Abstract
Genotyping-by-sequencing (GBS) has recently emerged as a promising genomic approach for exploring plant genetic diversity on a genome-wide scale. However, many uncertainties and challenges remain in the application of GBS, particularly in non-model species. Here, we present a GBS protocol we developed and use for plant genetic diversity analysis. It uses two restriction enzymes to reduce genome complexity, applies Illumina multiplexing indexes for barcoding and has a custom bioinformatics pipeline for genotyping. This genetic diversity-focused GBS (gd-GBS) protocol can serve as an easy-to-follow lab guide to assist a researcher through every step of a GBS application with five main components: sample preparation, library assembly, sequencing, SNP calling and diversity analysis. Specifically, in this presentation, we provide a brief overview of the GBS approach, describe the gd-GBS procedures, illustrate it with an application to analyze genetic diversity in 20 flax (Linum usitatissimum L.) accessions and discuss related issues in GBS application. Following these lab bench procedures and using the custom bioinformatics pipeline, one could generate genome-wide SNP genotype data for a conventional genetic diversity analysis of a non-model plant species.
Highlights
Plant genetic diversity analysis is an important component in studies of plant genetics, breeding, conservation and evolution
Major steps include DNA extraction and digestion, adapter ligation, PCR amplification, fragment size selection, library pooling, sequencing, data processing, single nucleotide polymorphism (SNP) calling and genetic diversity analysis. These steps may vary in restriction endonuclease (RE) use, next generation sequencing (NGS) platform and in bioinformatics analysis for SNP genotyping for different study objectives
Since 2009, we have explored workable GBS protocols for plant genetic diversity analysis based on the Roche 454 pyrosequencing platform [4,10,17] and Illumina HiSeq and MiSeq sequencing platforms
Summary
Plant genetic diversity analysis is an important component in studies of plant genetics, breeding, conservation and evolution. The GBS approach is based on genome reduction with restriction enzymes [9], does not require a reference genome for SNP discovery, is a combined one-step process of marker discovery and genotyping and provides a rapid, high-throughput and cost-effective tool for a genome-wide analysis of genetic diversity for a range of non-model species and germplasm sets [5,10]. These characteristics are advantageous and encouraging for genetic diversity analysis of plants with no informative markers available. It is our hope that researchers can start to take advantage of the power of whole genome sequencing to analyze genetic diversity, in non-model plants, with the help of the published gd-GBS protocol
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