倍性复杂植物的2种倍性鉴定方法的建立及应用(专题约稿）

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。

Establishment and application of two ploidy identification methods for ploidy complex plants

[Objective] Plant ploidy identification is the basis for research on biological problems such as classification and ploidy breeding. Ploidy identification of complex ploidy species has been the focus of researchers" attention. That identification has become a difficult task especially for plants with a large number of chromosomes, small morphology, dense cytoplasm, and complex chromosome ploidy of species within the genus, and it is of great importance to explore a rapid and effective ploidy identification method. [Methods] In this study, young leaves of different plants in the genus Kiwifruit were used as material for ploidy identification using two methods - flow cytometry and single nucleotide polymorphism (SNP) loci heterozygous allele depth ratio (HADR) distribution. We explored the factors affecting ploidy identification, such as the growth status of the sampled leaves, the concentration of PVP (polyvinylpyrrolidone), the number of filter mesh and the number of filters, and the adjustment of parameters for whole-genome SNP typing of different ploidy samples. [Results] The highest number of intact nuclei was obtained by taking unexpanded young leaves in the flow cytometry assay; a 5% PVP concentration was the most suitable for reducing the adhesion between nuclei, 2% was not effective, and 7% caused no significant change. The 500 mesh filter used three times gave the best results; more impurities were found after the use of the 200 mesh and 300 mesh filtrations. SNP genotyping is mainly related to the quality of the assembly of the simulated genome and the parameter settings for filtering the identified SNPs. [Conclusion] The use of young unexpanded leaves to ensure a sufficient number of intact nuclei and reduce the adhesion between nuclei is the key to ploidy identification by flow cytometry. Chromosome ploidy of the same plant material did not change before and after CO60 the irradiation treatment. The assembly of high quality reference genome is the basis of SNP typing and the guarantee of heterozygote frequency distribution estimation at SNP loci. The results of the 2 ploidy identification methods in ploidy complex plants were consistent and could be validated against each other.