Molecular Genetics and Genomics

, Volume 289, Issue 6, pp 1147–1156 | Cite as

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

  • Chunxian ChenEmail author
  • Clive H. Bock
  • Tom G. Beckman
Original Paper


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.


Microsatellite marker Sequence alignment Unigene Paralog Heterozygosity 



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).

Supplementary material

438_2014_875_MOESM1_ESM.pdf (115 kb)
Supplementary material 1 (PDF 115 kb)


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Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

  • Chunxian Chen
    • 1
    Email author
  • Clive H. Bock
    • 1
  • Tom G. Beckman
    • 1
  1. 1.USDA, ARS, Southeastern Fruit and Tree Nut Research LaboratoryByronUSA

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