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Expressing hidden traits

What if the traits a grower wanted to add to his favourite wheat variety were already stored in its germplasm, just waiting to be properly switched on?

October 22, 2009  By Carolyn King

What if the traits a grower wanted to add to his favourite wheat variety were already stored in its germplasm, just waiting to be properly switched on? That is the novel hypothesis behind a new project to create high-quality spring wheat lines with resistance to virulent forms of three diseases: stem rust, fusarium head blight and wheat streak mosaic. And, although it is early on in the process, the approach being used in this project could become an option for improving many traits in many crops.

The taller subline is similar in height to the original McKenzie but is resistant to wheat streak mosaic and fusarium head blight. The other subline shown here is resistant to both diseases.
Photos courtesy of Steve Haber/AAFC.

This project’s approach is based on a different set of assumptions than most crop improvement efforts. Typically, conventional crop improvement involves identifying a specific source of a desirable trait and then using crossing, backcrossing and repeated selection to develop a high quality-variety with that trait. This process can be complex and time-consuming if the desired trait is found only in plants with many poor agronomic qualities, because the initial crossing produces plants with a jumble of wanted and unwanted traits. And if the desired trait happens to be resistance to a rapidly spreading, devastating disease, then crop losses could be heavy before high-quality, resistant varieties are finally in place.

Dr. Steve Haber, a cereal virologist with Agriculture and Agri-Food Canada’s (AAFC) Cereal Research Centre in Winnipeg, is leading the new project. He explains his innovative approach: “Our starting set of assumptions is that many, perhaps most, of our already well-established, well-adapted advanced lines and varieties may already have information in them, if properly expressed, for the desirable traits that we’re interested in. So, rather than bring in a known gene or set of genes from another source and then have to sort that all out in repeated backcrossing, we start with something that is already well adapted agronomically, and we alter the expression of the information that is already there to get expression of desirable traits.”


De novo discovery
This intriguing approach grew out of work by Haber and Dr. Dallas Seifers of Kansas State University. Since 1999, they have been working together to identify new sources of resistance to wheat streak mosaic to use in conventional wheat breeding. This work involved exposing generations of wheat plants to the wheat streak mosaic virus. In the course of this work, they observed that some individual plants had traits that differed from the parent plant’s traits. By 2006 they had accumulated increasingly extensive and rigorous evidence that one could start with susceptible lines or varieties and derive lines with stable, heritable resistance from their direct descendants.


Although the original McKenzie (photo A) is only intermediate in reaction to fusarium head blight, the researchers were able to evolve FHB-resistant sublines (photo B).
A. Heads of the original McKenzie line, 20 days after spray-inoculation with FHB.


To explore this surprising finding, Haber grew many generations of a contemporary wheat variety called McKenzie, originally starting from a single seed. He exposed each generation to a protocol using the wheat streak mosaic virus to stress the plants.
McKenzie wheat is a doubled haploid (DH) variety, which means that, from a conventional genetic perspective, every generation should be genetically identical to the previous one. The researchers did not introduce genes from any other line or variety. For the early and intermediate generations, they grew fairly small numbers of plants under very tightly controlled conditions indoors, in part to ensure that no stray pollen was coming in that might introduce new traits.
And yet the generations of McKenzie included some individual plants with de novo traits, traits not seen in the original line. For instance, the original McKenzie is rather tall, susceptible to wheat streak mosaic, and only intermediate in reaction to fusarium head blight. But some individuals in the subsequent generations were much more susceptible to wheat streak mosaic while some were more resistant, and some were taller while others were shorter than the original line.
The stress seemed to cause the plants to produce progeny with differing traits, almost as if the stressed plants were unsure of what was the best response to the stress, so they produced offspring with various assortments of old and
de novo traits, hoping that at least some of their offspring would have the right combination of traits to allow them to survive and thrive.

From each generation, the researchers picked out the individual plants that best expressed one or more desired characteristics and used that seed for the next generation. In follow-up experiments, the researchers established that some of the variations were heritable expressions of traits. Once established, these traits behave just like conventional genetic traits, so breeders can use these sublines to cross, backcross and select traits in the conventional manner.
So Haber and his colleagues were able to derive McKenzie sublines that, in generation after generation, expressed traits not seen in the original McKenzie.
Next, Haber and his colleague Dr. Jeannie Gilbert, a plant pathologist at AAFC’s Cereal Research Centre, decided to try using the de novo approach to add resistance to fusarium head blight (FHB) to the McKenzie sublines.
FHB is a complex disease and it has taken plant breeders many years using conventional breeding techniques to develop FHB-resistant wheat varieties for the Prairies, such as AC Waskada and 5602HR. Gilbert outlines the main challenges for conventional breeding of FHB resistance: “The principle difficulty is that there is no one single gene that can be transferred into a wheat variety that will confer resistance. The trait is multigenic and therefore to get real resistance you need three or possibly more genes for resistance. The second difficulty is that the varieties that are resistant are Chinese in origin and have very different quality from the wheat that Canada is famous for. So when we use something like Sumai-3, which is probably the most widely used wheat variety to try to transfer fusarium head blight resistance, we often end up with wheat lines that exhibit yield loss, shattering and other poor qualities.”

