Host plant genetic control of associated fungal and insect species in a Populus hybrid cross

Abstract Plants employ a diverse set of defense mechanisms to mediate interactions with insects and fungi. These relationships can leave lasting impacts on host plant genome structure such as rapid expansion of gene families through tandem duplication. These genomic signatures provide important clues about the complexities of plant/biotic stress interactions and evolution. We used a pseudo‐backcross hybrid family to identify quantitative trait loci (QTL) controlling associations between Populus trees and several common Populus diseases and insects. Using whole‐genome sequences from each parent, we identified candidate genes that may mediate these interactions. Candidates were partially validated using mass spectrometry to identify corresponding QTL for defensive compounds. We detected significant QTL for two interacting fungal pathogens and three insects. The QTL intervals contained candidate genes potentially involved in physical and chemical mechanisms of host–plant resistance and susceptibility. In particular, we identified adjoining QTLs for a phenolic glycoside and Phyllocolpa sawfly abundance. There was also significant enrichment of recent tandem duplications in the genomic intervals of the native parent, but not the exotic parent. Tandem gene duplication may be an important mechanism for rapid response to biotic stressors, enabling trees with long juvenile periods to reach maturity despite many coevolving biotic stressors.

The gene-for-gene theory suggests a very simple dynamic for the genetic interactions that occur between two species. A gene in the host plant that is important in biotic relationships has a corresponding, coevolving gene from a pathogen/insect which can lead to resistance or susceptibility depending on the life history of the pathogen/insect (Flor, 1971;Friesen, Meinhardt, & Faris, 2007). Much of the evidence for these interactions has been found in crop systems where plant species often have dominant, single-gene resistance to feeding (Thompson, 1988). For example, there are over twenty different genes in wheat (Triticum aestivum L.) that each confers resistance to the Hessian fly, Mayetiola destructor (Thompson & Burdon, 1992). Exposure of Hessian fly populations to these resistant varieties of wheat created selection pressure that led to increased virulent gene combinations in the pest (Gallun, 1977;Panda & Khush, 1995). Similarly, in plant-fungal systems breeding for dominant resistance in cereal crops resulted in new selective forces that increased virulent gene frequencies in Puccinia spp. cereal rusts (Chen, 2005;Pretorius, Singh, Wagoire, & Payne, 2000). This in turn can lead to an evolutionary arms race between plants, insects, and fungi with the continual development of mechanisms to overcome both genetic defenses and virulent attacks (Bergelson, Kreitman, Stahl, & Tian, 2001; Thompson & Burdon, 1992).
The relationships of host plant genetics and biotic association can also be more complex than these crop breeding systems suggest, and they can leave a lasting impact on genome structure (Lefebvre & Chèvre, 1995). Host plant and biotic associations can lead to the expansion of gene families responsible for the host plant response to biotic stress. For example, the Kunitz protease inhibitors (KPIs) in Populus are important in defense responses against insects by inhibition of herbivore digestion (Haruta, Major, Christopher, Patton, & Constabel, 2001;Major & Constabel, 2008). The KPI gene family has greatly expanded in response to insect attack through tandem duplication events (Philippe, Ralph, Külheim, Jancsik, & Bohlmann, 2009). Similarly, plant resistance (R) genes, which are important in the defense response of plants to fungal pathogen attack, have also expanded through tandem and segmental duplication events due to biotic pressures (Hulbert, Webb, Smith, & Sun, 2001;Leister, 2004). Analyzing how the genome is structured in the host plant when it associates with fungi and insects is important for studying these relationships and understanding the complexities of their genetic interactions.
Given their rapid growth and vegetative reproduction, Populus species have become a focus for research into biofuel production making them a valuable commercial crop (Meilan et al., 2002;Stanton, Neale, & Li, 2010;Taylor, 2002). Populus has also become an important genetic model for research into a wide variety of ecological and adaptive traits , including interactions with the biotic community (Crutsinger et al., 2014;Whitham et al., 2006). In particular, interspecific hybrids of P. trichocarpa × P. deltoides segregate for a wide variety of traits including resistance to insect and fungal attack (Newcombe, 1998;Newcombe & Ostry, 2001). Such hybrid family crosses can be used to identify regions of the genome that are important in mediating biotic stress.
In this study, we investigated the genome composition of loci associated with insect and fungal species in an interspecific Populus family. We surveyed insects and fungal pathogens in a P. deltoides × P. trichocarpa pseudo-backcross family and used quantitative trait locus (QTL) analysis and comparative genomics to address three main questions: (a) Is there heritable, host genetic control of fungal and insect species? (b) What protein domains and gene ontology terms are enriched in the QTL intervals in the genomes of each Populus species? (c) What candidate genes in the intervals are unique to each species when comparing the P. trichocarpa and P. deltoides genomes?

