New sequencing technology called nanopore helps scientists reveal previously hidden features buried in plant genomes
Decoding the full genome for black mustard has helped advance breeding of oilseed mustard crops and improved breeding of wheat, canola and lentils.
“We were interested in black mustard because basically it’s half the genome of condiment mustard and carinata,” said Isobel Parkin, research scientist with Agriculture Canada and member of the Plant Phenotyping and Imaging Research Centre at the University of Saskatchewan.
“It was basically the only (brassica) genome that we didn’t have sequence for at the time and it was of interest because it has a lot of variation for traits.”
Commonly used in seed form as a cooking spice, black mustard (Brassica nigra) is grown on the Indian sub-continent and is closely related to mustard and canola grown in Canada.
It’s characteristics include abiotic stress tolerance and resistance to clubroot and blackleg.
Besides tracking down different sources of resistance, Parkin said the researchers were testing new genome sequencing technology called nanopore that enables direct, real-time analysis of long DNA and RNA fragments.
Changes to an electrical current are monitored as nucleic acids are passed through a protein nanopore, which is then decoded to provide the specific DNA or RNA sequence.
The nanopore technology conveys a more detailed image of the plant’s genes, allowing clearer views of which genes are responsible for which traits.
Consequently, the information advances crop breeding that up until recently was previously not available. Breeding is now more efficient because researchers can select genes more easily for specific desired traits.
“Scientists at PIRC (Plant Phenotyping and Imaging Research Centre) have been working on clubroot resistance for a long time. They’ve been utilizing black mustard as a source of resistance, capturing that resistance and trying to move it into canola is obviously a target,” she said.
Sequencing is well established, but technological changes and developments have been phenomenal during the past few years.
“You can actually generate gigabases of DNA in a couple of days. For example, the canola genome is approximately one gigabase in size and you can generate about 20 canola genomes in a couple of days,” she said.
“That’s a lot of data and that’s pretty fast. It’s lot faster than it used to be by I’d say a few hundred-fold. That’s one of the things that people in my line of work are generally struggling with now is the fact that you have so much data to store. So computer storage and computer processing power is becoming the most limiting thing in a lot of our analysis.”
Generally, what used to happen was sequencing very short fragments of DNA, which is a hundred letters or a hundred bases of DNA at a time.
However, that created massive sequence data, which became difficult to assemble in a coherent fashion.
“It became computationally quite challenging, especially in plants because plants have lots of duplicated things within the genome. There’s lots of repeats, so things are present multiple times. And so it’s quite complicated to put things together when you have very short sort of fragments of them,” she said.
The new technology now allows for tens of thousands of bases or letters, which makes it easier to extract information out of a genome.
“This means you’re sequencing entire genes rather than sequencing it in little chunks,” she said. With the development of smaller genome assembly for black mustard, nanopore sequencing technology has researchers quickly sequencing lines of canola and carinota, which is twice the size of black mustard.
She said digital plant breeding will continue accelerating as new tools are developed and integrated into software programs.
This will in turn create new crop varieties, more resilient to stresses of disease, insects and drought.
“It’s trying to link genotype to phenotype. So to speed things up for breeders, if you can identify which parts of the genome predict a particular trait then you can use molecular mechanisms to eliminate a couple of years or probably more of your breeding cycle. So you can actually generate germplasm and improve varieties much faster,” she said.