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

Original Paper

Abstract

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.

Keywords

Microsatellite marker Sequence alignment Unigene Paralog Heterozygosity 

Supplementary material

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

References

  1. Altschul SF, Madden TL, Schaffer AA, Zhang J, Zhang Z, Miller W, Lipman DJ (1997) Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. Nucleic Acids Res 25:3389–3402PubMedCentralPubMedCrossRefGoogle Scholar
  2. Chen C, Gmitter FG Jr (2013) Mining of haplotype-based expressed sequence tag single nucleotide polymorphisms in citrus. BMC Genomics 14:746PubMedCentralPubMedCrossRefGoogle Scholar
  3. Chen C, Zhou P, Choi YA, Huang S, Gmitter FG (2006) Mining and characterizing microsatellites from citrus ESTs. Theor Appl Genet 112:1248–1257PubMedCrossRefGoogle Scholar
  4. Chen C, Bowman KD, Choi YA, Dang PM, Rao MN, Huang S, Soneji JR, McCollum TG, Gmitter FG (2008) EST-SSR genetic maps for Citrus sinensis and Poncirus trifoliata. Tree Genet Genomes 4:1–10CrossRefGoogle Scholar
  5. Doyle JJ, Doyle JL (1987) A rapid DNA isolation procedure for small quantities of fresh leaf tissue. Phytochem Bul 19:11–15Google Scholar
  6. Gmitter FG Jr (2011) The haploid mandarin and diploid sweet orange genome sequences. Plant and Animal Genomes XIX Conference Town and Country Convention Center, San Diego, CA: W146Google Scholar
  7. Gordon D, Abajian C, Green P (1998) Consed: a graphical tool for sequence finishing. Genome Res 8:195–202PubMedCrossRefGoogle Scholar
  8. Gordon D, Desmarais C, Green P (2001) Automated finishing with autofinish. Genome Res 11:614–625PubMedCentralPubMedCrossRefGoogle Scholar
  9. Kapustin Y, Souvorov A, Tatusova T, Lipman D (2008) Splign: algorithms for computing spliced alignments with identification of paralogs. Biol Direct 3:20PubMedCentralPubMedCrossRefGoogle Scholar
  10. Kong Q, Zhang G, Chen W, Zhang Z, Zou X (2012) Identification and development of polymorphic EST-SSR markers by sequence alignment in pepper, Capsicum annuum (Solanaceae). Am J Bot 99:e59–e61PubMedCrossRefGoogle Scholar
  11. Miah G, Rafii MY, Ismail MR, Puteh AB, Rahim HA, Islam KhN, Latif MA (2013) A review of microsatellite markers and their applications in rice breeding programs to improve blast disease resistance. Int J Mol Sci 14:22499–22528PubMedCentralPubMedCrossRefGoogle Scholar
  12. Morgante M, Hanafey M, Powell W (2002) Microsatellites are preferentially associated with nonrepetitive DNA in plant genomes. Nat Genet 30:194–200PubMedCrossRefGoogle Scholar
  13. Narina SS, d’Orgeix CA, Sayre BL (2011) Optimization of PCR conditions to amplify microsatellite loci in the bunchgrass lizard (Sceloporus slevini) genomic DNA. BMC Res Notes 4:26PubMedCentralPubMedCrossRefGoogle Scholar
  14. Navarro E, Espinosa L (2000) Improving quality of expressed sequence tag (EST) databases: recovery of reversed, antisense cDNA sequences. Microb Comp Genomics 5:17–24PubMedCrossRefGoogle Scholar
  15. Rozen S, Skaletsky H (2000) Primer3 on the WWW for general users and for biologist programmers. Methods Mol Biol (Clifton, NJ) 132:365–386Google Scholar
  16. Saha MC, Mian MA, Eujayl I, Zwonitzer JC, Wang L, May GD (2004) Tall fescue EST-SSR markers with transferability across several grass species. Theor Appl Genet 109:783–791PubMedCrossRefGoogle Scholar
  17. Saha MC, Cooper JD, Mian MA, Chekhovskiy K, May GD (2006) Tall fescue genomic SSR markers: development and transferability across multiple grass species. Theor Appl Genet 113:1449–1458PubMedCrossRefGoogle Scholar
  18. Tang J, Vosman B, Voorrips RE, van der Linden CG, Leunissen JA (2006) QualitySNP: a pipeline for detecting single nucleotide polymorphisms and insertions/deletions in EST data from diploid and polyploid species. BMC Bioinformatics 7:438PubMedCentralPubMedCrossRefGoogle Scholar
  19. Thiel T, Michalek W, Varshney RK, Graner A (2003) Exploiting EST databases for the development and characterization of gene-derived SSR-markers in barley (Hordeum vulgare L.). Theor Appl Genet 106:411–422PubMedGoogle Scholar
  20. Varshney RK, Graner A, Sorrells ME (2005) Genic microsatellite markers in plants: features and applications. Trends Biotechnol 23:48–55PubMedCrossRefGoogle Scholar
  21. Wendel JF, Cronn RC, Alvarez I, Liu B, Small RL, Senchina DS (2002) Intron size and genome size in plants. Mol Biol Evol 19:2346–2352PubMedCrossRefGoogle Scholar
  22. You FM, Huo N, Gu YQ, Luo MC, Ma Y, Hane D, Lazo GR, Dvorak J, Anderson OD (2008) BatchPrimer3: a high throughput web application for PCR and sequencing primer design. BMC Bioinformatics 9:253PubMedCentralPubMedCrossRefGoogle Scholar
  23. Zhou S, Ji G, Liu X, Li P, Moler J, Karro JE, Liang C (2012) Pattern analysis approach reveals restriction enzyme cutting abnormalities and other cDNA library construction artifacts using raw EST data. BMC Biotechnol 12:16PubMedCentralPubMedCrossRefGoogle Scholar

Copyright information

© Springer-Verlag Berlin Heidelberg 2014

Authors and Affiliations

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

Personalised recommendations