| SNP genotyping and genetic map construction
SNP loci were selected from whole-genome resequencing data generated for the parent trees of the pedigree, focusing on loci that were fixed for different alleles in P. deltoides and P. trichocarpa. Sequencing was performed on the Illumina GAII system with single-end read lengths of 75 bp and a total depth of ~35× on average. Reads were aligned using MAQ, and SNPs were called using Samtools mpileup with a minimum quality of 30 and a minimum depth of 5 reads per allele, and a subset of loci was confirmed by Sanger sequencing (Slavov et al., 2012). Polymorphic loci were selected that were maternally informative for P. trichocarpa. These were incorporated into an Illumina Infinium Bead Array which was used to genotype 3,568 SNP loci in 692 of the progeny. These data were then used to create the genetic map which was composed of 19 linkage groups corresponding to 19 Populus chromosomes. Genotyping and map construction are described in more detail elsewhere (Muchero et al., 2015).

| Family 52-124 parent and progeny phenotyping
In order to identify regions of the Populus genome associated with biotic stress, a variety of fungal pathogens and insect herbivore species was surveyed in the WVU Morgantown and Westport Oregon plantation sites. Melampsora sp. rust was identified visually by local pathologist Dr. William MacDonald. Sphaerulina musiva was identified by sequencing of the ITS region (Verkley, Quaedvlieg, Shin, & Crous, 2013). Insect identification was completed using insect morphological features, feeding symptoms, and known hosts/species distributions for Phyllocolpa sp. (Kopelke, 2007;Smith & Fritz, 1996), Mordwilkoja vagabunda (Ignoffo & Granovsky, 1961a, 1961b, and Pemphigus populitransversus (Bird, Faith, Rhomberg, Riska, & Sokal, 1979;Faith, 1979). In early August 2017, at the Westport site full canopies were scored by counting galls of the leaf-folding sawfly Phyllocolpa sp.
( Figure 1f), on 534 unique genotypes and a total of 1,020 trees. To estimate productivity of individuals and confirm that availability of resources did not drive insect attraction or feeding, main stem diameter in millimeters was recorded for all trees to be used as a covariate in the analysis.

| Within-family broad-sense heritability (H 2 ) calculation
To prevent arbitrary score bias, Melampsora sp. and S. musiva values were normalized to have a mean of 0 and standard deviation of 1 (score.transform function of the CTT R package) by applying the inverse of the cumulative distribution function of the normal distribution to the sample percentile score (Gianola & Norton, 1981). Surveys of the Melampsora sp. leaf rust, S. musiva leaf spot, and counts of the Phyllocolpa sp. galls had within-garden microsite variation removed using thin-plate spline regression (fields R package). Residuals of the spatial correction were added to the mean of each survey dataset for each tree observation to spatially correct and rescale the values.
These corrected values were then used to calculate the proportion of variance in the fungal and insect distributions that were due to genotype using a linear-mixed model (lmer function of the lme4 R package) with insect counts and fungal scores as the response, tree genotype as the predictor, and either competing fungus score or stem diameter biomass estimates as a covariate where applicable.
Broad-sense heritability was calculated as H 2 = 2 g ∕( 2 g + 2 e ), where 2 g is the genetic variance due to genotype, and 2 e is the residual variance. Rapid, simulation-based exact likelihood ratio tests were used to evaluate the significance of variation due to genotype for each linear model (exactRLRT function of the RLRsim R package). SAS software version 9.4 (2013) was used to test for the normality of all datasets, and for all subsequent statistical analyses, transformations were conducted when necessary. Finally, best linear unbiased predictors (BLUPs) were extracted from these models to use in the QTL analysis.

