Contents 1034I.1034II.1035III.1037IV.1038V.1042VI.1043VII.1045References1045 Summary As temperatures warm and precipitation patterns shift as a result of climate change, interest in the identification of tree genotypes that will thrive under more arid conditions has grown. In this review, we discuss the multiple definitions of ‘drought tolerance’ and the biological processes involved in drought responses. We describe the three major approaches taken in the study of genetic variation in drought responses, the advantages and shortcomings of each, and what each of these approaches has revealed about the genetic basis of adaptation to drought in conifers. Finally, we discuss how a greater knowledge of the genetics of drought tolerance may aid forest management, and provide recommendations for how future studies may overcome the limitations of past approaches. In particular, we urge a more direct focus on survival, growth and the traits that directly predict them (rather than on proxies, such as water use efficiency), combining research approaches with complementary strengths and weaknesses, and the inclusion of a wider range of taxa and life stages.

Climate change is imposing new selective pressures on forest tree populations. Evolutionary responses may be important for trees, despite long generation times, since many populations harbor significant genetic variation in environmentally relevant traits. However, the heritability of climate responses (e.g., how much growth differs under drought) has been investigated in only one tree species previously. Here, we quantify heritable variation in environmental response traits of Pinus ponderosa using a greenhouse trial. Five seedlings from each maternal family were exposed to either wet or dry treatments for four months. Traits measured include height and root length, root:shoot mass ratios, and stomatal density and number of rows. Narrow‐sense heritability for all traits was estimated using a half‐sib analysis. Many traits varied by treatment, and some also differed along gradients of “home” climate. Dry treatment root‐to‐shoot dry mass ratio was associated with survival in that treatment. Estimated heritability for drought responsiveness ranged from 0.15 to 0.65, with the highest values occurring in height growth responses. Variation was particularly high for shoot growth under drought conditions. Our results suggest that increasingly arid conditions could result in strong responses to selection.

Background Single Nucleotide Polymorphism (SNP) markers have rapidly gained popularity due to their abundance in most genomes and their amenability to high-throughput genotyping techniques. Reduced-representation restriction-enzyme-based sequencing methods (GBS or RADseq) have been demonstrated to be robust and cost-effective genotyping methods. While previous studies have shown that alignment of the short-read fragments to a genome sequence results in better SNP calling than de novo approaches, only a few tree species - and few conifers in particular - have an annotated sequence. While these could be used to align sequence fragments from related species, sequence divergence might result in SNPs being missed if they are in fragments that don't align properly. Producing a new annotated genome sequence for every conifer species before SNP analyses are conducted is still prohibitive, as many conifer genomes are huge (> 19 GB) and include a large proportion of repeat sequences, making assembly difficult. Here we compare four bioinformatics pipelines, two of which require a reference genome (TASSEL-GBS V2 and Stacks), two of which are de novo pipelines (UNEAK and Stacks). We used Illumina sequence data from 94 ponderosa pines, with loblolly pine as the reference genome. Results The number of SNPs called was much lower without a reference genome (62–196 thousand vs. 2.1–2.7 million SNPs). UNEAK was the fastest overall and identified more SNPs than Stacks de novo. Stacks with a reference genome produced the highest number of SNPs with lowest proportion of paralogs, while SNPs identified by TASSEL-GBS V2 exhibited the highest heterozygosity, minor allele frequency, and proportion of paralogs. More SNPs were uniquely identified by Stacks than TASSEL, though there was high overlap between methods. Conclusion The present case study provides a comprehensive comparison between four commonly-used SNP calling pipelines, and identifies the Stacks reference-based approach as the best overall for conifers (or other species with large repetitive genomes) that do not have a published reference genome for the same species. However, all four pipelines had distinct benefits and limitations, with Stacks for instance being less user-friendly than some of the other pipelines. In addition, researchers studying other conifer species using similar approaches should be prepared to analyze very large numbers of SNPs.

Background Genotype-to-environment (G2E) association analysis coupled with genotype-to-phenotype (G2P) association analysis promises exciting advances towards discovering genes responsible for local adaptation. We combine G2E and G2P analysis with gene annotation in Pinus ponderosa (ponderosa pine), an ecologically and economically important conifer that lacks a sequenced genome, to identify genetic variants and gene functions that may be associated with local adaptation to drought. Results We identified SNP markers in 223 genotypes from across the Sierra Nevada by aligning GBS sequence fragments to the reference genome of Pinus taeda (loblolly pine). Focusing on SNPs in or near coding regions, we found 1458 associated with 5 largely-uncorrelated climate variables, with the largest number (1151) associated with April 1st snow pack. We also planted seeds from a subset of these trees in the greenhouse, subjected half of the seedlings to a drought treatment, and measured phenotypes thought to be associated with drought tolerance, including root length and stomatal density. 817 SNPs were associated with the control-condition values of six traits, while 1154 were associated with responsiveness of these traits to drought. Conclusions While no individual SNPs were associated with both the environmental variables and the measured traits, several categories of genes were associated with both, particularly those involved in cell wall formation, biotic and abiotic stress responses, and ubiquitination. However, functions of many of the associated genes have not yet been determined due to the lack of gene annotation information for trees and future studies are needed.

As climate changes, understanding the genetic basis of local adaptation in plants becomes an ever more pressing issue. Combining Genotype-Environment Association (GEA) with Genotype-Phenotype Association (GPA) analysis has an exciting potential to uncover the genetic basis of environmental responses. We use these approaches to identify genetic variants linked to local adaptation to drought in Pinus ponderosa. Over 4 million SNPs were identified using 223 individuals from across the Sierra Nevada of California. We found 1458 associated with five largely uncorrelated climate variables, with the largest number (1151) associated with April 1st snowpack. We also conducted a greenhouse study with various drought-tolerance traits measured in seedlings grown in control and drought treatments. 817 SNPs were associated with control-condition trait values, while 1154 were associated with responsiveness of these traits to drought. While no individual SNPs were associated with both the environmental variables and the measured traits, several annotated genes were associated with both, particularly those involved in cell wall formation, biotic and abiotic stress responses, and ubiquitination. However, the functions of many of the associated genes have not yet been determined due to the lack of gene annotation information for conifers. Future studies are needed to assess the developmental roles and ecological significance of these unknown genes.