Wrestling genes out of the giant barley and wheat genomes

How do you locate a gene associated with a specific trait when there are several billion base pairs to search through? This is the problem researchers working with barley and wheat genomes face. To tell us more about this and his recently published article in Genome Biology is wheat geneticist Brande Wulff.

Finding a gene associated with a discrete trait in the giant barley or wheat genomes can be a bit like paddling about in the Pacific Ocean looking for an atoll: at 5.5 billion base pairs the barley genome is huge, but even this is dwarfed by its wheat brethren, a behemoth of 17 billion base pairs.

Genetic recombination-mapping

The traditional solution has been to use genetic recombination-mapping to reduce the size of the genome into a bite-sized chunk delimited by molecular markers. This is followed by building a contiguous physical sequence across the interval by screening a whole genome shotgun library and fishing out those rare clones that help span the interval.

However, large tracks of the barley and wheat genomes are almost devoid of recombination. Therefore, two molecular markers that sit genetically neck-on-neck can be oceans apart on the physical DNA genome sequence – it can take years of grueling lab work to close the gap. And, more often than not, somehow every other gene in the interval can be made to fit the scientist’s expectations of the candidate gene!

Mutational genomics

Resequencing whole wheat genomes is at present impractical

“Mutational genomics” can overcome these obstacles and lead you directly to the gene. With mutagenesis you can obtain an allelic series of mutants in your gene of interest (providing the trait is relatively easy to screen for). By sequencing a handful of independently derived mutants and looking for a gene that is mutated in all individuals, a single candidate gene can be identified. Sayonara to recombination – we don’t depend on you any longer!

Yet, at the risk of drowning in data, resequencing whole barley genomes is at best cumbersome, while resequencing whole wheat genomes is at present impractical. The trick is to sequence just the fraction of the genome that matters. One approach makes use of exome capture. The capture can target the whole exome, or a particular gene family. The drawback is that an exome capture is biased by our own expectations and/or known, annotated genes, which are typically derived from a single reference genome. However, gene annotations often miss out genes, while a single reference genome may only represent 70% of the pan genome space of the species.

Chromosomes

Unlike a whole genome, a single chromosome is small enough to devour in one sitting

To overcome the bias imposed by exome capture, Sánchez-Martín and colleagues in this issue of Genome Biology turned to nature’s very own compartmentalization method: the barley genome is divided into seven chromosomes, while that of wheat is divided into 21 chromosomes. Thanks to recent improvements in chromosome flow sorting it is now possible to obtain highly pure preps of just about any barley or wheat chromosome independent of the cultivar. And, unlike a whole genome, a single chromosome is small enough to devour in one sitting. Finally, mapping a gene to a single chromosome is straightforward, and does not even require recombination – just Mendel’s good old rule of independent segregation!

Thus, in a method dubbed MutChromSeq, Sánchez-Martín and colleagues compared the sequences of mutant chromosomes to wildtype, and cloned two genes, one from barley and the other from wheat, in a fraction of the time and cost it would typically take by traditional map-based approximation.

A key advantage of MutChromSeq is that it is completely unbiased with regards to existing reference sequences or annotations. This makes it particularly attractive for accelerating gene discovery underpinning adaptive variation in non-reference cultivars.

Mutational genomics, in various guises, is poised to dramatically accelerate gene discovery in barley and wheat in the years to come. However, the poor agronomy of the wild relatives of these important crops imposes practical barriers to generating and screening large mutant populations. Just try threshing a few seeds from a wild goat grass – you quickly loose the will to live! It will require novel approaches to conquer this vast and wild frontier of genetics.

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