| Quantitative Trait Loci (QTL) analysis
The R software package R/qtl (Broman, Wu, Sen, & Churchill, 2003) was used for all QTL analyses described below. Composite interval mapping (CIM) was used to associate Melampsora sp. leaf rust, S. musiva leaf spot, and Phyllocolpa sp. to QTL positions (cim function).
An additional CIM QTL analysis was conducted for fungi surveyed in 2008 to further evaluate potential competitive bias of co-occurring fungal pathogens. This was done by subsetting individuals out of the full dataset to exclude trees with symptoms of both pathogens, leaving us with 90 individuals that only showed Melampsora sp. symptoms and 434 individuals only infected with S. musiva leaf spot. Single QTL mapping was used to associate the binary scores for the S. musiva canker, M. vagabunda, and P. populitransversus to QTL positions (scanone function). The method used for both mapping approaches was the expectation-maximization (EM) algorithm.
Estimation of QTL interval significance was completed by performing 1,000 permutations. Intervals with logarithm of odds (LOD) scores that were above the p-value threshold (alpha = 0.05), as determined from the permutation tests, were selected for further analysis. The percent variance explained by significant markers for fungal and insect surveys, that were mapped using CIM, were calculated by extracting significant marker positions and creating a fit QTL model (fitQTL function). The positive allele contributing to an increase in susceptibility to fungi and insects was found by generating effect plots for each phenotype and its significant marker position (effectplot function).

| Physical genome intervals
Physical genome intervals in the P. trichocarpa genome (v3.0) were examined for each significant QTL for biotic associations. The intervals were defined as 1 Mb regions centered on the marker with the highest LOD score. Fixed physical genome sizes were used rather than intervals defined based on LOD scores due to the large variation in magnitude of LOD observed for the significant QTL. For example, intervals of 1 LOD centered on the QTL ranged in size from 169 to 4,620 kb. Much of this variation was likely due to variation in marker density and local recombination rates, in addition to phenotyping and genotyping error. We believe that a fixed 1 Mb interval is a more consistent and conservative approach given the size of the family and the variation in strength of the QTL (Yin, DiFazio, Gunter, Riemenschneider, & Tuskan, 2004). On average, this represents approximately 6.34 cM, based on a total map size of 2,617 cM and a total assembled genome length of 420 Mb.
Orthologous intervals were identified in the P. deltoides clone WV94 reference genome (v2.1) obtained from Phytozome (Goodstein et al., 2012). Orthology was determined using a combination of protein sequence conservation and synteny using MCScanX (Wang et al., 2012). Briefly, all proteins were compared in all-vs-all searches using blastp both within genomes and between genomes. These were then chained into collinear segments using the MCScanX algorithm. Orthologous segments were identified based on the presence of large numbers of gene pairs in collinear order with high sequence identity (median blastp E score < 1e−180; Figure 2). Synonymous (Ks) and nonsynonymous (Ka) nucleotide substitution rates were calculated using the Bioperl DNAstatistics module (Stajich, 2002; Table S1), domain composition (Table S2), and Gene Ontology (GO) terms (Table S3) were obtained for each genome from Phytozome (v12.1). Intervals were customized for the grandparents of the pseudo-backcross progeny (clones 93-968 for P. trichocarpa and D124 for P. deltoides) by converting the respective reference genome based on alignment of short-read sequences derived from each species. Specifically, we generated 243 and 248 million 250 bp paired-end Illumina HiSeq sequences for 93-968 and D124, respectively. This yielded an average coverage of ~150× per genome. These were aligned to the respective reference genome for each species (Nisqually v3.0 for P. trichocarpa and WV94 v2.0 for P. deltoides) using bwa mem with default parameters. SNPs and small indels were identified using samtools mpileup and bcftools call with default multiallelic variant settings (Li, 2011;Li et al., 2009), and sequence depth was extracted using vcftools (Danecek et al., 2011). Sequences were converted using the vcftools utility vcf-consensus. Genes with no coverage in the alignments were excluded from the intervals for each species.

