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Xianzhou Nie and his colleagues are developing tools that take a few minutes instead of a few months to screen breeding lines for PVY resistance. (Photo: AAFC)

Fast-Tracking Extreme Resistance

Researchers are developing new markers for much more efficient selection of PVY-immune potato lines.

A key tool in the arsenal for controlling potato virus Y (PVY) is cultivar resistance. Now, researchers are developing better molecular markers for faster, more accurate screening of potato breeding lines for “extreme resistance” to PVY – equivalent to PVY immunity. That will enable breeders to more efficiently develop new varieties with total protection against this serious disease.

“PVY is one of the most widespread viruses and also one of the most economically important viruses infecting potatoes, not just in Canada but also worldwide,” says Xianzhou Nie, a research scientist at the Fredericton Research and Development Centre of Agriculture and Agri-Food Canada (AAFC). He is leading the AAFC-funded research to develop these new markers.

PVY is spread by infected seed potatoes and by aphids. Depending on the potato cultivar and the PVY strain, the virus can cause such symptoms as leaf mottling, stunted plants and tuber necrosis, and can result in up to 90 per cent yield reductions.

Fortunately, the Canadian potato industry is making progress in the fight against PVY. Nie notes, “In Canada, about four or five years ago, a very high percentage of seed potatoes were infected with PVY and it was really a major problem. But in recent years, because of the excellent work by researchers, extension personnel and growers, the occurrence of the virus has declined, which is good news.”

These efforts include using PVY-free seed potatoes and frequently spraying potato foliage with mineral oil, supplemented with insecticides, to reduce aphid-transmitted PVY. Many aphid species can spread PVY, so invasions of PVY-carrying aphids can peak several times during the growing season; as a result, spraying throughout the growing season is important for effective PVY control.

AAFC’s potato breeding program has already released a handful of potato lines with extreme resistance to PVY. Having more such cultivars will be important for growers.

“First of all, cultivars with extreme resistance could save a lot of money for potato growers. Every year, millions of dollars are spent applying mineral oil or a combination of mineral oil and insecticide on the seed crop,” explains Nie. “Secondly, resistance is more environmentally friendly. And thirdly, it is very effective protection – when you have a cultivar with extreme resistance to PVY, you don’t need to do anything to protect it from PVY infection.”

Cultivars with resistance to PVY have either extreme resistance or hypersensitive resistance. With extreme resistance, the plant cannot be infected by any strain of PVY. That’s really important because the virus has many strains and sub-strains.

“About 20 or 30 years ago, the most common strain was PVYO, ‘O’ meaning ordinary strain. But since the last 20 years or so, PVY strain dynamics have been changing,” says Nie. “Some recombinant strains, including PVYN:O, which is a recombinant strain from PVYN and PVYO, and PVYNTN have become the dominant strains in many parts of the world. And in Canada we’re seeing that trend as well, with more PVYN:O and PVYNTN in most of our potato-growing areas.”

Hypersensitive resistance helps the potato plant survive the virus, but it doesn’t provide complete protection. Nie explains that when the virus contacts a variety with a hypersensitive resistance gene, the plant will have an immediate reaction, triggering the formation of necrotic tissues. The virus will be trapped in those dying tissues and then will disappear with the tissue death.

“Hypersensitivity in most cases does not offer really strong protection. First, most of the genes for hypersensitive resistance provide resistance only to specific strains of the virus. Second, when the virus load is high, hypersensitive resistance doesn’t totally prevent infection.”

So Nie’s work is focusing on markers to select for extreme resistance. In this research, he is collaborating with his AAFC colleagues Agnes Murphy and David De Koeyer.

The new markers represent two big steps up in terms of selection efficiency, compared to traditional screening methods. “The traditional approach in the potato breeding process is to do a cross between two parents and then get thousands and thousands of true seeds. Then the seeds are grown in the greenhouse and the field,” he says.

“Next we inoculate each plant with PVY to see if the plant can be infected. So, first we do mechanical inoculation, and then we do graft inoculation [of any plants that are not infected after mechanical inoculation]. Any plants that are not infected even after graft inoculation are assumed to have extreme resistance.

