Development and characterisation of simple sequence repeat (SSR) markers for white clover (Trifolium repens L.)

Highly informative molecular markers, such as simple sequence repeats (SSRs ), can greatly accelerate breeding programs. The aim of this study was to d evelop and characterise a comprehensive set of SSR markers for white clover (Trifolium repens L.), which can be used to tag genes and quantitative tra it loci controlling traits of agronomic interest. Sequence analysis of 1123 clones from genomic libraries enriched for (CA)(n) repeats yielded 793 clo nes containing SSR loci. The majority of SSRs consisted of perfect dinucleo tide repeats, only 7% being trinucleotide repeats. After exclusion of redun dant sequences and SSR loci with less than 25 bp of flanking sequence, 397 potentially useful SSRs remained. Primer pairs were designed for 117 SSR lo ci and PCR products in the expected size range were amplified from 101 loci . These markers were highly polymorphic, 88% detecting polymorphism across seven white clover genotypes with an average allele number of 4.8. Four pri mer pairs were tested in an F-2 population revealing Mendelian segregation. Successful cross-species amplification was achieved in at least one out of eight legume species for 46 of 54 primer pairs. The rate of successful amp lification was significantly higher for Trifolium species when compared to species of other genera. The markers developed in this study not only provi de valuable tools for molecular breeding of white clover but may also have applications in related taxa.