High-throughput targeted SNP discovery using Next Generation Sequencing (NGS) in few selected candidate genes in Eucalyptus camaldulensis
© Hendre et al; licensee BioMed Central Ltd. 2011
Published: 13 September 2011
The present era of high throughput technologies offer immense promise and innovative applications for SNP discovery and high quality parallel genotyping [1, 2]. Using advancements in the next generation sequencing (NGS) technologies, the en masse SNP discovery for targeted genomic regions is possible for eucalypts. The river red gum or Eucalyptus camaldulensis (Ec) is a fast growing, hardy and highly adaptable eucalypt species acclimatized to Indian climatic conditions and these new advancements would aid in developing new tools and techniques for its improvement. In our knowledge, limited efforts have been undertaken to identify SNP markers in eucalypts either by employing RNA sequencing  or by using few genes available in the literature . Despite these miniscule efforts, useful SNP markers were discovered in Cinnamoyl CoA Reductase (CCR) gene with potential application . Using the recently released whole genome sequence of E. grandis (Eg), herein we describe targeted SNP discovery in 41 candidate genes by employing Illumina’s 72-bases paired end sequencing technology.
Materials and methods
A paired end library suitable for 72-bases read length was prepared and sequenced on an Illumina GAIIx sequencer and analyzed using bwa and samtools with appropriate parameters (outsourced to Genotypic Technologies Ltd, Bangalore). The SNP data was adjusted for read depth (1/10th SD) and rare allele frequency (<5%). Further approximate equal frequency (EF) blocks were manually estimated by nearest neighborhood (NN) analysis in MS Excel (MS Office 2007), wherein, a block of NN SNPs having frequency difference of less than 0.02-0.03 was considered as single EF block. Web-based gene prediction tool FGENESH (http://linux1.softberry.com) was used for identifying genic regions such as UTRs, exons and introns with Arabidopsis thaliana gene model.
Results and discussion
Results from SNP discovery in 41 candidate genes.
Predicted gene region
SNP frequency parameters
SNP count (range)
SNP Frequency in bases/SNP (range)
Total length in bp (range)
Herein, NGS (Illumina) platform was successfully used for identifying ~1,200 SNPs in 41 targeted genes in Ec which has shed important light on quantitative and qualitative distribution of SNPs. In addition, the analysis of EF blocks also provided important guidelines for selection of SNPs for genotyping.
The authors acknowledge valuable discussions with Dr. Navin Sharma, Dr. DS Gurumurthy, (both ITC R&D Centre, Bangalore, India) and Dr. BR Thumma, Dr. Simon Southerton, (both CSIRO-Plant Industry, Canberra, Australia) and also the Eucagen website (http://eucalyptusdb.bi.up.ac.za/gbrowse8x) for making the Eg sequence available.
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