Development and validation of new SSR markers from expressed regions in the garlic genome

Limited number of simple sequence repeat (SSR) markers is available for the genome of garlic (Allium sativum L.) although SSR markers have become one of the most preferred marker systems because they are typically co-dominant, reproducible, cross species transferable and highly polymorphic. In this study, in order to develop new EST-SSR markers for the garlic genome, garlic expressed sequence tags (EST) at publicly available GarlicESTdb were screened for the presence of SSR motifs. A total of 132 SSR motifs were identified in 7720 non-redundant garlic EST sequences. Among these SSR motifs, dinucleotide repeats (60, 45.45%) were the most abundant repeat motifs and 34 (56.66%) of the dinucleotide repeats were (AT/TA)n repeats. Primer pairs were designed for 53 SSR motifs and 24 of these primer pairs were selected as SSR markers based on their consistent PCR amplification pattern among the garlic genotypes, polymorphisms and low banding complexity. In addition, two EST-SSR markers were developed from the sequences of garlic cDNA-AFLP fragments. In order to test utility of 26 EST-SSR markers developed in this study, genetic relationships among 31 garlic genotypes were assessed using these markers. Twenty-six EST-SSR markers amplified 130 polymorphic DNA fragments and number of polymorphic alleles per SSR marker ranged from 2 to 13 with an average of 5 alleles. Heterozygosity and polymorphic information content (PIC) of the EST-SSR markers were between 0.23 and 0.88, and 0.20 and 0.84, respectively. Six EST-SSR markers were present more than one copy in the diploid genome of garlic. Twenty-one of the garlic genotypes used in this study were analyzed in a previous study using AFLP markers and the garlic genotypes clustered together with AFLP markers were also grouped together with EST-SSR markers demonstrating the concordance between the dendrograms of AFLP and EST-SSR markers. Our results indicated that polymorphic EST-SSR markers developed in this study can be utilized for diversity and breeding studies for expressed regions of garlic genome effectively.