基于甘蔗热带种(LA-purple)全基因组序列的SSR开发及特征分析

Development and Characterization of SSR Markers from the Whole Genome Sequences of Saccharum officinarum(LA-purple)

现代甘蔗栽培品种(2n=100~130)是由甘蔗热带种(2n=80)与割手密(2n=40~128)种间杂交而来,形成异源多倍体、非整倍体作物,使得甘蔗栽培品种中80%~90%的染色体来源于热带种。开发热带种基因组SSR分子标记,有助于甘蔗遗传多样性分析、分子标记辅助选择、遗传图谱的构建等。本研究基于热带种LA-purple的全基因组测序数据的255 398个预测基因序列(累计总长为1 029 222 285 bp),利用Perl程序与生物信息学软件结合,发掘SSR位点,获得了153 150个SSR位点,平均每1.67个基因有1个SSR位点,其中二、三核苷酸重复基序分别为39 556个和50 072个,占总SSR位点数的58.5%。在二核苷酸重复基序中,TA/AT所占比例最高,占41.4%,CG/GC所占比例最低,占4.6%;在三核苷酸碱基重复基序中,TGT/ACA所占比例最高,为15.6%。在TA/AT重复类型中选取100个基序重复次数在60~90之间的SSR位点,进行引物设计与合成,在12个甘蔗属材料中进行PCR扩增分析,从中筛选出52对具有多态性SSR引物,其中有27对引物在研究的2个甘蔗栽培品种间表现为多态。这些基因组SSR标记的开发,不仅可以用于甘蔗栽培品种DNA指纹图谱分析,而且为甘蔗属不同种的遗传图谱构建、遗传多样性分析和重要性状的遗传机制解析奠定基础,为甘蔗分子育种研究提供重要支撑。

Modern sugarcane cultivars(2n=100–130)are derived from interspecific hybridization and backcross breeding between Saccharum officinarum(2n=80)and Saccharum spontaneum(2n=40–128),forming polyploid and aneuploid crops.The main components(80%–90%)of the sugarcane cultivars’genome are originated from S.officinarum.The development and mining of genomic SSR molecular marker of S.officinarum,will benefit sugarcane genetic diversity analysis,molecular marker assisted selection,and construction of genetic maps.In this study,we explored the SSR loci from 255 398 predicted gene sequences(with a cumulative length of 1 029 222 285 bp)derived from the whole genome sequencing project of a S.officinarum clone LA-purple,by combining Perl program with bioinformatics software.A total of 153 150 SSR loci,with an average of 1.67 genes per SSR locus,were identified,of which 39 556(25.8%)were dinucleotide repeat motifs and 50 072(32.7%)were tri-nucleotide repeat motifs.Among the dinucleotide repeat motifs,TA/AT had the highest proportion,accounting for 41.4%,while CG/GC had the lowest proportion,accounting for only 4.6%.Among the trinucleotide repeat motifs,TGT/ACA had the highest proportion,accounting for 15.6%.One hundred SSR loci with 60–90 repeats of TA/AT motifs were selected and analyzed by PCR amplification in 12 representative Saccharum clones,of which 52 were polymorphic among the 12 clones and 27 were polymorphic between the tested two modern sugarcane cultivars.The genome-wide development of these gene-based SSR markers will not only facilitate the DNA fingerprinting analysis of sugarcane cultivars,but also help to construct the genetic maps,analyze the genetic diversity,study the genetic mechanism of important traits in Saccharum species,and provide important support to the molecular breeding in sugarcane.