Development of a fingerprinting database and assembling an SSR reference kit for genetic diversity analysis of wheat

Abstract

Understanding of the current and expanded genetic diversity is very important for raising the yield of wheat. Genetic diversity based on molecular markers has been studied in plants for over thirty decades. SSR is the currently most popular marker system in wheat. In order to utilize the diversity held in NARS (National Agricultural Research Station) and CGIAR (Consultative Group on International Agricultural Research) germplasm collections, one of the GCP’s (Generation Challenge Program) premier capacity building activities is to build databases that contain traditional and molecular data on germplasm so that scientists all over the world can access information with relevance to their region on traits, genes, and sequences. In the present study, a fingerprinting database was established containing 134 SSR primers and 2 457 wheat genotypes with the data from CIMMYT and three collaborators: ICARDA, Agropolis, and CAAS. On the base of the database, a SSR reference kit for wheat genetic diversity analysis was developed, which will facilitate the use of this data in new projects and cross-laboratory comparisons. In total, 46 SSR primers with comparatively high polymorphism were selected as the reference markers to constitute the standard allele kit, 334 genotypes fingerprinted within the GCP tier 1 project “Genotyping a composite germplasm set in wheat” were chosen to represent the SSR allele kit consisting of 794 alleles amplified by 46 SSR markers. Genotypes were originally selected and DNA extracted by CIMMYT and 3 additional collaborators: ICARDA, Agropolis, and CAAS. These 334 genotypes, when taken as a group, amplify every allele seen for the wheat genotypes for all the 46 SSRs. The Genotypes were originally amplified with 26 SSRs at CIMMYT, with 8 SSRs at INRA, France, and with 12 SSRs at CAAS. Genotypes were amplified with all SSRs at CIMMYT again to confirm the results. For reamplification at CIMMYT, forward primers were labeled at the 5’ end with either one of three phosphoramidite fluorescent dyes 6-carboxyflouresein (6-Fam), tetrachloro-6-carboxyflouresein (Tet) or hexachloro-6- carboxyflouresein (Hex). PCR-reactions were carried out in an MJ-Research thermocycler model PTC225. Amplification products were separated on an ABI-Prism SequencerTM377 using 4.5% polyacrylamide denaturing gels. Fragment sizes were calculated semi-automatically with the computer software GeneScan 3.1 by comparing to fragments of an internal size standard (GeneScan 350 or 500) labeled with N,N,N,N, -tetramethyl-6-carboxyrhodamine (Tamra). GeneScan fragments were assigned to alleles using the category function of the software Genotyper 2.1. The two genotypes, Opata and Synthetic were run in each gel as controls. Finally, a SSR reference kit was assembled which includes 46 pairs of SSR primers, protocols (PCR condition, detection of polymorphisms, etc), description of polymorphism in the reference samples, reference DNA samples for a complete range of repeatable 592 alleles, methods for comparing new to preexisting data, and classification of various genotypes. The database and the reference kit will provide a powerful tool for genetic diversity studies of wheat germplasm worldwide.