Periodic Reporting for period 1 - SAMEY (Stabilising autopolyploid meiosis for enhanced yield)
Periodo di rendicontazione: 2023-08-01 al 2026-01-31
Hybrid breeding has been one of the biggest contributors to yield increase of the last century. Hybrids are individuals which have genetically different parents, which results in a “hybrid vigour” effect. This hybrid vigour effect can confer advantages over inbred parent lines in growth, yield, and resilience and tolerance to different types of environmental stresses, important for securing agricultural production in a changing climate. An even greater hybrid vigour effect is possible in autopolyploids, which can have up to four different copies of each chromosome, than in diploids, which have can only have up to two different copies of each chromosome. In effect, “double hybrids” can be made in autopolyploids, with up to four different parents contributing to hybrid vigour in a single individual. However, to date the double hybrid effect has almost never been used for breeding. Most of our crops are not autopolyploids. We can induce autopolyploidy through chromosome doubling, but this causes meiotic instability, where multiple crossovers occur between the four chromosome copies during meiosis. This pairing disruption leads to potential loss of chromosomes and chromosome fragments essential for seed fertility and viability. Although induced autopolyploids are meiotically unstable, this is not the case for established autopolyploids. In the majority of established autopolyploids, a maximum of one crossover per two homologous chromosomes during meiosis is strictly enforced, thus achieving 100% pairing and correct segregation of chromosomes into daughter cells. In this project, we aim to stabilise meiosis in induced autopolyploid Brassica by knock-out of crossover promoting genes, over-expression of crossover suppressing genes and selection of natural genetic variants. Stable autopolyploids will be used to produce double-hybrid lines, which will be evaluated for hybrid vigour for yield-related traits.
At the start of the project we checked the literature to identify autopolyploid taxa with polysomic inheritance (random assortment of four homologous chromosomes). We found that only around 40 naturally occurring polysomic polyploids have been confirmed so far, and unexpectedly found also that several taxonomic allopolyploids (originating via hybridisation between species) also seem to show polysomic inheritance, which is usually thought to be characteristic of taxonomic autopolyploids. We published these results as well as a summary of the methodologies (including modern genomic methods) which can be used to determine whether a specific species is undergoing disomic or polysomic inheritance in a review paper.
We first characterised meiosis in a set of 20 autotetraploid B. rapa ssp. rapa using whole genome sequencing and a set of cytogenetic probes allowing unique identification of every B. rapa chromosome. All lines showed high frequencies of tetravalent formation, with minor variation between genotypes. All chromosomes also showed tetravalent formation, some with higher frequencies than others. The tetravalents persisted from diakinesis to metaphase I, and resulted in the formation of aneuploid progeny (approximately 50% of individual plants with one or more missing and/or additional chromosomes). Despite this, individual accessions maintained approximately 40 chromosomes and high fertility, possibly due to self-incompatibility preventing fixation of deleterious chromosome complements (aneuploidy events) coupled with high tolerance to minor changes in chromosome dosage (3 or 5 copies instead of 4). These results have been submitted as a preprint and are currently in review.
We then checked TILLING lines (with SNP mutations) for major crossover-related meiosis gene HEI10 and its related transcription factor TAF4b for changes in crossover frequency in diploid B. rapa. HEI10 mutants did not show significant reduction in crossover frequency, but TAF4b mutants did. One TAF4b mutant in particular showed univalents at meiosis and a general reduction in crossover frequency. However, on closer examination, all mutants (heterozygous and homozygous) were also completely sterile, with no pollen or seed production, indicating other effects of TAF4b on fertility-related genes and not just on meiosis.
Investigation of a wider variety of Brassica diploids and tetraploids for natural variation in crossover frequency is ongoing. Genotype-specific differences in crossover frequency have been confirmed, and fascinatingly all B. oleracea tetraploid genotypes investigated so far, in contrast to B. rapa, show regular meiosis and mostly resolve the (few) tetravalents that form between diakinesis and metaphase I as well as produce self-pollinated seeds. We are currently further investigating this effect, and will plan to use these stable B. oleracea genotypes to produce different multi-parental combinations to test our hypotheses related to hybrid vigour.
