利用流式细胞仪快速鉴定棉花倍性的方法比较

doi:10.13560/j.cnki.biotech.bull.1985.2022-0783

摘要/Abstract

摘要：

利用流式细胞仪快速检测棉花DNA倍性，为棉花的倍性鉴定和育种研究提供基础数据。以陆地棉珂字棉312（Gossypium hirsutum Linn.）的老叶和嫩叶为试材，用WPB和LB01解离液提取细胞核，经PI染色后使用流式细胞仪检测细胞倍性。结果发现，用WPB解离液提取棉花嫩叶获得的细胞核用于倍性检测效果更好，表现为主峰离子团清晰而集中、背景碎片少，可以得到明显的主峰、杂峰更少，且收集到的细胞核数目更多。初步建立了利用流式细胞仪快速检测棉花DNA倍性的方法。

关键词: 棉花, 流式细胞术, 细胞核DNA含量, 染色体倍性

Abstract:

Rapid detection of cotton DNA ploidy by flow cytometry provides basic data for cotton ploidy identification and breeding research. The old and young leaves of Gossypium hirsutum Linn. Coker 312 were used as test material，and cell nuclei were extracted using WPB and LB01 dissociation solution and stained with PI for the ploidy detection by flow cytometry. The result showed that nuclei extracted from the cotton young leaves using WPB dissociation solution were more effective for ploidy detection，which had clear and focused peak particle，little background debris and obtained clear peak，less miscellaneous peak，less cellular debris and more nuclei. In conclusion，a method for rapid detection of cotton DNA ploidy by flow cytometry is initially established.

Key words: Gossypium hirsutum Linn., flow cytometry, nuclear DNA content, chromosome ploidy

王娅丽, 周利利, 王娜, 程红梅. 利用流式细胞仪快速鉴定棉花倍性的方法比较[J]. 生物技术通报, 2022, 38(12): 144-148.

WANG Ya-li, WANG Na, CHENG Hong-mei. Comparison of Methods for Rapid Determination of Cotton Ploidy by Flow Cytometry[J]. Biotechnology Bulletin, 2022, 38(12): 144-148.

