Genotyping By Sequencing in Fruit Tree Species

Genotyping-by-sequencing (GBS) applies Next Generation Sequencing to large genomes that have been reduced in complexity and hence enables genotyping of mass individuals in a cost effective manner. GBS is now replacing other techniques such as microsatellites and SNP arrays as a method of choice for genetic markers identification for a range of traits.  However reduced-representation sequencing has a degree of technical uncertainty which can result in uneven sequencing of samples and consequently missing data issues in GBS analysis. In this study, we investigate the effects of using different restriction enzymes for library construction. We report the consequences on the efficiency and reproducibility of the GBS approach in a range of fruit crops, including apple, pear, and kiwifruit (Actinidia chinensis). In apple we have constructed three GBS libraries using BamHI, ApeKI and MspI enzymes for a set of 61 apple DNAs. For pear we detected more than 205 million raw SNP sites, among which 29,865 SNP sites were without ambiguity in 13 taxa.  In kiwifruit, we employed GBS data from 48 taxa of a bi-parental population segregating for tolerance to Pseudomonas syringae pv actinidiae (Psa) to construct a draft genetic map consisting of thirty-one Linkage Groups (LGs) in each parent, with a LOD 4 for grouping with at least 10 markers per LG.  We have employed this map to identify putative markers for a QTL conferring tolerance to Psa.  We are also utilizing GBS data for  genome-wide selection and genome-wide association studies.