Application Of SSR Markers To Variety Identification Of Chinese Winter Wheat Cultivars (Lines)

Simple sequence repeats (SSRs), or microsatellite, are streches of DNA consiting of tandemly repeating 1-5 nucleotide units. They have been shown to be ubiquitous in most eukaryotic genomes. SSRs are valuable as genetic markers because they are abundant, informative, co-dominant and easily assayed by polymerase chain reaction (PCR). In wheat genomes, SSRs have been found significantly more polymorfic than RFLPs. SSR markers are gaining widespread use in such applications as variety identification, genetic diversity and genetic relatedness studies.In this study, (1) two DNA molecular weight standards were used to investigate relationship between migrating distances of DNA fragments and their sizes in denaturing polyacrylamide gel (PAGE) so that an allele sizing method could be found; (2) the allele diversity at 8 SSR loci among 71 Chinese winter wheat cultivars (lines) was evaluated for the purpose of variety identification and plant breeder's rights protection, and genetic relatedness of 53 winter wheat varieties were analysed. The main results from this study are as follows:1. In 6% denaturing PAGE, the relationship between the sizes of DNA fragments (in base pairs) and then- corresponding migrating distances roughly follows power function y=axb, where y is the fragment size and x the migrating distance, and vice versa. As the range of fragment sizes narrows, fragment sizes can be approximated using a cubic regression curve y=ax3+bx2+cx+d fitted to data of DNA molecular weight standards, where y is the fragment size and x the migrating distance.2. A method of allele sizing was developed based on the cubic regression curves fitted to the data of molecular weight standard. The allele profiles of 71 Chinese cultivars (lines) at 8 loci have been set up using this method.3. Among 71 cultivars (lines), 75 alleles in total were detected, ranging from 6 (gwm186) to 15 (gwm174) per set of primer pairs, with an average of 9.4. The PIC values of the SSRloci were calculated, which varied from the lowest of 0.6552 (gwm186) to the highest of 0.8739 (gwm174), averaging 0.7968. The study showed that all the 71 cultivars (lines) could be uniquely identified and distinguished from each other using the 8 SSR markers. The siblings were at least one allele different from each other. SSR markers are especially suitable for identifying cultivars (lines) that are developed through conventional hybridization breeding method.4. A cluster analysis of 53 cultivars (lines) based on the allele diversity at 8 loci was performed. The result was consistent with the pedigree information Several closely related cultivars (lines) according to pedigree were not grouped together. The possible reasons for the seeming discrepancy between cluster analysis result and pedigree information in this study and others were suggested as follows: firstly, some highly informative SSR markersamong commercial cultivars may implicate they are not closely linked to agronomically important traits and were subjected to lesser selection pressure during breeding process, more or less random segregations of SSR alleles resulted among the cultivars sharing same or similar pedigrees; secondly, the different selection criterion applied to individual plants may cause significant genetic diversity among siblings; thirdly, given the complexity of wheat genome, a small number of SSR loci may not be sufficiently representative of the whole genome, which may lead to a degree of deviation in cluster results. Compared with pedigree information, SSR markers can reveal more genetic variation among genotypes.