Genotyping-by-sequencing (GBS), an ultimate marker-assisted selection (MAS) tool to accelerate plant breeding

Abstract

Marker-assisted selection (MAS) refers to the use of molecular markers to assist phenotypic selections in crop improvement. Several types of molecular markers, such as single nucleotide polymorphism (SNP), have been identified and effectively used in plant breeding. The application of next-generation sequencing (NGS) technologies has led to remarkable advances in whole genome sequencing, which provides ultra-throughput sequences to revolutionize plant genotyping and breeding. To further broaden NGS usages to large crop genomes such as maize and wheat, genotyping-by-sequencing (GBS) has been developed and applied in sequencing multiplexed samples that combine molecular marker discovery and genotyping. GBS is a novel application of NGS protocols for discovering and genotyping SNPs in crop genomes and populations. The GBS approach includes the digestion of genomic DNA with restriction enzymes followed by the ligation of barcode adapter, PCR amplification and sequencing of the amplified DNA pool on a single lane of flow cells. Bioinformatic pipelines are needed to analyze and interpret GBS datasets. As an ultimate MAS tool and a cost-effective technique, GBS has been successfully used in implementing genome-wide association study (GWAS), genomic diversity study, genetic linkage analysis, molecular marker discovery and genomic selection under a large scale of plant breeding programs.

Keywords: genomic selection (GS); genotyping-by-sequencing (GBS); marker-assisted selection (MAS); next-generation sequencing (NGS); single nucleotide polymorphism (SNP).

FIGURE 1

Schematic steps of the genotyping-by-sequencing (GBS) protocol for plant breeding. Panel (A): tissue is obtained from any plant species as depicted here a young triticale plant; Panel (B): ground leaf tissues for DNA isolation, quantification and normalization. At this step it is important to prevent any cross-contamination among samples; Panel (C): DNA digestion with restriction enzymes; Panel (D): ligations of adaptors (ADP) including a bar coding (BC) region in adapter 1 in random PstI-MseI restricted DNA fragments; Panel (E): representation of different amplified DNA fragments with different bar codes from different biological samples/lines. These fragments represent the GSB library; Panel (F): analysis of sequences from library on a NGS sequencer; Panel (G): bioinformatic analysis of NGS sequencing data; Panel (H): possible application of GBS results.