Association genetics of acetophenone defence against spruce budworm in mature white spruce.
ABSTRACT: BACKGROUND:Outbreaks of spruce budworm (SBW, Choristoneura fumiferana Clem.) cause major recurrent damage in boreal conifers such as white spruce (Picea glauca [Moench] Voss) and large losses of forest biomass in North America. Although defensive phenolic compounds have recently been linked to chemical resistance against SBW, their genetic basis remains poorly understood in forest trees, especially in conifers. Here, we used diverse association genetics approaches to discover genes and their variants that may control the accumulation of acetophenones, and dissect the genetic architecture of these defence compounds against SBW in white spruce mature trees. RESULTS:Out of 4747 single nucleotide polymorphisms (SNPs) from 2312 genes genotyped in a population of 211 unrelated individuals, genetic association analyses identified 35 SNPs in 33 different genes that were significantly associated with the defence traits by using single-locus, multi-locus and multi-trait approaches. The multi-locus approach was particularly effective at detecting SNP-trait associations that explained a large fraction of the phenotypic variance (from 20 to 43%). Significant genes were regulatory including the NAC transcription factor, or they were involved in carbohydrate metabolism, falling into the binding, catalytic or transporter activity functional classes. Most of them were highly expressed in foliage. Weak positive phenotypic correlations were observed between defence and growth traits, indicating little or no evidence of defence-growth trade-offs. CONCLUSIONS:This study provides new insights on the genetic architecture of tree defence traits, contributing to our understanding of the physiology of resistance mechanisms to biotic factors and providing a basis for the genetic improvement of the constitutive defence of white spruce against SBW.
Project description:Periodic outbreaks of spruce budworm (SBW) affect large areas of ecologically and economically important conifer forests in North America, causing tree mortality and reduced forest productivity. Host resistance against SBW has been linked to growth phenology and the chemical composition of foliage, but the underlying molecular mechanisms and population variation are largely unknown. Using a genomics approach, we discovered a ?-glucosidase gene, Pg?glu-1, whose expression levels and function underpin natural resistance to SBW in mature white spruce (Picea glauca) trees. In phenotypically resistant trees, Pg?glu-1 transcripts were up to 1000 times more abundant than in non-resistant trees and were highly enriched in foliage. The encoded Pg?GLU-1 enzyme catalysed the cleavage of acetophenone sugar conjugates to release the aglycons piceol and pungenol. These aglycons were previously shown to be active against SBW. Levels of Pg?glu-1 transcripts and biologically active acetophenone aglycons were substantially different between resistant and non-resistant trees over time, were positively correlated with each other and were highly variable in a natural white spruce population. These results suggest that expression of Pg?glu-1 and accumulation of acetophenone aglycons is a constitutive defence mechanism in white spruce. The progeny of resistant trees had higher Pg?glu-1 gene expression than non-resistant progeny, indicating that the trait is heritable. With reported increases in the intensity of SBW outbreaks, influenced by climate, variation of Pg?glu-1 transcript expression, Pg?GLU-1 enzyme activity and acetophenone accumulation may serve as resistance markers to better predict impacts of SBW in both managed and wild spruce populations.
Project description:Insect herbivory may drive evolution by selecting for trees with heritable resistance against defoliation. The spruce budworm (Choristoneura fumiferana, SBW) is a highly damaging forest insect pest that can affect population structure of white spruce (Picea glauca) in North America. Resistance against SBW was recently described in white spruce and was linked to three constitutive resistance biomarkers: the phenolic compounds piceol and pungenol, and expression of a beta-glucosidase encoding gene (Pgβglu-1). We investigated the phenotypic variability and heritability of these resistance biomarkers and of picein, the precursor of piceol, in the foliage of 874 trees belonging to 33 full-sib families and 71 clonal lines under evaluation in seven field locations in Eastern Canada. We aimed to (i) determine their genetic control, (ii) estimate the genetic and phenotypic correlations among defense biomarkers, and (iii) determine whether their constitutive levels are associated with detrimental trade-offs on growth. Quantitative genetics analyses indicated that all four traits are moderately to highly heritable. The full-sib and clonal analyses showed that additive and non-additive genetic effects play major and minor roles, respectively. Positive genetic and phenotypic correlations between resistance biomarkers and primary growth indicated that there is no trade-off between total height and height increment and resistance traits, contradicting the GDBH (Growth Differentiation Balance Hypothesis). Our findings about the predominant additive genetic basis of the resistance biomarkers show that adaptive evolution of white spruce natural populations to resist to SBW is possible and that potentially important gains could also be expected from artificial selection.
