4 DISCUSSION
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. 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) were important for resistance to Melampsora sp. fungus, M. vagabunda galls, and Phyllocolpa sp. oviposition. In contrast, the interval associated with S. musivacanker symptoms was the only case in which susceptibility to the fungus was dominant and inherited from the non-coevolved host (P. trichocarpa ), as has been previously observed (Muchero et al., 2018; George Newcombe & Ostry, 2001a).
We detected a major QTL for Melampsora sp. resistance on Chr04. This region is known to contain the MXC3 locus which confers resistance to infection of many species of the Melampsora leaf rusts (George Newcombe, Stirling, & Bradshaw, 2001; Yin 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 theP. 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 theP. deltoides genome. Several of these 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 (H. 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 a false association on Chr04 with the S. musiva leaf spot score. 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). In our case, we had two very different pathogens that were utilizing the leaf tissue in vastly dissimilar ways. S. musiva is a necrotrophic fungus, which requires dead tissue to reproduce, andMelampsora sp. is biotrophic, requiring living tissue to reproduce. The presence of S. musiva on the trees had a much larger effect on the occurrence of Melampsora sp. infection. This may indicate that the S. musiva fungus had a competitive advantage over Melampsora sp. in utilization of thePopulus leaf tissue at our site. Further supporting this finding was the complete loss of Melampsora sp. symptoms at the plantation site over the course of ten years and the continual presence of the S. musiva leaf spot. A similar interaction has been reported in wheat between the biotroph Blumeria graminis  f.sp. tritici  (Bgt ), the powdery mildew pathogen, and necrotroph Zymoseptoria tritici , the cause ofSeptoria tritici blotch (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 pathogen-pathogen interactions can be more direct rather than relying solely on the host plant genetics (Orton & Brown, 2016).
The S. musiva stem canker symptoms were found to be associated with a QTL interval on Chr16. In this study, susceptibility to the necrotroph appeared to be originating from the presence of P. trichocarpa alleles in the progeny. Previous work on a similarPopulus hybrid cross supports this with susceptibility toS. musiva stem canker originating primarily from dominant alleles derived from P. trichocarpa (G. Newcombe, 1998; George Newcombe & Ostry, 2001a). Interestingly, the loci conferring susceptibility in family 52-124 did not overlap with the four loci that were uncovered in a previous genome-wide association study of S. musivasusceptibility 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 andP. 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, 1961). 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 as well as 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 (F. 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 (F. 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 was 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). Diversification of their function through tandem duplication due to gene-for-gene coevolution has also been demonstrated in many plant-fungal pathosystems (Leister, 2004). Although R-genes have not been recognized as frequently in plant-insect interactions, those that have been discovered typically mediate interactions with piercing-sucking insects such as aphids and whiteflies (Kaloshian, 2004).
In addition to the R-genes, there were a series of genes encoding cytochrome P450 family proteins in both species as well as 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 galls, much like the woody structures M. vagabunda leaves behind on branches once they have finished feeding.
We detected two QTL for Phyllocolpa sp. with P. trichocarpa alleles being positively associated with leaf fold occurrence in both cases. The P. trichocarpa genomic interval corresponding to the Chr10 QTL was enriched for several domains and GO terms that could be involved in gall development. For example, the interval was enriched for sugar transporters as well as GO terms for sucrose transporter activity. Often in the case of galling insects plant tissue 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). Previous research in Salix (closely related to Populus ) has shown that common phenolic glycosides in leaf tissue are important in host choice in both the free-feedingNematus oligospilus and galling Euura amerinae specialist sawflies (Fernández et al., 2019; Kolehmainen, Roininen, Julkunen-Tiitto, & Tahvanainen, 1994). We therefore pursued QTL mapping of metabolites to determine if phenolic compounds might be important determinants of the potential ovipositional relationship.
We detected three significant QTL for gentisyl alcohol 5-O-glucoside levels. One of these overlapped with the Phyllocolpa sp. leaf fold QTL on Chr10. The QTL on Chr14 also overlapped with a suggestive QTL peak for Phyllocolpa sp . (LOD=3.96). This is the first case of an association of gentisyl alcohol 5-O -glucoside with a specialist arthropod in Populus , and is, in fact, the first report of this metabolite in Populus . Salirepin (gentisyl alcohol 2-O -glucoside), a closely related metabolite, is a well-known constituent in Populus sp, (Busov et al., 2006; Tschaplinski et al., 2019; Veach et al., 2018) and leaves of P. deltoides andP. trichocarpa x deltoides have a lower abundance, later eluting metabolite with a nearly identical fragmentation pattern to salirepin that we tentatively identify as gentisyl alcohol 2-O -glucoside. The QTL contains three putative candidate genes, including an aldehyde dehydrogenase 5F1 (Podel.10G175800) that may be involved in the reduction of gentisyl aldehyde to the alcohol, and two UDP-glycosyltransferases (Podel.10G184800, 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 two-fold increase in arthropod species richness and around a four-fold 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 key to connecting Populusgenetics to the assemblage of the surrounding communities of organisms.
A striking finding in this study was enrichment 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 thatP. deltoides has been co-evolving 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; George 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.
Our study has demonstrated that many recent tandem duplications, found across biotic stress QTL intervals, have functional annotations that are involved in physical, chemical, and tolerance mechanisms of host plant resistance as well as a few that may be implicated in host plant susceptibility. The enrichment of recent tandem duplications may be a signature of gene-for-gene interactions, and a mechanism that protects long-lived plants such as trees, enabling them to reach maturity despite many coevolving biotic stressors. In addition to the direct effect of the host plant genetics on associated organisms, we have also demonstrated an indirect community effect that was mediated by thePopulus progeny (Whitham et al., 2006). The fungal pathogen succession highlights the complexities of how host plant-pathogen coevolution can affect multiple species interactions in more subtle ways than expected in a natural environment.