Envirotyping within a multi-environment trial allowed identifying genetic determinants of winter oilseed rape yield stability.

KEY MESSAGE: A comprehensive environmental characterization allowed identifying stable and interactive QTL for seed yield: QA09 and QC09a were detected across environments; whereas QA07a was specifically detected on the most stressed environments. A main challenge for rapeseed consists in maintaining seed yield while adapting to climate changes and contributing to environmental-friendly cropping systems. Breeding for cultivar adaptation is one of the keys to meet this challenge. Therefore, we propose to identify the genetic determinant of seed yield stability for winter oilseed rape using GWAS coupled with a multi-environmental trial and to interpret them in the light of environmental characteristics. Due to a comprehensive characterization of a multi-environmental trial using 79 indicators, four contrasting envirotypes were defined and used to identify interactive and stable seed yield QTL. A total of four QTLs were detected, among which, QA09 and QC09a, were stable (detected at the multi-environmental trial scale or for different envirotypes and environments); and one, QA07a, was specifically detected into the most stressed envirotype. The analysis of the molecular diversity at QA07a showed a lack of genetic diversity within modern lines compared to older cultivars bred before the selection for low glucosinolate content. The results were discussed in comparison with other studies and methods as well as in the context of breeding programs.

Genomic and phenomic predictions help capture low-effect alleles promoting seed germination in oilseed rape in addition to QTL analyses.

KEY MESSAGE: Phenomic prediction implemented on a large diversity set can efficiently predict seed germination, capture low-effect favorable alleles that are not revealed by GWAS and identify promising genetic resources. Oilseed rape faces many challenges, especially at the beginning of its developmental cycle. Achieving rapid and uniform seed germination could help to ensure a successful establishment and therefore enabling the crop to compete with weeds and tolerate stresses during the earliest developmental stages. The polygenic nature of seed germination was highlighted in several studies, and more knowledge is needed about low- to moderate-effect underlying loci in order to enhance seed germination effectively by improving the genetic background and incorporating favorable alleles. A total of 17 QTL were detected for seed germination-related traits, for which the favorable alleles often corresponded to the most frequent alleles in the panel. Genomic and phenomic predictions methods provided moderate-to-high predictive abilities, demonstrating the ability to capture small additive and non-additive effects for seed germination. This study also showed that phenomic prediction estimated phenotypic values closer to phenotypic values than GEBV. Finally, as the predictive ability of phenomic prediction was less influenced by the genetic structure of the panel, it is worth using this prediction method to characterize genetic resources, particularly with a view to design prebreeding populations.

Genetic control of root morphology in response to nitrogen across rapeseed diversity.

Rapeseed (Brassica napus L.) is an oil-containing crop of great economic value but with considerable nitrogen requirement. Breeding root systems that efficiently absorb nitrogen from the soil could be a driver to ensure genetic gains for more sustainable rapeseed production. The aim of this study is to identify genomic regions that regulate root morphology in response to nitrate availability. The natural variability offered by 300 inbred lines was screened at two experimental locations. Seedlings grew hydroponically with low or elevated nitrate levels. Fifteen traits related to biomass production and root morphology were measured. On average across the panel, a low nitrate level increased the root-to-shoot biomass ratio and the lateral root length. A large phenotypic variation was observed, along with important heritability values and genotypic effects, but low genotype-by-nitrogen interactions. Genome-wide association study and bulk segregant analysis were used to identify loci regulating phenotypic traits. The first approach nominated 319 SNPs that were combined into 80 QTLs. Three QTLs identified on the A07 and C07 chromosomes were stable across nitrate levels and/or experimental locations. The second approach involved genotyping two groups of individuals from an experimental F2 population created by crossing two accessions with contrasting lateral root lengths. These individuals were found in the tails of the phenotypic distribution. Co-localized QTLs found in both mapping approaches covered a chromosomal region on the A06 chromosome. The QTL regions contained some genes putatively involved in root organogenesis and represent selection targets for redesigning the root morphology of rapeseed.

Do nitrogen- and sulphur-remobilization-related parameters measured at the onset of the reproductive stage provide early indicators to adjust N and S fertilization in oilseed rape (Brassica napus L.) grown under N- and/or S-limiting supplies?

