中国玉米审定品种标准SSR指纹库的构建

摘要

[目的]对数量庞大的已知玉米品种构建可共享的作物品种标准DNA指纹库.[方法]基于荧光毛细管电泳检测平台和植物品种DNA指纹库管理系统,利用筛选的40对SSR核心引物对3998份中国玉米审定品种标准样品进行建库,通过多实验室、多检测平台进行建库数据的质量评估.[结果]绘制了40个玉米建库引物的等位基因频率分布图作为每个引物的特征图谱,在建库试验中发挥了相当于参照样品的作用.形成了一套十重荧光毛细管电泳组合,并在SSR指纹分析器中建立了一套系统默认PANEL作为不同实验室建库时的标准PANEL.统计玉米审定品种指纹库构建的试验情况,每份样品具有2—5套原始试验数据及对应的指纹图谱,其中61%的样品做了2组独立试验,33%的样品做了3组独立试验,最终建成的标准指纹库累计缺失和差异位点仅占0.2%,数据完整性达到99.8%.在不同实验室、不同电泳检测平台的评估结果表明,同一荧光电泳检测平台上获得SSR指纹数据一致性高,而不同的电泳检测平台获得的数据存在一定偏差,为实现不同实验室的SSR指纹数据共享,需要统一荧光引物、分析软件及电泳检测平台.对所有审定品种指纹数据进行整体两两比较,表明中国玉米审定品种之间差异比较大,品种间差异位点百分比集中在80%—95%(占78.28%),品种间差异位点百分比在50%以上的已达到99.21%,而低于20%的只有0.09%;对玉米审定品种杂合率水平进行分析,平均品种杂合率达到64%,主要集中在50%—80%(占89%).通过玉米品种标准指纹比对服务平台(网址:http://www.maizedna.org/)实现了指纹库的共享.[结论]形成了构建作物品种SSR指纹库的标准化程序,构建了3998份玉米审定品种的SSR指纹库,通过多实验室联合比较试验,保证建库数据的准确性和数据库的可共享性;建立了玉米品种标准指纹比对服务平台网站,实现玉米审定品种指纹库在全国种子检验系统的共享.%[Objective] It is of great importance to construct a shareable high-quality crop variety standard DNA fingerprint database for effectively managing the huge number of known varieties.[Method] Based on fluorescence capillary electrophoresis detection platforms and the plant variety DNA fingerprint database management system, a database containing 3998 maize authorized accessions was built with 40 SSR primer pairs. Multi-laboratories and multi-detecting platforms were used to conduct the quality evaluation of the database.[Result] Allele frequency distribution graphs of the 40 corn primers were plotted as characteristic spectrums of each primer, which played the role of the similarity of reference samples in the database construction. A decuplet fluorescent capillary electrophoresis combination was formed and a set of system default PANEL was established in the SSR analyzer. Statistics were conducted on the experimental conditions of the database construction. Of the total samples, 61% of them were subjected to two group independent trials and 33% of them were subjected to three group independent trials. Each sample had 2-5 sets of original experimental data and the corresponding fingerprint maps. The accumulated loss and variable sites of the final built standard fingerprint database accounted for only 0.2%, the data integrity reached 99.8%. The assessment results in different laboratories and different electrophoresis platforms showed that the SSR fingerprint data obtained high agreement on the same electrophoresis fluorescence detection platform, but showed a certain bias in different electrophoretic detection platforms. In order to realize the sharing of SSR fingerprint data in different laboratories, a unified fluorescent primer, analysis software and electrophoresis detection platform were needed. Overall pairwise comparisons were conducted on all the fingerprint data, the results showed that there existed a relative big overall difference among the certification varieties of corn in China. Percentage of different sites among the authorized varieties was mainly concentrated between 80% and 95% (accounted for 78.28%), the percentage of different sites with more than 50% reached 99.21%, and less than 20% of only 0.09%. The average hybrid rate of the authorized varieties reached 64% and mainly concentrated between 50% and 80% (accounted for 89%). By using the corn variety standard fingerprint matching service platform (URL: http: //www.maizedna.org/), a shared fingerprint database is realized. [Conclusion] A standard procedure in constructing crop variety SSR fingerprint database was formed in this study and the SSR fingerprint database was constructed with a scale of nearly 4000 corn authorized varieties. Through joint multi-laboratory comparison tests, the accuracy of database building and the sharing property of database were ensured. A service platform website for corn variety standard fingerprint matching was established in this study, thus achieving sharing of corn authorized variety fingerprint database in national seed identification system, and providing an important reference for other crop species in building high-quality SSR fingerprint database.