Abstract
BackgroundNext Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP) discovery in Acacia auriculiformis and Acacia mangium. Like other non-model species, SNP detection and genotyping in Acacia 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 A. auriculiformis x A. mangium hybrids.ResultsWe identified a total of 37,786 putative SNPs by aligning short read transcriptome data from four parents of two Acacia hybrid mapping populations using Bowtie against 7,839 de novo transcriptome contigs. Given a set of 10 validated SNPs from two lignin genes, our in silico SNP detection approach is highly accurate (100%) compared to the traditional in vitro 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 Acacia contigs using Medicago truncatula 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 A. auriculiformis and A. mangium natural germplasms, respectively.ConclusionWe have successfully discovered a large number of SNP markers in A. auriculiformis x A. mangium 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 Acacia. 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.
Highlights
Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP) discovery in Acacia auriculiformis and Acacia mangium
There is no study that compare the genetic diversity of both species, the lower SNP frequency observed in the transcriptome of A. mangium [12] suggested that A. mangium has lower genetic diversity than A. auriculiformis
A total of 7,839 contigs with lengths ranging from 200-15,266 bp where 6,771 and 1,068 contigs came from AA6 and AM20 de novo transcriptome assemblies, respectively were selected as reference sequences for SNP detection
Summary
Generation Sequencing has provided comprehensive, affordable and high-throughput DNA sequences for Single Nucleotide Polymorphism (SNP) discovery in Acacia auriculiformis and Acacia mangium. The main objective of this study is to develop the first high-throughput SNP genotyping assay for linkage map construction of A. auriculiformis x A. mangium hybrids. Marker-assisted breeding is a promising approach for selection of superior trees with improved wood and pulp properties for the establishment this end, Single Nucleotide Polymorphism (SNP) is the ideal marker because it provides affordable and highthroughput genotyping compared to other markers [4]. SNPs can be used in genetic diversity assessment of natural germplasms, estimation of outcrossing rate in natural germplasms and seed orchards, and more importantly, clone and hybrid identification in breeding program of both species. There is no study that compare the genetic diversity of both species, the lower SNP frequency observed in the transcriptome of A. mangium [12] suggested that A. mangium has lower genetic diversity than A. auriculiformis
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