黄花菜染色体制片及荧光原位杂交技术体系的建立  

Establishment of chromosome preparation and of fluorescence in situ hy bridization technique system of Hemerocallis citrina Barni

作　　者：武江 李森[1] 李子瑜 武丹阳 Wu Jiang;Li Sen;Li Ziyu;Wu Danyang(College of Horticulture,Shanxi Agricultural University,Taigu 030801,China)

机构地区：[1]山西农业大学园艺学院,山西太谷030801

出　　处：《山西农业大学学报（自然科学版）》2020年第6期13-20,共8页Journal of Shanxi Agricultural University(Natural Science Edition)

基　　金：山西省科技厅软科学项目(2019041030)。

摘　　要：[目的]黄花菜(Hemerocallis citrina Barni)是我国特有的经济型蔬菜,目前急需加速分子育种进程,开发遗传学平台。本文旨在优化黄花菜染色体制片技术,构建黄花菜荧光原位杂交体系,为遗传图谱与物理图谱的整合提供技术基础。[方法]以大同黄花菜(H.cv.‘DatongHuanghua’)为研究材料,分别对中期根尖细胞和粗线期花粉母细胞的制片方法进行优化,同时以拟南芥45S rDNA和5S rDNA序列为探针,筛选和优化黄花菜荧光原位杂交体系技术参数。[结果]中期染色体制片最佳体系:2.9μg·mL-1浓度的八羟基喹啉溶液处理4 h,混合酶液(纤维素酶∶果胶酶=2∶1)37℃酶解160 min;粗线期染色体制片最佳体系:采集4~5 mm龄段花蕾,卡诺固定液4℃固定24 h后,混合酶液(纤维素酶∶果胶酶=2∶1)37℃酶解220 min,使用改良火焰干燥法,均可得到理想制片。采用直接标记法对45S rDNA和5S rDNA探针序列进行荧光标记,结果显示杂交信号清晰稳定,背景干扰小。[结论]本研究构建并优化了黄花菜染色体制片技术及荧光原位杂交体系,可为黄花菜细胞遗传学研究和基因组研究的深入开展提供技术基础。[Objective]Hemerocallis citrina Barni is a unique economical vegetable in China.There is an urgent need to accelerate the molecular breeding process and develop several genetic platforms.The purpose of this research was to optimize the chromosome preparation technology of H.citrina establish the fluorescence in situ hybridization(FISH)system,and to provide technical basis for the integration of genetic and physical maps.[Methods]The preparation methods of root tips cells at metaphase stage and pollen mother cells at pachytene stage were optimized using Hcitrine.cv.‘DatongHuanghua’as the research material.The system of FISH technical parameters were screened with two probes of Arabidopsis 45 S and 5 S rDNA sequences.[Results]The optimal preparation system for metaphase chromosome production was determined as following:the material was treated with 8-hydroxyquinoline solution at concentration of 2.9μg·mL-1 for 4 h,then kept in mixed enzyme solution(cellulase∶pectinase=2∶1)for 160 min at 37℃.The optimal system for chromosome production at pachytene stage:collected 4-5 mm length flower buds,fixed in Carnot fixation solution for 24 h at 4℃,then mixed with enzyme solution(cellulase∶pectinase=2∶1)and kept at 37℃for 220 min,and desiccated with the improved flame drying method.The 45 SrDNA and 5 SrDNA probe sequences were synthesized by direct labeling method,and the results showed that the hybridization signals were stable and clear with little background interference.[Conclusion]This research constructed and optimized the chromosome preparation method and FISH technical system of H.citrina,and provided a technical basis for the further study of cytogenetics and genomics on this species.

关 键 词：黄花菜 染色体制片 根尖中期细胞 花粉母细胞粗线期 荧光原位杂交 

分 类 号：S682[农业科学—观赏园艺]