Ecophyto Maturation - Ecophyto Maturation

Breeding oilseed rape Resistance to INsects: understanding Genetic and chemIcal deTerminants Of Sinapis alba resistaNce to the pollen beetle – BRING IT ON

BRING IT ON: Breeding oilseed rape resistance to insects

Breeding oilseed rape resistance to insects: understanding genetic and chemical determinantes of Sinapis alba resistance to the pollen beetle

General objectives

BRING IT ON aims at proposing a varietal solution by improving oilseed rape resistance to one of its main insect pests, the pollen beetle. Currently, there is no resistant variety against pollen beetle, which is mainly controlled by pesticides. <br /><br />We have identified some resistance in the white mustard, a close relative to oilseed rape. BRING IT ON will focus on obtaining the tools to transfer this resistance to oilseed rape, through identifying its genetic and biochemical bases.

BRING IT ON aims at determining the genetic and biochemical components of the resistance from three different sources: INN_Sa_PB_001, INN_Sa_PB_002 and INN_Sa_PB_003.
To address the genetic components, each source was crossed with a suscpetible accession, INN_Sa_PB_004 to develop a segregating population through SSD, first to the F3 stage (around 250 individuals per population), then to the F6 stage (between 100 and 150 individuals per population). The F2 and F5 plants that generated the F3 and F6 seed lots respectively, were genotyped with 10 000 SNP that are specific to Sinapis alba and covering the entire genome. Phenoptyping the resistance to pollen beetle in the F3 and F6 seed lots was done in controlled conditions by placing four pollen beetles per primary inflorescence of 10 plants per F3 or F6 lot (Bellec et al 2022). A QTL analysis by Composite Interval Mapping was performed, confronting the genotypic and phenotypic data.
The biochemical components were studied using a bioassay allowing to observe the feeding behaviour of pollen beetles on a synthetic medium. Two pollen beetles, starved for 72h, were exposed to two agar disks with differing elements. They were followed by videaotracking for three hours to determine the attractiveness or rejection for feeding, over 30 repetitions. The pollen beetles were exposed to 10 repetitions of a total extract of perianth components from each resistant accessions in comparison to the susceptible accessions, then on fractions or sub-fractions of these extracts, generated using different solvents (hexane, dichloromethane, methanol, and combinations). A UPLC-MS analyses was then performed on the discriminant sub-fractions to determine their composition and thus potential candidate compounds.
The confirmation of the QTL effects will be evaluated through the study of independent materials, notably a panel of lines or HIFs, segregating for the QTL alleles, in controlled conditions but also five field trials. Likewise, the effect of the candidate compounds will be confirmed through tests with pure or combinations of compounds.
The identification of diagnostic molecular markers will be supported by the genomic sequence of each source of resistance. For this, genome assemblies have been generated using long read sequencing and optical mapping. A bio-analysis of the regions corresponding to the QTLs as well as the biosynthetic pathways for the candidate compounds, will allow to access candidate genes. These features as well as all the genomic and metabolomic data publicly available on the Brassicaceae will allow the identification of possible leads to speed up the transfer of resistance to oilseed rape.

BRING IT ON has met, during the first half of the project, its first successes, both at the genetic and the biochemical levels.
The first QTL analyses, with two F3 populations, have shown three QTLs, two derived from INN_Sa_PB_001, on chromosomes S06 and S09, while INN_Sa_PB_002 seemed to hold one QTL, also on chromosome S06. The genomic position also indicated that the regions identified on the S06 chromosome co-localized. Thus, this portion of resistance might be shared by both sources of resistance.
The biochemical approach, using the bio-guided fractionation has been performed on INN_Sa_PB_001 and INN_Sa_PB_002. Thus, a repellent effect has been observed for a common sub-fraction between the two sources, and four other sub-fractions seemed active in the most resistant accession, INN_Sa_PB_001. Metabolomic analyses showed seven candidate metabolites, four of whom could be identified.