| Tandem duplications
Tandemly duplicated genes were identified using all-vs-all blastp searches within each genome for biotic stress-associated intervals (Table S4). Genes with blastp E scores < 1e−180 that were located within 500 kb of one another were considered to be recent tandem duplications. The window size was determined by testing a range of values and choosing a window size at which the number of newly discovered tandem duplicates began to decline ( Figure 3). The E score cutoff was chosen because P. trichocarpa and P. deltoides orthologs also have a median blastp E score in this range, suggesting that these tandem duplications mostly occurred after these species diverged from a common ancestor. This should focus the analysis primarily on genes that are recently duplicated and therefore potentially differentially duplicated between the species. The QTL intervals were tested for significant enrichment of tandem duplicates by using a Monte Carlo simulation. Sets of contiguous genes equal in number to those contained in each QTL interval were randomly selected from the whole genome, and the number of sampled tandem duplications was counted for each iteration. This was repeated F I G U R E 2 Collinear genes in P. deltodies (Pd) and P. trichocarpa

| Heritability of fungal and insect associations
Clonal repeatability (or within-family broad-sense heritability) was estimated for each categorical survey trait (Table 1)

| QTL mapping of leaf metabolites
Only one tested compound, gentisyl alcohol 5-O-glucoside levels,

| Interaction between Melampsora sp. and S. musiva leaf infection
Melampsora sp. leaf rust infection severity was dependent upon the disease severity of S. musiva leaf spot symptoms (F = 35.2, pvalue = .0001; Table 3). The severity of Melampsora sp. infection for individuals with no S. musiva leaf spot was significantly higher than for individuals with S. musiva leaf spot symptoms (Figure 6).
S. musiva leaf spot infection severity was significantly lower for individuals with a Melampsora sp. leaf spot score of 3 compared to individuals with less severe Melampsora sp. leaf spot symptoms ( Figure 6).

| PFAM, GO term and tandem duplication enrichment
The total number of genes present in the QTL intervals of each parental species was 174 on Chr04, 81 on Chr16, 161 on Chr10, 445 on Chr13, and 156 on Chr05 (  Table S3). Finally, seven of the genes in these intervals showed evidence of positive selection based on the ratio of nonsynonymous to synonymous nucleotide substitutions between the P. deltoides and P. trichocarpa orthologs (Table 5).
In total, there were 107 recent tandem duplicates in the P. trichocarpa intervals and 195 in the P. deltoides intervals for all biotic QTLs (Figure 7, Table 6). This includes 23 and 41 recent tandem duplicates for the Melampsora sp. intervals; 6 and 10 for the S. musiva intervals; 32 and 96 for the Phyllocolpa sp. intervals; and 47 and 46 for the M. vagabunda intervals, for P. trichocarpa and P. deltoides, respectively. The total number of recent tandem duplicates was significantly enriched relative to tandem counts for random intervals of the same size as the QTL intervals for the P. deltoides grandparent (p-value = .0118) but not for the P. trichocarpa grandparent (p-value = .1191).

