大豆EST-SNP的挖掘、鉴定及其CAPS标记的开发

采用生物信息学方法将大豆EST序列联配到大豆基因组序列上,挖掘到大豆EST-SNP位点537个。对其靶向基因进行功能注释分析,发现他们主要参与亚细胞定位、蛋白质结合与催化以及代谢等与大豆重要农艺性状形成相关的生物过程。同时开发了简便易行的SNP检测方法,利用EMBOSS软件筛选导致酶切位点改变的EST-SNP,分别以大豆绥农14、合丰25、Acher、Evans、Peking、PI209332、固新野生大豆、科丰1号、南农1138-2的DNA及其混合的DNA为模板,设计引物进行PCR扩增,发现44个PCR产物中有36个测序峰图在预期的EST-SNP位点表现出多态性。酶切分析发现26个PCR产物具有酶切多态性,可以作为CAPS标记。结果表明该EST-SNP挖掘体系及其CAPS标记转化系统具有高效率、低成本等优点,有利于促进大豆的遗传育种研究。

The Mining of Citrus EST-SNP and Its Application in Cultivar Discrimination

Single nucleotide polymorphisms （SNPs） are the most abundant sequence variations found in plant genomes and are widely used as molecular genetic markers in cultivar identification and genetic diversity studies. The objective of this study was to identify SNP markers useful for discrimination of citrus cultivars, since large numbers of expressed sequence tags （ESTs） of sweet orange are available from the National Center for Biotechnology Information （NCBI）. We now have the opportunity to discover SNP markers suitable for determining the haplotypes with which to distinguish very closely related cultivars and to assess genetic diversity within or between related species of citrus. SNPs and small insertions/deletions （Indels） from ESTs of sweet orange and satsuma were identified by the in silico SNP discovery strategy. 55 296 EST sequences of sweet orange and 2 575 of satsuma retrieved from the NCBI repository were mined for potential SNPs. Cleaved amplified polymorphic sequences （CAPS） and sequencing approaches were used to validate putative SNPs in a sample of 30 citrus accessions. A total of 3 348 putative SNPs were identified based on the abundance of sequences and haplotype cosegregation. Of these 3 348 SNPs, the transitions, transversions and Indels ratios were 47.9, 36.1 and 16.0%, respectively. The SNPs occurred on average at a frequency of 1 per 164 bp in the coding region of citrus. 14 SNPs were randomly selected and genotyped according to 30 citrus accessions including 23 accessions of sweet orange; 11 SNPs displayed polymorphism with an average polymorphism information content （PIC） of 0.20 among 30 citrus accessions. The genetic diversity present in sweet orange was low, so the 14 SNP markers failed to discriminate different cultivars of sweet orange, but they did succeed in distinguishing accessions of inter-species of citrus. In this study, SNPs were mined from EST sequences of sweet orange and satsuma, which displayed potential capability as molecular markers to discriminate inter-species accessions of citrus. It is anticipated that these putative SNPs could be applied in citrus genetics research and breeding.

柑橘CAPS标记和AS-PCR引物的开发

以从GenBank中下载的93 955条甜橙[Citrus sinensis(L.)Osbeck]EST序列为源序列,利用QualitySNP软件包从中挖掘出1 521个单核苷酸多态性(single nucleotide polymorphism,SNP)候选位点。进一步利用酶切位点查找软件WatCut进行分析,发现其中125个SNP为酶切扩增多态性序列(cleavedamplified polymorphic sequences,CAPS)位点。随机挑选40个CAPS候选位点设计引物,对12个含多种类型的柑橘品种进行分型,以此验证各标记的有效性。同时,还挑选了25个经生物信息学预测可导致其编码蛋白氨基酸序列改变的SNP位点,分别设计出一组等位基因特异PCR(allele specific PCR,AS-PCR)引物,以6个不同类型柑橘品种的DNA为模板进行PCR扩增,扩增产物采用琼脂糖凝胶电泳进行检测,筛选多态性引物。结果显示,40个CAPS候选位点中有26个位点具有酶切扩增多态性,且分型结果稳定,可作为CAPS标记。此外,还筛选获得15组在不同柑橘品种间检测到多态性的AS-PCR引物,重复性好,可有效用于柑橘品种的SNP分型。