Genome-wide characterization and selection of expressed sequence tag simple sequence repeat primers for optimized marker distribution and reliability in peach
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Simple sequence repeats (SSR) in Prunus expressed sequence tags (EST) were mined, and flanking primers designed and used for genome-wide characterization and selection of primers to optimize marker distribution and reliability in peach. A total of 4,770 and 9,029 SSRs were identified from 12,618 contigs and 34,238 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) against the peach reference genome at 9e-03 resulted in 23,553 hits (96,621 alignments) with 16,885 queries, and “no hits found” (NHF) for the remaining 4,203 queries. A majority of aligned primers had only one 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 eight 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 rate of 97.2 %. The average number of alleles detected in the four cultivars was 3.84. The polymorphism information content (PIC) values suggested that a majority of the 288 primers had a high rate of allele polymorphism among the four peach cultivars. The advantages of genome-wide analysis of EST-SSR primers and options to improve the polymorphism rate are discussed.
KeywordsMicrosatellite Short tandem repeat (STR) Marker-assisted selection (MAS) Variety authentication Reference genome
The authors thank Bryan Blackburn, Luke Quick, and Minling Zhang for their technical assistance. The research is partially supported by the USDA National Program of Plant Genetic Resources, Genomics and Genetic Improvement (Project number 6606-21000-004-006) and an USDA National Institute of Food and Agriculture Specialty Crop Research Initiative project (2009-51181- 06036).
Data archiving statement
All Prunus EST sequences and accession numbers are available at the National Center for Biotechnology Information EST database (http://www.ncbi.nlm.nih.gov/nucest/?term=Prunus). The peach (Prunus persica) reference genome assembly (version 1.0) is available at the Genome Database for Rosaceae (http://www.rosaceae.org/species/Prunus_persica/genome_v1.0), so is the mined Prunus EST-SSR primer information (http://www.rosaceae.org/node/336118). The 10545 EST-SSR forward and reverse primers and the selected 288 primers are attached as ESM Tables 2 and 3, respectively.
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