MAIN CONCLUSION: Specific combinations of physiological and molecular parameters associated with N and S remobilization measured at the onset of flowering were predictive of final crop performances in oilseed rape. Oilseed rape (Brassica napus L.) is a high nitrogen (N) and sulphur (S) demanding crop. Nitrogen- and S-remobilization processes allow N and S requirements to reproductive organs to be satisfied when natural uptake is reduced, thus ensuring high yield and seed quality. The quantification of physiological and molecular indicators of early N and S remobilization could be used as management tools to correct N and S fertilization. However, the major limit of this corrective strategy is to ensure the correlation between final performances-related variables and early measured parameters. In our study, four genotypes of winter oilseed rape (OSR) were grown until seed maturity under four nutritional modalities combining high and/or low N and S supplies. Plant final performances, i.e., seed production, N- and S-harvest indexes, seed N and S use efficiencies, and early parameters related to N- or S-remobilization processes, i.e., photosynthetic leaf area, N and S leaf concentrations, leaf soluble protein and leaf sulphate concentrations, and leaf RuBisCO abundance at flowering, were measured. We demonstrated that contrasting final performances existed according to the N and S supplies. An optimal N:S ratio supply could explain the treatment-specific crop performances, thus justifying N and S concurrent managements. Specific combinations of early measured plant parameters could be used to predict final performances irrespective of the nutritional supply and the genotype. This work demonstrates the potential of physiological and molecular indicators measured at flowering to reflect the functioning of N- and S-compound remobilization and to predict yield and quality penalties. However, because the predictive models are N and S independent, instant N and S leaf analyses are required to further adjust the adequate fertilization. This study is a proof of a concept which opens prospects regarding instant diagnostic tools in the context of N and S mineral fertilization management.

Resolution of quantitative resistance to clubroot into QTL-specific metabolic modules.

Plant disease resistance is often under quantitative genetic control. Thus, in a given interaction, plant cellular responses to infection are influenced by resistance or susceptibility alleles at different loci. In this study, a genetic linkage analysis was used to address the complexity of the metabolic responses of Brassica napus roots to infection by Plasmodiophora brassicae. Metabolome profiling and pathogen quantification in a segregating progeny allowed a comparative mapping of quantitative trait loci (QTLs) involved in resistance and in metabolic adjustments. Distinct metabolic modules were associated with each resistance QTL, suggesting the involvement of different underlying cellular mechanisms. This approach highlighted the possible role of gluconasturtiin and two unknown metabolites in the resistance conferred by two QTLs on chromosomes C03 and C09, respectively. Only two susceptibility biomarkers (glycine and glutathione) were simultaneously linked to the three main resistance QTLs, suggesting the central role of these compounds in the interaction. By contrast, several genotype-specific metabolic responses to infection were genetically unconnected to resistance or susceptibility. Likewise, variations of root sugar profiles, which might have influenced pathogen nutrition, were not found to be related to resistance QTLs. This work illustrates how genetic metabolomics can help to understand plant stress responses and their possible links with disease.

Genetic basis of nitrogen use efficiency and yield stability across environments in winter rapeseed.

BACKGROUND: Nitrogen use efficiency is an important breeding trait that can be modified to improve the sustainability of many crop species used in agriculture. Rapeseed is a major oil crop with low nitrogen use efficiency, making its production highly dependent on nitrogen input. This complex trait is suspected to be sensitive to genotype × environment interactions, especially genotype × nitrogen interactions. Therefore, phenotyping diverse rapeseed populations under a dense network of trials is a powerful approach to study nitrogen use efficiency in this crop. The present study aimed to determine the quantitative trait loci (QTL) associated with yield in winter oilseed rape and to assess the stability of these regions under contrasting nitrogen conditions for the purpose of increasing nitrogen use efficiency. RESULTS: Genome-wide association studies and linkage analyses were performed on two diversity sets and two doubled-haploid populations. These populations were densely genotyped, and yield-related traits were scored in a multi-environment design including seven French locations, six growing seasons (2009 to 2014) and two nitrogen nutrition levels (optimal versus limited). Very few genotype × nitrogen interactions were detected, and a large proportion of the QTL were stable across nitrogen nutrition conditions. In contrast, strong genotype × trial interactions in which most of the QTL were specific to a single trial were found. To obtain further insight into the QTL × environment interactions, genetic analyses of ecovalence were performed to identify the genomic regions contributing to the genotype × nitrogen and genotype × trial interactions. Fifty-one critical genomic regions contributing to the additive genetic control of yield-associated traits were identified, and the structural organization of these regions in the genome was investigated. CONCLUSIONS: Our results demonstrated that the effect of the trial was greater than the effect of nitrogen nutrition levels on seed yield-related traits under our experimental conditions. Nevertheless, critical genomic regions associated with yield that were stable across environments were identified in rapeseed.