| D ISCUSS I ON
The goal of our research was to utilize QTL analysis as a tool to identify regions of the Populus genome that were important in mediating biotic interactions. Upon identification of these regions, we were able to directly compare the parental genomes of the hybrid cross to look for similarity in content and potential gene-for-gene interactions reflected in recent tandem duplication expansion. We found that the host plant genotype had a significant effect on fungi and insects in our study. Additionally, the progeny segregated for varying resistance to fungal and insect pathogens and pests that were inherited from the grandparents and parents of the 52-124 family cross.
Novel alleles (i.e., those from the grandparent that was not native to the region where the trials occurred) appear to be important for resistance to Melampsora sp. fungus, M. vagabunda galls, and Phyllocolpa sp. oviposition. This is not the first case of potential novel alleles being important in resistance to biotic stress. In a P. trichocarpa GWAS population, three genes have been found that confer novel resistance to S. musiva canker infection and an additional single gene that, when inherited, can suppress that resistance (Muchero et al., 2018). Although cases such as this are still under investigation to understand mechanisms of novel resistance, it can also originate from a variety of simple traits inherent to a non-native species such as delayed emergence due to phenology (Mercader, Aardema, & Scriber, 2009) or changes in secondary metabolites that are important cues in insect recognition of the host important in oviposition (Nahrstedt, 1989). In contrast, the interval associated with S. musiva canker symptoms was the only case in which susceptibility to the fungus was dominant and inherited from the noncoevolved host (P. trichocarpa), as has been previously observed (Muchero et al., 2018;Newcombe & Ostry, 2001).
We detected a major QTL for Melampsora sp. resistance on Chr04. Although the specific strain that infected the trees is unknown, this genomic interval is known to contain the MXC3 locus which confers resistance to infection of multiple species of the Melampsora leaf rusts (Newcombe, Stirling, & Bradshaw, 2001;Yin, DiFazio, Gunter, Jawdy, et al., 2004). Based on mean parental infection scores and the allelic effects at the QTL, progeny in family 52-124 inherited this resistance from the P. trichocarpa grandmother 93-968. Furthermore, the QTL interval contained two tandem repeats of stigma-specific proteins (Stig1) in both the P. trichocarpa genome and the orthologous interval in the P. deltoides genome. Several of these genes showed evidence of positive selection based on Ka/Ks ratios (Table 5). Stigma-specific proteins, specifically Stig1, have been found to be associated with female sterility in tobacco (Nicotiana tabacum) and petunia (Petunia hybrida; Goldman, Goldberg, & Mariani, 1994;Verhoeven et al., 2005). Stig1 is known to mediate secretion of exudate lipids in the intercellular spaces and high expression of the protein inhibits pollen grains from penetrating style tissue preventing fertilization (Verhoeven et al., 2005). Most lipid transfer proteins, such as Stig1, are important in plant cell wall loosening, and their expression can prevent penetration of plant tissues (Nieuwland et al., 2005). Diversification of the protein family containing Stig1 may play an important role in lowering fungal infection in Populus by providing a physical barrier to resist the Melampsora sp. hyphae.
Another protein domain that was found to be enriched in the P.
