Development of B. carinata with super-high erucic acid content through interspecific hybridization.

KEY MESSAGE: Disomic alien chromosome addition Brassica carinata lines with super-high erucic acid content were developed through interspecific hybridization with B. juncea and characterized using molecular, cytological and biochemical techniques. Brassica carinata [A.] Braun (BBCC, 2n = 34) is a climate-resilient oilseed. Its seed oil is high in erucic acid (> 40%), rendering it well suited for the production of biofuel and other bio-based applications. To enhance the competitiveness of B. carinata with high erucic B. napus (HEAR), lines with super-high erucic acid content were developed through interspecific hybridization. To this end, a fad2B null allele from Brassica juncea (AABB, 2n = 36) was introgressed into B. carinata, resulting in a B. carinata fad2B mutant with erucic acid levels of over 50%. Subsequently, the FAE allele from B. rapa spp. yellow sarson (AA, 2n = 20) was transferred to the fad2B B. carinata line, yielding lines with erucic acid contents of up to 57.9%. Molecular analysis using the Brassica 90 K Illumina Infinium™ SNP genotyping array identified these lines as disomic alien chromosome addition lines, with two extra A08 chromosomes containing the BrFAE gene. The alien chromosomes from B. rapa were clearly distinguished by molecular cytogenetics in one of the addition lines. Analysis of microspore-derived offspring and hybrids from crosses with a CMS B. carinata line showed that the transfer rate of the A08 chromosome into male gametes was over 98%, resulting in almost completely stable transmission of an A08 chromosome copy into the progeny. The increase in erucic acid levels was accompanied by changes in the proportions of other fatty acids depending on the genetic changes that were introduced in the interspecific hybrids, providing valuable insights into erucic acid metabolism in Brassica.

Stable, fertile lines produced by hybridization between allotetraploids Brassica juncea (AABB) and Brassica carinata (BBCC) have merged the A and C genomes.

Many flowering plant taxa contain allopolyploids that share one or more genomes in common. In the Brassica genus, crop species Brassica juncea and Brassica carinata share the B genome, with 2n = AABB and 2n = BBCC genome complements, respectively. Hybridization results in 2n = BBAC hybrids, but the fate of these hybrids over generations of self-pollination has never been reported. We produced and characterized B. juncea × B. carinata (2n = BBAC) interspecific hybrids over six generations of self-pollination under selection for high fertility using a combination of genotyping, fertility phenotyping, and cytogenetics techniques. Meiotic pairing behaviour improved from 68% bivalents in the F1 to 98% in the S5 /S6 generations, and initially low hybrid fertility also increased to parent species levels. The S5 /S6 hybrids contained an intact B genome (16 chromosomes) plus a new, stable A/C genome (18-20 chromosomes) resulting from recombination and restructuring of A and C-genome chromosomes. Our results provide the first experimental evidence that two genomes can come together to form a new, restructured genome in hybridization events between two allotetraploid species that share a common genome. This mechanism should be considered in interpreting phylogenies in taxa with multiple allopolyploid species.

Hybrids between Brassica napus and B. nigra show frequent pairing between the B and A/C genomes and resistance to blackleg.

High frequencies of homoeologous and even non-homologous chromosome recombination in Brassica hybrids can transfer useful traits between genomes, but also destabilise synthetic allopolyploids. We produced triploid hybrids (2n = 3x = ABC) from the cross B. napus (rapeseed, 2n = 4x = AACC) × B. nigra (black mustard, 2n = 2x = BB) by embryo rescue and allohexaploid hybrids (2n = 6x = AABBCC = 54) by chromosome doubling of the triploids. These hybrids demonstrated resistance to blackleg disease (causal agent: Leptosphaeria maculans) inherited from their B. nigra parent. In order to assess the possibility of transfer of this resistance between the B genome and the A and C subgenomes of B. napus, as well as to assess the genomic stability of allohexaploids from the cross B. napus × B. nigra, frequencies of non-homologous chromosome pairing in these hybrids were assessed using classical cytogenetics and genomic in-situ hybridization. Meiosis was highly irregular, and non-homologous chromosome pairing between the B genome and the A/C genomes was common in both triploid hybrids (observed in 38% of pollen mother cells) and allohexaploid hybrids (observed in 15% of pollen mother cells). Our results suggest that introgression of blackleg resistance from the B genome into the A or C genomes should be possible, but that allohexaploids from this genome combination are likely unstable.

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.