植物品种SSR指纹分析专用软件SSR Analyser的研发

doi:10.3864/j.issn.0578-1752.2018.12.003

摘要/Abstract

摘要： 【目的】开发适用于植物品种SSR指纹分析的软件工具，实现植物品种SSR指纹分析的自动化和标准化，解决SSR标记在实际应用中存在的数据采集效率较低、数据共享难度较大等问题。【方法】在商业化软件GeneMarker®的基础上，针对植物品种SSR指纹分析的特殊性，在SSR指纹处理、panel设计、数据库对接等方面进行算法开发或优化，形成植物品种定制化软件SSR Analyser，并在玉米等多种作物上测试其分析效果。【结果】在SSR指纹处理功能上，软件通过先用系统计算的矩阵进行弱消除，再用Pull-up峰匹配算法进行单峰消除的方案实现了对pull-up峰准确自动消除；通过优化N+1峰、连续多峰等特殊峰型读取的算法，解决了特异峰不识别、读不准、误读等问题，对2 bp重复类型SSR标记为主的植物品种指纹采集更加精准；通过完善邻峰过滤、高低峰过滤和二倍体过滤算法，解决了植物品种混合样品的有效峰采集问题。在Panel设计功能上，软件兼顾了Panel设计的灵活性、方便性和统一性，在保证数据采集标准化的前提下，更加适应复杂的试验情况：提高了标记参数设置的灵活性，根据不同参数作用范围，标记参数设置既有针对特定物种、Panel一次性固定设置的参数，也有针对每个引物位点、每块电泳板单独设定或微调的参数；实现了从已有标记中快速重新组合形成新panel的功能；保证了panel的统一调用和同步更新。通过将软件与指纹库管理系统的无缝对接，实现了样品准备到指纹采集全流程的自动化、标准化，形成的指纹库具有直观可追溯的优势。将SSR Analyser在玉米大规模建库中试用，表明该软件的指纹分析更加简单高效，比原软件分析效率提高了10倍以上；将SSR Analyser扩大到水稻、大豆、黄瓜、西瓜、大白菜等多种二倍体作物和小麦、棉花等多倍体作物中试用，表明在二倍体作物上使用效果较好；在多倍体作物上，对其中的二倍体化的标记使用效果较好，但对非二倍体化的标记仍需进一步完善过滤算法。【结论】开发的SSR Analyser软件具有数据分析程序简单高效、对特殊峰型的SSR标记指纹采集精准、适合基于混合样品的植物品种SSR指纹采集、与指纹库管理系统无缝对接的优点，大大改善了SSR标记在植物品种鉴定中的应用效果。

关键词: 植物品种, SSR, DNA指纹, 荧光毛细管电泳, 软件开发

Abstract: 【Objective】Develop software tools for plant variety identification by SSR fingerprinting to realize the automatic and standardized analysis of plant variety identification, solving problems of low efficiency of data collection and hard for data sharing et al for SSR markers in practice. 【Method】Based on commercialized software GeneMarker®, develop and optimize algorithms to deal with the specialty of SSR fingerprinting analysis for data analysis, panel design, database synchronization et al. SSR Analyser is generated as personalized software and tested on maize and other crops for its effectiveness. 【Result】From the perspective of processing function, the software is able to first weakly eliminate pull-up peak using matrix by system calculation. Then, use matching algorithm with single peak removal method to completely remove pull-up peak automatically and correctly. By optimizing the reading algorithm of N+1 peak, stutter peak et al, unrecognized/inaccurate/mis-reading of special peak is solved. Therefore, it is more accurate for Dinucleotide SSR markers’ fingerprinting. By completing the filtering algorithm of neighboring peak, high and low peak, and diploid crops, it solves the problem of effective peak collection for blended samples. From the perspective of panel design, the software balances the flexibility, convenience and uniformity. On the premise of standardized data collection, the software is more suitable for complicated experiment: enhances the flexibility of marker parameter settings, which include setting parameters at one time for specific species using panels, or setting parameters individually for electrophoresis gel for each primer locus; realizes that generating new panels from existing markers; ensures the unification by calling panels and its synchronous updating. By seamless connecting the software and fingerprinting database management system, it realizes the automation and standardization from sample preparation to fingerprint collection, which makes the database more intuitive and traceable. Using the SSR Analyser for building database of maize indicates that the software is more efficient than the original software, which is 10 times of efficiency; After expanding the SSR Analyser’s application scope to rice, soybean, cucumber, watermelon, Chinese cabbage and other diploid crops, and polyploidy crops such as wheat and cotton, suggests that it is more applicable to diploid crops for polyploidy crops, markers developed based on diploid principle have better application. However, other types of markers need optimization on filtering algorithm. 【Conclusion】SSR Analyser is simple and effective in data analysis, which can accurately collect special peak for SSR markers. It is suitable for fingerprint collection of mixed samples and seamless connects with fingerprint database system, which greatly improves the application of SSR markers in plant variety identification.

Key words: plant varieties, SSR, DNA fingerprinting, fluorescent capillary electrophoresis, software development

王凤格，李欣，杨扬，易红梅，江彬，张宪晨，霍永学，朱丽，葛建镕，王蕊，任洁，王璐，田红丽，赵久然. 植物品种SSR指纹分析专用软件SSR Analyser的研发[J]. 中国农业科学, 2018, 51(12): 2248-2262.

WANG FengGe, LI Xin, YANG Yang, YI HongMei, JIANG Bin, ZHANG XianChen, HUO YongXue, ZHU Li, GE JianRong, WANG Rui, REN Jie, WANG Lu, TIAN HongLi, ZHAO JiuRan. SSR Analyser：A Special Software Suitable for SSR Fingerprinting of Plant Varieties[J]. Scientia Agricultura Sinica, 2018, 51(12): 2248-2262.

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