Development of high-throughput SNP-based genotyping in <it>Acacia auriculiformis</it> x <it>A</it>. <it>mangium</it> hybrids using short-read transcriptome data

• Main Authors: Wong Melissa ML, Cannon Charles H, Wickneswari Ratnam
• Format: Article
• Language: English
• Published: BMC 2012-12-01
• Series: BMC Genomics
• Online Access: http://www.biomedcentral.com/1471-2164/13/726

<p>Abstract</p> <p>Background</p> <p>Next Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP) discovery in <it>Acacia auriculiformis</it> and <it>Acacia mangium</it>. Like other non-model species, SNP detection and genotyping in <it>Acacia</it> are challenging due to lack of genome sequences. The main objective of this study is to develop the first high-throughput SNP genotyping assay for linkage map construction of <it>A</it>. <it>auriculiformis</it> x <it>A</it>. <it>mangium</it> hybrids.</p> <p>Results</p> <p>We identified a total of 37,786 putative SNPs by aligning short read transcriptome data from four parents of two <it>Acacia</it> hybrid mapping populations using Bowtie against 7,839 <it>de novo</it> transcriptome contigs. Given a set of 10 validated SNPs from two lignin genes, our <it>in silico</it> SNP detection approach is highly accurate (100%) compared to the traditional <it>in vitro</it> approach (44%). Further validation of 96 SNPs using Illumina GoldenGate Assay gave an overall assay success rate of 89.6% and conversion rate of 37.5%. We explored possible factors lowering assay success rate by predicting exon-intron boundaries and paralogous genes of <it>Acacia</it> contigs using <it>Medicago truncatula</it> genome as reference. This assessment revealed that presence of exon-intron boundary is the main cause (50%) of assay failure. Subsequent SNPs filtering and improved assay design resulted in assay success and conversion rate of 92.4% and 57.4%, respectively based on 768 SNPs genotyping. Analysis of clustering patterns revealed that 27.6% of the assays were not reproducible and flanking sequence might play a role in determining cluster compression. In addition, we identified a total of 258 and 319 polymorphic SNPs in <it>A</it>. <it>auriculiformis</it> and <it>A</it>. <it>mangium</it> natural germplasms, respectively.</p> <p>Conclusion</p> <p>We have successfully discovered a large number of SNP markers in <it>A</it>. <it>auriculiformis</it> x <it>A</it>. <it>mangium</it> hybrids using next generation transcriptome sequencing. By using a reference genome from the most closely related species, we converted most SNPs to successful assays. We also demonstrated that Illumina GoldenGate genotyping together with manual clustering can provide high quality genotypes for a non-model species like <it>Acacia</it>. These SNPs markers are not only important for linkage map construction, but will be very useful for hybrid discrimination and genetic diversity assessment of natural germplasms in the future.</p>