Development and Use of a High Density SNP Array to Evaluate Genetic Diversity in Alfalfa

A large set of genome wide markers able to be assayed with a high-throughput genotyping platform can accelerate molecular breeding applications such as genome wide association mapping, fine mapping, marker-assisted selection, and genomic selection. By sequencing transcriptomes of 27 diverse alfalfa (M. sativa) genotypes, we discovered 0.9 million SNPs. Approximately 10,000 of these SNPs were utilized to develop an Illumina Infinium array. A total of 576 genotypes, including cultivated tetraploid alfalfa, wild diploid alfalfa, and other related species, were evaluated with this array. Of the 10,000 SNPs, 85% passed the quality control and gave clear genotype scores for alfalfa. For several related species, 85% of the SNPs could be genotyped for M. arborea, 78% for M. truncatula and M. lupulina, and 48% for Trifolium repens and T. pratense. For a given SNP, up to five clusters (AAAA, AAAB, AABB, ABBB, and BBBB), representing five alternative genotypes (nulliplex, simplex, duplex, triplex, and quadruplex), could be present in tetraploid alfalfa. Various software programs including GenomeStudio, beadarrayMSV, and fitTetra were evaluated to assess their accuracy in genotype calling and ability to distinguish allele dosage. From the resulting data, we evaluated the genetic diversity present among a set of alfalfa germplasm spanning the fall dormancy spectrum. We will present the development and application of this high-throughput SNP genotyping array and discuss potential uses of the array for genome wide association studies and genomic selection in alfalfa.