trichocarpa Chr04 interval was the malonyl-CoA decarboxylase C-terminal domain. Similarly, the gene ontology function for malonyl-CoA decarboxylase activity was also enriched in P. trichocarpa in the same interval. Genes which are capable of transforming malonyl-CoA could be beneficial in resistance to Melampsora sp. as it is an important precursor in the production of several pathogen defensive compounds, such as isoprenoids in the Mevalonate pathway (Chen, Kim, Weng, & Browse, 2011;Dixon, 2001).
Interestingly, an overlapping interval on Chr04 was found to be associated with the activity of the leaf spot symptoms of the S. musiva fungus. Upon further investigation, we found that trees that were not infected by the S. musiva leaf spot had more severe symptoms of the Melampsora sp. leaf rust. This suggests that a competitive interaction occurred between the two pathogens in the field during the year of survey and was reflected in an inflated association on Chr04 with the S. musiva leaf spot score. Furthermore, when individuals with presence of Melampsora sp. infection were removed from the S. musiva analysis, the association with Chr04 is no longer significant, and a new QTL appeared on Chr06 (Table S5). Competition among fungal pathogens is not uncommon in field conditions with most examples focused on different genotypes within the same fungal species (Abdullah et al., 2017). The outcome of within host tissue colonization of multiple strains or species often relies upon the genetic similarity of the pathogens (Abdullah et al., 2017;Koskella, Giraud, & Hood, 2006 (Orton & Brown, 2016). Similarly, the outcome of the interaction of the two pathogens in wheat was competitive. However, the necrotroph was found to actually be capable of reducing the reproductive capability of the biotroph which indicates pathogenpathogen interactions can be more direct rather than relying solely on the host plant genetics (Orton & Brown, 2016).
The S. musiva leaf spot and stem canker symptoms were found to be associated with QTL intervals on Chr06 and Chr16, respectively. In this study, susceptibility to the necrotrophic fungi appeared to be originating from the presence of P. trichocarpa alleles in the progeny for both symptoms. Previous work on a P. trichocarpa x deltoides intercross supports this with susceptibility to S. musiva necrotrophic fungi originating primarily from dominant alleles derived from P. trichocarpa (Newcombe, 1998;Newcombe & Ostry, 2001). The Chr06 leaf spot association contained numerous genes with methyltransferase activity which was higher in the P. deltoides interval. Methyltransferase enzymes are important in plant secondary metabolism and have been found to be key in the production of a variety of antimicrobial compounds (Noel, Dixon, Pichersky, Zubieta, & Ferrer, 2003). Interestingly, the loci conferring susceptibility in family 52-124 in the Chr16 canker interval did not overlap with the four loci that were uncovered in a previous genome-wide association study of S. musiva susceptibility in P. trichocarpa (Muchero et al., 2018), suggesting that different mechanisms may be involved in hybrid interactions with this pathogen. However, the QTL did contain a tandem repeat of a G-type lectin receptor-like protein kinase that was expanded in P. deltoides. This protein could play a similar role to a receptor-like kinase from the same family (Yang et al., 2016) that was associated with susceptibility to S. musiva in the P. trichocarpa study (Muchero et al., 2018).
M. vagabunda has been recorded completing its life cycle on several species of Populus including P. deltoides and P. tremuloides (Floate, 2010). Although the aphid's life history has been documented, little is known about the influence of host plant genetics on gall formation or resistance to feeding (Floate, 2010;Ignoffo & Granovsky, 1961a).