“In our experience, it takes about three months to use this approach to fully assess about 200 plants in the greenhouse for extreme resistance to PVY.”

Using marker-assisted selection is a major advance beyond this traditional approach. With molecular markers, you can screen the seed in the lab for a specific fragment of the DNA associated with a resistance gene, instead of having to take months to grow seeds into plants and test them.

Nie explains that regular marker-assisted selection processes involve first “amplifying” the relevant DNA fragment, creating thousands or millions of copies of it. Then the DNA is extracted using a process called gel electrophoresis, and the resulting pattern is analyzed. This process takes about one to three weeks to assess about 200 plants.

So gel electrophoresis is much faster than traditional screening processes, but it has still some limitations. Nie says, “Even at the best capacity, we could only run about 80 samples per gel if we want good results. The gel itself takes about three hours. And sometimes the marker is difficult to use because it does not separate [the target fragments from non-target fragments] very well. So just reading the gel can be a real challenge.”

So Nie and his colleagues are taking PVY-resistance markers to the next level. They are developing markers using a process called high-resolution melting DNA analysis (HRM).

HRM uses the same amplification process as regular markers, but then the amplified samples are simply placed in a HRM machine. The machine heats up the samples and records the melting temperature for each sample; the melting temperature is affected by very small – but very important – differences in the DNA.

Nie explains that HRM analysis is better than gel electrophoresis in a couple of ways. “After the amplification, it only takes about two or three minutes to run a plate, which can hold 96 samples. And with the machine there is no human error, unlike when you read a gel.” So it takes only a few minutes to accurately and reliably analyze about 200 samples, compared to weeks with gel electrophoresis.

The researchers started working on HRM markers for PVY resistance about two years ago, and they have already developed and tested a HRM marker for one resistance gene.

Genes that confer extreme resistance to PVY are called Ry genes – ‘R’ for resistance and ‘y’ for PVY. Three different Ry genes are used in potato breeding programs. Rysto is from a wild potato species called Solanum stoloniferum. Ryadg is from the potato species Solanum tuberosum subspecies andigena. And Rychc is from Solanum chacoense, another wild species.

The new HRM marker detects Rysto. To develop this marker, the researchers converted a gel electrophoresis-based marker to HRM. “German researchers found that [gel-based] marker, which is very nicely linked to the R gene; however, it is very difficult to use. So we analyzed the DNA sequence area that harbours the marker, and we found the specific position in the DNA that is linked to the R gene. Based on that information, we designed the primers to amplify that specific region in both the susceptible and resistant varieties. Then we put the samples on the HRM machine,” says Nie.

If a sample has the Rysto gene, then the melting temperature will be lower because the DNA fragment in the resistant lines is about 20 nucleotides shorter than in the susceptible ones. (Nucleotides are the building blocks of DNA.)

Nie and his colleagues recently published this HRM Rysto marker in an international journal. “The HRM technology has been used for other crops and including potatoes, but we are the first to report using it for PVY markers to my knowledge,” Nie notes.

AAFC potato breeders are already intending to use the new Rysto marker, and Nie hopes other breeders around the world will use it.

“PVY is always a challenge to potato production worldwide. In developed countries, such as in Europe and North America, we have very stringent phytosanitary measures and inspection systems to safeguard our potato production systems. But some other countries do not have such systems or their systems aren’t good enough. Extreme resistance is an important opportunity, not just for Canadian potatoes but for others in the global potato industry.”

In the coming months, Nie and his colleagues expect to officially release a new HRM marker for Ryadg. And they are planning to start work on an HRM Rychc marker. “If we use all three Ry genes in AAFC’s potato breeding program, that will really increase the future usage of new varieties,” he notes.

For the future, Nie hopes potato varieties with extreme resistance to PVY will become more acceptable and widely planted, as breeders continue to add more and more of the other key agronomic and quality traits into new PVY-immune varieties. In the meantime, he suggests growers continue to follow the practices recommended by extension personnel such as using virus-free seed potatoes and regularly spraying mineral oil.

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