Advances in understanding Norway spruce natural resistance to needle bladder rust infection: transcriptional and secondary metabolites profiling.

BACKGROUND: Needle rust caused by the fungus Chrysomyxa rhododendri causes significant growth decline and increased mortality of young Norway spruce trees in subalpine forests. Extremely rare trees with enhanced resistance represent promising candidates for practice-oriented reproduction approaches. They also enable the investigation of tree molecular defence and resistance mechanisms against this fungal disease. Here, we combined RNA-Seq, RT-qPCR and secondary metabolite analyses during a period of 38 days following natural infection to investigate differences in constitutive and infection-induced defence between the resistant genotype PRA-R and three susceptible genotypes. RESULTS: Gene expression and secondary metabolites significantly differed among genotypes from day 7 on and revealed already known, but also novel candidate genes involved in spruce molecular defence against this pathogen. Several key genes related to (here and previously identified) spruce defence pathways to needle rust were differentially expressed in PRA-R compared to susceptible genotypes, both constitutively (in non-symptomatic needles) and infection-induced (in symptomatic needles). These genes encoded both new and well-known antifungal proteins such as endochitinases and chitinases. Specific genetic characteristics concurred with varying phenolic, terpene, and hormone needle contents in the resistant genotype, among them higher accumulation of several flavonoids (mainly kaempferol and taxifolin), stilbenes, geranyl acetone, α-ionone, abscisic acid and salicylic acid. CONCLUSIONS: Combined transcriptional and metabolic profiling of the Norway spruce defence response to infection by C. rhododendri in adult trees under subalpine conditions confirmed the results previously gained on artificially infected young clones in the greenhouse, both regarding timing and development of infection, and providing new insights into genes and metabolic pathways involved. The comparison of genotypes with different degrees of susceptibility proved that several of the identified key genes are differently regulated in PRA-R, and that the resistant genotype combines a strong constitutive defence with an induced response in infected symptomatic needles following fungal invasion. Genetic and metabolic differences between the resistant and susceptible genotypes indicated a more effective hypersensitive response (HR) in needles of PRA-R that prevents penetration and spread of the rust fungus and leads to a lower proportion of symptomatic needles as well as reduced symptom development on the few affected needles.

RNA-Seq and secondary metabolite analyses reveal a putative defence-transcriptome in Norway spruce (Picea abies) against needle bladder rust (Chrysomyxa rhododendri) infection.

BACKGROUND: Norway spruce trees in subalpine forests frequently face infections by the needle rust fungus Chrysomyxa rhododendri, which causes significant growth decline and increased mortality of young trees. Yet, it is unknown whether trees actively respond to fungal attack by activating molecular defence responses and/or respective gene expression. RESULTS: Here, we report results from an infection experiment, in which the transcriptomes (via RNA-Seq analysis) and phenolic profiles (via UHPLC-MS) of control and infected trees were compared over a period of 39 days. Gene expression between infected and uninfected ramets significantly differed after 21 days of infection and revealed already known, but also novel candidate genes involved in spruce molecular defence against pathogens. CONCLUSIONS: Combined RNA-Seq and biochemical data suggest that Norway spruce response to infection by C. rhododendri is restricted locally and primarily activated between 9 and 21 days after infestation, involving a potential isolation of the fungus by a hypersensitive response (HR) associated with an activation of phenolic pathways. Identified key regulatory genes represent a solid basis for further specific analyses in spruce varieties with varying susceptibility, to better characterise resistant clones and to elucidate the resistance mechanism.

Association genetics of phenolic needle compounds in Norway spruce with variable susceptibility to needle bladder rust.

KEY MESSAGE: Accumulation of phenolic needle metabolites in Norway spruce is regulated by many genes with small and additive effects and is correlated with the susceptibility against fungal attack. Norway spruce accumulates high foliar concentrations of secondary phenolic metabolites, with important functions for pathogen defence responses. However, the molecular genetic basis underlying the quantitative variation of phenolic compounds and their role in enhanced resistance of spruce to infection by needle bladder rust are unknown. To address these questions, a set of 1035 genome-wide single nucleotide polymorphisms (SNPs) was associated to the quantitative variation of four simple phenylpropanoids, eight stilbenes, nine flavonoids, six related arithmetic parameters and the susceptibility to infection by Chrysomyxa rhododendri in an unstructured natural population of Norway spruce. Thirty-one significant genetic associations for the flavonoids gallocatechin, kaempferol 3-glucoside and quercetin 3-glucoside and the stilbenes resveratrol, piceatannol, astringin and isorhapontin were discovered, explaining 22-59% of phenotypic variation, and indicating a regulation of phenolic accumulation by many genes with small and additive effects. The phenolics profile differed between trees with high and low susceptibility to the fungus, underlining the importance of phenolic compounds in the defence mechanisms of Norway spruce to C. rhododendri. Results highlight the utility of association studies in non-model tree species and may enable marker-assisted selection of Norway spruce adapted to severe pathogen attack.