The perspectives for the second half of the project include the finalization of discovery activities, notably the preparation of the F6 materials, the genotyping and phenotyping of this material, then the refined QTL analyses, but also the identification of active fractions from the third source of resistance, INN_Sa_PB_003 and the identification of candidate compounds.
The validation activities will be started. On the biochemical aspect, the effect of candidate compounds will be tested through a bioassay. On the genetic side, the preparation of dedicated materials has already started, with the fixation of a panel of 80 accessions. The preparation of HIF materials from the F5 individuals has been reported, as the size of the QTL are currently too large to select the F5 individuals. This material will be evaluated in the field and in controlled conditions.
Finally, both discovery and validation data will be used in bio-analyses to obtain a list of candidate genes in Sinapis alba, involved in resistance, but also to a list of metabolites present in species closer to oilseed rape.

The results from BRING IT ON have not been published yet. Nevertheless, they will be presented at the International Rapeseed Conference in September 2023 in Sidney. Some of the results have also been presented in the PhD manuscript of Laura Bellec: «Un tango à trois: combiner l'écologie nutritionnelle et chimique à la génétique quantitative pour élucider les déterminants de la sélection de la plante hôte chez un insecte phytophage«, successfully defended on June 9th, 2023.

The BRING IT ON project aims at bringing a new solution to breed oilseed rape (Brassica napus) for resistance to one of its major insect pests, the pollen beetle (Brassicogethes aeneus). Thus, it is targeted to the “Conception of more resilient systems to bio-aggressors” principal theme, with a varietal resistance to the pest that could be used in different crop management approaches. Our results show that such resistance exists in the close relative Sinapis alba and we have identified and confirmed contrasted accessions in both field and controlled conditions. With no other option for resistance breeding in oilseed rape, our solution is currently at TRL4. However, to reach TRL5, we need to ensure the ability to transfer efficiently the resistance to oilseed rape. Understanding the genetic and chemical determinants will give tools to achieve this transfer and accelerate resistant oilseed rape breeding by many years. Indeed, oilseed rape is heading for a dead end as a crop if solutions to escape the dependency to insecticides are not found. Finally, although specific to the resistance to pollen beetle, our project will generate resources that could be used for other oilseed rape insect pests and methods for other crop/insect interactions.
BRING IT ON aims at making use of the diversity of resistance present in S. alba to identify the genetic determinants of this resistance and identify the chemicals compounds produced by S. alba resistant accessions to provide a solution for oilseed rape resistance breeding. Genetic and genomic resources in S. alba will be constructed from three different sources of resistance and QTL analyses will be performed to identify genomic regions involved in resistance. In parallel, previous results have shown that the resistance is expressed after contacts between the pests and the plant, thus, identifying chemical compounds produced by the plant that deter the pollen beetle will point towards biosynthetic pathways to improve by breeding. The combination of the two approaches will help prioritizing regions to target for validation and then use in breeding.
Thus, the main user for this solution will be breeding companies that will be able to take up the solution quickly to generate resistant varieties. From the expected outcomes of BRING IT ON, marker assisted selection from the QTL regions or candidate genes will be used to generate new varieties in less than 10 years.
In line with the Ecophyto plan, our solution will help decrease insecticide treatments. Thus, it will be beneficial to growers from both an economic and health perspectives. Reducing pesticide treatments in the spring at flowering time, a crucial period for pollinators, will also impact populations of beneficials insects, whether bees or biocontrol agents for other insect pests. According to a survey conducted in 2020 by Terres Inovia, resistant varieties, coupled with crop management strategies could help avoiding an average of 1.2 insecticide application per year.
BRING IT ON has built a complementary consortium from 1) a private company (INNOLEA, Partner1) dedicated to the identification and understanding of beneficial alleles for oilseed crop breeding, in line with 3 major plant breeding companies in France (Lidea, Limagrain and RAGT) and bringing a strong genetic expertise, 2) a public institute (IGEPP, Partner2) specialized in developing innovative and sustainable solutions for plant production and protection and bringing a strong chemical ecology expertise and 3) an applied research institute (Terres Inovia, Partner3) for oil and protein crops and hemp with an objective to help growers in the diversification and sustainability of their production, with a strong expertise in field evaluation.

Project coordination

Sébastien Faure (Innolea)

The author of this summary is the project coordinator, who is responsible for the content of this summary. The ANR declines any responsibility as for its contents.

Partner

IGEPP Institut de Génétique Environnement et Protection des Plantes
Terres Inovia
Innolea Innolea

Help of the ANR 449,931 euros
Beginning and duration of the scientific project: December 2021 - 36 Months

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