Using wild relatives and related species to build climate resilience in Brassica crops.

Climate change will have major impacts on crop production: not just increasing drought and heat stress, but also increasing insect and disease loads and the chance of extreme weather events and further adverse conditions. Often, wild relatives show increased tolerances to biotic and abiotic stresses, due to reduced stringency of selection for yield and yield-related traits under optimum conditions. One possible strategy to improve resilience in our modern-day crop cultivars is to utilize wild relative germplasm in breeding, and attempt to introgress genetic factors contributing to greater environmental tolerances from these wild relatives into elite crop types. However, this approach can be difficult, as it relies on factors such as ease of hybridization and genetic distance between the source and target, crossover frequencies and distributions in the hybrid, and ability to select for desirable introgressions while minimizing linkage drag. In this review, we outline the possible effects that climate change may have on crop production, introduce the Brassica crop species and their wild relatives, and provide an index of useful traits that are known to be present in each of these species that may be exploitable through interspecific hybridization-based approaches. Subsequently, we outline how introgression breeding works, what factors affect the success of this approach, and how this approach can be optimized so as to increase the chance of recovering the desired introgression lines. Our review provides a working guide to the use of wild relatives and related crop germplasm to improve biotic and abiotic resistances in Brassica crop species.

A novel deletion in FLOWERING LOCUS T modulates flowering time in winter oilseed rape.

KEY MESSAGE: A novel structural variant was discovered in the FLOWERING LOCUS T orthologue BnaFT.A02 by long-read sequencing. Nested association mapping in an elite winter oilseed rape population revealed that this 288 bp deletion associates with early flowering, putatively by modification of binding-sites for important flowering regulation genes. Perfect timing of flowering is crucial for optimal pollination and high seed yield. Extensive previous studies of flowering behavior in Brassica napus (canola, rapeseed) identified mutations in key flowering regulators which differentiate winter, semi-winter and spring ecotypes. However, because these are generally fixed in locally adapted genotypes, they have only limited relevance for fine adjustment of flowering time in elite cultivar gene pools. In crosses between ecotypes, the ecotype-specific major-effect mutations mask minor-effect loci of interest for breeding. Here, we investigated flowering time in a multiparental mapping population derived from seven elite winter oilseed rape cultivars which are fixed for major-effect mutations separating winter-type rapeseed from other ecotypes. Association mapping revealed eight genomic regions on chromosomes A02, C02 and C03 associating with fine modulation of flowering time. Long-read genomic resequencing of the seven parental lines identified seven structural variants coinciding with candidate genes for flowering time within chromosome regions associated with flowering time. Segregation patterns for these variants in the elite multiparental population and a diversity set of winter types using locus-specific assays revealed significant associations with flowering time for three deletions on chromosome A02. One of these was a previously undescribed 288 bp deletion within the second intron of FLOWERING LOCUS T on chromosome A02, emphasizing the advantage of long-read sequencing for detection of structural variants in this size range. Detailed analysis revealed the impact of this specific deletion on flowering-time modulation under extreme environments and varying day lengths in elite, winter-type oilseed rape.

Long-read sequencing reveals widespread intragenic structural variants in a recent allopolyploid crop plant.

Genome structural variation (SV) contributes strongly to trait variation in eukaryotic species and may have an even higher functional significance than single-nucleotide polymorphism (SNP). In recent years, there have been a number of studies associating large chromosomal scale SV ranging from hundreds of kilobases all the way up to a few megabases to key agronomic traits in plant genomes. However, there have been little or no efforts towards cataloguing small- (30-10 000 bp) to mid-scale (10 000-30 000 bp) SV and their impact on evolution and adaptation-related traits in plants. This might be attributed to complex and highly duplicated nature of plant genomes, which makes them difficult to assess using high-throughput genome screening methods. Here, we describe how long-read sequencing technologies can overcome this problem, revealing a surprisingly high level of widespread, small- to mid-scale SV in a major allopolyploid crop species, Brassica napus. We found that up to 10% of all genes were affected by small- to mid-scale SV events. Nearly half of these SV events ranged between 100 bp and 1000 bp, which makes them challenging to detect using short-read Illumina sequencing. Examples demonstrating the contribution of such SV towards eco-geographical adaptation and disease resistance in oilseed rape suggest that revisiting complex plant genomes using medium-coverage long-read sequencing might reveal unexpected levels of functional gene variation, with major implications for trait regulation and crop improvement.

Transcriptomics reveal high regulatory diversity of drought tolerance strategies in a biennial oil crop.

