基于流式细胞术和SNP分型的猕猴桃倍性鉴定分析

Ploidy Identification Analysis of Kiwifruit Basedon Flow Cytometry and SNP Typing

研究以猕猴桃属内不同植物的幼嫩叶片为材料,利用流式细胞术和全基因组SNP(single nucleotide polymorphism)位点杂合子等位基因深度比率(heterozygous allele depth ratio)分布2种方法进行猕猴桃倍性鉴定。对取样叶片的生长状态、防止细胞核黏连的PVP(聚乙烯吡咯烷酮)浓度、滤网目数及过滤次数、不同倍性样本全基因组SNP分型的参数调整等因素进行探索。结果表明,流式细胞术检测中取未展开的幼嫩叶片获得完整细胞核的数目最多;5%PVP对减少细胞核之间的黏连最适宜;500目滤网过滤3次效果最好。SNP的分型主要与模拟基因组的组装质量和过滤识别SNP的参数设置有关。流式细胞术鉴定倍性的关键技术是使用未展开的幼嫩叶片以保证足够数量的完整细胞核及减少细胞核之间的黏连。同一植物材料的染色体倍性在60Co-γ辐照处理前后未发生改变。全基因组SNP位点杂合子频率分布图判断的倍性与流式细胞术鉴定结果一致。2种鉴定结果可以相互验证,使倍性的判断变得更加准确,为加快猕猴桃育种提供了基础。

In this study,young leaves of different plants within the genus Kiwifruit were used as material for the identification of kiwifruit ploidy using two methods:Flow cytometry and single nucleotide polymorphism(SNP)loci heterozygous allele depth ratio distribution.The growth status of the sampled leaves,the concentration of PVP(polyvinylpyrrolidone)to prevent nuclear adhesion,the number of filter mesh and the number of filters,and the adjustment of parameters for genome-wide SNP typing of different ploidy samples were explored.The results showed that the highest number of intact nuclei was obtained from young unexpanded leaves in the flow cytometry assay;5% PVP was the most suitable for reducingthe adhesion between nuclei;500 mesh filter was the best for three times;SNP typing was mainly relatedto the quality of mock genome assembly and the parameter setting of filtering to identify SNPs. The keytechnique for ploidy identification by flow cytometry was to use young, unexpanded leaves to ensure a sufficientnumber of intact nuclei and to reduce adhesions between nuclei. Chromosomal ploidy of the sameplant material did not change before and after 60Co-γ irradiation treatment. The ploidy determined by thegenome-wide SNP locus heterozygote frequency distribution map was consistent with the flow cytometryresults, and the two identification methods could be verified against each other, making the determinationof ploidy more accurate and providing a basis for accelerating kiwifruit breeding.