Localization of QTLs for in vitro plant regeneration in tomato.

BACKGROUND: Low regeneration ability limits biotechnological breeding approaches. The influence of genotype in the regeneration response is high in both tomato and other important crops. Despite the various studies that have been carried out on regeneration genetics, little is known about the key genes involved in this process. The aim of this study was to localize the genetic factors affecting regeneration in tomato. RESULTS: We developed two mapping populations (F2 and BC1) derived from a previously selected tomato cultivar (cv. Anl27) with low regeneration ability and a high regeneration accession of the wild species Solanum pennellii (PE-47). The phenotypic assay indicated dominance for bud induction and additive effects for both the percentage of explants with shoots and the number of regenerated shoots per explant. Two linkage maps were developed and six QTLs were identified on five chromosomes (1, 3, 4, 7 and 8) in the BC1 population by means of the Interval Mapping and restricted Multiple QTL Mapping methods. These QTLs came from S. pennellii, with the exception of the minor QTL located on chromosome 8, which was provided by cv. Anl27. The main QTLs correspond to those detected on chromosomes 1 and 7. In the F2 population, a QTL on chromosome 7 was identified on a similar region as that detected in the BC1 population. Marker segregation distortion was observed in this population in those areas where the QTLs of BC1 were detected. Furthermore, we located two tomato candidate genes using a marker linked to the high regeneration gene: Rg-2 (a putative allele of Rg-1) and LESK1, which encodes a serine/threonine kinase and was proposed as a marker for regeneration competence. As a result, we located a putative allele of Rg-2 in the QTL detected on chromosome 3 that we named Rg-3. LESK1, which is also situated on chromosome 3, is outside Rg-3. In a preliminary exploration of the detected QTL peaks, we found several genes that may be related to regeneration. CONCLUSIONS: In this study we have identified new QTLs related to the complex process of regeneration from tissue culture. We have also located two candidate genes, discovering a putative allele of the high regeneration gene Rg-1 in the QTL on chromosome 3. The identified QTLs could represent a significant step toward the understanding of this process and the identification of other related candidate genes. It will also most likely facilitate the development of molecular markers for use in gene isolation.

Drought Sensitivity of Norway Spruce at the Species' Warmest Fringe: Quantitative and Molecular Analysis Reveals High Genetic Variation Among and Within Provenances.

Norway spruce (Picea abies) is by far the most important timber species in Europe, but its outstanding role in future forests is jeopardized by its high sensitivity to drought. We analyzed drought response of Norway spruce at the warmest fringe of its natural range. Based on a 35-year old provenance experiment we tested for genetic variation among and within seed provenances across consecutively occurring strong drought events using dendroclimatic time series. Moreover, we tested for associations between ≈1,700 variable SNPs and traits related to drought response, wood characteristics and climate-growth relationships. We found significant adaptive genetic variation among provenances originating from the species' Alpine, Central and Southeastern European range. Genetic variation between individuals varied significantly among provenances explaining up to 44% of the phenotypic variation in drought response. Varying phenotypic correlations between drought response and wood traits confirmed differences in selection intensity among seed provenances. Significant associations were found between 29 SNPs and traits related to drought, climate-growth relationships and wood properties which explained between 11 and 43% of trait variation, though 12 of them were due to single individuals having extreme phenotypes of the respective trait. The majority of these SNPs are located within exons of genes and the most important ones are preferentially expressed in cambium and xylem expansion layers. Phenotype-genotype associations were stronger if only provenances with significant quantitative genetic variation in drought response were considered. The present study confirms the high adaptive variation of Norway spruce in Central and Southeastern Europe and demonstrates how quantitative genetic, dendroclimatic and genomic data can be linked to understand the genetic basis of adaptation to climate extremes in trees.