Why old duplicated genes are not thrown away in (paleo)polyploids? Example from the petC gene in Brassica napus.

Duplication of genes at different time period, through recurrent and frequent polyploidization events, have played a major role in plant evolution, adaptation and diversification. Interestingly, some of the ancestral duplicated genes (referred as paleologs), have been maintained for millions of years, and there is still a poor knowledge of the reasons of their retention, especially when testing the phenotypic effect of individual copies by using functional genetic approaches. To fill this gap, we performed functional genetic (CRISPR-Cas9), physiological, transcriptomic and evolutionary studies to finely investigate this open question, taking the example of the petC gene (involved in cytochrome b6/f and thus impacting photosynthesis) that is present in four paleologous copies in the oilseed crop Brassica napus. RNA-Seq and selective pressure analyses suggested that all paleologous copies conserved the same function and that they were all highly transcribed. Thereafter, the Knock Out (K.O.) of one, several or all petC copies highlighted that all paleologous copies have to be K.O. to suppress the gene function. In addition, we could determine that phenotypic effects in single and double mutants could only be deciphered in high light conditions. Interestingly, we did not detect any significant differences between single mutants K.O. for either the A03 or A09 copy (despite being differentially transcribed), or even between mutants for a single or two petC copies. Altogether, this work revealed that petC paleologs have retained their ancestral function and that the retention of these copies is explained by their compensatory role, especially in optimal environmental conditions.

Epigenomic and structural events preclude recombination in Brassica napus.

Meiotic recombination is a major evolutionary process generating genetic diversity at each generation in sexual organisms. However, this process is highly regulated, with the majority of crossovers lying in the distal chromosomal regions that harbor low DNA methylation levels. Even in these regions, some islands without recombination remain, for which we investigated the underlying causes. Genetic maps were established in two Brassica napus hybrids to detect the presence of such large nonrecombinant islands. The role played by DNA methylation and structural variations in this local absence of recombination was determined by performing bisulfite sequencing and whole genome comparisons. Inferred structural variations were validated using either optical mapping or oligo fluorescence in situ hybridization. Hypermethylated or inverted regions between Brassica genomes were associated with the absence of recombination. Pairwise comparisons of nine B. napus genome assemblies revealed that such inversions occur frequently and may contain key agronomic genes such as resistance to biotic stresses. We conclude that such islands without recombination can have different origins, such as DNA methylation or structural variations in B. napus. It is thus essential to take into account these features in breeding programs as they may hamper the efficient combination of favorable alleles in elite varieties.

Untangling structural factors driving genome stabilization in nascent Brassica napus allopolyploids.

Allopolyploids have globally higher fitness than their diploid progenitors; however, by comparison, most resynthesized allopolyploids have poor fertility and highly unstable genome. Elucidating the evolutionary processes promoting genome stabilization and fertility is thus essential to comprehend allopolyploid success. Using the Brassica model, we mimicked the speciation process of a nascent allopolyploid species by resynthesizing allotetraploid Brassica napus and systematically selecting for euploid individuals over eight generations in four independent allopolyploidization events with contrasted genetic backgrounds, cytoplasmic donors, and polyploid formation type. We evaluated the evolution of meiotic behavior and fertility and identified rearrangements in S1 to S9 lineages to explore the positive consequences of euploid selection on B. napus genome stability. Recurrent selection of euploid plants for eight generations drastically reduced the percentage of aneuploid progenies as early as the fourth generation, concomitantly with a decrease in number of newly fixed homoeologous rearrangements. The consequences of homoeologous rearrangements on meiotic behavior and seed number depended strongly on the genetic background and cytoplasm donor. The combined use of both self-fertilization and recurrent euploid selection allowed identification of genomic regions associated with fertility and meiotic behavior, providing complementary evidence to explain B. napus speciation success.

A Modified Meiotic Recombination in Brassica napus Largely Improves Its Breeding Efficiency.

Meiotic recombination is the main tool used by breeders to generate biodiversity, allowing genetic reshuffling at each generation. It enables the accumulation of favorable alleles while purging deleterious mutations. However, this mechanism is highly regulated with the formation of one to rarely more than three crossovers, which are not randomly distributed. In this study, we showed that it is possible to modify these controls in oilseed rape (Brassica napus, AACC, 2n = 4x = 38) and that it is linked to AAC allotriploidy and not to polyploidy per se. To that purpose, we compared the frequency and the distribution of crossovers along A chromosomes from hybrids carrying exactly the same A nucleotide sequence, but presenting three different ploidy levels: AA, AAC and AACC. Genetic maps established with 202 SNPs anchored on reference genomes revealed that the crossover rate is 3.6-fold higher in the AAC allotriploid hybrids compared to AA and AACC hybrids. Using a higher SNP density, we demonstrated that smaller and numerous introgressions of B. rapa were present in AAC hybrids compared to AACC allotetraploid hybrids, with 7.6 Mb vs. 16.9 Mb on average and 21 B. rapa regions per plant vs. nine regions, respectively. Therefore, this boost of recombination is highly efficient to reduce the size of QTL carried in cold regions of the oilseed rape genome, as exemplified here for a QTL conferring blackleg resistance.

Genome Size Variation and Comparative Genomics Reveal Intraspecific Diversity in Brassica rapa.

Traditionally, reference genomes in crop species rely on the assembly of one accession, thus occulting most of intraspecific diversity. However, rearrangements, gene duplications, and transposable element content may have a large impact on the genomic structure, which could generate new phenotypic traits. Comparing two Brassica rapa genomes recently sequenced and assembled using long-read technology and optical mapping, we investigated structural variants and repetitive content between the two accessions and genome size variation among a core collection. We explored the structural consequences of the presence of large repeated sequences in B. rapa 'Z1' genome vs. the B. rapa 'Chiifu' genome, using comparative genomics and cytogenetic approaches. First, we showed that large genomic variants on chromosomes A05, A06, A09, and A10 are due to large insertions and inversions when comparing B. rapa 'Z1' and B. rapa 'Chiifu' at the origin of important length differences in some chromosomes. For instance, lengths of 'Z1' and 'Chiifu' A06 chromosomes were estimated in silico to be 55 and 29 Mb, respectively. To validate these observations, we compared using fluorescent in situ hybridization (FISH) the two A06 chromosomes present in an F1 hybrid produced by crossing these two varieties. We confirmed a length difference of 17.6% between the A06 chromosomes of 'Z1' compared to 'Chiifu.' Alternatively, using a copy number variation approach, we were able to quantify the presence of a higher number of rDNA and gypsy elements in 'Z1' genome compared to 'Chiifu' on different chromosomes including A06. Using flow cytometry, the total genome size of 12 Brassica accessions corresponding to a B. rapa available core collection was estimated and revealed a genome size variation of up to 16% between these accessions as well as some shared inversions. This study revealed the contribution of long-read sequencing of new accessions belonging to different cultigroups of B. rapa and highlighted the potential impact of differential insertion of repeat elements and inversions of large genomic regions in genome size intraspecific variability.