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Capturing ancestral diversity

Sifting through the genetic variation in canola’s progenitors for traits to help the crop withstand challenging conditions.

February 14, 2024  By Carolyn King


Young Brassica napus roots grown in soil rhizobox. PhotoS courtesy of Deven Guenter and Ian Fiedelleck, AAFC.

A Saskatoon research team aims to increase the genetic diversity available in canola germplasm so breeders can develop Prairie canola crops that are more robust and resilient. The team’s strategy is to tap into a profusion of traits – from the roots up – in canola’s two progenitor species.

 “Canola, or Brassica napus, is what is called an allotetraploid. It was formed by the fusion of two smaller diploid plants, basically a turnip [Brassica rapa] and a cabbage [Brassica oleracea],” notes Isobel Parkin, a research scientist with Agriculture and Agri-Food Canada (AAFC) in Saskatoon. Diploid means the plant has two complete sets of chromosomes. Brassica rapa has 10 pairs of chromosomes, and Brassica oleracea has nine pairs. And Brassica napus has 10 rapa pairs plus nine oleracea pairs. 

Parkin explains, in nature, this hybridization of Brassica rapa and Brassica oleracea is extremely unusual. Almost all of the time, each species would only cross with others of its own species. The resulting allotetraploid was not only rare but also lucky for farmers. “It was then selected for because you got a bigger, beefier plant that produced more seed,” she says. 

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“However, since this hybridization event didn’t happen very often, you have immediately a genetic bottleneck where you have created a new species from a very small number of lines. In that way, you’ve reduced the amount of diversity or variation that is present.” As well, further genetic narrowing of the Brassica napus germplasm pool occurred through selection for particular characteristics desired in the cultivated crop.

 “Canola’s diploid parents have carried on evolving in their own separate ways and in multitudes of environments. And there are a lot more representatives of the diploids than there are of the napus lines.” So, Brassica rapa and Brassica oleracea are great sources of new variations for canola breeding.

Resynthesizing napus
One key technique that Parkin’s research team and other scientists use to access the genetic variation in canola’s two parental species is known as resynthesis. In essence, they recreate that hybridization event in the lab, crossing a Brassica rapa plant and a Brassica oleracea plant to get a Brassica napus plant.

 “We cross them and then we do what is called embryo rescue. As the embryos begin to form in the pods, we take them out and tissue culture them, and baby them along to get plants,” she explains.

 “The other issue is that the fertility of the resynthesized lines can sometimes be a little less than what you would hope for. But usually through several generations you can increase the fertility, or you can cross them back to natural napus, which is what we’ll be doing to stabilize the fertility.”

 Parkin is currently leading a Brassica napus resynthesis project with a major focus on root traits for sustainable canola production. In addition, the project is on the lookout for other traits in the diploids like resistance to key disease and insect threats.

Crazy and useful diversity
“We have been doing canola resynthesis for many years in my lab, but we haven’t done it quite so extensively with so many lines as in this project,” Parkin notes. 

This project includes a very large collection of about 600 Brassica rapa lines from Plant Gene Resources of Canada’s national collection of seed germplasm in Saskatoon. 

“We have fewer Brassica oleracea types. The reason for that is that there are a lot more oilseed rapa types than oilseed oleracea types – both produce oil, but rapa types produce more oil than most oleracea types,” she explains. Brassica oleracea has been bred mainly for use as vegetables, including crops like broccoli, Brussels sprouts, cauliflower and cabbage.

Fortunately, Parkin’s team has access to a remarkable collection of Brassica oleracea material from the University of Warwick in the United Kingdom.

 “The University of Warwick has made a very interesting collection not only of Brassica oleracea material, but they have also crossed their oleracea lines with many wild relatives of oleracea. Those wild relatives are carrying even more crazy variations than the vegetable types that have been bred for a long time,” she says.

 “As a result, we have this collection of about 150 lines that have mostly oleracea but then they have bits of genome from these really wild species. So the lines carry all this massive genetic variation.

 “We’ve been growing the lines over the last few months, and they have variations of everything. For instance, they have variations in leaf morphology, with some lines having velvety leaves with tiny hairs on them. And they have various colours – they contain lots of carotenoids [yellows, oranges, reds, blues] and anthocyanins [purples, blues, reds, blacks], so they’ve got purple pods and so on. They are really quite funky.” 

