Broadening the genetic diversity underpinning seed quality and yield related traits in mustard rape and oilseed rape

Vegetable oils are an important part of the human diet, providing essential fatty acids. Amongst the most important oilseed crops, second in global production only to soybean, are the oilseed Brassica crops. These are principally the species B. napus (oilseed rape; cultivated widely in the UK) and B. juncea (mustard rape; cultivated widely in India). Both are recently-formed allotetraploids (containing the Brassica A and C genomes in the case of B. napus and the A and B genomes in the case of B. juncea), so both suffer from the complications arising from polyploidy and a lack of genetic diversity in breeding material. Broadening the genetic bases of the crops and identifying the genes underpinning trait variation are important objectives in both species.

In addition to being important crops, the Brassica species represent an excellent system in which to study the evolution of genomes and the quantitative control of traits during the "diploidisation" process following polyploidy. All three diploid Brassica genomes (A, B and C) have been sequenced (those of B and C genomes are unpublished, but available under collaboration agreements). Powerful new technologies, based on Illumina transcriptome sequencing have been developed and applied to B. napus, enabling the development of a detailed gene-based genomic framework and the capability of associating, across diversity panels, trait variation with both gene sequence variation and gene expression variation.

In the proposed research, we aim to understand the principle genetic bases underpinning quantitative variation of an initial set of traits of importance for the commercial production of both mustard rape and oilseed rape. In doing this, we will: (1) establish genomic resources for B. juncea to match those already developed by the group of IB for B. napus, (2) analyse the evolution of the B genome relative to those of the A and C genomes, (3) establish functional genotypes for a B. juncea diversity panel and an expanded B. napus panel, (4) identify candidate genes and alleles for crop improvement and (5) promote knowledge exchange with the breeding industries to facilitate crop improvement.

The proposed research builds upon ongoing activities of the applicants, involves the sharing of genetic resources and includes a strong training element. The transcriptome corresponding to the B genome will be assembled to form a unigene set using Illumina mRNAseq data obtained from B. nigra. Leaf transcriptome sequencing will be undertaken for a B. juncea DH mapping population and used, in combination with unigenes corresponding to the A and B genomes for the construction of a detailed SNP linkage map. The linkage map will be used to organise genome sequence scaffolds of the A and B genomes into pseudomolecules that will be used to define hypothetical orders of unigenes in the A and B genomes of B. juncea. The evolutionary histories of the Brassica A, B and C genomes will be analysed, as observed in the diploid species and the allotetraploids B. juncea and B. napus, since their divergence from a common ancestor. Leaf transcriptome sequencing will be undertaken for a B. juncea diversity panel and additional lines of a B. napus diversity panel, SNP variation will be scored across the respective panels and transcripts quantified across all accessions. The diversity panels will be analysed, in suitable environments, for the variation of traits prioritised in consultation with the breeding and farming industries in India and the UK and used for Associative Transcriptomics analysis. The traits are anticipated to include seed composition traits analysed in the UK (content of oil, protein, glucosinolates, erucic acid, linolenic acid, tocopherols) and resistance to various biotic and abiotic stresses analysed in India. Knowledge of the genetic bases for trait variation and markers for beneficial alleles will be generated and made available for crops.

We aim to understand the principle genetic bases underpinning quantitative variation of an initial set of traits of importance for the commercial production of both mustard rape and oilseed rape, including:

(1) Establish genomic resources for B. juncea to match those already developed for B. napus. This will involve assembly of the transcriptome of B. nigra, construction of a high density (>20,000 marker) transcriptome SNP linkage map of B. juncea and the development of DNA sequence pseudomolecules for the Brassica A and B genomes as represented in B. juncea.

(2) Analyse the evolution of the B genome relative to those of the A and C genomes. This will involve comparative sequence-level analysis of sequence pseudomolecules for the Brassica A, B and C genomes as represented in B. juncea (A and B) and, from studies completed previously, B. napus (A and C).

(3) Establish functional genotypes for a B. juncea diversity panel and an expanded B. napus panel. This will involve undertaking mRNAseq (using RNA from leaves) from panels of genetically diverse B. juncea and B. napus accessions, quantification of transcript abundance and SNP genotyping based on the sequence data.

(4) Identify candidate genes and alleles for crop improvement. This will involve phenotypic analysis of the B. juncea and B. napus diversity panels for seed composition and yield, and Associative Transcriptomics to identify candidate genes and alleles controlling the traits and conversion of validated associations to molecular markers to assist breeding.

(5) Promote knowledge exchange with the breeding industries to facilitate crop improvement. This will involve engagement with breeding, farming and processing industries in both India and UK, leading to new opportunities for adding value to oilseed Brassica crops (via improved seed composition) and improving their yield.

The outcome of project will be new opportunities for adding value to oilseed Brassica crops (via improved seed composition) and improving their yield potential. These opportunities will be realised by breeders of oilseed/mustard rape, who will be able to design crossing strategies within each species and select alleles using molecular markers developed for polymorphisms identified in the project as being associated with the control of trait variation. Exploitation in B. napus of alleles present in the B genome of B. juncea, and exploitation in B. juncea of alleles present in the C genome of B. napus is more complex, but achievable by inter-specific crossing and marker-assisted selection of alleles captured by homoeologous recombination. In this way, knowledge and genetic resources can be exploited for crop improvement in both countries.

The primary route for dissemination, knowledge exchange and commercialization will be via the lead partners in India and the UK. In the UK this will include engagements with organisations such as HGCA and via community meetings involving breeders, such as UK-BRC and OREGIN Stakeholder meetings. Contact will also be made with active UK-based companies working downstream of the breeders, such as farmers (e.g. Velcourt ltd) and processors (such as Cargill), with a view to early sharing of insights into progress and opportunities. In India, the knowledge generated through this project will be passed on to the breeders working in public sector organizations and private sector companies working on varietal development with value added products in oilseed brassicas which will ultimately benefit the farming community

Once decisive insights into the first-order genetic control of key traits have been obtained, further refinement will be necessary in order to identify minor (but still important) components. This work can be undertaken by the private sector and both public and private sector in India, with advice from the academic partners in the project. This will require ramping up the genetic analysis to several hundreds of accessions. Where natural genetic variation of the type required for optimum performance has not been identified (e.g. where a gene knock-out or over-expression variant is required, but no such alleles are present in the diversity panel), induction of the necessary variation (for example by a mutagenesis approach followed by molecular selection) could be undertaken. Rapeseed breeders can undertake directly or subcontract trait analyses. Where activities such as sequence data processing are already routine, such as mRNAseq SNP identification and transcript quantification, commercial services are available, for example from the Cambridge-based company Eagle Genomics as a collaboration with U. York. A commercial service provider (Tag Genetics Ltd.) is available to undertake further association analyses for the breeders, if required.