An Effective Strategy for Analyzing Pooled Genome Sequencing Data in Outcrossing Woody Species: A Case Study to Localize the Weeping (W) Locus in Malus

The development of next generation sequencing (NGS) technologies has drastically renovated approaches in genomics studies. Implementing the NGS-enabled whole genome sequencing in bulk segregant analysis (BSA), a methodology used widely in genetic mapping, is one of such examples. Several mapping strategies in pooled genome sequencing data analysis have been proposed and demonstrated with successful applications. To perform similar pooled genome sequencing data analysis in woody plants of heterogeneously heterozygous genomes, such as Malus species, these mapping strategies need to be adapted as DNA variants from these species are of complex inheritance patterns and their phases are often unknown. In this study, an effective strategy that includes two approaches, dubbed mutant allele frequency density (MAFD) mapping and allele frequency directional difference and density (AFDDD) mapping, is proposed and shown to be useful in mapping the weeping phenotype to chromosome 13 in Malus. To be principally sound, the genetic basis for these approaches were investigated and confirmed. Both MAFD and AFDDD mapping approaches can be performed without prior knowledge of the parental genotypes.