Allele segregation analysis of F1 hybrids between independent Brassica allohexaploid lineages.

In the Brassica genus, we find both diploid species (one genome) and allotetraploid species (two different genomes) but no naturally occurring hexaploid species (three different genomes, AABBCC). Although hexaploids can be produced via human intervention, these neo-polyploids have quite unstable genomes and usually suffer from severe genome reshuffling. Whether these genome rearrangements continue in later generations and whether genomic arrangements follow similar, reproducible patterns between different lineages is still unknown. We crossed Brassica hexaploids resulting from different species combinations to produce five F1 hybrids and analyzed the karyotypes of the parents and the F1 hybrids, as well as allele segregation in a resulting test-cross population via molecular karyotyping using SNP array genotyping. Although some genomic regions were found to be more likely to be duplicated, deleted, or rearranged, a consensus pattern was not shared between genotypes. Brassica hexaploids had a high tolerance for fixed structural rearrangements, but which rearrangements occur and become fixed over many generations does not seem to show either strong reproducibility or to indicate selection for stability. On average, we observed 10 de novo chromosome rearrangements contributed almost equally from both parents to the F1 hybrids. At the same time, the F1 hybrid meiosis produced on average 8.6 new rearrangements. Hence, the increased heterozygosity in the F1 hybrid did not significantly improve genome stability in our hexaploid hybrids and might have had the opposite effect. However, hybridization between lineages was readily achieved and may be exploited for future genetics and breeding purposes.

Challenges and prospects for a potential allohexaploid Brassica crop.

The production of a new allohexaploid Brassica crop (2n = AABBCC) is increasingly attracting international interest: a new allohexaploid crop could benefit from several major advantages over the existing Brassica diploid and allotetraploid species, combining genetic diversity and traits from all six crop species with additional allelic heterosis from the extra genome. Although early attempts to produce allohexaploids showed mixed results, recent technological and conceptual advances have provided promising leads to follow. However, there are still major challenges which exist before this new crop type can be realized: (1) incorporation of sufficient genetic diversity to form a basis for breeding and improvement of this potential crop species; (2) restoration of regular meiosis, as most allohexaploids are genetically unstable after formation; and (3) improvement of agronomic traits to the level of "elite" breeding material in the diploid and allotetraploid crop species. In this review, we outline these major prospects and challenges and propose possible plans to produce a stable, diverse and agronomically viable allohexaploid Brassica crop.

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.

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.

Functional analysis of the Brassica napus L. phytoene synthase (PSY) gene family.

Phytoene synthase (PSY) has been shown to catalyze the first committed and rate-limiting step of carotenogenesis in several crop species, including Brassica napus L. Due to its pivotal role, PSY has been a prime target for breeding and metabolic engineering the carotenoid content of seeds, tubers, fruits and flowers. In Arabidopsis thaliana, PSY is encoded by a single copy gene but small PSY gene families have been described in monocot and dicotyledonous species. We have recently shown that PSY genes have been retained in a triplicated state in the A- and C-Brassica genomes, with each paralogue mapping to syntenic locations in each of the three "Arabidopsis-like" subgenomes. Most importantly, we have shown that in B. napus all six members are expressed, exhibiting overlapping redundancy and signs of subfunctionalization among photosynthetic and non photosynthetic tissues. The question of whether this large PSY family actually encodes six functional enzymes remained to be answered. Therefore, the objectives of this study were to: (i) isolate, characterize and compare the complete protein coding sequences (CDS) of the six B. napus PSY genes; (ii) model their predicted tridimensional enzyme structures; (iii) test their phytoene synthase activity in a heterologous complementation system and (iv) evaluate their individual expression patterns during seed development. This study further confirmed that the six B. napus PSY genes encode proteins with high sequence identity, which have evolved under functional constraint. Structural modeling demonstrated that they share similar tridimensional protein structures with a putative PSY active site. Significantly, all six B. napus PSY enzymes were found to be functional. Taking into account the specific patterns of expression exhibited by these PSY genes during seed development and recent knowledge of PSY suborganellar localization, the selection of transgene candidates for metabolic engineering the carotenoid content of oilseeds is discussed.