Spring droughts are expected to become more frequent in Central Europe as a result of climate change. Their coincidence with flowering of biennial crops like winter oilseed rape (Brassica napus) can cause major impact for yield development. However, no data is available on the diversity of genetic regulation of drought tolerance during this stage under realistic conditions. Here, we assessed the phenotypic plasticity of drought response for eight diverse B. napus accessions under field-like conditions and linked their stress response to gene and miRNA expression during early and late stress. We observed highly diverse responses, both on the phenotypic and on the gene expression level. Our data suggest that drought tolerant accessions have more effective molecular protection mechanisms like ROS scavenging, source/sink ratio and regulation of developmental timing, compared to otherwise phenotypically similar accessions. Bna.MAP3K13.C05 expression was found to be protective independently of the tolerance mechanism, indicating cross-talk to nitrogen signaling. Moreover, we identified putative miRNA genes in the B. napus genome which respond to stress and may also be involved in protective mechanisms, representing possible breeding targets.

The vernalisation regulator FLOWERING LOCUS C is differentially expressed in biennial and annual Brassica napus.

Plants in temperate areas evolved vernalisation requirement to avoid pre-winter flowering. In Brassicaceae, a period of extended cold reduces the expression of the flowering inhibitor FLOWERING LOCUS C (FLC) and paves the way for the expression of downstream flowering regulators. As with all polyploid species of the Brassicaceae, the model allotetraploid Brassica napus (rapeseed, canola) is highly duplicated and carries 9 annotated copies of Bna.FLC. To investigate whether these multiple homeologs and paralogs have retained their original function in vernalisation or undergone subfunctionalisation, we compared the expression patterns of all 9 copies between vernalisation-dependent (biennial, winter type) and vernalisation-independent (annual, spring type) accessions, using RT-qPCR with copy-specific primers and RNAseq data from a diversity set. Our results show that only 3 copies - Bna.FLC.A03b, Bna.FLC.A10 and to some extent Bna.FLC.C02 - are differentially expressed between the two growth types, showing that expression of the other 6 copies does not correlate with growth type. One of those 6 copies, Bna.FLC.C03b, was not expressed at all, indicating a pseudogene, while three further copies, Bna.FLC.C03a and Bna.FLC.C09ab, did not respond to cold treatment. Sequence variation at the COOLAIR binding site of Bna.FLC.A10 was found to explain most of the variation in gene expression. However, we also found that Bna.FLC.A10 expression is not fully predictive of growth type.

"Doubled-haploid" allohexaploid Brassica lines lose fertility and viability and accumulate genetic variation due to genomic instability.

Microspore culture stimulates immature pollen grains to develop into plants via tissue culture and is used routinely in many crop species to produce "doubled haploids": homozygous, true-breeding lines. However, microspore culture is also often used on material that does not have stable meiosis, such as interspecific hybrids. In this case, the resulting progeny may lose their "doubled haploid" homozygous status as a result of chromosome missegregation and homoeologous exchanges. However, little is known about the frequency of these effects. We assessed fertility, meiosis and genetic variability in self-pollinated progeny sets (the MDL2 population) resulting from first-generation plants (the MDL1 population) derived from microspores of a near-allohexaploid interspecific hybrid from the cross (Brassica napus × B. carinata) × B. juncea. Allelic inheritance and copy number variation were predicted using single nucleotide polymorphism marker data from the Illumina Infinium 60K Brassica array. Seed fertility and viability decreased substantially from the MDL1 to the MDL2 generation. In the MDL2 population, 87% of individuals differed genetically from their MDL1 parent. These genetic differences resulted from novel homoeologous exchanges between chromosomes, chromosome loss and gain, and segregation and instability of pre-existing karyotype abnormalities. Novel karyotype change was extremely common, with 2.2 new variants observed per MDL2 individual. Significant differences between progeny sets in the number of novel genetic variants were also observed. Meiotic instability clearly has the potential to dramatically change karyotypes (often without detectable effects on the presence or absence of alleles) in putatively homozygous, microspore-derived lines, resulting in loss of fertility and viability.

Inherited allelic variants and novel karyotype changes influence fertility and genome stability in Brassica allohexaploids.

Synthetic allohexaploid Brassica hybrids (2n = AABBCC) do not exist naturally, but can be synthesized by crosses between diploid and/or allotetraploid Brassica species. Using these hybrids, we aimed to identify how novel allohexaploids restore fertility and normal meiosis after formation. Chromosome inheritance, genome structure, fertility and meiotic behaviour were assessed in three segregating allohexaploid populations derived from the cross (B. napus × B. carinata) × B. juncea using a combination of molecular marker genotyping, phenotyping and cytogenetics. Plants with unbalanced A-C translocations in one direction (where a C-genome chromosome fragment replaces an A-genome fragment) but not the other (where an A-genome fragment replaces a C-genome fragment) showed significantly reduced fertility across all populations. Genomic regions associated with fertility contained several meiosis genes with putatively causal mutations inherited from the parents (copies of SCC2 in the A genome, PAIR1/PRD3, PRD1 and ATK1/KATA/KIN14a in the B genome, and MSH2 and SMC1/TITAN8 in the C genome). Reduced seed fertility associated with the loss of chromosome fragments from only one subgenome following homoeologous exchanges could comprise a mechanism for biased genome fractionation in allopolyploids. Pre-existing meiosis gene variants present in allotetraploid parents may help to stabilize meiosis in novel allohexaploids.

