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Sequence analysis reveals genomic factors affecting EST-SSR primer performance and polymorphism


This study was to explore genomic factors affecting the performance and polymorphism of 340 randomly selected EST-SSR (expressed sequence tag-simple sequence repeat) primers through BLAST of primer sequences to a reference genome. Genotyping showed 111 failed and 229 succeeded. The failed types included “no peaks” (NP, 69 primers), “weak peaks” (WP, 30), and “multiple peaks” (MP, 12). The successful types were divided into HM (homozygous between two selected parents, 78 primers) and HT (heterozygous at least in one parent, 151 primers). The BLAST revealed primer alignment status, genomic amplicon size (GAS), and genomic and expressed amplicon size difference (ASD). The alignment status was categorized as: “no hits found” (NHF); “multiple partial alignments” (MPA); “single partial alignment” (SPA); “multiple full alignments” (MFA); and “single full alignment” (SFA). NHF and partial alignment (PA) mainly resulted from discrepant nucleotides in contig-derived primers. The ASD separated 247 non-NHF primers into: “deletion”, “same size”, “insertion”, “intron (GAS ≤500)”, “intron (GAS >500)”, and “error” categories. Most SFA primers were successful. About 88 % “error”, 53 % NHF primers, and 47 % “intron (GAS >500)” failed. The “deletion” and “insertion” primers had the higher HT rates, and the “same size” had the highest HM rate. Optimized primer selection criteria are discussed.

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The authors thank Dr. Fred Gmitter for the published genotyping data that was used for categorization of primer performance and genotypes in this study. The research is partially supported by the USDA national program of plant genetic resources, genomics and genetic improvement (Project Number: 6606-21000-004-006).

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Correspondence to Chunxian Chen.

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This article reports the results of research only. Mention of a trademark or proprietary product is solely for the purpose of providing specific information and does not constitute a guarantee or warranty of the product by the US Department of Agriculture and does not imply its approval to the exclusion of other products that may also be suitable.

Communicated by S. Hohmann.

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Chen, C., Bock, C.H. & Beckman, T.G. Sequence analysis reveals genomic factors affecting EST-SSR primer performance and polymorphism. Mol Genet Genomics 289, 1147–1156 (2014).

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  • Microsatellite marker
  • Sequence alignment
  • Unigene
  • Paralog
  • Heterozygosity