与聚类分析结果一致。同时,基于EST-SSR标记的聚类分析结果显示,辣椒种质间的遗传相似性与果实性状、熟性和辣味等表型数据之间的相关性不大。更多还原

【Abstract】 The amount of EST derived form pepper in public available sequence database grows dramatically, which offers abundant sequence resource for the development of SSR markers. While, the utilization fo pepper EST sequence information was very limited. In the study, a set of polymorphic EST-SSR loci markers was identified and subsequently validated using about120000pepper EST sequences in NCBI.Firstly, the bioinformatic tools and data mining methods were employed to analyze the occurring and distribution characteristics of EST-SSR in pepper. A total of30759Unigenes covering23.12Mbp were obtained after process and assembly of118060pepper ESTs retrieved from NCBI.3758EST-SSR loci were identified on the Unigenes with the occurring frequency of1/6.15kb. The amount of EST-SSR loci with dinucleotide motif and trinucleotide motif took account of70.7%and26.8%of the total respectively. There were83.5%EST-SSR loci with the repeat number no more than10times. AG/TC, taking account of43.8%of the total, was the dominant motif type. AAG/TTC was the most frequent occurring trinucleotide motif, accounting for6.8%of the total. The distribution density of EST-SSR with dinucleotide motif was20/10kb in5’UTR, which was higher than that of8/20kb in CDS. However, the EST-SSR with trinucleotide motif, the distribution density was4/10kb, which was comparable with the5/10kb in CDS. The results offered valuable information for SSR marker development in pepper.Secondly, the characters of redundancy and heterozygosity of EST sequences in public sequence database were used to identify a set of polymorphic EST-SSR loci by sequence alignment and a set of polymorphic EST-SSR markers were subsequently validated. The contigs containing the redundant EST sequences were screened and a total of68polymorphic SSR loci were identified,65of which had suitable sequence length for primer design. The polymorphisms of the putative polymorphic SSR loci were validated by31pepper genotypes, resulting in33polymorphic EST-SSR loci. The repeating number of the motif for the polymorphic loci ranged from2to10times. There were18loci with the motif repeating number less than5times, accounting for55%of the total. There are27EST-SSR loci having significant homology with the genes with known functions, which covered the physiological proceedings of seed maturity, stress response and so on. There were7,18and15EST-SSR that could transfer to eggplant, tomato and potato respectively. A total of14EST-SSR markers had the transferability on all the corps of eggplant, tomato and potato. Meanwhile, there were6EST-SSR markers that could not transfer to either of eggplant, tomato and potato. The results not only offered a set of SSR markers locating in the transcript regions for pepper genetic breeding researches, but also provided experimental evidences for further mining the sequence information in public database and developing the SSR markers with the low repeating number.Finally, the molecular genetic diversity of31pepper varieties was analyzed by using33polymorphic EST-SSR markers.91alleles were amplified by the33polymorphic markers. The maximum of6and average of2.76alleles per locus were detected respectively. The average observed and expected heterozygosities were0.28and0.39respectively. The polymorphic information content (PIC) ranged from0.03to0.74with the means of0.38. The cluster analysis showed that those varieties could be divided into2major groups according to0.48Jaccard’s similarity. The first group had5varieties,most of which were Capsicum Chinense Jacquin, and the second group had26varieties which could be divided into6subgroup. The results of principal coordinates analysis were basically consistent with the UPGMA cluster. Furthermore, the cluster analysis based on EST-SSR markers revealed that the genetic similarity was not correlation with the morphologic traits of the fruit, such as fruit type, maturity, hot taste, etc.更多还原