Metabotyping: a new approach to investigate rapeseed (Brassica napus L.) genetic diversity in the metabolic response to clubroot infection.

Clubroot disease affects all Brassicaceae spp. and is caused by the obligate biotroph pathogen Plasmodiophora brassicae. The development of galls on the root system is associated with the establishment of a new carbon metabolic sink. Here, we aimed to deepen our knowledge of the involvement of primary metabolism in the Brassica napus response to clubroot infection. We studied the dynamics and the diversity of the metabolic responses to the infection. Root system metabotyping was carried out for 18 rapeseed genotypes displaying different degrees of symptom severity, under inoculated and noninoculated conditions at 42 days postinoculation (dpi). Clubroot susceptibility was positively correlated with clubroot-induced accumulation of several amino acids. Although glucose and fructose accumulated in some genotypes with minor symptoms, their levels were negatively correlated to the disease index across the whole set of genotypes. The dynamics of the metabolic response were studied for the susceptible genotype 'Yudal,' which allowed an "early" metabolic response (established from 14 to 28 dpi) to be differentiated from a "late" response (from 35 dpi). We discuss the early accumulation of amino acids in the context of the establishment of a nitrogen metabolic sink and the hypothetical biological role of the accumulation of glutathione and S-methylcysteine.

Multi-Omic Investigation of Low-Nitrogen Conditional Resistance to Clubroot Reveals Brassica napus Genes Involved in Nitrate Assimilation.

Nitrogen fertilization has been reported to influence the development of clubroot, a root disease of Brassicaceae species, caused by the obligate protist Plasmodiophora brassicae. Our previous works highlighted that low-nitrogen fertilization induced a strong reduction of clubroot symptoms in some oilseed rape genotypes. To further understand the underlying mechanisms, the response to P. brassicae infection was investigated in two genotypes "Yudal" and HD018 harboring sharply contrasted nitrogen-driven modulation of resistance toward P. brassicae. Targeted hormone and metabolic profiling, as well as RNA-seq analysis, were performed in inoculated and non-inoculated roots at 14 and 27 days post-inoculation, under high and low-nitrogen conditions. Clubroot infection triggered a large increase of SA concentration and an induction of the SA gene markers expression whatever the genotype and nitrogen conditions. Overall, metabolic profiles suggested that N-driven induction of resistance was independent of SA signaling, soluble carbohydrate and amino acid concentrations. Low-nitrogen-driven resistance in "Yudal" was associated with the transcriptional regulation of a small set of genes, among which the induction of NRT2- and NR-encoding genes. Altogether, our results indicate a possible role of nitrate transporters and auxin signaling in the crosstalk between plant nutrition and partial resistance to pathogens.

Identification and Quantification of Glucosinolates and Phenolics in a Large Panel of Brassica napus Highlight Valuable Genetic Resources for Chemical Ecology and Breeding.

Glucosinolate (GLS) and phenolic contents in Brassicaceae contribute to biotic and abiotic stress responses. Breeding crop accessions harboring agroecologically relevant metabolic profiles require a characterization of the chemical diversity in Brassica germplasm. This work investigates the diversity of specialized metabolites in 281 accessions of B. napus. First, an LC-HRMS2-based approach allowed the annotation of 32 phenolics and 36 GLSs, revealing 13 branched and linear alkyl-GLSs and 4 isomers of hydroxyphenylalkyl-GLSs, many of which have been rarely reported in Brassica. Then, quantitative UPLC-UV-MS-based profiling was performed in leaves and roots for the whole panel. This revealed striking variations in the content of 1-methylpropyl-GLS (glucocochlearin) and a large variation of tetra- and penta-glucosyl kaempferol derivatives among accessions. It also highlighted two main chemotypes related to sinapoyl-O-hexoside and kaempferol-O-trihexoside contents. By offering an unprecedented overview of the phytochemical diversity in B. napus, this work provides a useful resource for chemical ecology and breeding.

