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)
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1.陕西理工大学;2.西北大学生命科学学院生物学专业 陕西西安

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本研究由陕西理工大学秦巴生物资源与生态环境省部共建重点实验室(培育)“市校共建”科研专项(SXC-2102) 和陕西省科学技术厅项目(2021NY-085)共同资助


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

    【目的】植物倍性鉴定是分类、倍性育种等生物学问题研究的基础,对于复杂倍性物种的倍性鉴定一直是研究人员关注的焦点,特别是对于具有染色体数目多、形态较小、细胞质较浓、属内物种染色体倍性复杂等特点的植物的倍性鉴定成为难点,探索出快速有效的倍性鉴定方法具有重要意义。【方法】本研究以猕猴桃属内不同植物的幼嫩叶片为材料,利用2种方法: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)分布鉴定倍性。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分型的参数调整等影响倍性鉴定的因素进行探索。【结果】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%的PVP对减少细胞核之间的黏连最适宜、2%效果不佳、7%无明显变化;500目滤网过滤3次效果最好,200目和300目过滤后杂质较多。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。【结论】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。

    Abstract:

    [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.

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吴栋,王瑀,周希希,等.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)[J].西北植物学报,2023,43(8):1276-1285

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  • 收稿日期:2023-01-30
  • 最后修改日期:2023-05-20
  • 录用日期:2023-05-26
  • 在线发布日期: 2023-09-08
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