基于甘蔗AP85-441和R570基因组参考序列的微卫星位点鉴定和SSR标记开发

徐志军 赵胜 胡小文 孔冉 苏俊波 刘洋

摘  要：分子標记缺乏是制约甘蔗分子标记技术发展的重要因素。本研究利用甘蔗AP85-441和R570基因组参考序列，使用MISA软件分别鉴定出512 835和97 839个微卫星位点，分别占割手密基因组（AP85-441）和甘蔗基因组（R570）高质量参考序列的0.32%和0.35%。在2个基因组序列中，优势重复单元均为单核苷酸、二核苷酸和三核苷酸，各重复单元均以AT富集的基元为主。割手密和甘蔗基因组序列中分别有472 117和89 748个位点可以开发SSR标记。对割手密、甘蔗与高粱基因进行同源性分析，分别鉴定出16 691个和13 271个对应到高粱1～10号染色体上的同源基因，利用基因序列中的SSR位点，开发出13 224和7624对SSR引物。对开发的引物分别以割手密和甘蔗基因组为模板进行e-PCR检测，这些引物在割手密基因组中以多位点扩增为主，在2个基因组中的有效标记比例分别为79.35%和36.13%、79.01%和93.36%。部分SSR引物在基因组中表现出特异性扩增，有1368对仅在AP85-441中单扩增，有1420对仅在R570序列中单扩增，共有752对SSR引物可在2个基因组中单扩增，且这些SSR的扩增位点和来源基因均分布于所在基因组的全部染色体上。在禾本科作物基因组中e-PCR检测表明，开发的单扩增SSR标记具有较好的特异性。本研究鉴定的SSR位点，有助于进一步丰富甘蔗的分子标记;开发的3540对SSR引物对于栽培种甘蔗遗传图谱构建中遗传来源区分和同源连锁群确定具有重要的参考意义。

关键词：甘蔗;微卫星序列;同源基因;SSR标记;特异扩增

中图分类号：S566.1;S188+.1      文献标识码：A

Abstract： Lacking of molecular markers is an important factor restricting the development of marker-based genetic and breeding research. In this study， 512 835 and 97 839 microsatellite sequence， which accounted for 0.32% and 0.35% of the genome， were identified based AP85-441 and R570 genomic reference sequences respectively， using the MISA software. In the genomes， the dominant repeat units were single nucleotide， dinucleotide and trinucleotide， and each repeat unit was dominated by AT-enriched elements. 472 117 and 89 748 loci of the genomes could be used to develop SSR markers. 16 691 and 13 271 homologous genes corresponding to sorghum chromosome 1–10 were identified by homology analysis on the genes of Saccharum spontaneum， sugarcane and sorghum， and 13 224 and 7624 pairs of SSR primers were developed by the gene sequence. In silico PCR analysis of these SSR markers revealed that the markers were mostly multilocus amplified in Saccharum spontaneum genome， and the effective markers in the two genomes were 79.35% and 36.13%， 79.01% and 93.36%， respectively. Part of the SSR markers showed specific amplification in the genomes. 1368 and 1420 SSR were specific single-locus amplification in AP85-441 and R570， respectively. 752 SSR were single-locus amplification in the two genomes， and the amplification sites and source genes of these SSR were distributed on all chromosomes in the genome. In this study， the SSR loci identified would be conducive to enriching the molecular markers of sugarcane， and the 3540 pairs of SSR primers developed will provide support for the determination of homologous linkage groups and group genetic origin in the construction of sugarcane genetic map.

Keywords： sugarcane; microsatellite sequence; homologous gene; SSR marker; specific amplification

DOI： 10.3969/j.issn.1000-2561.2020.04.013

微卫星序列（Microsatellite sequence），又称简单重……