Association genetics of drought tolerance in ponderosa pine (Pinus ponderosa)

Drought stress is a major cause of tree mortality in Mediterranean coniferous forests. This study aims to investigate the genetics of drought tolerance in ponderosa pine (Pinus ponderosa), a highly valuable species in the western United States. Genotype-to-environment (G2E) association investigates the statistical association between genetic variation at individual loci and the environment, while genotype-to-phenotype (G2P) association identifies loci linked to a particular phenotype by correlating genotypes at SNPs with the variation in certain traits. By combining G2E and G2P association genetics, this study can identify both the loci and traits that may explain variation in drought tolerance in this pine species. Single Nucleotide Polymorphism (SNP) markers have rapidly gained popularity due to their abundance in most genomes and their amenability to high-throughput genotyping techniques. Genotyping-by-sequencing (GBS) has been demonstrated to be a robust and cost-effective genotyping method. We first compared the performance of four GBS bioinformatics pipelines, two of which require a reference genome (TASSEL-GBS V2 and Stacks), two of which are de novo pipelines (UNEAK and Stacks), on this large-genome non-model organism. Stacks with a reference genome produced the highest number of SNPs with lowest proportion of paralogs. Over 4 million SNPs were identified with 223 ponderosa pine individuals using this method and the reference genome of loblolly pine (Pinus taeda). Then I ran a G2E analysis with these SNPs and five chosen climatic variables using LFMM2, which controls for the effects of demographic processes and population structure on the distribution of genetic variation. I found 213 SNPs strongly associated with mean maximum temperature of summer, 335 with mean minimum temperature of winter, 1798 with April 1st snow pack, and 120 SNPs with mean climatic water deficit. Protein functions linked to associated SNPs include ubiquitination, the abscisic acid (ABA) signaling pathway, cell division or growth of roots or shoots, cell wall organization, seed dormancy. The G2P analysis was carried out based on greenhouse experiment data. Seeds from 48 genotyped mother trees were planted in both dry and wet treatments. Eight phenotypic traits were measured during or after the greenhouse experiment. Six were drought-responsive, including root length, root-shoot dry mass ratio, stomata density on adaxial side, and number of stomatal rows on abaxial side (all higher in dry treatment), as well as shoot weight and height growth (lower). I found 153 SNPs strongly associated with root length, 80 with shoot weight, 145 with height growth, 42 with adaxial stomatal density, 85 with abaxial stomatal rows, and 1530 with root-to-shoot ratio. The identified SNPs reside in genes with a wide variety of functions, including ubiquitination, abscisic acid (ABA) signaling pathway, cell division or growth of roots or shoots, cell wall organization, which overlap with most of the identified protein functions in the G2E analysis. Potentially, future studies can develop molecular tools based on the associated genetic markers to assist breeders and gene resource managers in developing and managing adapted populations