Project description:Variation in insect herbivory can lead to population structure in plant hosts as indicated by defence traits. In annual herbaceous, defence traits may vary between geographic areas but evidence of such patterns is lacking for long-lived species. This may result from the variety of selection pressures from herbivores, long distance gene flow, genome properties, and lack of research. We investigated the antagonistic interaction between white spruce (Picea glauca) and spruce budworm (SBW, Choristoneura fumiferana) the most devastating forest insect of eastern North America in common garden experiments. White spruces that are able to resist SBW attack were reported to accumulate the acetophenones piceol and pungenol constitutively in their foliage. We show that levels of these acetophenones and transcripts of the gene responsible for their release is highly heritable and that their accumulation is synchronized with the most devastating stage of SBW. Piceol and pungenol concentrations negatively correlate with rate of development in female SBW and follow a non-random geographic variation pattern that is partially explained by historical damage from SBW and temperature. Our results show that accumulation of acetophenones is an efficient resistance mechanism against SBW in white spruce and that insects can affect population structure of a long-lived plant.
Project description:BACKGROUND: In conifers, terpene synthases (TPSs) of the gymnosperm-specific TPS-d subfamily form a diverse array of mono-, sesqui-, and diterpenoid compounds, which are components of the oleoresin secretions and volatile emissions. These compounds contribute to defence against herbivores and pathogens and perhaps also protect against abiotic stress. RESULTS: The availability of extensive transcriptome resources in the form of expressed sequence tags (ESTs) and full-length cDNAs in several spruce (Picea) species allowed us to estimate that a conifer genome contains at least 69 unique and transcriptionally active TPS genes. This number is comparable to the number of TPSs found in any of the sequenced and well-annotated angiosperm genomes. We functionally characterized a total of 21 spruce TPSs: 12 from Sitka spruce (P. sitchensis), 5 from white spruce (P. glauca), and 4 from hybrid white spruce (P. glauca × P. engelmannii), which included 15 monoterpene synthases, 4 sesquiterpene synthases, and 2 diterpene synthases. CONCLUSIONS: The functional diversity of these characterized TPSs parallels the diversity of terpenoids found in the oleoresin and volatile emissions of Sitka spruce and provides a context for understanding this chemical diversity at the molecular and mechanistic levels. The comparative characterization of Sitka spruce and Norway spruce diterpene synthases revealed the natural occurrence of TPS sequence variants between closely related spruce species, confirming a previous prediction from site-directed mutagenesis and modelling.
Project description:Microsatellites or simple sequence repeats (SSRs) are highly informative molecular markers for various biological studies in plants. In spruce (Picea) and other conifers, the development of single-copy polymorphic genomic microsatellite markers is quite difficult, owing primarily to the large genome size and predominance of repetitive DNA sequences throughout the genome. We have developed highly informative single-locus genomic microsatellite markers in black spruce (Picea mariana) and red spruce (Picea rubens) using a simple but efficient method based on a combination of AFLP and microsatellite technologies.A microsatellite-enriched library was constructed from genomic AFLP DNA fragments of black spruce. Sequencing of the 108 putative SSR-containing clones provided 94 unique sequences with microsatellites. Twenty-two of the designed 34 primer pairs yielded scorable amplicons, with single-locus patterns. Fourteen of these microsatellite markers were characterized in 30 black spruce and 30 red spruce individuals drawn from many populations. The number of alleles at a polymorphic locus ranged from 2 to 18, with a mean of 9.3 in black spruce, and from 3 to 15, with a mean of 6.2 alleles in red spruce. The polymorphic information content or expected heterozygosity ranged from 0.340 to 0.909 (mean?=?0.67) in black spruce and from 0.161 to 0.851 (mean?=?0.62) in red spruce. Ten SSR markers showing inter-parental polymorphism inherited in a single-locus Mendelian mode, with two cases of distorted segregation. Primer pairs for almost all polymorphic SSR loci resolved microsatellites of comparable size in Picea glauca, P. engelmannii, P. sitchensis, and P. abies.The AFLP-based microsatellite-enriched library appears to be a rapid, cost-effective approach for isolating and developing single-locus informative genomic microsatellite markers in black spruce. The markers developed should be useful in black spruce, red spruce and other Picea species for various genetics, genomics, breeding, forensics, conservation studies and applications.
Project description:BACKGROUND: To explore the potential value of high-throughput genotyping assays in the analysis of large and complex genomes, we designed two highly multiplexed Illumina bead arrays using the GoldenGate SNP assay for gene mapping in white spruce (Picea glauca [Moench] Voss) and black spruce (Picea mariana [Mill.] B.S.P.). RESULTS: Each array included 768 SNPs, identified by resequencing genomic DNA from parents of each mapping population. For white spruce and black spruce, respectively, 69.2% and 77.1% of genotyped SNPs had valid GoldenGate assay scores and segregated in the mapping populations. For each of these successful SNPs, on average, valid genotyping scores were obtained for over 99% of progeny. SNP data were integrated to pre-existing ALFP, ESTP, and SSR markers to construct two individual linkage maps and a composite map for white spruce and black spruce genomes. The white spruce composite map contained 821 markers including 348 gene loci. Also, 835 markers including 328 gene loci were positioned on the black spruce composite map. In total, 215 anchor markers (mostly gene markers) were shared between the two species. Considering lineage divergence at least 10 Myr ago between the two spruces, interspecific comparison of homoeologous linkage groups revealed remarkable synteny and marker colinearity. CONCLUSION: The design of customized highly multiplexed Illumina SNP arrays appears as an efficient procedure to enhance the mapping of expressed genes and make linkage maps more informative and powerful in such species with poorly known genomes. This genotyping approach will open new avenues for co-localizing candidate genes and QTLs, partial genome sequencing, and comparative mapping across conifers.
