皱纹盘鲍基因组单核苷酸多态标记开发与应用

IOCAS-IR > 海洋环流与波动重点实验室

	皱纹盘鲍基因组单核苷酸多态标记开发与应用
其他题名	Development and application of single nucleotide polymorphism markers in the Pacific abalone, Haliotis discus hannai Ino
	亓海刚
学位类型	博士
	2008-12-31
学位授予单位	中国科学院海洋研究所
学位授予地点	海洋研究所
关键词	皱纹盘鲍 Est 单核苷酸多态标记测序as-pcr
摘要	本研究利用皱纹盘鲍的EST序列进行单核苷酸多态（SNP）标记开发；对等位基因特异性PCR（AS-PCR）方法进行了优化，使之适合SNP基因型分析；对一个作图家系开发基因相关SNP标记，并对标记在子代个体中的分离情况进行了分析，探讨了借助SNP在遗传连锁图谱上定位功能基因的方法。对大约5800条EST序列进行拼接，共获得含有4条以上序列的contig 150个，在86个含有单碱基突变位点的contigs中发现SNP 302个，碱基置换类型A/G（C/T）、A/C（G/T）、A/T、C/G的位点数目分别为147、90、21、16个。50个contigs在BLASTx分析后具有匹配（E值小于1E-5），其中的220个SNPs全部为同义cSNPs，位于遗传密码子的第三简并位。粗略估算，皱纹盘鲍核基因组中SNP的平均分布密度不低于1%。通过扩增DNA pool后产物直接测序共验证了28个SNP。PCR直接测序法操作简单，结果可靠，一次测序可能验证多个位点，有时还可以发现新的突变位点。通过重点研究引物3’端不同错配对PCR扩增的影响，对AS-PCR的引物设计原则及反应条件进行了探讨及优化，发现在AS引物的3’端倒数第二位引入额外的强错配后，AS-PCR检测SNP的特异性会得到很大的提高，从而使AS-PCR可以满足小规模的SNP基因型分析。根据EST-contig或者单一的EST序列设计PCR引物74组，其中43组可以特异扩增皱纹盘鲍基因组DNA。用这些引物扩增一个作图家系的父母本，并对PCR产物纯化后分别进行双向直接测序，在18个基因片段中发现了86个SNP，其中82个是新SNP，并且绝大多数SNP位于内含子序列中。这些SNP在父母本中的基因型，在多数情形下，是一方为纯合（AA），而另一方为杂合（AB）；父母本均为杂合和均为纯合的形式则较少。在父母本中杂合形式的SNP位点，理论上可以在子代中发生分离。在每个基因的内含子中选择父母本基因型为AA×AB或者AB×AA的SNP位点，设计AS-PCR分型引物并检测123个子代个体的基因型。在对9个基因中的11个SNP位点（包括5个母本标记和6个父本标记）进行子代基因型分析后发现，2个位点不符合孟德尔1：1分离（P < 0.05），符合预期分离比率的9个位点存在较大可能定位于遗传连锁图谱。通过分析两组父母本标记（F142和M142，F459和M459）发现，在根据父母本序列设计SNP分型引物时，可以设计指向同一基因的成对的父母本标记，从而使两个单亲标记可作为一个共同标记使用。
其他摘要	Single nucleotide polymorphism (SNP) markers were discovered from expressed sequences (ESTs) of the Pacific abalone (Haliotis discus hannai). An allele-specific polymerase chain reaction (AS-PCR) was developed and optimized for SNPs genotype scoring. Gene-associated SNP markers were exploited for a mapping family and analyzed in 123 progenies. The method of locating functional genes on genetic linkage maps was also discussed in the paper. About 150 EST-contigs containing four or more sequences were obtained by assembling 5800 ESTs and 302 SNPs were discovered from 86 EST-contigs. The numbers of A/G (C/T), A/C (G/T), A/T and C/G SNPs were 147, 90, 21 and 16, respectively. Fifty-two contigs had a positive match with functional ESTs in the public database after BLASTx analysis (E-value <=1E-5), among which there were 220 SNPs but all were synonymous cSNPs. Roughly estimated, the SNP frequency was not less than 1% in the genome of H. discus. Twenty-eight SNPs were validated by PCR direct sequencing (PCR-DS) of pooled DNA. PCR-DS was simple and accurate, could detect several loci by one reaction and discover new SNPs sometimes. The principle for AS-primer design was confirmed by evaluating the impacts of primer-template mismatch on PCR amplifications. The AS-PCR was suitable for small-scale SNPs genotyping as the specificity of detection was enhanced by adding an additional strong mismatch in the penultimate base of the 3’end of AS-primers. The PCR products of the parents of a mapping family from forty-three pairs of EST-derived primers were directly sequenced in both directions. Eighty-six SNPs were found among which 82 were new SNPs and most were in introns. In most cases the SNP genotype of one parent was homozygous (AA) and the genotype of another parent was heterozygous (AB), which were theoretically segregable in the progeny. The cases were fewer that SNP genotypes of both parents were homozygous or heterozygous. Eleven sets of AS-primers of nine genes were designed for genotyping 11 SNPs in 123 progenies, of which 5 were maternal markers and 6 were paternal markers. Chi-square test indicated that 2 loci showed significant segregation distortion (P < 0.05) from Mendelian ratio. The other 9 loci can probably be located on the genetic linkage map being constructed. Besides, the maternal and the paternal SNP markers from two adjacent loci of one gene could be used as one marker in genomic mapping.
页数	124
语种	中文
文献类型	学位论文
条目标识符	http://ir.qdio.ac.cn/handle/337002/907
专题	海洋环流与波动重点实验室
推荐引用方式 GB/T 7714	亓海刚. 皱纹盘鲍基因组单核苷酸多态标记开发与应用[D]. 海洋研究所. 中国科学院海洋研究所,2008.

条目包含的文件
文件名称/大小	文献类型	版本类型	开放类型	使用许可
10001_20051800684107（1090KB）			限制开放	--	浏览