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Background  Next 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.    Results  We 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.    Conclusion  We 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.

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