DNA Markers … with a twist for improved bean breeding

Agricultural Research, May, 2003 by Jan Suszkiw

Americans eat nearly 8 pounds of dry beans each year in soups, salads, and other dishes and more than 7 pounds of frozen, canned, or fresh snap beans. But only the best bean cultivars make the cut when it comes to traits that will help growers profit and satisfy consumer tastes.

One cultivar trait farmers prize most is disease resistance, especially against bean common mosaic virus and bean common mosaic necrosis virus. Severe outbreaks of these aphid-borne viruses can cause yield losses of up to 60 percent and threaten the $512 million dry bean crop of California, Colorado, Idaho, Michigan, Nebraska, North Dakota, and Washington. Also at risk is the $190 million snap bean crop of Florida, Illinois, New York, Oregon, Wisconsin, and other states.

Forefront in the war on such viruses is marker-assisted selection (MAS). It's a biotech approach some breeders are using to rapidly identify resistant plants by confirming the presence of certain genes rather than by observing actual disease symptoms induced in a greenhouse. Now, an advance by ARS scientists in Prosser, Washington, could make MAS even more useful by enabling it to do what it often couldn't before: distinguish homozygous from heterozygous plants. Homozygous plants carry the desirable gene on each of the two chromosomes. Heterozygous plants carry the beneficial gene on one chromosome, while the other chromosome contains a slightly different version of the gene that does not confer the desirable trait.

The advance, called co-dominant interpretation, should save breeders substantial time, labor, and money spent ensuring that promising cultivars are homozygous, or "true breeding," meaning their offspring will consistently show the same traits over many generations.

In beans, the gene for resistance to the two viruses is dominant, say George Vandemark and Phillip Miklas, plant geneticists at ARS' Vegetable and Forage Crops Production Research Unit. When a gene is dominant, a single copy in a heterozygous plant will confer the desirable trait, making the plant visually indistinguishable from the true-breeding, homozygous plant. This presents an obvious problem in excluding heterozygous individuals from the pool of plants used for seed generation, say the scientists.

X Marks the Spot

In some quarters, classical plant breeding techniques have given way to MAS because it detects certain traits more easily. The markers themselves are specific sequences of DNA inside the plant's chromosomes. A marker serves as a road sign that says a desired gene is close by.

Key to finding markers is the polymerase chain reaction (PCR). It's the same technology that forensics experts use to identify DNA fingerprints in blood, hair, or other biological samples. But bean breeders use it to search for plants that possess a specific virus-resistance gene called bc-[1.sup.2]. This gene, which confers resistance to most bean mosaic virus strains, is often bred into pinto, navy, and other dry beans as well as snap beans.

A breeder prefers plants with two identical copies (homozygous) of the virus-resistance gene because the plants will be true breeding, meaning all their offspring will be virus resistant. But if the plant is heterozygous, it will have one copy of the bc-[1.sup.2] resistance gene and one copy of an alternate gene called bc-1, which confers susceptibility to most bean mosaic virus strains. Heterozygous plants won't breed true, and some of their offspring will be susceptible to virus attack.

Breeders want to exclude the bc-1 gene from their breeding programs, but this is difficult with existing PCR detection methods, Miklas says. Breeders now rely on a process called progeny testing, where the plants' offspring (progeny) are tested for resistance to the virus in a greenhouse. Such testing allows breeders to detect heterozygous plants and eliminate them from the breeding program. This process can take 6 months to 1 year and adds considerably to the cost of breeding programs.

Making a Good Biotech Tool Better

Use of DNA markers typically begins with PCR to identify plants having the bc-[1.sup.2] resistance gene. Until recently, the PCR technique was able to detect the bc-[1.sup.2] gene but not the number of copies present.

Vandemark and Miklas overcame this problem by using a newer PCR method that can accurately tell how much marker DNA is present by labeling it with a fluorescent compound and then measuring the fluorescence. "Since our quantitative PCR assay is specific for the bc-[1.sup.2] gene, we can expect that homozygous plants will fluoresce twice as much as heterozygous plants," says Vandemark.

In trials, the scientists used a small reference group of known heterozygous plants to create a confidence interval (CI). The CI establishes the amount of fluorescence expected from heterozygous plants. "We then said that any plant that shows marker fluorescence greater than the CI is homozygous," Vandemark says. They validated the technique by performing progeny testing on all the plants, showing that it was 99 percent accurate in determining which ones had one copy or two of the bc-[1.sup.2] gene.