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

item Chen, Chunxian
item Bock, Clive
item Beckman, Thomas

Submitted to: Molecular Genetics and Genomics
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 5/29/2014
Publication Date: 6/4/2014
Publication URL:
Citation: Chen, C., Bock, C.H., Beckman, T.G. 2014. Sequence analysis reveals genomic factors affecting EST-SSR primer performance and polymorphism. Molecular Genetics and Genomics. 289(6): 1147-1156.

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: Search for simple sequence repeat (SSR) motifs and design of flanking primers in expressed sequence tag (EST) sequences can be easily done at a large scale using bioinformatics programs. However, failed amplification and/or detection, along with lack of polymorphism, is often seen among randomly selected EST-SSR primers. This study is to look into genomic factors in primers and amplicons potentially associated with these failures. 340 genotyped forward (F) and reverse (R) primers were BLAST to a reference genome to verify the primer sequence alignment statuses and determine the genomic features in amplicons. According to the genotyping results, 69 primers yielded “no peaks” (NP), 30 “weak peaks” (WP), and 12 “multiple peaks” (MP), which were all categorized into the failed types. Of the 239 successful primers, 49 were homozygous with no polymorphism between the two parents (the II-II type); 29 were homozygous and polymorphic (aa-pp); and 161 were others, heterozygous (HT) in at least one parent. Introns appeared in the amplicons of 23 successful primers. Based on the BLAST alignment scenarios at the cutoff e-value, 2e-02 (0.02), 56 F and 52 R primers had “no hits found” (NHF); 22 F and 11 R “multiple partial alignments” (MPA); 43 F and 38 R “single partial alignment” (SPA); 58 F and 56 R “multiple full alignments” (MFA); and 161 F and 183 R “single full alignment” (SFA), which were involved in 93, 30, 76, 101, and 239 loci, respectively. Among them, 15, 3, 5, 13, and 105 were paired in the same category respectively, and the remaining 199 F and 199 R in total had their corresponding R and F in a different category. Primers of partial alignment mainly derived from contig consensus regions containing discrepant nucleotides. Excluding 93 NHF primers, the other primers were also categorized: 82 “deletion”, 68 “same size”, 31 “insertion”, 41 “intron” included in the amplicons if the presumable intron cutoff size was set 20bp, and 25 “error” in which each F and R primer was aligned two far away to form an amplicon. Among the highest rates of all the three failed types together were 53% NHF and 37% MPA, and 80% “error” and 43% “intron (amp>500). Among the highest producer of the II or aa-pp types were the “same size” (68%) and “insertion” (58%) primers. Optimized primer design and selection criteria, along with the causes of different alignment scenarios, were discussed.