Project description:Through the use of genome-wide association studies (GWAS) mapping it is possible to establish the genetic basis of phenotypic trait variation. Our GWAS study presents the first such effort in Norway spruce (Picea abies (L). Karst.) for the traits related to wood tracheid characteristics. The study employed an exome capture genotyping approach that generated 178 101 Single Nucleotide Polymorphisms (SNPs) from 40 018 probes within a population of 517 Norway spruce mother trees. We applied a least absolute shrinkage and selection operator (LASSO) based association mapping method using a functional multi-locus mapping approach, with a stability selection probability method as the hypothesis testing approach to determine significant Quantitative Trait Loci (QTLs). The analysis has provided 30 significant associations, the majority of which show specific expression in wood-forming tissues or high ubiquitous expression, potentially controlling tracheids dimensions, their cell wall thickness and microfibril angle. Among the most promising candidates based on our results and prior information for other species are: Picea abies BIG GRAIN 2 (PabBG2) with a predicted function in auxin transport and sensitivity, and MA_373300g0010 encoding a protein similar to wall-associated receptor kinases, which were both associated with cell wall thickness. The results demonstrate feasibility of GWAS to identify novel candidate genes controlling industrially-relevant tracheid traits in Norway spruce.
Project description:Constitutive defense mechanisms are critical to the understanding of defense mechanisms in conifers because they constitute the first barrier to attacks by insect pests. In interior spruce, trees that are putatively resistant and susceptible to attacks by white pine weevil (Pissodes strobi) typically exhibit constitutive differences in traits such as resin duct size and number, bark thickness, and terpene content. To improve our knowledge of their genetic basis, we compared globally the constitutive expression levels of 17,825 genes between 20 putatively resistant and 20 putatively susceptible interior spruce trees from the British Columbia tree improvement program. We identified 54 upregulated and 137 downregulated genes in resistant phenotypes, relative to susceptible phenotypes, with a maximum fold change of 2.24 and 3.91, respectively. We found a puzzling increase of resistance by downregulated genes, as one would think that "procuring armaments" is the best defense. Also, although terpenes and phenolic compounds play an important role in conifer defense, we found few of these genes to be differentially expressed. We found 15 putative small heat-shock proteins (sHSP) and several other stress-related proteins to be downregulated in resistant trees. Downregulated putative sHSP belong to several sHSP classes and represented 58% of all tested putative sHSP. These proteins are well known to be involved in plant response to various kinds of abiotic stress; however, their role in constitutive resistance is not yet understood. The lack of correspondence between transcriptome profile clusters and phenotype classifications suggests that weevil resistance in spruce is a complex trait.
Project description:In plants, relationships between resistance to herbivorous insect pests and growth are typically controlled by complex interactions between genetically correlated traits. These relationships often result in tradeoffs in phenotypic expression. In this study we used genetical genomics to elucidate genetic relationships between tree growth and resistance to white pine terminal weevil (Pissodes strobi Peck.) in a pedigree population of interior spruce (Picea glauca, P. engelmannii and their hybrids) that was growing at Vernon, B.C. and segregating for weevil resistance. Genetical genomics uses genetic perturbations caused by allelic segregation in pedigrees to co-locate quantitative trait loci (QTLs) for gene expression and quantitative traits. Bark tissue of apical leaders from 188 trees was assayed for gene expression using a 21.8K spruce EST-spotted microarray; the same individuals were genotyped for 384 SNP markers for the genetic map. Many of the expression QTLs (eQTL) co-localized with resistance trait QTLs. For a composite resistance phenotype of six attack and oviposition traits, 149 positional candidate genes were identified. Resistance and growth QTLs also overlapped with eQTL hotspots along the genome suggesting that: 1) genetic pleiotropy of resistance and growth traits in interior spruce was substantial, and 2) master regulatory genes were important for weevil resistance in spruce. These results will enable future work on functional genetic studies of insect resistance in spruce, and provide valuable information about candidate genes for genetic improvement of spruce.
Project description:White pine weevil is a major pest of conifers in North America, especially for Spruce trees. Constitutive defenses are important in understanding defense mechanisms because they constitute the initial barrier to attacks by weevils and other pests. Resistant and susceptible trees exhibit constitutive differences in spruce. To improve our knowledge of their genetic basis, we compared the constitutive expression levels of 17,825 genes between 20 resistant and 20 susceptible trees in interior spruce (Picea glauca). Overall design: Twenty hybridizations were performed to compare untreated bark of resistant and susceptible trees.RNA isolated from each of the 20 individual untreated resistant trees was compared directly against the 20 individual untreated susceptble trees using two hybridizations with a dye flip for each tree pair.