Study on the Identification of Potato Ploidy Using Flow Cytometry Based on Liquid Nitrogen Grinding Method

doi:10.13560/j.cnki.biotech.bull.1985.2020-0478

Abstract

Abstract:

The ploidy identification of potato chromosomes is an important part in the evaluation of potato germplasm resources. Flow cytometry can quickly and accurately determine the nuclear DNA content of cells,which is widely used to identify plant’s chromosome ploidy. A high-throughput flow cytometry system that is suitable for potato ploidy identification is established for providing evidences for potato breeding. Twenty pieces of potato cooperative 88,progeny of an induced parthenogenesis,were used as materials,a liquid nitrogen grinding method was used to prepare leaf cell nuclear suspension,which was then compared with the leaf cell nuclear suspension by traditional blade shredding method,and chromosome ploidy of the known tetraploid potato cooperative 88 and diploid potato IVP101 was determined. It was found that there was no significant difference between the ploidy identification results by the two methods,but the liquid nitrogen grinding method was simple and less time-consuming. The flow cytometry based on the liquid nitrogen grinding method quickly and accurately identified potato ploidy. In addition,fluorescent staining time（from 15 min to 12 h）of the nucleus suspension prepared by the liquid nitrogen grinding method did not affect the ploidy determination results,which is convenient for researchers to flexibly choose the staining time in actual operation.

Key words: liquid nitrogen grinding method, potato, ploidy identification, flow cytometry

ZHENG Ying-zhuan, LÜ Yan, YANG Dong-xu, LI Guo-wei, WANG Hong-yang, LI Can-hui. Study on the Identification of Potato Ploidy Using Flow Cytometry Based on Liquid Nitrogen Grinding Method[J]. Biotechnology Bulletin, 2021, 37(1): 282-288.

Figures/Tables 4

References 17

[1]	Brown M, Wittwer C. Flow Cytometry:Principles and clinical applications in hematology[J]. Clinical Chemistry, 2000,46(8):1221-1229.
[2]	Miyashita T, Araki H, Hoshino Y. Ploidy distribution and DNA content variations of Lonicera caerulea(Caprifoliaceae)in Japan[J]. Journal of Plant Research, 2011,124(1):1-9. URL pmid: 20422248
[3]	Mallón R, Rodríguez-Oubiña J, González ML. In vitro propagation of the endangered plant Centaurea ultreiae:assessment of genetic stability by cytological studies, flow cytometry and RAPD analysis[J]. Plant Cell, Tissue and Organ Culture, 2010,101(1):31-39.
[4]	弓娜, 田新民, 周香艳, 等. 流式细胞术在植物学研究中的应用——检测植物核DNA含量和倍性水平[J]. 中国农学通报, 2011,27(9):21-27.
	Gong N, Tian XM, Zhou XY, et al. Applications of flow cytometry in plant research——Analysis of nuclear DNA content and ploidy level in plant cells[J]. Chinese Agricultural Science Bulleti, 2011,27(9):21-27.
[5]	Sandoval A, Hocher V, Verdeil JL. Flow cytometric analysis of the cell cycle in different coconut palm(Cocos nucifera L.)tissues cultured in vitro[J]. Plant Cell Reports, 2003,22(1):25-31.
[6]	孟芝君, 武文明. 流式细胞仪倍体分析的原理及其应用进展[J]. 检验医学与临床, 2011,8(18):2249-2251.
	Meng ZJ, Wu WM. The principle and application progress of ploidy analysis by flow cytometry[J]. Laboratory Medicine and Clinic, 2011,8(18):2249-2251.
[7]	Watanabe K. Potato genetics, genomics, and applications[J]. Breeding Science, 2015,65(1):53-68.
[8]	陶抵辉, 李小红, 王利群, 等. 植物染色体倍性鉴定方法研究进展[J]. 生命科学研究, 2009,13(5):453-458.
	Tao DH, Li XH, Wang LQ, et al. Progresses on determination of cell chromosome ploidy level of plants[J]. Life Science Research, 2009,13(5):453-458.
[9]	王婷婷, 张光海, 蔡汶玻, 等. 利用流式细胞法鉴定马铃薯倍性方法的优化[J]. 中国马铃薯, 2019,33(1):1-7.
	Wang TT, Zhang GH, Cai WB, et al. Optimization of potato ploidy identification method using flow cytometry[J]. Chinese Potato Journal, 2019,33(1):1-7.
[10]	裴丹, 葛孟清, 董天宇, 等. 208个葡萄品种染色体倍性的流式细胞分析[J]. 中外葡萄与葡萄酒, 2019(5):21-28.
	Pei D, Ge MQ, Dong TY, et al. Analysis of ploidy of 208 grape varieties by FCM[J]. Sino-overseas Grapeving and Wine, 2019(5):21-28.
[11]	吕顺, 任毅, 王芳, 等. 利用流式细胞术快速鉴定169份香蕉种质资源的染色体倍性[J]. 果树学报, 2018,35(6):668-684.
	Lv S, Ren Y, Wang F, et al. Ploidy identification of 169 Musa germplasms by flow cytometry[J]. Journal of Fruit Science, 2018,35(6):668-684.
[12]	赖彪, 吴传龙, 秦永华, 等. 流式细胞仪测定荔枝倍性和基因组大小的细胞核提取液筛选与应用[J]. 果树学报, 2019,36(7):939-946.
	Lai B, Wu CL, Qin YH, et al. Nuclear isolation buffer optimization and the determination of ploidy level and genome size of litchi using flow cytometry[J]. Journal of Fruit Science, 2019,36(7):939-946.
[13]	龚思, 梁少华, 严静怡, 等. 西瓜未受精子房离体培养技术[J]. 中国瓜菜, 2019,32(5):17-21.
	Gong S, Liang SH, Yan JY, et al. Study on the technology of unfertilized ovary culture in watermelon[J]. Zhongguo Gua-cai, 2019,32(5):17-21.
[14]	田路明, 齐丹, 曹玉芬, 等. 利用流式细胞仪鉴定梨种质资源染色体倍性[J]. 中国果树, 2018(3):29-32.
	Tian LM, Qi D, Cao YF, et al. Ploidy identification of pear germplasm by flow cytometer[J]. Zhongguo Guo-shu, 2018(3):29-32.
[15]	朱道圩, 杨宵, 郅玉宝, 等. 用流式细胞仪鉴定中华猕猴桃的多倍体[J]. 植物生理学通讯, 2007,43(5):905-908.
	Zhu DX, Yang X, Zhi YB, et al. Identification of polyploidy in Actinidia chinensis Planch. by flow cytometer[J]. Plant Physiology Communications, 2007,43(5):905-908.
[16]	高友汉, 韩冰. 利用流式细胞仪鉴定9个居群蒙古冰草的染色体倍性[J]. 畜牧与饲料科学, 2013,34(6):1-3.
	Gao YH, Han B. Chromosome ploidy determination of 9 populations of Agropyron mongolicum keng by flow cytometry[J]. Animal Husbandry and Feed Science, 2013,34(6):1-3.
[17]	柳斌, 任红剑, 李美荣, 等. 利用流式细胞仪鉴定槭树染色体倍性的方法探究[J]. 山东林业科技, 2019,49(5):18-21.
	Liu B, Ren HJ, Li MR, et al. Research on the method of measuring the ploidy of Acer L. by flow cytometry[J]. Shandong Forestry Science and Technology, 2019,49(5):18-21.