基于荧光检测技术的小麦品种SSR鉴定体系的建立

【目的】建立基于SSR荧光标记的小麦品种DNA指纹鉴定体系，为中国小麦育成品种鉴定提供高通量技术手段。【方法】收集已定位到小麦21条染色体上的SSR标记引物，通过PCR扩增和变性聚丙烯酰胺凝胶电泳检测技术，筛选在中国小麦育成品种中多态性高的标记，对筛选出的引物5′末端利用6-FAM、HEX、ROX和TAMRA 4种荧光染料之一进行标记,利用DNA分析仪对扩增产物的峰型进行评价并检测不同等位变异的扩增片段大小，选择峰型简单易读、多态性高、较均匀分布到21条染色体上的标记，确定不同位点等位变异的大小及相应的参照品种，建立基于荧光SSR标记的高通量小麦品种鉴定体系。【结果】利用2 438对SSR标记对8份植物学性状差异大的小麦育成品种进行初步筛选，共筛选出260对多态性较高、扩增稳定的SSR引物。利用上述260对引物对48份小麦品种继续进行复筛，选出130对扩增稳定、多态性高、带型好的SSR引物。对这些引物的正向5′末端分别标记6-FAM、HEX、ROX和TAMRA荧光后，利用DNA分析仪进行检测评价，最终选择了42个PCR扩增稳定、峰图简单、多态性高、连锁群分布均匀的SSR荧光标记。根据DNA分析仪检测到的每个标记的不同等位变异大小，为相应位点的不同等位变异进行了命名，并为每个等位变异选取了相应参照品种。根据每个引物标记的荧光和扩增的片段大小范围对42对引物进行合理搭配，在同一毛细管内对多个荧光标记进行检测，提高了DNA指纹数据采集效率,降低了检测成本。利用该体系对1 625份小麦育成品种进行DNA指纹数据采集，在42个位点中共检测到434个等位变异，每个位点的等位变异个数在3—23，平均10.3个；每个位点的PIC值范围为0.240—0.829，平均0.610。利用1 625份小麦育成品种DNA指纹数据,构建了中国小麦育成品种的DNA指纹数据库。【结论】筛选出42对SSR标记引物，建立了基于荧光SSR标记的小麦品种鉴定体系，可用于高通量小麦品种DNA指纹鉴定。

Development of a Wheat Variety Identification System Based on Fluorescently Labeled SSR Markers

【Objective】In this study, a wheat variety identification system based on fluorescently labeled SSR markers was developed to provide a high-throughput DNA profiling means for identification of Chinese wheat varieties.【Method】Information on SSR markers which have been mapped to the 21 pairs of wheat chromosomes was collected. Highly polymorphic markers among China released wheat varieties were screened by PCR amplification followed by denaturing polyacrylamide gel electrophoresis. Markers selected were labeled at the 5′ end of forward primer using one of the four fluorescent dyes: TAMRA, HEX, ROX and 6-FAM. DNA Analyzer was employed to detect the fluorescently labeled PCR-amplified fragments. Peak patterns, polymorphism and distribution on linkage maps of these markers were evaluated and makers with simple peak pattern, high polymorphism and even distribution were chosen. The sizes of the alleles at each marker locus were determined and reference variety corresponding to each allele was assigned. A wheat variety identification system based on these selected fluorescently labeled SSR markers was developed.【Result】Eight morphologically diversified China released wheat varieties were PCR amplified using 2 438 mapped SSR markers for screening polymorphic and stably amplifying primer pairs. Two hundred and sixty primer pairs met the criterion. After further screening, 130 out of 260 mapped SSR markers were chosen based on distribution on wheat linkage map, PCR amplification stability and polymorphism revealed in 48 wheat varieties released in China. These 130 markers were labeled with four fluorescent dyes and further screened using DNA Analyzer. Finally, 42 SSR markers with relatively even distribution on linkage map, simple peak patterns, high polymorphism and stable PCR amplification were chosen. Sizes of alleles at each marker locus were determined using DNA Analyzer. Each allele was named according to its DNA Analyzer determined size. Reference variety corresponding to each major allele was assigned to eliminate systemic error arising between batches of samples. To profile wheat varieties cost-efficiently, 42 markers was divided into seven groups according to the fluorescent type of the markers and range of allele sizes at each marker locus. As many as 9 markers can be analyzed in the same capillary tube. DNA profiling was conducted on 1 625 wheat cultivars using this system. A total of 434 alleles were detected. The number of alleles varied between 3 and 23, with an average of 10.3 per locus. The PIC values ranged from 0.291 to 0.829, with an average of 0.610. A DNA profile database of China released wheat varieties was constructed based on the DNA profiling information of 1 625 wheat cultivars.【Conclusion】 A wheat variety identification system based on fluorescently labeled 42 SSR markers was established, which will provide a high-throughput DNA profiling alternative for identification of wheat varieties.