Location: Fruit and Nut ResearchTitle: Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach Author
|Beckman, Thomas - Tom|
Submitted to: Tree Genetics and Genomes
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 6/3/2014
Publication Date: 6/5/2014
Publication URL: http://link.springer.com/article/10.1007/s11295-014-0759-4
Citation: Chen, C., Bock, C.H., Okie, W., Gmitter, F.J., Jung, S., Main, D., Beckman, T.G., Wood, B.W. 2014. Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach. Tree Genetics and Genomes. 10(5): 1271-1279. Interpretive Summary: Expressed sequence tag (EST) simple sequence repeat (SSR) markers have been widely used in linkage analysis, gene mapping, variety authentication and other genetic studies. However, marker distribution, amplification/detection reliability, and polymorphism status of randomly selected primers are unknown until primers are synthesized and tested. These primers that fail in amplification/detection are very wasteful, and worth a better approach to lead to optimal primer selection. A look into genomic factors in EST-SSR primers and amplicons potentially associated with these failures and/or polymorphisms can provide helpful guidance on selection of primers with better successful rate and polymorphism. Optimal selection also appears to help improve the polymorphism rate.
Technical Abstract: Expressed sequence tag (EST) simple sequence repeats (SSRs) in Prunus were mined, and flanking primers designed and used for genome-wide characterization and selection of primers to optimize marker distribution and reliability. A total of 12,618 contigs were assembled from 84,727 ESTs, along with 34,238 singlets. 4770 SSRs were identified from the 12,618 contigs, and 9,029 SSRs were from singlets, from which 3,695 and 6,849 primers were designed, respectively. Alignment of the 10,544 forward and reverse primer sequences (21,088 queries total) against the peach reference genome, resulted in 23,553 hits with 96,621 alignments with 16,885 queries at a preset e value of 9e-03 (0.009), with “no hits found” (NHF) for the remaining 4,203 queries. A majority of aligned primers had only 1 hit/alignment on the peach scaffolds, and the distribution of the 5,500 singly aligned primers (pairs) on each 500 kb genome interval was determined. The average number of ESR-SSR primers per 500 kb interval was 10.8. The primers were categorized into 8 subgroups based on the difference between the genome amplicon size and expressed amplicon size of each primer, with 288 primers of optimized distribution and reliability selected for genotype evaluation. Only 2 of the 288 primers failed in all 4 peach cultivars screened, with an overall successful primer/sample (amplification and detection) rate of 97.2%. The average number of alleles detected in the 4 cultivars was 3.84. Of the 288 primers, 110 scored zero for heterozygosity (H). The calculated polymorphism information content (PIC) and gene diversity values suggested the majority of the 288 primers had a high rate of allele polymorphism among the four peach cultivars. The validated primers that had good H and PIC values will be used for genetic mapping and other marker applications. The advantages of genome-wide analysis of EST-SSR primers and the options to improve the heterozygosity and polymorphism rate are discussed.