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

Identifieur interne : 000272 ( PubMed/Curation ); précédent : 000271; suivant : 000273

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

Auteurs : Chunxian Chen ; Clive H. Bock ; Tom G. Beckman

Source :

Molecular genetics and genomics : MGG [ 1617-4623 ] ; 2014.

RBID : pubmed:24948352

English descriptors

KwdEn :
- Citrus sinensis (genetics), DNA Primers (chemistry), Expressed Sequence Tags, Genome, Plant, Genomics, Genotype, Microsatellite Repeats, Polymerase Chain Reaction, Polymorphism, Genetic, Sequence Alignment.
MESH :
- chemical , chemistry : DNA Primers.
- genetics : Citrus sinensis.
- Expressed Sequence Tags, Genome, Plant, Genomics, Genotype, Microsatellite Repeats, Polymerase Chain Reaction, Polymorphism, Genetic, Sequence Alignment.

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.

DOI: 10.1007/s00438-014-0875-8
PubMed: 24948352

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Chunxian Chen

<affiliation><nlm:affiliation>USDA, ARS, Southeastern Fruit and Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA, 31008, USA, chunxian.chen@ars.usda.gov.</nlm:affiliation>
<wicri:noCountry code="subField">chunxian.chen@ars.usda.gov.</wicri:noCountry>
</affiliation>

Le document en format XML

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<author><name sortKey="Chen, Chunxian" sort="Chen, Chunxian" uniqKey="Chen C" first="Chunxian" last="Chen">Chunxian Chen</name>
<affiliation><nlm:affiliation>USDA, ARS, Southeastern Fruit and Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA, 31008, USA, chunxian.chen@ars.usda.gov.</nlm:affiliation>
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<front><div type="abstract" xml:lang="en">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.</div>
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<Abstract><AbstractText>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.</AbstractText>
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Sequence analysis reveals genomic factors affecting EST-SSR primer performance and polymorphism.

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

Source :

English descriptors

Abstract

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