High-throughput and parallel SNP discovery in selected candidate genes in Eucalyptus camaldulensis using Illumina NGS platform

Abstract

Next generation sequencing (NGS) technologies have revolutionized the pace and scale of genomics- and transcriptomics-based SNP discovery across different plant and animal species. Herein, 72-base paired-end Illumina sequencing was employed for high-throughput, parallel and large-scale SNP discovery in 41 growth-related candidate genes in Eucalyptus camaldulensis. Approximately 100 kb of genome from 96 individuals was amplified and sequenced using a hierarchical DNA/PCR pooling strategy and assembled over corresponding E. grandis reference. A total of 1191 SNPs (minimum 5% other allele frequency) were identified with an average frequency of 1 SNP/83.9 bp, whereas in exons and introns, it was 1 SNP/108.4 bp and 1 SNP/65.6 bp, respectively. A total of 75 insertions and 89 deletions were detected of which approximately 15% were exonic. Transitions (Tr) were in excess than transversions (Tv) (Tr/Tv: 1.89), but exceeded in exons (Tr/Tv: 2.73). In exons, synonymous SNPs (Ka) prevailed over the non-synonymous SNPs (Ks; average Ka/Ks ratio: 0.72, range: 0-3.00 across genes). Many of the exonic SNPs/indels had potential to change amino acid sequence of respective genes. Transcription factors appeared more conserved, whereas enzyme coding genes appeared under relaxed control. Further, 541 SNPs were classified into 196 'equal frequency' (EF) blocks with almost similar minor allele frequencies to facilitate selection of one tag-SNP/EF-block. There were 241 (approximately 20%) 'zero-SNP' blocks with absence of SNPs in surrounding ±60 bp windows. The data thus indicated enormous extant and unexplored diversity in E. camaldulensis in the studied genes with potential applications for marker-trait associations.