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Submitted to: Plant and Animal Genome Conference
Publication Type: Abstract Only Publication Acceptance Date: 12/3/2014 Publication Date: 1/11/2015 Publication URL: http://pag14.mapyourshow.com/5_0/sessions/sessiondetails.cfm?ScheduledSessionID=1EAECD Citation: 2015. Sequence alignment status and amplicon size difference affecting EST-SSR primer performance and polymorphism. Plant and Animal Genome Conference. 22:224. Interpretive Summary: Expressed sequence tag (EST) simple sequence repeat (SSR) markers have been widely used in trait linkage analysis, gene mapping, variety authentication and other genetic studies. However, it is wasteful because some randomly selected EST-SSR primers fail to detect and/or lack polymorphism. A look into genomic factors in EST-SSR primers and amplicons potentially associated with these failures and/or polymorphisms can provide helpful guidance on selection of primers with better successful rate and polymorphism. Technical Abstract: Little attention has been given to failed, poorly-performing, and non-polymorphic expressed sequence tag (EST) simple sequence repeat (SSR) primers. This is due in part to a lack of interest and value in reporting them but also because of the difficulty in addressing the causes of failure on a primer-by-primer basis. However, failed amplification and/or detection, along with a lack of polymorphism, is often observed among randomly selected EST-SSR primers. In this study, primer sequence alignment status and genomic amplicon sizes were found to be strongly associated with EST-SSR primer performance and polymorphism, according to the BLAST output of 340 EST-SSR primers to a reference genome. Summarized from the genotyping results and observed primer performance, 69 primers yielded “no peaks” (NP), 30 yielded “weak peaks” (WP), and 12 yielded “multiple peaks” (MP), all of which were categorized as failed primers. Of the 229 primers that were identified as successful, 49 were homozygous with no polymorphism between the two parents used (the II-II type), 29 were homozygous and polymorphic (aa-pp), and the remaining 151 were heterozygous (HT) in at least one parent. Introns were visualized in the amplicons of 23 successful primers. Based on the BLAST alignment on a reference genome using a cutoff e-value of 2e-02 (0.02), the primer alignment status and the genomic amplicon-derived features were determined and categorized. The alignment status of 340 F and 340 R primers were classified into five categories: 1) 56 F and 52 R primers in the “no hits found” (NHF) category; 2) 22 F and 11 R in the “multiple partial alignments” (MPA) category; 3) 43 F and 38 R in the “single partial alignment” (SPA) category; 4) 58 F and 56 R in the “multiple full alignments” (MFA) category; and 5) 161 F and 183 R in the “single full alignment” (SFA) category. In the five alignment categories, only 15, 3, 5, 13, and 105 F and R were paired respectively, and the remaining 199 F had their corresponding R in a different category (or vice versa). Individual primers with partial alignments were derived mainly from contig consensus regions containing discrepant nucleotides. Based on the difference between the genomic amplicon size (GAS) and expressed amplicon size (EAS), the 247 primers were categorized as: “deletion” (82 primers), “same size” (68 primers), “insertion” (31 primers), “intron (GAS<=500)” (17 primers), “intron (GAS>500)” (24 primers), and “error” (25 primers). It was found that 88% of the “error” primers, 63% of the “intron (GAS>500)” primers, 53% of the NHF primers, and 37% of the MPA primers in their respective category had failed. As expected, most primers in the SFA category were successful. In addition, successful primers in the “deletion”, “intron (GAS<=500) and “insertion” categories had the higher HT rates, whereas successful primers in the “same size” category had the highest rate of less useful II-II and aa-pp genotypes. Optimized primer design and selection criteria, and causes of different alignment characteristics, are discussed. |
