SSCP-SNP in pearl millet - a new marker system for comparative genetics
SSCP-SNP in pearl millet - a new marker system for comparative genetics
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DOI：
10.1007/s00122-005-1981-0
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发表时间：
2005-05-01
影响因子：
5.4
通讯作者：
Gale, MD
中科院分区：
文献类型：
--
作者：
Bertin, I;Zhu, JH;Gale, MD
珍珠粟中有相当多的基因组资源，标记辅助选择已经在ICRISAT的公共育种方案中使用。本文介绍了实验，以扩展这些公开可用的资源，单核苷酸多态性（SNP）为基础的标记系统。利用水稻基因组序列，建立了一种新的分子标记系统--单链构象多态性（SSCP）-SNP，以预测小米表达序列标签（ESTs）中内含子-外显子的边界，并设计引物，使其能够跨越内含子进行扩增。充足的小米EST可供我们鉴定299个同源的单拷贝水稻基因，其中内含子的位置可以精确预测。然后设计PCR引物以扩增含有内含子的约500-bp基因组片段。这些片段的SSCP凝胶分析显示相当多的多态性。一个详细的DNA序列分析的变化在4个SSCP-SNP位点的8个近交基因型的面板显示复杂的变异模式，约一个SNP或插入缺失（插入缺失），每59 bp的内含子，但在外显子少得多。大约三分之二的变异来自SNP，三分之一来自插入缺失。SSCP检测到了大部分单倍型。作为一种标记系统，SSCP-SNP的开发成本低于简单重复序列（SSR），因为大部分工作都是在计算机上进行的，并且具有类似的部署成本和吞吐量潜力。多态性的比率较低，但可用，与平均PIC为0.49相对于0.72的SSR在我们的8个近交基因型面板屏幕。该系统的主要优势在于比较应用。同线信息可用于将SSCP-SNP标记靶向特定染色体区域，或者相反，SSCP-SNP标记可用于解开基因组特定部分中的详细同线关系。最后，初步分析表明，小米SSCP-SNP引物在其他谷物中扩增的成功率约为50%。通过更特异地设计引物以精确匹配模型基因组序列，也有相当大的潜力将SSCP-SNP促进到COS（保守的正向同源组）标记系统以用于跨物种的应用。
A considerable array of genomic resources are in place in pearl millet, and marker-aided selection is already in use in the public breeding programme at ICRISAT. This paper describes experiments to extend these publicly available resources to a single nucleotide polymorphism (SNP)-based marker system. A new marker system, single-strand conformational polymorphism (SSCP)-SNP, was developed using annotated rice genomic sequences to initially predict the intron-exon borders in millet expressed sequence tags (ESTs) and then to design primers that would amplify across the introns. An adequate supply of millet ESTs was available for us to identify 299 homologues of single-copy rice genes in which the intron positions could be precisely predicted. PCR primers were then designed to amplify approximately 500-bp genomic fragments containing introns. Analysis of these fragments on SSCP gels revealed considerable polymorphism. A detailed DNA sequence analysis of variation at four of the SSCP-SNP loci over a panel of eight inbred genotypes showed complex patterns of variation, with about one SNP or indel (insertion-deletion) every 59 bp in the introns, but considerably fewer in the exons. About two- thirds of the variation was derived from SNPs and one-third from indels. Most haplotypes were detected by SSCP. As a marker system, SSCP-SNP has lower development costs than simple sequence repeats (SSRs), because much of the work is in silico, and similar deployment costs and through-put potential. The rates of polymorphism were lower but useable, with a mean PIC of 0.49 relative to 0.72 for SSRs in our eight inbred genotype panel screen. The major advantage of the system is in comparative applications. Syntenic information can be used to target SSCP-SNP markers to specific chromosomal regions or, conversely, SSCP-SNP markers can be used to unravel detailed syntenic relationships in specific parts of the genome. Finally, a preliminary analysis showed that the millet SSCP-SNP primers amplified in other cereals with a success rate of about 50%. There is also considerable potential to promote SSCP-SNP to a COS (conserved orthologous set) marker system for application across species by more specifically designing primers to precisely match the model genome sequence.