新疆本地梨品种基因组大小及倍性的流式细胞术检测优化  

Optimization of Flow Cytometry for the Determination of Genome Size and Ploidy of Local Pear(Pyrus)Varieties in Xinjiang

作　　者：牛莹莹[1] 周伟权 陈湘颖 丁想 董胜利 章世奎[3] 廖康[1] NIU Ying-Ying;ZHOU Wei-Quan;CHEN Xiang-Ying;DING Xiang;DONG Sheng-Li;ZHANG Shi-Kui;LIAO Kang(Research Centre of Characteristic Fruit Tree/College of Horticulture and Forestry,Xinjiang Agricultural University,Urumqi 830052,China;Turpan Institute of Agricultural Sciences,Xinjiang Academy of Agricultural Sciences,Turpan 838000,China;Luntai National Fruit Germplasm Resources Garden,XinjiangAcademy ofAgricultural Sciences,Luntai 841600,China)

机构地区：[1]新疆农业大学林学与园艺学院/特色果树研究中心,乌鲁木齐830052 [2]新疆农业科学院吐鲁番农业科学研究所,吐鲁番838000 [3]新疆农业科学院轮台果树资源圃,轮台841600

出　　处：《农业生物技术学报》2020年第5期933-944,共12页Journal of Agricultural Biotechnology

基　　金：新疆维吾尔自治区园艺学重点学科基金(2016-10758-3)。

摘　　要：优化测定新疆本地梨(Pyrus)品种基因组大小及染色体倍性的流式细胞检测技术,可对新疆本地梨品种的基因组大小及倍性进行更为高效的检测和鉴定。本研究以二倍体’砀山酥梨’为参照,采用优化后的流式细胞术检测方法对29份新疆本地梨品种进行基因组大小估测及染色体倍性的鉴定。检测流程:以一年生枝水培嫩叶为试材,用蒸馏水、去离子水各冲洗2~3次,采用木本植物缓冲液(woody plant buffer, WPB)进行解离,先加入1 m L,切碎后再加入1 mL;500目滤膜过滤1次;4℃冰箱孵育5 min;4℃,1 000 r/min离心5 min;弃上清液至0.1 mL,分别加入200μL的WPB解离液及碘化丙啶(propidium iodide, PI)染液,避光染色10 min后上机检测。结果表明,检测的29个新疆本地梨品种基因组大小均值为(566.54±125.17) Mb,检测出1个嵌合体,3个三倍体及1个四倍体。其中,’也历克阿木特’基因组大小为(666.80±154.56) Mb,同时出现二倍体和三倍体染色体峰,为嵌合体;’可特阿木特’、’艾温切可’、’黑酸梨’基因组大小分别为(721.12±20.87) Mb、(791.33±36.84) Mb、(725.16±76.40) Mb,均为三倍体;’沙01’基因组大小为(1032.61±49.41) Mb,为四倍体。24个二倍体品种的基因组大小在(480.95±16.24)^(599.14±38.36) Mb之间,相差120 Mb;3个三倍体品种的基因组大小在(721.12±20.87)^(791.33±36.84) Mb之间,相差70 Mb。本研究优化了新疆本地梨品种基因组大小估测和染色体倍性鉴定的流式细胞术检测方法,操作简单、耗时短,不足之处是染色体特征信息无法获取。本研究结果可为今后梨倍性育种及基因组学研究提供技术支持。Optimizing a flow cytometry method technical scheme for determining genome size and ploidy of local pear(Pyrus)varieties in Xinjiang can detect and identify the genome size and ploidy more efficiently.Different source materials(dry method to store new shoot tender leaves,annual branch hydroponic tender leaves,autumn shoots tender leaves,seed cultivation cotyledon and seed cultivation tender leaves)of local pear varieties in Xinjiang,different mechanical dissociation methods(blade chopped method,grinding method),membrane filtration times(500 mesh filter once and filter twice)and propidium iodide(PI)dyeing liquid dosage(200 and 300μL)were compared based on the detection results that taken diploid'Dangshansuli'as reference.The results showed that the genomic size estimation and chromosome ploidy identification of 29 local pear varieties in Xinjiang were carried out by using the optimized detection method.The optimization method as follow:Young leaves which from annual branches hydroponic tender leaves in spring were collected 0.1 g,washed with distilled water and deionized water for 2 to 3 times,respectively.Then wipe it dry and put into a culture dish which had been added with 1 mL precooled woody plant buffer(WPB)dissociation solution,and chopped up quickly with a sharp blade.After chopping,add 1 mL WPB dissociation solution,mix and stand for 2~5 min.The mixture liquid was filtered with 500 mesh membrane into 2.0 mL centrifuge tube and incubated at 4℃for 5 min.The single cell nuclear suspension were got from the treated mixture liquid by centrifuge at 4℃at 1000 r/min for 5 min.Retain 0.1 mL supernate at bottom and add 200μL precooled WPB,200μL precooled PI,mix well.Place it in refrigerator at 4℃and dye for 10 min without light.After staining,detect it with flow cytometer,and collect 5000 mononuclear particle granules.Using this method,the results showed that the mean genome size of local pear varieties in Xinjiang detected was(566.5±125.17)Mb.The genome size of'Yilikamut'was(666.80±154.56)Mb,and it was

关 键 词：新疆本地梨品种 流式细胞术 基因组大小 倍性 

分 类 号：S661.2[农业科学—果树学]