Haber and Gilbert thought the de novo approach might be a way around these hurdles. That is, perhaps it might be easier to help the plant muster its own defences, than for breeders to add the resistant genes to the plant.
So in 2007, they took about 30 of the McKenzie sublines to the FHB nursery at Glenlea, Manitoba. By using the de novo protocol to stress the plants, they were able develop sublines with much better or much worse resistance to FHB, as well as having resistance to wheat streak mosaic and being either much taller or shorter than the original McKenzie.
In 2008, Haber and his colleagues took the most interesting sublines and confirmed their results in indoor studies and field experiments.
They have conducted further experiments to better understand and confirm what is happening in the  de novo approach. They have found that exposure to virus infection is not the only type of stress that causes the de novo response; stresses like excessive heat or cold can evoke similar responses. They also have established that the approach works for other types of wheat, like winter and durum varieties. For instance, using a winter wheat variety that is very susceptible to most types of leaf rust and susceptible to wheat streak mosaic, Seifers and Haber evolved lines with heritable resistance to both diseases.

Developing resistance to three diseases
Haber’s new project is building on this previous work in order to create wheat lines with resistance to wheat streak mosaic virus, fusarium head blight and recently emerging races of stem rust.
Wheat varieties with resistance to all three diseases would be very useful to Prairie wheat growers. FHB is the most significant cereal disease in Canada. It causes yield loss and grade loss, and produces toxins that limit the use of infected grain for feed or food. FHB is very difficult to control once it becomes established in an area.
Wheat streak mosaic is caused by a virus transmitted by the wheat curl mite. Damage from the disease can range from reduced yields to crop failure. No commercial wheat varieties in Canada have resistance to the virus, and no pesticides are available to control the mites or the virus. The mite needs a “green bridge”: that is, it needs to be on living wheat or some closely related living grass to survive, so the disease is a special concern in areas where both winter and spring wheat are grown. These days, more Prairie producers are choosing to add winter wheat to their rotations as breeders provide better winter wheat varieties. So the risk of wheat streak mosaic is increasing.
Stem rust is of great interest these days because of the Ug99 race of the disease. This very virulent type of stem rust emerged in Uganda in 1999 and is now affecting wheat crops .in East Africa and the Middle East. If Ug99 arrives in North America, it could cause serious yield losses here. Most wheat varieties now grown in Canada are susceptible
to it.
In this new project, Haber and his research team will use the de novo approach to further-improve FHB resistance in the McKenzie sublines that already have shorter stature, resistance to wheat streak mosaic and some resistance to FHB. The researchers will also apply the approach more widely to improve FHB resistance in other high-quality wheat lines and varieties.  In addition, they will use the improved McKenzie sublines to evolve lines that also have stem rust resistance.
Haber says, “We have demonstrated that our approach is bringing about stable, heritable resistance for two known disease problems: wheat streak mosaic and fusarium head blight. We have already demonstrated as an academic proposition that we can bring about resistance for leaf rust, so there is no reason why it should not be tried for
stem rust.”
This project is funded by Western Grains Research Foundation. Some of Haber’s related research into the de novo approach has received funding from Manitoba’s Agri-Food Research and Development Initiative and from the Brockhouse Prize through the Natural Sciences and Engineering Research Council.

Powerful potential for crop improvement?

Further follow-up experiments are needed to determine if Haber’s de novo approach works well in most situations. However, the results so far suggest that this approach might offer a crop improvement method that could sometimes be an easier and quicker option than conventional breeding methods.

For instance, the de novo approach may be better at retaining the high-quality characteristics of the original germplasm in situations where a conventional breeding approach would require crossing a well-adapted variety with a poorly adapted variety. The researchers have carried out some initial small scale-testing of the best McKenzie sublines to explore this possibility, and the results suggest that quality is maintained or improved slightly using the de novo approach. They now have enough seed from these sublines for Dr. Stephen Fox, a wheat breeder with AAFC, to more rigorously evaluate the sublines’ agronomic quality at a larger scale.

