Abstract:

【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.

Key words: common wheat, SSR, fluorescently labeled markers, variety identification, DNA profiling