Nitrogen Uptake Efficiency, Mediated by Fine Root Growth, Early Determines Temporal and Genotypic Variations in Nitrogen Use Efficiency of Winter Oilseed Rape.

Maintaining seed yield under low N inputs is a major issue for breeding, which requires thoroughly exploiting the genetic diversity of processes related to Nitrogen Use Efficiency (NUE). However, dynamic analysis of processes underlying genotypic variations in NUE in response to N availability from sowing to harvest are scarce, particularly at the whole-plant scale. This study aimed to dynamically decipher the contributions of Nitrogen Uptake Efficiency (NUpE) and Nitrogen Utilization Efficiency (NUtE) to NUE and to identify traits underlying NUpE genetic variability throughout the growth cycle of rapeseed. Three experiments were conducted under field-like conditions to evaluate seven genotypes under two N conditions. We developed NUE_DM (ratio of total plant biomass to the amount of N available) as a new proxy of NUE at harvest, valid to discriminate genotypes from the end of inflorescence emergence, and N conditions as early as the beginning of stem elongation. During autumn growth, NUpE explained up to 100% of variations in NUE_DM, validating the major role of NUpE in NUE shaping. During this period, under low N conditions, up to 53% of the plant nitrogen was absorbed and NUpE genetic variability resulted not from differences in Specific N Uptake but in fine-root growth. NUtE mainly contributed to NUE_DM genotypic variation during the reproductive phase under high-N conditions, but NUpE contribution still accounted for 50-75% after flowering. Our study highlights for the first time NUpE and fine-root growth as important processes to optimize NUE, which opens new prospects for breeding.

Using probe genotypes to dissect QTL × environment interactions for grain yield components in winter wheat.

Yield is known to be a complex trait, the expression of which interacts strongly with environmental conditions. Understanding the genetic basis of these genotype × environment interactions, particularly under limited input levels, is a key objective when selecting wheat genotypes adapted to specific environments. Our principal objectives were thus: (1) to identify genomic regions [quantitative trait loci (QTL)] involving QTL × environment interactions (QEI) and (2) to develop a strategy to understand the specificity of these regions to certain environments. The two main components of yield were studied: kernel number (KN) and thousand-kernel weight (TKW). The Arche × Récital doubled-haploid population of 222 lines was grown in replicated field trials during 2000 and 2001 at three locations in France, under two nitrogen levels. The 12 environments were characterized in terms of water deficit, radiation, temperature and nitrogen stress based on measurements conducted on the four-probe genotypes: Arche, Récital, Ritmo and Soissons. A four-step strategy was developed to explain QTL specificity to some environments: (1) the detection of QTL for KN and TKW in each environment; (2) the estimation of genotypic sensitivities as the factorial regression slope of KN and TKW to environmental covariates and the detection of QTL for these genotypic sensitivities; (3) study of the co-locations of QTL for KN and TKW and of the QTL for sensitivities; in the event of a co-location partitioning the QEI, appropriate covariates were employed; (4) a description of the environments where QTL were detected for KN and TKW using the environmental covariates. A total of 131 QTL were found to be associated with KN, TKW and their sensitivity to environmental covariates across the 12 environments. Four of these QTL, for both KN and TKW, were located on linkage groups 1B, 2D1, 4B and 5A1, and displayed pleiotropic effects. Factorial regression explained from 15.1 to 83.2% of the QEI for KN and involved three major environmental covariates: cumulative radiation-days ±3 days at meiosis, cumulative degree-days >25°C ±3 days at meiosis and nitrogen stress at flowering. For TKW, 13.5-81.8% of the effect of the QEI was partitioned and involved three major environmental covariates: water deficit from flowering to the milk stage, cumulative degree-days >0°C from the milk stage to maturity and soil water deficit at maturity. A comparative analysis was then performed on the QTL detected during this and previous studies published on QEI and some interacting QTL may be common to different genetic backgrounds. Focusing on these QTL common to different genetic backgrounds would give some guidance to understand genotype × environment interaction.

Clubroot Symptoms and Resting Spore Production in a Doubled Haploid Population of Oilseed Rape (Brassica napus) Are Controlled by Four Main QTLs.