图/表 1

参考文献 23

[1]	Okubazghi KW, Li XN, Cai XY, et al. Genome-wide assessment of genetic diversity and fiber quality traits characterization in Gossypium hirsutum races[J]. J Integr Agric, 2017, 16(11):2402-2412. doi: 10.1016/S2095-3119(17)61671-X URL
[2]	赵孟良, 任延靖, 田闵玉, 等. 基于流式细胞仪鉴定菊芋倍性方法的建立及应用[J]. 西北农林科技大学学报:自然科学版, 2021, 49(3):138-146.
	Zhao ML, Ren YJ, Tian MY, et al. Establishment and application of aploidy method for identification of Helianthus tuberosus L. by flow cytometry[J]. J Northwest A ＆ F Univ Nat Sci Ed, 2021, 49(3):138-146.
[3]	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 Sci Res, 2009, 13(5):453-458.
[4]	马国斌, 王鸣, 郑学勤. 西瓜体细胞无性系变异初探[J]. 上海农业学报, 2003, 19(3):9-12.
	Ma GB, Wang M, Zheng XQ. Preliminary studies on somaclonal variation of watermelon[J]. Acta Agric Shanghai, 2003, 19(3):9-12.
[5]	郑思乡, 雷小云, 董志渊, 等. 离体培养条件下金鱼草多倍体诱导研究[J]. 云南林业科技, 2003, 32(4):80-83.
	Zheng SX, Lei XY, Dong ZY, et al. A study of polyploid induction of Antirrhinum majus by in vitro culture[J]. Yunnan For Sci Technol, 2003, 32(4):80-83.
[6]	杨晓明, 王翠玲. 葡萄多倍体诱导及其特征特性研究[J]. 甘肃农业大学学报, 2005, 40(6):741-744.
	Yang XM, Wang CL. Studies on induction and characteristics of grape polyploid[J]. J Gansu Agric Univ, 2005, 40(6):741-744.
[7]	刘文革, 阎志红, 张红梅, 王鸣. 不同倍性西瓜发芽种子成苗过程中的耐盐性研究[J]. 中国西瓜甜瓜, 2002, 15(3):1-2.
	Liu WG, Yan ZH, Zhang HM, et al. The Study of Salt-Tolerance in Germinating Seeds and Seedling of Different Ploidy Watermelon[J]. China Watermelion Muskmelon, 2002, 15(3):1-2.
[8]	Mustafa NS, Liu F, Odongo MR, et al. Effects of colchicine treatments on chromosome doubling in three diploid cotton species[J]. JMEST, 2017, 4(4):7140-7145.
[9]	Wang KB, Song GL, Wang CY, et al. FISH-based karyotype of Gossypium herbaceum generated with 45S rDNA and gDNA of Gossypium raimondii as probes[J]. 棉花学报, 2008, 20(4):264-273.
	Wang KB, Song GL, Wang CY, et al. FISH-based karyotype of Gossypium herbaceum generated with 45S rDNA and gDNA of Gossypium raimondii as probes[J]. Cotton Sci, 2008, 20(4):264-273.
[10]	王坤波, 王文奎, 王春英, 等. 海岛棉原位杂交及核型比较[J]. 遗传学报, 2001, 28(1):69-75, 98.
	Wang KB, Wang WK, Wang CY, et al. Studies of FISH and karyotype of Gossypium barbadense[J]. Acta Genet Sin, 2001, 28(1):69-75, 98.
[11]	Hao L, Song GL, Li BL, et al. Karyotype analysis of Gossypium arboreum × G. bickii by genome in situ hybridization[J]. Acta Genet Sin, 2006, 33(6):565-572. doi: 10.1016/S0379-4172(06)60086-3 URL
[12]	Favoreto FC, Carvalho CR, Lima ABP, et al. Genome size and base composition of Bromeliaceae species assessed by flow cytometry[J]. Plant Syst Evol, 2012, 298(6):1185-1193. doi: 10.1007/s00606-012-0620-x URL
[13]	Nath S, Mallick SK, Jha S. An improved method of genome size estimation by flow cytometry in five mucilaginous species of Hyacinthaceae[J]. Cytometry A, 2014, 85(10):833-840. doi: 10.1002/cyto.a.22489 URL
[14]	张宁宁, 杨静, 孙卫邦. 利用流式细胞仪测定荚蒾属植物的基因组大小[J]. 植物分类与资源学报, 2014, 36(6):730-736.
	Zhang NN, Yang J, Sun WB. Genome size estimation of Viburnum(Adoxaceae)species by using flow cytometry[J]. Plant Divers Resour, 2014, 36(6):730-736.
[15]	柳觐, 李开雄, 孔广红, 等. 云南芒果种质基因组大小测定与变异分析[J]. 热带亚热带植物学报, 2015, 23(4):386-390.
	Liu J, Li KX, Kong GH, et al. Genome size and variation analysis of mango(Mangifera indica L. )germplasms in Yunnan by flow cytometry[J]. J Trop Subtrop Bot, 2015, 23(4):386-390.
[16]	杨转英, 吴传龙, 丰锋, 等. 不同株系菠萝蜜染色体倍性及基因组大小分析[J]. 果树学报, 2015, 32(4):567-571.
	Yang ZY, Wu CL, Feng F, et al. Determination and analysis of ploidy and genome size of different jackfruit lines[J]. J Fruit Sci, 2015, 32(4):567-571.
[17]	张志珂, 王永清, 林顺权, 等. 借助细胞流式仪进行枇杷基因组学测序材料的倍性鉴定[J]. 果树学报, 2012, 29(3):498-504.
	Zhang ZK, Wang YQ, Lin SQ, et al. Ploidy identification of loquats for genome sequencing project by flow cytometry[J]. J Fruit Sci, 2012, 29(3):498-504.
[18]	闫东玲, 赵丽娜, 王贺新, 等. 利用流式细胞术鉴定东北地区主要越桔属植物染色体倍性[J]. 分子植物育种, 2019, 17(4):1257-1263.
	Yan DL, Zhao LN, Wang HX, et al. Identification of chromosome ploidy of Vaccinium in the northeast of China by flow cytometry[J]. Mol Plant Breed, 2019, 17(4):1257-1263.
[19]	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. Identification of chromosomal ploidy of Pyrus germplasm resources by flow cytometry[J]. China Fruits, 2018(3):29-32.
[20]	闫蕊, 阮成江, 吴波, 等. 油茶种质倍性的SSR-PAGE和SSRseq鉴定[J]. 分子植物育种, 2018, 16(19):6390-6397.
	Yan R, Ruan CJ, Wu B, et al. SSR-PAGE and SSRseq identification of ploidy on Camellia oleifera germplasm[J]. Mol Plant Breed, 2018, 16(19):6390-6397.
[21]	闫佼. 香蕉种质资源基因组类型的分子标记及倍性鉴定[D]. 南昌: 江西农业大学, 2016.
	Yan J. Ploidy and genome type identification in Musa germplasm by molecular markers and flow cytometry[D]. Nanchang: Jiangxi Agricultural University, 2016.
[22]	Peng RH, Liu F, Song GL, et al. An efficient method of nuclei extraction in cotton[J]. Agric Sci ＆ Technol, 2009, 10(3):19-21.
[23]	刘凤霞, 李京一, 王志刚, 等. 适合流式细胞仪分析的梨叶片细胞核提取缓冲液筛选[J]. 农业生物技术学报, 2018, 26(6):1034-1042.
	Liu FX, Li JY, Wang ZG, et al. The screening of the optimum buffer to produce pear(Pyrus)leaf nuclear suspensions for flow cytometry analysis[J]. J Agric Biotechnol, 2018, 26(6):1034-1042.