Concurrently we have been trialling and optimising Brassica transformation protocols. B. rapa has proven extremely recalcitrant to regeneration in culture, but we have managed to produce transformants via a spray protocol (modified floral dip), and have applied a genome-editing construct designed to cause promotor-level variation in the A6 HEI10 gene copy of B. rapa. Screening is in progress.
We first characterised meiosis in a set of 20 autotetraploid B. rapa ssp. rapa using whole genome sequencing and a set of cytogenetic probes allowing unique identification of every B. rapa chromosome. All lines showed high frequencies of tetravalent formation, with minor variation between genotypes. All chromosomes also showed tetravalent formation, some with higher frequencies than others. The tetravalents persisted from diakinesis to metaphase I, and resulted in the formation of aneuploid progeny (approximately 50% of individual plants with one or more missing and/or additional chromosomes). Despite this, individual accessions maintained approximately 40 chromosomes and high fertility, possibly due to self-incompatibility preventing fixation of deleterious chromosome complements (aneuploidy events) coupled with high tolerance to minor changes in chromosome dosage (3 or 5 copies instead of 4). These results have been submitted as a preprint and are currently in review.
We then checked TILLING lines (with SNP mutations) for major crossover-related meiosis gene HEI10 and its related transcription factor TAF4b for changes in crossover frequency in diploid B. rapa. HEI10 mutants did not show significant reduction in crossover frequency, but TAF4b mutants did. One TAF4b mutant in particular showed univalents at meiosis and a general reduction in crossover frequency. However, on closer examination, all mutants (heterozygous and homozygous) were also completely sterile, with no pollen or seed production, indicating other effects of TAF4b on fertility-related genes and not just on meiosis.
Investigation of a wider variety of Brassica diploids and tetraploids for natural variation in crossover frequency is ongoing. Genotype-specific differences in crossover frequency have been confirmed, and fascinatingly all B. oleracea tetraploid genotypes investigated so far, in contrast to B. rapa, show regular meiosis and mostly resolve the (few) tetravalents that form between diakinesis and metaphase I as well as produce self-pollinated seeds. We are currently further investigating this effect, and will plan to use these stable B. oleracea genotypes to produce different multi-parental combinations to test our hypotheses related to hybrid vigour.
Concurrently we have been trialling and optimising Brassica transformation protocols. B. rapa has proven extremely recalcitrant to regeneration in culture, but we have managed to produce transformants via a spray protocol (modified floral dip), and have applied a genome-editing construct designed to cause promotor-level variation in the A6 HEI10 gene copy of B. rapa. Screening is in progress.
To the best of our knowledge (also based on our published literature review), no induced autopolyploid has yet been confirmed to have immediately stable meiosis, polysomic inheritance and resolution of tetravalents between diakinesis and metaphase I. However, we have found that Brassica oleracea, a close relative of B. rapa, shows exactly this phenotype across four genotypes tested so far, as well as good seed fertility and self-compatibility. This result suggests that whole species may be already cytologically pre-adapted to polyploidy, which has major implications for our understanding of the barriers facing polyploid species establishment. We will confirm this result and then publish it.
A technical advance that has arisen out of the project is the optimisation of a spray-based transformation protocol for B. rapa, avoiding the substantial challenges associated with tissue culture-based regeneration in this species required for classical transformation methods. Many groups have tried and failed to adapt floral dip methods to the crop Brassica species – those who have succeeded universally report extremely low success rates (usually <0.5%); our current results suggest a much better success rate. We will confirm these results and publish, hopefully to the benefit of the research and breeding community.
A technical advance that has arisen out of the project is the optimisation of a spray-based transformation protocol for B. rapa, avoiding the substantial challenges associated with tissue culture-based regeneration in this species required for classical transformation methods. Many groups have tried and failed to adapt floral dip methods to the crop Brassica species – those who have succeeded universally report extremely low success rates (usually <0.5%); our current results suggest a much better success rate. We will confirm these results and publish, hopefully to the benefit of the research and breeding community.