Clubroot, caused by Plasmodiophora brassicae Woronin, is one of the most important diseases of oilseed rape (Brassica napus L.). The rapid erosion of monogenic resistance in clubroot-resistant (CR) varieties underscores the need to diversify resistance sources controlling disease severity and traits related to pathogen fitness, such as resting spore production. The genetic control of disease index (DI) and resting spores per plant (RSP) was evaluated in a doubled haploid (DH) population consisting of 114 winter oilseed rape lines, obtained from the cross 'Aviso' × 'Montego,' inoculated with P. brassicae isolate "eH." Linkage analysis allowed the identification of three quantitative trait loci (QTLs) controlling DI (PbBn_di_A02, PbBn_di_A04, and PbBn_di_C03). A significant decrease in DI was observed when combining effects of the three resistance alleles at these QTLs. Only one QTL, PbBn_rsp_C03, was found to control RSP, reducing resting spore production by 40%. PbBn_rsp_C03 partially overlapped with PbBn_di_C03 in a nucleotide-binding leucine-rich repeat (NLR) gene-containing region. Consideration of both DI and RSP in breeding for clubroot resistance is recommended for the long-term management of this disease.

Using genotype x nitrogen interaction variables to evaluate the QTL involved in wheat tolerance to nitrogen constraints.

Lower market prices and environmental concerns now orientate wheat (Triticum aestivum L.) breeding programs towards low input agricultural practices, and more particularly low nitrogen (N) input management. Such programs require knowledge of the genetic determination of plant reaction to N deficiency. Our aim was to characterize the genetic basis of N use efficiency and genotype x N interactions. The detection of QTL for grain yield, grain protein yield and their components was performed on a mapping population of 222 doubled haploid lines (DH), obtained from the cross between an N stress tolerant variety and an N stress sensitive variety. Experiments on the population were carried out in seven different environments, and in each case under high (N(+)) and low (N(-)) N supplies. In total, 233 QTL were detected for traits measured in each combination of environment and N supply, for "global" interaction variables (N(+)-N(-) and N(-)/N(+)), for sensitivity to N stress and for performance under N-limited conditions which were assessed using factorial regression parameters. The 233 QTL were detected on the whole genome and clustered into 82 genome regions. The dwarfing gene (Rht-B1), the photoperiod sensitivity gene (Ppd-D1) and the awns inhibitor gene (B1) coincided with regions that contained the highest numbers of QTL. Non-interactive QTL were detected on linkage groups 3D, 4B, 5A1 and 7B2. Interactive QTL were revealed by interaction or factorial regression variables (2D2, 3D, 5A1, 5D, 6A, 6B, 7B2) or by both variables (1B, 2A1, 2A2, 2D1, 4B, 5A2, 5B). The usefulness of QTL meta-analysis and factorial regression to study QTL x N interactions and the impact of Rht-B1, Ppd-D1 and B1, are discussed.

Estimation of genetic parameters of a DH wheat population grown at different N stress levels characterized by probe genotypes.

Low market prices and environmental concerns in Europe favor lower input wheat production systems. To efficiently breed for new varieties adapted to low input management while maintaining high yield levels, our objective was to characterize the heritability and its components for yield and nitrogen traits under different nitrogen levels. Two hundred and twenty-two doubled-haploid (DH) lines from the cross between Arche (tolerant) and Récital (sensitive) were tested in France at four locations in 2000, and three in 2001, under high (N+) and low (N-) nitrogen supplies. The response of yield to the environment of four probe genotypes, the parents and two controls, were tested and used as descriptors of these environments. Grain yield (GY), its components, and grain and straw nitrogen, called nitrogen traits, were studied. A factorial regression was performed to assess the sensitivity (slope) of the DH lines to nitrogen stress and their performance to low nitrogen supply. An index based on the nitrogen nutrition index at flowering of the probe genotype Récital was the best descriptor of the environment stress. Heritabilities of yield and nitrogen traits for both nitrogen supplies were always above 0.6. When nitrogen stress increased, heritabilities decreased and genotype x nitrogen interaction variances increased. The decrease in heritability was mainly explained by a decrease in genetic variance. Genetic variation for sensitivity to nitrogen stress and performance under low nitrogen supply were shown in the population. GY decreased from 278 to 760 g/m2 per unit of nitrogen stress index increase and GY under moderate nitrogen stress varied from 340 to 613 g/m2. Those contrasted reactions revealed specific lines to include in breeding programs for improving GY under low nitrogen supply.