USDA, ARS, Southeastern Fruit and Tree Nut Research Laboratory, 21 Dunbar Road, Byron, GA, 31008, USA,
Mol Genet Genomics. 2014 Dec;289(6):1147-56. doi: 10.1007/s00438-014-0875-8. Epub 2014 Jun 20.
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.
本研究通过引物序列与参考基因组的 BLAST 来探索影响 340 个随机选择的 EST-SSR（表达序列标签-简单重复序列）引物性能和多态性的基因组因素。基因分型显示 111 个失败，229 个成功。失败的类型包括“无峰”（NP，69 个引物）、“弱峰”（WP，30 个）和“多峰”（MP，12 个）。成功的类型分为 HM（两个选定亲本之间的纯合，78 个引物）和 HT（至少在一个亲本中杂合，151 个引物）。BLAST 揭示了引物的比对状态、基因组扩增子大小（GAS）和基因组与表达扩增子大小差异（ASD）。比对状态分为：“未发现命中”（NHF）；“多个部分比对”（MPA）；“单个部分比对”（SPA）；“多个完全比对”（MFA）和“单个完全比对”（SFA）。NHF 和部分比对（PA）主要是由于源自连续引物的差异核苷酸。ASD 将 247 个非 NHF 引物分为：“缺失”、“相同大小”、“插入”、“内含子（GAS≤500）”、“内含子（GAS＞500）”和“错误”类别。大多数 SFA 引物是成功的。约 88%的“错误”、53%的 NHF 引物和 47%的“内含子（GAS＞500）”失败。“缺失”和“插入”引物的 HT 率较高，而“相同大小”的 HM 率最高。讨论了优化的引物选择标准。
