流式细胞术估测枣染色体倍性和基因组大小方法的建立及应用  

Establishment and Application of a Method for Chromosome Ploidy Identification and Genome Size Estimation Using Flow Cytometry in Ziziphus jujuba

作　　者：王利虎 吕晔[1,2] 罗智 刘平 刘孟军[1,2] 

机构地区：[1]河北农业大学中国枣研究中心,保定071001 [2]河北省枣产业技术研究院,保定071001 [3]河北农业大学林学院,保定071001

出　　处：《农业生物技术学报》2018年第3期511-520,共10页Journal of Agricultural Biotechnology

基　　金：国家自然科学基金(No.31372029);国家科技支撑计划(No.2013BAD14B03);河北省自然科学基金(No.C2014204047);河北省人才培养工程项目(No.A201501026);河北省枣产业技术研究院建设引导经费(No.16967645D);河北省2017年省级研究生创新项目(No.CXZZBS2017072);北京市2011协同创新中心项目(No.PXM2017_014207_000043)

摘　　要：流式细胞术广泛应用于植物的染色体倍性鉴定和基因组大小估测。本研究拟建立一套适于枣(Ziziphus jujuba)的流式细胞术方法,为枣倍性育种和基因组分析提供技术支持。以二倍体临猗梨枣和其同源四倍体辰光为材料,系统比较了细胞裂解液种类、叶片成熟度、叶片保存方式及碘化丙啶(propidium Iodide,PI)染液用量对倍性检测效果的影响。结果表明,Tris-Mg Cl2裂解液的裂解效果最好,木本植物缓冲液(woody plant buffer,WPB)的裂解效果最差;幼嫩叶片检测效果优于成熟叶片,老化叶片不能产生清晰的主峰;在4℃和-20℃条件下,检测效果随着保存天数的增加而降低,4℃保存1~3 d或-20℃保存3~6个月后检测效果未发生明显变化,4℃保存7 d或-20℃保存12个月仍可进行测定;综合考虑成本和检测效果,PI染液适宜用量为30μL。利用建立的枣流式细胞术倍性检测方法进行倍性检测,确定枣和酸枣(Z.acidojujuba)三倍体和二倍体的荧光强度比值为1.43~1.60,四倍体和二倍体荧光强度比值为1.70~2.11,六倍体和二倍体荧光强度比值为2.90~3.27。估测出的冬枣(组培苗)基因组大小为449.94±3.60 Mb,与其基因组测序结果误差仅为1.33%;同时估测出了献县酸枣、台南1号毛叶枣(Z.mauritiana)(多倍体)、滇刺枣(野生二倍体毛叶枣)基因组大小分别为404.25±2.33,1 349.73±8.22,462.97±8.72 Mb。本研究建立的利用流式细胞术鉴定枣属植物染色体倍性和估测枣属植物基因组大小的方法,操作简单、准确、省时省力,1人1 d内可完成对200余个样品的测定,该方法还适用于梨(Pyrus bretschneideri)、葡萄(Vitis vinifera)、杨树(Populus)和白菜(Brassica rapa)等植物。本研究结果可为枣属植物倍性育种和组学研究提供技术支撑,同时为其他植物提供借鉴参考。Flow cytometry has been widely used in chromosome ploidy identification and genome size estimation in plants. This research is to establish a flow cytometry method suitable for jujube （Ziziphus jujuba） in order to provide technological supports for jujube ploidy breeding and genome analysis. Chinese jujube ＇Linyilizao＇ （diploid） and its autotetraploid ＇Chenguang＇ were employed as materials to systematically compare the ploidy detection effects of cell lysis solution types, maturity level of leaves, preserved way of leaves, and dosage of propidium iodide （PI）. The results showed that cell lysis solution of Tris-MgCl2, woody plant buffer （WPB） and general purpose buffer （GPB） had clear peak when the ＇Linyilizao＇ was as the test material. The GPB and Tris-MgCl2 had clear peak, while WPB not showed peak when the ＇Chenguang＇ was as the test material. In general, cell lysis solution of Tris-MgCl2 showed better results than cell lysis solution of WPB and GPB. So the cell lysis solution of Tris-MgCl2 was the best for cell lysis and WPB was the worst. Young leaves showed better result than mature leaves, and senescent leaves could not make clear peak. With the increase of preserved period of leaves, the testing effects slightly decreased under 4 ℃ and -20 ℃. There was no significant difference among the test effects of leaves preserved 1～3 d at 4 ℃ or 3～6 months at -20 ℃. And the leaves preserved 7 d at 4 ℃ or 12 months at -20 ℃ could still be used for ploidy identification. In consideration of cost and detection effect of different dosages of PI, 30 μL PI was optimal. By using the above established method for ploidy identification, the ratio of fluorescence intensity of triploid to diploid in Chinese jujube and sour jujube was in the range of 1.43～1.60. In addition, the ratio of fluorescence intensity of tetraploid to diploid was in the range of 1.70～2.11, while the ratio of hexaploid to diploid was 2.90～3.27. The genome size for Chinese jujube ＇Dongzao＇ was

关 键 词：枣 流式细胞术 倍性鉴定 基因组大小 

分 类 号：S665.1[农业科学—果树学]