基于核桃参考基因组的SSR位点鉴定分析和单态性标记开发

Genome-Wide Analysis of Microsatellite Loci and Specific Monomorphism SSR Marker Development in Walnut (Juglans regia L.) Reference Genome

[目的]为了阐明核桃(Juglans regia L.)(2n=2x=32)全基因组不同染色体上SSR位点数量及其分布特征,开发并验证单态性SSR引物。[方法]基于核桃品种‘中牧查一’的高质量参考基因组序列图谱,使用MISA软件筛选并分析SSR位点,利用Primer 3.0进行引物设计,通过电子PCR进行引物多态性分析,筛选并合成单态性SSR引物并通过真实PCR实验验证其有效性。[结果](1)在全基因组16条染色体上鉴定得到了共计357629个SSR位点,分布密度为662.28 SSRs·Mb^(−1),其中,10~30 bp长度的短序列占95.00%以上,优势重复单元以A/T碱基为主;不同染色体上SSR位点数量差异较大,其中,Chr1染色体上SSR位点数量最多,Chr16染色体上最少,SSR重复单元的数量及种类与染色体长度呈极显著正相关,进一步统计分析获得的644种稀有SSR单元中六核苷酸重复基元占多数;(2)基于不同染色体上SSR重复单元的类型和数量构建分布矩阵,聚类分析发现,以遗传距离0.075为阈值可将所有染色体分为4组,其中,第4组中成员最多(11条),而第1组中仅有Chr10染色体,总体看,Chr10染色体自成一个主枝,显示其可能经历了较为保守的进化过程;(3)利用SSR位点侧翼的保守序列设计出SSR引物303009对,通过电子PCR筛选并随机合成了32对单态性引物进行验证,其中,30对(93.75%)在6个核桃品种中扩增出了清晰的目标产物,28对(87.50%)的PCR扩增结果与电子PCR评估结果一致。[结论]本研究鉴定了‘中牧查一’核桃参考基因组不同染色体序列中的SSR位点,发现其数量和重复类型变化较大,且与染色体长度呈极显著正相关,单碱基重复的SSR是最常见的类型。建立了电子PCR与传统方法联用的引物开发流程,同时验证了其有效性,为核桃SSR引物的个性化快速开发提供了有效策略。筛选获得的28对单态性引物可为分子辅助育种中杂交子代“私生检测”等研究提供科学借鉴与参考。

[Objective]To identify the number and distribution of SSR loci on different chromosomes in the whole genome of walnut(Juglans regia L.,2n=2x=32),and to develop and validate the monomorphic SSR primers.[Method]In this study,walnut whole genome sequencing data were used as experimental materials,and the whole genome microsatellites were screened and analyzed by bioinformatics software MISA.Primer 3.0 was employed to design monomorphic SSR primers.SSR primers were evaluated by electronic PCR and some of the monomorphic ones were synthesized randomly to detect their usefulness and verify the effectiveness of the method.[Result](1)A total of 357629 SSR loci were identified in the walnut genome,with a distribution density of 662.28 SSRs/MB.The dominant repeat units were mainly A/T bases,showing significant base preference.These SSR sequences were mainly short sequences with a length of 10~30 bp,up to more than 95.00%.The number of SSR loci on different chromosomes varied greatly.Among them,the number of SSR loci on chromosome 1 was the largest,and the numer of SSR loci on chromosome 16 was the least.The number and type of SSRs showed positively correlated with chromosome sequence length.Most of the 644 rare SSR units were hexa-nucleotides.(2)Based on cluster analysis,all the 16 chromosomes could be divided into 4 groups,of which the number of members in group 4 was the most(11),and there was only chromosome 10 in group 1.In general,chromosome 10 forms a main branch,indicating that it may have experienced a relatively conservative evolutionary history.(3)303009 pairs of SSR primers were designed by using the conservative sequence flanking the SSR locus.And then 32 pairs of monomorphic primers clarified by electronic PCR were randomly screened and synthesized for wet PCR experiments,of which 30 pairs(93.75%)were amplified in 6 walnut varieties.The PCR amplification results of 28 pairs(87.50%)were consistent with that of electronic PCR.[Conclusion]In this study,SSR loci in different chromosome sequences of‘Zhongmucha-1’walnut reference genome are identified.Their amounts and repeat types are found to be highly variable among different chromosome sequences and show a highly significant positive correlation with chromosome length.Mono-nucleotide repeat SSRs are the most common type.A novel protocol combining electronic PCR and traditional screening methods are established and validated,which provide an effective strategy for the personalized and rapid development of walnut SSR primers.The developed 28 pairs of monomorphic primers can provide scientific basis for“Illegitimacy Testing”of hybrid offspring in molecular marker assisted breeding.