Flexible and scalable genotyping-by-sequencing strategies for population studies

BMC Genomics

Stephen L Dellaporta²  Joe Tohme¹  Hongyu Zhao⁵  John P Mottinger³  John D Overton⁴  Maria A Moreno²  Christopher A Fragoso⁵  Christopher Heffelfinger² 

[1] Agrobiodiversity Research Area, Centro Internacional de Agricultura Tropical (CIAT), A.A. 6713, Cali, Colombia;Department of Molecular, Cellular, and Developmental Biology, Yale University, New Haven, CT 06511, USA;Department of Cell and Molecular Biology, University of Rhode Island, Kingston, RI 02881, USA;Current: Regeneron Genetics Center, Regeneron, Tarrytown, NY 10591, USA;Department of Computational Biology and Bioinformatics, Yale University, New Haven, CT 06520-8034, USA

关键词: Agricultural genomics;    Plant breeding;    Trait mapping;    Population genomics;    Reduced representation sequencing;    GBS;    Genotyping;   

Others  :  1092451
DOI  :  10.1186/1471-2164-15-979

 received in 2014-05-28, accepted in 2014-10-23,  发布年份 2014

【 摘 要 】

Background

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. 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.

Results

A 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.

Conclusions

This 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.

【 授权许可】

   
2014 Heffelfinger et al.; licensee BioMed Central Ltd.

【 图 表 】

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