Linkage mapping in tetraploid blackberry (Rubus spp.) using high-throughput genomic sequencing and restriction-site associated DNA sequencing (RAD-seq)

Genomic molecular marker research and linkage mapping in cultivated polyploid species of Rubus, in particular blackberry, has lagged behind diploid members of the genus due to the complex analyses required to deduce allele dosage among homologous and homeologous chromosomes. Traditional techniques for studying linkage in tetraploid species, including Rubus, have made use of limited numbers of scorable markers – such as expressed sequence tag simple sequence repeats (EST-SSRs) or amplified fragment length polymorphisms (ALFPs) – and large mapping populations essential for the observation of recombination events among dominant markers. More recently, high-throughput genomic sequencing has enabled the discovery of an abundance of co-dominant, single nucleotide polymorphic (SNP) markers, at orders of magnitude greater than previous marker discovery methods. The purposes of this research are 1) to derive a linkage map of single nucleotide polymorphisms (SNPs) in tetraploid blackberry (Rubus spp.) using the reduced-representation, restriction-site associated genomic DNA sequencing method; 2) to discover quantitative trait loci (QTL) associated with traits of economic importance to blackberry breeders and producers; and 3) to characterize the structure and diversity within the blackberry genome, especially through comparisons against its diploid cousin Rubus idaeus, the red raspberry. The techniques developed in the present study extend the utility of restriction site associated DNA sequencing (RAD-Seq) to tetraploid species, and will be of interest to others working with polyploid species. This research generated high-density maps for two blackberry cultivars, ‘Chester Thornless’ and ‘Prime-Jim’ (‘APF-12’), consisting of 2,118 markers in 29 linkage groups spanning a total map distance of 1,059.1 cM and 1,759 markers in 31 linkage groups spanning 1,024.6 cM, respectively. The primocane-fruiting locus F was determined to be heterozygous duplex in ‘Chester Thornless’, and supporting evidence for the positioning of the thornless locus S at one end of the fourth linkage group was uncovered. Furthermore, the within-genome heterozygosity was determined to be 1.915% for ‘Chester Thornless’ and 1.749% for ‘Prime-Jim’. Double reduction was observed in the data to occur at a minimum rate of 7.94% across all heterozygous loci. This value enabled the calculation of observation-based estimates for the inbreeding coefficients for both cultivars: 0.04554 for ‘Chester Thornless’ and 0.04235 for ‘Prime-Jim’ at a predicted maximum