Manipulating recombination in crop polyploids

Term: 4 years ending April 2024
Status: Complete
Researcher(s): Isobel Parkin, AAFC
SaskCanola Investment: $266,896
Total Project Cost: $1,170,605
Funding Partners: SWDC, AAFC/NRC

Project Summary

There is a pressing need to improve productivity of crops, in order to maximize yield without further expanding arable land. The ability to make further crop improvements relies on the introduction of novel allelic variation, one such source being related species; however, interspecific barriers to recombination limit the transfer of new variation into crops. Most polyploid species including wheat and canola have evolved mechanisms to prevent chromosome pairing between their progenitor genomes, to ensure fertility in subsequent generations. This includes the wheat Ph1 (pairing homoeologous 1) locus that when deleted causes high levels of aberrant chromosome pairing between the progenitor genomes, and enhanced levels of crossovers in interspecific hybrids; and genes controlling this process have been proposed. However, in canola, the control mechanism was thought to be somewhat different, and the gene(s) controlling the process had yet to be identified.  Identifying and manipulating gene(s) involved in controlling the specificity of recombination in either of these important crop species would provide tools for crop breeders to allow the capture of novel allelic variation.

The project aimed to identify key genes involved in controlling recombination in wheat and canola and to manipulate the function of those genes in order to understand their role. Genetic analyses of a canola was used to identify genomic regions controlling homoeologous recombination. Gene expression was studied in specialized cells (meiocytes) of canola where recombination takes place. Using the cumulative information, a number of genes were targeted for gene editing in both wheat and canola in order to study their impact on recombination.

Genetic analyses of canola identified three quantitative trait loci (QTL) controlling homologous recombination, but importantly one locus provided almost 50% of the observed variation, suggesting it was the canola equivalent of the wheat Ph1 locus.  This study was the first to assess homoeologous recombination and map associated QTLs in allotetraploid B. napus. Gene expression data from the extracted meiocytes of two canola lines that differed at the Brassica Ph1 locus followed the expression of potential candidate genes throughout recombination, in addition to providing an unprecedented picture of gene expression during this process in canola. Three candidate genes within the Brassica Ph1 locus were targeted for gene editing using CRISPR/Cas9 in canola and plants with homozygous mutations were identified. In wheat, three genes that had been associated with homologous recombination in canola and showed gene expression in wheat meiocytes were identified and edited. The gene edited lines have been characterized at the molecular level and basic phenotypic differences catalogued. Recombination in the gene edited lines is still being assessed.

The Brassica Ph1 gene has applications for both increasing stability in canola, which ensures yield performance, and disrupting recombination in order to introduce new genetic variation. Resolution of the QTL region defines a key target for canola breeding and confirmation of the causative gene is ongoing. The gene edited lines of both crops provide novel platforms for studying recombination.  Notably, some of the gene edited wheat lines display phenotypic variation, such as reduced stature, that could be used directly in crop improvement. Exploring the deeper characteristics of these mutants promises a wealth of understanding regarding chromosome dynamics - how they pair up, segregate, and undergo crossover events. Investigating their impact on recombination will provide avenues for creating novel allelic configurations (in instances of increased recombination) or stabilizing favored gene combinations (reduced recombination).

The project identified important targets for manipulation in the crops, but unfortunately, the phenotyping of the gene edited lines could not be finalized by project end. Finding suitable protocols for routinely measuring recombination proved difficult and alternative methods such as cytology are labour intensive and not sufficiently high-throughput. In hindsight, protocols should have been established for high-throughput sequencing of gametes; yet, at the project start the available sequencing technologies were not sufficiently advanced to make this feasible.

The gene edited lines represent a valuable resource and should be fully phenotyped for levels and specificity of homologous recombination. In tandem, routine methods for measuring homologous recombination in the crops should be established. These methods would have utility for multiple additional purposes; for example, the impact of abiotic stress on recombination has long been implicated, which often leads to a reduction in fertility in stressed lines, thus identifying genotypes which are less affected would be of value for breeding more yield resilient crops.

Final Report PDF: Manipulating recombination in crop polyploids