TA B L E 6
Tandem duplication profiles for genetic intervals. Number of copies next to species gene name indicates the size of tandem expansion for the gene We detected a QTL on Chr05 in which alleles inherited from P. deltoides were positively associated with gall occurrence. Genes conferring lipoxygenase and oxidoreductase activity were enriched in both the P. deltoides and the P. trichocarpa QTL intervals. Lipoxygenase genes are known to be associated with the Populus response to both abiotic and biotic stressors (Cheng et al., 2006;Ralph et al., 2006).
They are often upregulated in the presence of mechanical damage, fungal pathogen invasion, and exposure to simulated insect feeding (Chen, Liu, Tschaplinski, & Zhao, 2009;Cheng et al., 2006). The lipoxygenases are important in the formation of jasmonic acid, the signaling molecule that upregulates plant defenses against herbivore feeding (Chen et al., 2009).
The M. vagabunda QTL interval on Chr05 also contained a tandem array of resistance genes (R-genes) that encoded disease resistance proteins (TIR-NBS-LRR class) that were greatly expanded in P. trichocarpa compared to P. deltoides, as well as repeats of the leucine-rich repeat protein kinase family proteins in both species. These protein families are well known for their roles in the recognition and upregulation of host plant defenses against bacterial and fungal infection (Bergelson et al., 2001;Martin, Bogdanove, & Sessa, 2003).
Neofunctionalization of R-genes through tandem duplication due to gene-for-gene coevolution has also been demonstrated in many plant-fungal pathosystems (Leister, 2004). Although R-genes have been more frequently related to plant-fungal interactions, there is increasing evidence that they are also important in mediating plantinsect interactions, especially in insects that utilize piercing-sucking feeding (Harris et al., 2003;Kaloshian, 2004).
In addition to the R-genes, there were a series of genes encoding cytochrome P450 family proteins in both species and a unique tandem set that was only present in the P. trichocarpa genome. P450 enzymes are important in the production of many classes of secondary metabolites such as furanocoumarins and terpenoids which are highly toxic to insects (Keeling & Bohlmann, 2006;Schuler, 2011).
Alternatively, cytochrome P450's may also be implicated in the susceptibility of the host plant to galling aphids. They are important in the synthesis of fatty acids and production of suberin in plant tissues (Höfer et al., 2008;Pinot & Beisson, 2011). Typically, suberin is important in separation of different tissues as well as in the establishment of apoplastic barriers that restrict nutrient/water loss as well as pathogen invasion (Höfer et al., 2008;Qin & LeBoldus, 2014). Several insects are known to produce suberized spherical galls on leaves including hymenopteran pests of Rosacea species and dipteran pests of Fabaceae (Krishnan & Franceschi, 1988;Oliveira et al., 2016). If aphids induce the suberization mechanism of the plant genome, it may lead to the increased toughening of aphid is modified in such a way as to act as a sugar sink, thereby enhancing its nutritional value to larvae (Larson & Whitham, 1991;Nyman, Widmer, & Roininen, 2000;Wool, 2004). The presence of these combinations of sugar transporter genes may be mediating a similar interaction between the Phyllocolpa sp. female sawflies and their chosen Populus hosts.
Phyllocolpa sp. galls are formed early in the season when a female sawfly selects a leaf and injects the longitudinal fold with small amounts of fluid on the underside of young leaves (Fritz & Price, 1988;Kopelke, 2007). The adult sawfly will proceed to oviposit near the base of the leaf and after 1-2 days the leaf fold forms, and the newly hatched larvae feeds on the inside of the gall (Smith & Fritz, 1996). The Phyllocolpa sp. galls were a unique biotic phenotype to this study as they were an estimate of female sawfly ovipositional choice rather than feeding success. Host selection for oviposition is initially driven by visual cues and reinforced by females assessing the nutrition and chemical cues of foliage (Boeckler, Gershenzon, & Unsicker, 2011;Panda & Khush, 1995 Podel.10G185000) that may be involved in the gentisyl alcohol conjugation to glucose. Specific substrates have yet to be determined for these genes, but a previous report suggests aldehyde dehydrogenase 5F1 genes are likely involved in the basic metabolism of Populus (Tian et al., 2015).
Phyllocolpa sp. sawflies are considered a keystone species as the abandoned or unused leaf folds are often used as a habitat for many other species such as aphids and spiders (Bailey & Whitham, 2007). The presence of folds in aspen forests is associated with a twofold increase in arthropod species richness and around a fourfold increase in arthropod abundance relative to forests where the insect is absent (Bailey & Whitham, 2003). This in turn makes the host plant and sawfly relationship important in examining how shifts in the genes of a population ultimately structure whole communities, effectively linking ecology and evolutionary biology. Further investigation of this potential relationship could be a key to connecting Populus genetics to the assemblage of the surrounding communities of organisms.
A striking finding in this study was an elevated number of recent tandem duplications in the P. deltoides genome but not the P. trichocarpa genome for the biotic QTL intervals. Out of the six chromosomes that yielded significant QTL results, four were associated with phenotypes that were fungi and insects native to the distribution of P. deltoides, but not P. trichocarpa. Given that P. deltoides has been coevolving with the majority of the surveyed fungi and insects, it was not unexpected that there were more recent tandem duplicates in biotic intervals in its genome as there is more selective pressure on the native species to overcome biotic stress (Constabel & Lindroth, 2010;Newcombe, Martin, & Kohler, 2010).
However, given the high amount of novel resistance occurring in the progeny, recent tandem duplication may also be important in naïve host resistance.
In our study, we demonstrated how host plant genetics directly affect associated fungi and insects in the field, as well as how Populus progeny indirectly structured interactions between pathogens (Whitham et al., 2006). The competition between Melampsora sp. and S. musiva highlights the complexities of how hybrid genetics are capable of strongly mediating multiple species interactions, which can result in inflated genetic associations. Finally, we have shown that many recent tandem duplications, found across biotic stress QTL intervals, have functional annotations that are involved in host plant physical/chemical resistance and tolerance as well as a few that may be implicated in host plant susceptibility. The enrichment of recent tandem duplications is a signature of gene-for-gene interactions and a mechanism that is essential to protect long-lived plants such as trees, enabling them to reach maturity despite many coevolving biotic stressors.