RNA-Seq reveals adaptive genetic potential of the rare Torrey pine (Pinus torreyana) in the face of Ips bark beetle outbreaks

The ability of tree species to adapt to water stress and increased frequency of bark beetle outbreaks with climate change may increase with population size and standing genetic variation, calling into question the resilience of small, rare plant populations.  The Torrey pine (Pinus torreyana) is a rare, genetically depauperate conifer that occurs naturally in a mainland and island population in southern California.  Due to recent declines in the mainland population coinciding with drought and Ips paraconfusus bark beetle outbreaks, the species would benefit from an assessment of adaptive genetic diversity.  Here, we use RNA-Seq to survey gene-coding diversity across 40 individuals to 1) characterize patterns of genetic diversity in the species and 2) test for genetic differentiation between trees that succumbed to beetle attack or survived following an outbreak.  Consistent with previous studies, we found few genetic variants, with most SNPs occurring as fixed differences between populations.  However, we found structure within the mainland and polymorphisms segregating in both populations.  Interestingly, we found differentiation in genotypes between attacked and surviving trees and 11 SNPs associated with survival status, several of which had defense-related functions.  While low diversity suggests limited adaptive capacity, genetic associations with survival in functionally relevant genes suggest adaptive potential for bark beetle defense.  This initial study prompts future research to explore the genetic basis of putative resistance and suggests conservation efforts should protect surviving genotypes and the full spectrum of genetic diversity across populations to preserve the evolutionary potential of the species.

Methods
Our dataset consisted of 40 Torrey pine trees, including 32 cases and controls that succumbed to attack or survived following a bark beetle outbreak, respectively, and eight additional trees for diversity estimates.  We filtered RNA-seq fastq files using the HTStream pre-processing pipeline (UCDavis) and aligned reads to the Pinus taeda genome v2.01 downloaded from the TreeGenes database.  Due to the large and fragmented nature of the genome assembly, we sorted contigs by size and concatenated them into 222 ~100 Mb ‘pseudocontigs’ with contigs separated by 10 N’s.  We then aligned processed Torrey pine reads to the P. taeda pseudocontigs using two-pass mode with the splice aware aligner STAR v2.6.0.  We used Samtools v1.9 to retain only uniquely mapped reads and to remove PCR/optical duplicates, secondary/supplementary alignments, reads failing QC, and unpaired reads.  Refer to the manuscript for further data processing details.