Exploring DNA variant segregation types in pooled genome sequencing enables effective mapping of weeping trait in Malus.

To unlock the power of next generation sequencing-based bulked segregant analysis in allele discovery in out-crossing woody species, and to understand the genetic control of the weeping trait, an F1 population from the cross 'Cheal's Weeping' × 'Evereste' was used to create two genomic DNA pools 'weeping' (17 progeny) and 'standard' (16 progeny). Illumina pair-end (2 × 151 bp) sequencing of the pools to a 27.1× (weeping) and a 30.4× (standard) genome (742.3 Mb) coverage allowed detection of 84562 DNA variants specific to 'weeping', 92148 specific to 'standard', and 173169 common to both pools. A detailed analysis of the DNA variant genotypes in the pools predicted three informative segregation types of variants: <lm×mm> (type I) in weeping pool-specific variants, and <lm×ll> (type II) and <hk×hk> (type III) in variants common to both pools, where the first allele is assumed to be weeping linked and the allele shown in bold is a variant in relation to the reference genome. Conducting variant allele frequency and density-based mappings revealed four genomic regions with a significant association with weeping: a major locus, Weeping (W), on chromosome 13 and others on chromosomes 10 (W2), 16 (W3), and 5 (W4). The results from type I variants were noisier and less certain than those from type II and type III variants, demonstrating that although type I variants are often the first choice, type II and type III variants represent an important source of DNA variants that can be exploited for genetic mapping in out-crossing woody species. Confirmation of the mapping of W and W2, investigation into their genetic interactions, and identification of expressed genes in the W and W2 regions provided insight into the genetic control of weeping and its expressivity in Malus.