The de novo approach might sometimes be faster, as well. Haber says, “In those cases where the system that we apply brings about a range of variations that, in the first few generations, start leading in the direction of the expression of desirable traits, then the process can be quite rapid.” In that case, a breeder could take a high-quality variety and directly address some of its weaknesses, and have improved lines in perhaps five years, rather than the 10 or 15 years it takes using a conventional breeding approach.
But Haber cautions, “At this point we have no way of knowing in advance if a particular line is going to be quickly amenable to the approach or is going to take many more cycles to start yielding variants with stable, heritable altered expression of the desired traits.”

The de novo approach might be well suited to creating resistance to complex diseases, as the experiments to develop FHB-resistant McKenzie lines suggest. Gilbert notes, “We’re hoping to be able to continue doing some work on leaf spot diseases because that’s another group of diseases for which there’s no quick fix when using a conventional breeding approach. It’s a complex of four or five diseases and they are all under different genetic control. So if we could use this method, where the plant itself is sort of pushed to find a way to resist the pathogens, that would be a much more efficient way of creating disease-resistant plants.”

Overall, the de novo approach seems to offer a tantalizing prospect. Haber says, “I’ve had some very good discussions with breeders about this. The way I put it to them was: If we can establish that this approach works reasonably well, most of the time and for most materials, then as a breeder you could say, I want this number of days to maturity, resistance to this disease and so forth. That is, you could rough in what you want the crop to be like.”
He adds, “What is also very appealing about this approach is that it doesn’t require you to do it instead of other approaches. At any time, you can take the lines generated by our approach and use them as a conventional genetic resource.”
Gilbert notes that some of the biggest benefits from the de novo approach could be for developing countries. “This approach does not require the huge numbers of plants that breeders generally have to handle; it can be done with much smaller populations. It doesn’t require a lot of technical knowledge. And it doesn’t require a lot of expensive equipment.”

Figuring out the underlying mechanism

The de novo approach seems to be a surprisingly simple process to quickly yield heritable changes. “As I tell my assistants, the most high-tech piece of apparatus that we use routinely is a digital camera,” says Haber.
But how does it work?
That stressed plants can so easily produce progeny with de novo traits seems to suggest genetic inheritance might be less rigid than what was taught decades ago in high school biology. Gilbert says, “We used to think you take your resistant line and cross it with a susceptible one and end up with expected ratios of resistant and susceptible individuals. But it seems as though plants have actually developed a means by which they can make sure that they survive to the next generation when put under stress. The stress might be drought or insect attack or something else that will put survival at risk. In a way it seems as if the variation is the plant’s attempt to make sure that some of its seeds will survive and produce the next generation.”

Haber says, “I’m fairly confident that the variation in traits is not a mutation in the manner of a rare, accidental mutation because we don’t need very large numbers or very many cycles to get this process going. If we start from scratch, usually we don’t need populations larger than 50 to 100 plants to get it going. I think it’s more in the nature of awakening or making visible natural mechanisms that are at work all the time in higher plant populations.”
There is some evidence for Haber’s hypothesis that the de novo traits arise from altering the expression of genetic information already in the plant. He says, “Saber Golkari, in the doctoral work that he carried out under supervision of Jeannie Gilbert, took Sumai-3, a Chinese wheat variety with very good resistance to fusarium head blight, and using quite sophisticated techniques, compared it with what are called near-isogenic lines, meaning that genetically, they are almost identical except they are susceptible to fusarium head blight. The outcome was, to put it very simply, that there was no difference; the susceptible closely related lines not only had the genes for resistance, but the expression of those genes was also triggered, just not in the right way, perhaps not the correct co-ordination or correct time.”

Haber and his colleagues are now searching for the underlying mechanism for the de novo approach through several studies. Dr. Brent McCallum, a leaf rust specialist with AAFC, is leading one of these studies. Using the leaf rust-resistant lines from Haber and Seifers, McCallum is looking into questions like whether the process reactivates old genes or perhaps causes completely new traits to appear.
Haber and Dr. Ken Standing of the Department of Physics and Astronomy at the University of Manitoba are exploring the possible mechanism from another angle. They are using a tool developed by Standing called time-of-flight mass spectrometry. It is able to make very rapid, detailed and sensitive examinations of the differences in the proteins between the original McKenzie and the new sublines.
Interestingly, in Haber and Standing’s very preliminary results, the proteins that have immediately shown up as different between the original McKenzie and the sublines are histones. Histones are known to be involved in regulating the expression of genes. Haber says, “It’s extremely early days, of course, but that looks intriguing.”


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