南京林业大学学报（自然科学版）2024，Vol.48Issue(2)：61-68,8.DOI:10.12302/j.issn.1000-2006.202302014
流式细胞仪鉴定青钱柳倍性方法的建立及其应用
Establishment and application of a flow cytometry method for chromosome ploidy identification of Cyclocarya paliurus
摘要
Abstract
[Objective]To provide technical support and basic data for the germplasm identification and genetic breeding of Cyclocarya paliurus,this study determined the chromosome ploidy using flow cytometry.[Method]C.paliurus leaves were used as study materials to compare the ploidy detection effects of different nuclei isolation buffers,centrifugation treatments,and leaf preservation methods.The ploidy identification method for C.paliurus using flow cytometry was as follows:0.50-1.00 cm2 leaves of the reference sample and the test sample were mixed and chopped with 1 mL of mGb buffer.After filtration,20 μL PI was added for staining for 1 min.The ploidy of 1 395 C.paliurus germplasm resources was determined by the established method.[Result]The optimal choice for isolating nuclei and achieving a clear peak in the resulting nucleus suspension was the use of Modified Gitschier buffer(mGb).Nuclear suspensions could be directly stained after filtration without centrifugation treatment.The optimal detection effect was obtained for leaves preserved at 4 ℃,and the most suitable storage time was 7 d.Drying leaves with silica gel yielded superior test results compared to freezing them,and the most suitable storage time for silica gel drying was 150 d.The coefficients of variation of 100 test samples ranged from 2.13％to 5.04％.If the estimated value of ploidy was 1.80-2.40,the sample was identified as diploid.If the estimated value of ploidy was 3.60-4.20,the sample was identified as tetraploid.When the estimated value of ploidy was 3.00±0.40,the reference sample with the same ploidy as the initial judgment was used for the second detection.Using this method for the ploidy identification of 1 395 germplasm resources 104 diploids and 1 291 tetraploids were detected.[Conclusion]Samples with an abnormal estimated value of ploidy could be identified quickly by the second detection with the same ploidy standard.The method is simple,efficient,and accurate,and provides an effective method for ploidy identification of C.paliurus germplasm.关键词
青钱柳/倍性鉴定/流式细胞术/细胞核裂解液/内标法Key words
Cyclocarya paliurus/ploidy identification/flow cytometry/nuclei isolation buffer/internal standard method分类
农业科技引用本文复制引用
宋子琪,卞国良,林峰,胡凤荣,尚旭岚..流式细胞仪鉴定青钱柳倍性方法的建立及其应用[J].南京林业大学学报（自然科学版）,2024,48(2):61-68,8.基金项目
国家自然科学基金项目(31971642,32271959) （31971642,32271959）
江苏省重点研发计划(现代农业)重点项目(BE2019388). （现代农业）
