Abstract
BackgroundMany areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Genotyping-by-sequencing (GBS) is a one such method highly suited to non-human organisms. In the GBS protocol, genomic DNA is fractionated via restriction digest, then reduced representation is achieved through size selection. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population. This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. We have modified the GBS technology in a number of ways. Custom, enzyme specific adaptors have been replaced with standard Illumina adaptors compatible with blunt-end restriction enzymes. Multiplexing is achieved through a dual barcoding system, and bead-based library preparation protocols allows for in-solution size selection and eliminates the need for columns and gels.ResultsA panel of eight restriction enzymes was selected for testing on B73 maize and Nipponbare rice genomic DNA. Quality of the data was demonstrated by identifying that the vast majority of reads from each enzyme aligned to restriction sites predicted in silico. The link between enzyme parameters and experimental outcome was demonstrated by showing that the sequenced portion of the genome was adaptable by selecting enzymes based on motif length, complexity, and methylation sensitivity. The utility of the new GBS protocol was demonstrated by correctly mapping several in a maize F2 population resulting from a B73 × Country Gentleman test cross.ConclusionsThis technology is readily adaptable to different genomes, highly amenable to multiplexing and compatible with over forty commercially available restriction enzymes. These advancements represent a major improvement in genotyping technology by providing a highly flexible and scalable GBS that is readily implemented for studies on genome-wide variation.Electronic supplementary materialThe online version of this article (doi:10.1186/1471-2164-15-979) contains supplementary material, which is available to authorized users.
Highlights
Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms
The key modification was that by choosing restriction enzymes that generated blunt ends fragments rather than ones with staggered ends, the custom enzyme-specific adaptors used in the original protocol [18] could be replaced with standard Illumina Y-adaptors
The results of these modifications were significant reduction in cost, compatibility with a variety of blunt-end restriction enzymes, and a streamlined protocol that was adaptable to high throughput population genomic applications
Summary
Many areas critical to agricultural production and research, such as the breeding and trait mapping in plants and livestock, require robust and scalable genotyping platforms. Since many restriction sites are conserved across a species, the sequenced portion of the genome is highly consistent within a population This makes the GBS protocol highly suited for experiments that require surveying large numbers of markers within a population, such as those involving genetic mapping, breeding, and population genomics. Genome resequencing has emerged as the principal means for identifying both the genotypes of single individuals and genetic variation within populations or species Methods such as whole genome and whole exome sequencing can generate data on large numbers of common and rare variants and discover previously uncharacterized variants. The amount of sequencing required to achieve high coverage, especially in large eukaryotic genomes such as many plants, can be prohibitively expensive This restricts the application of high-coverage WGS-based genotyping. WGS methods that rely on 20× to 30× coverage are preferred when attempting to identify sample specific variation or very limited numbers of samples in a population are available
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