NEW YORK (GenomeWeb) – Researchers from several European institutions — the University of Zürich, the John Innes Centre, and the Institute of Experimental Botany in the Czech Republic — have developed a method to reduce the complexity of studying specific genes in large genomes.
Dubbed MutChromSeq, the method combines mutagenesis, chromosome flow sorting, and sequencing to break up these genomes by chromosome and make looking at them more manageable. In a paper published today in Genome Biology, the researchers described using the technique to identify specific gene mutations in the barley and wheat genomes.
Weighing in at 5.5 billion base pairs and 17 billion base pairs, respectively, the barley and wheat genomes are difficult genetic subjects. "It's too much of a challenge to resequence whole genomes," Brande Wulff, co-corresponding author on the paper and researcher at the John Innes Centre, told GenomeWeb. "It's cost prohibitive and it's impractical due to the very large amount of data that you generate."
"Nature has already divided [whole genomes] into 21 bite-sized chunks that reduces it to something that we can manage," Wulff added. The challenge was to find a way to get a hold of that "bite-sized chunk" of the genome and analyze the regions of interest, he said.
Traditionally, researchers have used genetic recombination-mapping followed by contiguous physical sequencing to try to break the genome up into these chunks and fish out the genes or regions of interests. However, large tracks of the barley and wheat genomes are almost devoid of recombination, making this approach ineffective.
Wulff and some of his colleagues resolved this problem by taking a combined mutational genomics and exome capture approach which allowed them to focus on very small fraction of the genome, work that they published in Nature Biotechnology in April.
Wulff specifically wanted to focus on the area that encodes intercellular plant immune receptors in wheat since he is studying disease resistance to wheat stain rust, a pathogen that is deadly to wheat and can infect up to 80 percent of commercial wheat varieties. Intercellular plant immune receptors reside in an area in the wheat genome where 80 or 90 percent of disease resistance genes have been defined. "We demonstrated with this technology that we could now clone these very important immune receptor genes in plants," he said.
However, this method had an important drawback: it's biased. Their exome capture method was based upon what they know about the genes they are targeting — resistance genes, in Wulff's case. "You can be pretty sure going into it that your gene is going to look like one [that's already been defined by a previous study]," Wulff said. "Sometimes people have found that their disease resistance turns out to be a kinase or it turns out to be an extracellular type of receptor. We wanted to come up with a method that would allow us to clone these other types of disease resistance genes."
They used the same basic idea that they described in the Nature Biotechnology paper, and refocused it to look at chromosome sections instead of only sorting for genes with similar structures.
The researchers began by sending one wild type sample and six mutants each of the barley Eceriferum-q gene and wheat Pm2 genes to a laboratory at the Institute of Experimental Biology run by Jaroslav Doležel, who specializes in chromosome flow sorting. His laboratory sent back purified chromosome suspensions for each sample which Wulff and his colleagues sequenced. The barley libraries were constructed following the Broad Institute's DISCOVAR protocol and sequenced on the Illumina HiSeq 2500, while the wheat libraries were constructed using Illumina's TruSeq protocol and sequenced on an Illumina HiSeq 2000 platform. They then mapped the reads and analyzed single nucleotide variants.
"We have used MutChromSeq to successfully reclone the barley Eceriferum-q gene and clone de novo the wheat Pm2 gene," the researchers stated in their Genome Biology paper.
Wulff and his colleagues believe that while this method may not be necessary for smaller genomes, such as the tomato genome which is only 1 billion base pairs, it's really useful to reduce the complexity of the pea, oat, or rye genomes, which are similarly large and difficult to sequence.
"It's possible that three or four years from now, the cost of sequencing has dropped even further and computing powers have increased even more that perhaps at that stage you wouldn't even bother [with MutChromSeq]. You would just resequence whole wheat genomes," Wulff said. But at the moment it's a technology that many of his colleagues are now using to try and clone specific genes in wheat, he said.
Currently, the technique is dependent upon expertise in chromosome flow sorting which exists in only a few labs in the world, including Doležel's lab. However, Wulff noted that Doležel published a paper recently in The Plant Journal that showed that instead of purifying large amounts of a single chromosome with chromosome flow sorting, it is also possible to isolate individual chromosomes in the genome and amplify them to identify individual genes.
"That obviates the need to do chromosome flow sorting. All you have to be able to do is just make a chromosome spread and pick single chromosomes and amplify them," Wulff said. "And that's technically a lot less demanding."
While this technique has the potential to be widely used in the plant genetics community, the existing services that labs like Doležel offers and the difficulty involved in creating facilities designed for the process creates no motivation to commercialize it. Wulff said that the researchers that specialize in chromosome flow sorting do it well and have been happy to work with the community to meet their needs so far.
In the end, the research that Wulff and other plant scientists are conducting could identify genes that help make the plant breeding process better and more efficient, and translates to farmers getting better crops.