Genome assembly and association tests identify interacting loci associated with vigor, precocity, and sex in interspecific pistachio rootstocks

Understanding the basis of hybrid vigor remains a key question in crop breeding and improvement, especially for rootstock development where F 1 hybrids are extensively utilized. Full-sibling UCB-1 F 1 seedling rootstocks are widely planted in commercial pistachio orchards that are generated by crossing two highly heterozygous outbreeding parental trees of Pistacia atlantica (female) and P. integerrima (male). This results in extensive phenotypic variability, prompting costly removal of low-yielding small trees. To identify the genetic basis of this variability, we assembled chromosome-scale genome assemblies of the parental trees of UCB-1. We genotyped 960 UCB-1 trees in an experimental orchard for which we also collected multi-year phenotypes. We genotyped an additional 1,358 rootstocks in six commercial pistachio orchards and collected single-year tree size data. Genome-wide single marker association tests identified loci associated with tree size and shape, sex, and precocity. In the experimental orchard, we identified multiple trait-associated loci and a strong candidate for ZZ/ZW sex chromosomes. We found significant marker associations unique to different traits and to early vs. late phenotypic measures of the same trait. We detected two loci strongly associated with rootstock size in commercial orchards. Pseudo-testcross classification of markers demonstrated that the trait-associated alleles for each locus were segregating in the gametes of opposite parents. These two loci interact epistatically to generate the bimodal distribution of tree size with undesirable small trees observed by growers. We identified candidate genes within these regions. These findings provide a foundational resource for marker development and genetic selection of vigorous pistachio UCB-1 rootstock.