Mining of haplotype-based expressed sequence tag single nucleotide polymorphismsin citrus

Abstract

Background: Single nucleotide polymorphisms (SNPs), the most abundant variations in agenome, have been widely used in various studies. Detection andcharacterization of citrus haplotype-based expressed sequence tag (EST) SNPswill greatly facilitate further utilization of these gene-basedresources.Results: In this paper, haplotype-based SNPs were mined out of publicly availablecitrus expressed sequence tags (ESTs) from different citrus cultivars(genotypes) individually and collectively for comparison. There were a totalof 567,297 ESTs belonging to 27 cultivars in varying numbers andconsequentially yielding different numbers of haplotype-based quality SNPs. Sweet orange (SO) had the most (213,830) ESTs, generating 11,182 qualitySNPs in 3,327 out of 4,228 usable contigs. Summed from all the individuallymining results, a total of 25,417 quality SNPs were discovered -15,010 (59.1%) were transitions (AG and CT), 9,114 (35.9%) weretransversions (AC, GT, CG, and AT), and 1,293 (5.0%) wereinsertion/deletions (indels). A vast majority of SNP-containing contigsconsisted of only 2 haplotypes, as expected, but the percentages of 2haplotype contigs varied widely in these citrus cultivars. BLAST of the25,417 25-mer SNP oligos to the Clementine reference genome scaffoldsrevealed 2,947 SNPs had "no hits found", 19,943 had 1 unique hit/ alignment, 1,571 had one hit and 2+ alignments per hit, and 956 had 2+hits and 1+ alignment per hit. Of the total 24,293 scaffold hits, 23,955(98.6%) were on the main scaffolds 1 to 9, and only 338 were on 87 minorscaffolds. Most alignments had 100% (25/25) or 96% (24/25) nucleotideidentities, accounting for 93% of all the alignments. Considering almost allthe nucleotide discrepancies in the 24/25 alignments were at the SNP sites, it served well as in silico validation of these SNPs, in additionto and consistent with the rate (81%) validated by sequencing and SNaPshotassay.Conclusions: High-quality EST-SNPs from different citrus genotypes were detected, andcompared to estimate the heterozygosity of each genome. All the SNP oligosequences were aligned with the Clementine citrus genome to determine theirdistribution and uniqueness and for in silico validation, inaddition to SNaPshot and sequencing validation of selected SNPs. © 2013 Chen and Gmitter; licensee BioMed Central Ltd.