Drought stress has transgenerational effects on seeds and seedlings in winter oilseed rape (Brassica napus L.).

BACKGROUND: Drought stress has a negative effect on both seed yield and seed quality in Brassica napus (oilseed rape, canola). Here we show that while drought impairs the maternal plant performance, it also increases the vigour of progeny of stressed maternal plants. We investigated the transgenerational influence of abiotic stress by detailed analysis of yield, seed quality, and seedling performance on a growth-related and metabolic level. Seeds of eight diverse winter oilseed rape genotypes were generated under well-watered and drought stress conditions under controlled-environment conditions in large plant containers. RESULTS: We found a decrease in seed quality in seeds derived from mother plants that were exposed to drought stress. At the same time, the seeds that developed under stress conditions showed higher seedling vigour compared to non-stressed controls.This effect on seed quality and seedling vigour was found to be independent of maternal plant yield performance. CONCLUSIONS: Drought stress has a positive transgenerational effect on seedling vigour. Three potential causes for stress-induced improvement of seedling vigour are discussed: (1) Heterotic effects caused by a tendency towards a higher outcrossing rate in response to stress; (2) an altered reservoir of seed storage metabolites to which the seedling resorts during early growth, and (3) inter-generational stress memory, formed by stress-induced changes in the epigenome of the seedling.

Homoeologous exchange is a major cause of gene presence/absence variation in the amphidiploid Brassica napus.

Homoeologous exchanges (HEs) have been shown to generate novel gene combinations and phenotypes in a range of polyploid species. Gene presence/absence variation (PAV) is also a major contributor to genetic diversity. In this study, we show that there is an association between these two events, particularly in recent Brassica napus synthetic accessions, and that these represent a novel source of genetic diversity, which can be captured for the improvement of this important crop species. By assembling the pangenome of B. napus, we show that 38% of the genes display PAV behaviour, with some of these variable genes predicted to be involved in important agronomic traits including flowering time, disease resistance, acyl lipid metabolism and glucosinolate metabolism. This study is a first and provides a detailed characterization of the association between HEs and PAVs in B. napus at the pangenome level.

Flowering Time Gene Variation in Brassica Species Shows Evolutionary Principles.

Flowering time genes have a strong influence on successful reproduction and life cycle adaptation. However, their regulation is highly complex and only well understood in diploid model systems. For crops with a polyploid background from the genus Brassica, data on flowering time gene variation are scarce, although indispensable for modern breeding techniques like marker-assisted breeding. We have deep-sequenced all paralogs of 35 Arabidopsis thaliana flowering regulators using Sequence Capture followed by Illumina sequencing in two selected accessions of the vegetable species Brassica rapa and Brassica oleracea, respectively. Using these data, we were able to call SNPs, InDels and copy number variations (CNVs) for genes from the total flowering time network including central flowering regulators, but also genes from the vernalisation pathway, the photoperiod pathway, temperature regulation, the circadian clock and the downstream effectors. Comparing the results to a complementary data set from the allotetraploid species Brassica napus, we detected rearrangements in B. napus which probably occurred early after the allopolyploidisation event. Those data are both a valuable resource for flowering time research in those vegetable species, as well as a contribution to speciation genetics.

Mapping of homoeologous chromosome exchanges influencing quantitative trait variation in Brassica napus.

Genomic rearrangements arising during polyploidization are an important source of genetic and phenotypic variation in the recent allopolyploid crop Brassica napus. Exchanges among homoeologous chromosomes, due to interhomoeologue pairing, and deletions without compensating homoeologous duplications are observed in both natural B. napus and synthetic B. napus. Rearrangements of large or small chromosome segments induce gene copy number variation (CNV) and can potentially cause phenotypic changes. Unfortunately, complex genome restructuring is difficult to deal with in linkage mapping studies. Here, we demonstrate how high-density genetic mapping with codominant, physically anchored SNP markers can detect segmental homoeologous exchanges (HE) as well as deletions and accurately link these to QTL. We validated rearrangements detected in genetic mapping data by whole-genome resequencing of parental lines along with cytogenetic analysis using fluorescence in situ hybridization with bacterial artificial chromosome probes (BAC-FISH) coupled with PCR using primers specific to the rearranged region. Using a well-known QTL region influencing seed quality traits as an example, we confirmed that HE underlies the trait variation in a DH population involving a synthetic B. napus trait donor, and succeeded in narrowing the QTL to a small defined interval that enables delineation of key candidate genes.