Dr Evans Lagudah says the new gene isolation technique overcomes obstacles that were hindering gene-discovery technology.
PHOTO: Brad Collis
Australian researchers have opened yet another new chapter in the unending global pursuit of rust-resistant wheat, with the development of a new technique that makes it easier and faster to isolate resistance genes, including from all-important wild relatives.
The researchers believe they now have the tools to realise a long-held dream – to build much more robust forms of rust resistance for commercial varieties by carefully selecting the most appropriate combination of individual genes from a bank of previously isolated genes.
Working with collaborators in the UK and US, they have developed a technique called MutRenSeq.
This technique’s capabilities were put on show when the collaborators announced that MutRenSeq was used to isolate two particularly important stem rust resistance genes – Sr22 and Sr45.
Sr22 confers resistance to commercially important races of the stem rust pathogen, including the Ug99 race group. Sr45, originally discovered in goatgrass (Aegilops tauschii), provides resistance against stem rust pathogen races from parts of Africa, India and Australia.
The project is part of a large international effort involving collaborators at the John Innes Centre and Sainsbury Laboratory in England, the USDA-ARS Cereal Disease Laboratory in the US, the University of Sydney with financial support from the Two Blades Foundation, the BGRI Durable Rust Resistance in Wheat project, the UK’s Biotechnology and Biological Sciences Research Council and the GRDC.
The project was jointly led by Dr Brande Wulff at the Norwich Research Park in the UK, where the initial resistance gene enrichment technology was developed, and Dr Evans Lagudah at CSIRO in Canberra.
Dr Lagudah says that a technical advance was needed because resistance genes tend to be organised in the genome in ways that thwart existing gene-discovery technology.
“Resistance genes are found in close proximity in plant genomes, structured into gene families that can vary – in terms of gene number and gene sequence – within a population,” Dr Lagudah says.
“That means their exact position in the genome is somewhat fuzzy, which is a problem for current methods that need to pinpoint a gene’s exact position before it can be isolated.”
The new method, however, exploits the observation that disease-resistance genes Gene team tightens science’s grip on rust resistance frequently encode proteins that contain a similar structural motif, called a ‘nucleotide-binding leucine-rich repeat’ (or NLR). As such, their genes contain sequence similarities.
“A typical plant genome contains hundreds of NLR-encoding genes,” Dr Lagudah says. “Many reside in linked clusters. With the new technique we invented a way to first capture the NLR-rich fragments of a genome. That results in a pool of DNA that is enriched for resistance genes. We then sequenced that DNA.”
For the proof-of-concept study, the sequence was used to design 60,000 ‘NLR-bait’ markers (DNA sequences that recognise NLR-containing genes). These markers were designed to detect cereal NLR genes present in barley, bread wheat, durum, red wild einkorn, domesticated einkorn, and three goatgrass species (Aegilops tauschii, Ae. sharonensis and Ae. speltoides).
The markers made it possible to isolate NLR-enriched genomic DNA fragments from two comparable sources.
The first was bread wheat known to contain a well-characterised stem-rust-resistance gene, Sr33, as an internal control for the procedure. The second source was the same wheat line, but mutated with ethyl methane sulphonate.
These normal and mutated pools of NLR-enriched DNA sequences could then be compared, with the loss of resistance in the mutated pool – and the presence of mutated DNA – giving away the presence of a resistance gene.
First stem rust-resistant gene
The approach successfully identified the control gene, Sr33 – the first stem-rust-resistance gene isolated in the world. It was cloned and sequenced in Dr Lagudah’s laboratory by Dr Sambasivam ‘Sam’ Periyannan, who also played a key role in this study.
The technique was then used to isolate Sr22. Dr Periyannan explains that Sr22 confers resistance to commercially important races of the stem rust pathogen, including the Ug99 race group (which threatens wheat production in Africa), and is one of the few resistance genes effective against Yemeni and Ethiopian Ug99 isolates. As such, it is an important gene for global food security.
Historically, Sr22 was first detected in wild relatives of wheat (Triticum boeoticum and T. monococcum) in the 1970s, but its use in breeding programs was hampered due to carry-over into commercial varieties of genes that in addition to rust resistance also caused a yield penalty and a delay in heading.
To avoid the carry-over of deleterious traits, researchers tried to isolate Sr22 for a number of years but failed. In contrast, the new technique worked and has allowed the entire gene to be sequenced.
Goat grass link
A similar approach was used to isolate Sr45, a resistance gene originally discovered in goatgrass (Ae. tauschii), which provides resistance against stem rust pathogen races from parts of Africa, India and Australia (but not Canada).
“Sr22 belongs to a small gene family, with three members, while Sr45 was found to belong to a larger multi-gene family with eight to 12 genes,” Dr Periyannan says. “Therefore, in the case of Sr45 this method successfully identified a gene belonging to a complex multi-gene family.”
Sr22 and Sr45 now join Sr33 and Sr35 as the four major dominant stem-rust-resistance genes cloned to date from wheat. “All four genes are effective against the Ug99 race group, while Sr22 and Sr33 confer broad-spectrum resistance to multiple races at all plant developmental stages,” Dr Periyannan says.
In all, about 60 stem rust genes have been described to date in wheat and the MutRenSeq technique now offers the opportunity to isolate the remaining genes quickly.
“Having many resistance genes at our disposal means we can introduce clusters of genes as a single transgene,” Dr Lagudah says. “Multiple genes make it harder for pathogens to break down resistance. Having all the resistance genes at one location then means all the genes are inherited together, making it easy to track their progression through a breeding cycle.”
Dr Evans Lagudah,
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