Parkin notes, “Some of these variations may sound a bit crazy, but some of these wild species have been shown to have resistance to some of the diseases and insects that affect brassica crops. So these lines could be a source of other interesting variation, as well as variation in the root morphology that we’re looking at in this project.”

Revealing root traits, and more
“We are focusing on root morphology because of an emphasis on adaptation to problematic environments,” Parkin explains. She and her team want to identify root characteristics that will help with things like improving water-use efficiency to reduce drought impacts and increasing nutrient uptake so growers could reduce fertilizer inputs. Root architecture is also important for traits like resistance to lodging. And root characteristics may play a role in the plant’s ability to fight certain root diseases or insect pests, and in storing carbon in the soil.

Clubroot galls growing on the roots of a clubroot-susceptible Brassica napus line, in a soil rhizobox.

As you can imagine, roots are tricky to phenotype, especially if you are evaluating hundreds of lines. One option is to dig up the plants, wash off the roots, and then measure various root characteristics. As Parkin points out, that approach is really labor-intensive, and it damages the roots.

 Her project is using an image-based method for high-throughput screening of the root characteristics of the different lines grown in what are called soil rhizoboxes. “We’re growing the plants between two glass plates in a thin layer of soil. Most roots as they grow, even in the soil, will cling onto things. So, they cling onto the glass, and you can see the roots,” she explains.

 “The different lines will have very different root morphologies. For instance, they may have really big tap roots and very limited lateral roots or vice versa.” They will use various types of computer software to extract particular root details from the images. Then, at the end of the experiment, they’ll remove the glass, wash the roots and measure other characteristics like root mass. 

Parkin’s team will also be collecting data on how variations in root characteristics relate to the plant’s performance regarding traits like water-use efficiency. 

They hope to identify Brassica oleracea and Brassica rapa lines with optimal root architecture. Then they’ll use the more promising oleracea and rapa lines to create resynthesized Brassica napus lines.

 Next, they’ll screen those resynthesized lines for the desired root characteristics. And then they’ll cross the most promising resynthesized lines with elite canola lines to create pre-breeding materials.

 The diploid, resynthesized and pre-breeding lines will be evaluated under multiple growing conditions in the greenhouse, and the pre-breeding lines will also be tested in the field. 

In addition, the team will be genotyping the different lines. Then, by combining the genotype and phenotype data, they will identify the regions of the genome associated with the desired root traits and develop molecular markers for those traits.

 Along with all this root work, Parkin and her team will be capturing some of the other interesting variations in the oleracea and rapa lines. “We’ll screen the diploids to see if we can find novel resistance for diseases like clubroot, blackleg and sclerotinia. And we’ll also probably screen them for resistance to flea beetles and various other common problems of Prairie canola crops.”

 As part of this, Parkin’s team will be using the root imaging method to track clubroot gall formation on the roots between the glass plates. The team hopes to create a method to rapidly assess the lines for their response to clubroot.

Toward yield stability
This three-year project just began in 2023 so it is still pretty new. “We’ve started to explore the diversity within the diploids, growing all these interesting oleracea and rapa lines. And we have been doing testing of the root phenotyping methodology to get that going, and that is working quite well,” says Parkin. They have also been developing their clubroot screening method and are now doing a little fine-tuning so it will work perfectly every time.

 The phenotype data, genotype data, markers and pre-breeding lines resulting from this project will be very important resources for canola breeders and researchers, helping in the development of improved varieties for more sustainable canola production. 

“Overall, we’re hoping to identify genetic variation that will be useful in the Prairies,” says Parkin. “We’re focusing on some clear, useful traits, like clubroot resistance, and on identifying lines that are going to be more robust in tricky environments, like low nutrient or low moisture, but still yield well at the end of the season. That is the ultimate goal – to identify traits that will give canola growers much more yield stability.” 

Parkin’s collaborators on this project include Hossein Borhan, a molecular pathologist at AAFC in Saskatoon, and Mark Eramian, a professor in Computer Science at the University of Saskatchewan. This project is funded by the Canola Agronomic Research Program and the Western Grains Research Foundation. 

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