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The second generation of apple trees bred with  resistance to blue mold from a wild ancestor are  growing in the U.S. Department of Agriculture’s  Appalachian Fruit Research Laboratory in Kearneysville, West Virginia. DNA tests developed through RosBREED and apples genetically engineered to flower early are helping researchers introduce the disease resistance  into high-quality cultivars faster. <b>(Courtesy Jay Norelli, USDA Appalachian Fruit Research Laboratory)</b>

The second generation of apple trees bred with resistance to blue mold from a wild ancestor are growing in the U.S. Department of Agriculture’s Appalachian Fruit Research Laboratory in Kearneysville, West Virginia. DNA tests developed through RosBREED and apples genetically engineered to flower early are helping researchers introduce the disease resistance into high-quality cultivars faster. (Courtesy Jay Norelli, USDA Appalachian Fruit Research Laboratory)

Over generations, as breeders have selected apple trees with the best flavor, size and color, resistance to many common diseases was lost.

But genes for resistance are often still lurking in wild apple ancestors, and new DNA tools are giving breeders the power to return those key genes to domestic apple varieties in a matter of years, not decades.

In the case of blue mold — the most significant postharvest disorder globally — scientists found resistance hiding in the genome of Malus sieversii, the wild Eurasian apple from which the domestic species was derived. Now, researchers with the U.S. Department of Agriculture’s Appalachian Fruit Research Station in Kearneysville, West Virginia, are breeding that wild resistance back into an elite breeding parent.

New tools are helping them to do it fast: Cultivars are expected to be ready for breeders in just a few more years, said Jay Norelli, the plant pathologist leading the project. “We are tapping into the latest advantages that have been made in genomics science to really advance the efficiency of apple breeding,” Norelli said.

But while some of the tools used to expedite breeding are the result of genetic engineering, Norelli stressed that the process is not creating genetically modified apples.

The final, blue-mold resistant cultivar will have no genetically modified DNA. That’s very important to growers because some consumers have been wary of genetically modified crops, he said.

Breeding teamwork

All the genomics tools are available thanks to RosBREED — a national team of scientists seeking to improve the quality and disease resistance of apple, blackberry, peach, pear, rose, strawberry and sweet and tart cherry crops — and to a sister effort in Europe known as FruitBreedomics.

The American project was funded by the U.S. Department of Agriculture first in 2009 with a $14 million grant to look at fruit quality traits, then re-upped in 2015 for a $10 million focus on disease resistance.

From the start, RosBREED has been clear that it was not seeking to genetically engineer better crops, but rather to use DNA analysis tools to inform and improve conventional crossbreeding, said Cameron Peace, RosBREED co-director and horticulture professor at Washington State University.

Every generation, Norelli sends samples from his new seedlings to Peace’s lab at WSU, where RosBREED’s DNA-informed breeding programs for apple and cherries are based.

The lab focuses on translating discoveries from genomics research into strategies breeders can use, Peace said.

So far, his lab is developing DNA markers for disease resistance, fruit color, acidity levels and other desirable traits so that breeders can test and select seedlings without waiting for fruit.

That saves breeders the expense and time of growing a nursery full of trees that lack the desired genes in search of the perfect fruit. Eventually, RosBREED aims to help commercial service providers offer the tests it develops to breeders, expanding access to the tools, Peace said.

Reining in resistance

One-year-old apple trees are fruiting, thanks to an early-flowering gene that accelerates the crossbreeding process. These trees are the second generation of a cross to bring blue mold resistance from a wild apple ancestor into a modern cultivar. This fruit will be exposed to blue mold to verify that the DNA test scientists are using in the breeding program is accurate. <b>(Courtesy Jay Norelli, USDA Appalachian Fruit Research Laboratory)</b>

One-year-old apple trees are fruiting, thanks to an early-flowering gene that accelerates the crossbreeding process. These trees are the second generation of a cross to bring blue mold resistance from a wild apple ancestor into a modern cultivar. This fruit will be exposed to blue mold to verify that the DNA test scientists are using in the breeding program is accurate. (Courtesy Jay Norelli, USDA Appalachian Fruit Research Laboratory)

The challenge for breeders is that the wild ancestors carrying resistance also come with lots of undesirable traits that have been bred out of modern apples.

Keeping only the key resistance gene traditionally required four or five decades of back-crossing with high-quality cultivars to get rid of that wild DNA. Now, DNA-markers and genetically engineered tools have dramatically improved the pace.

Locating the blue mold resistance marker involved developing a genetic map of a cross between the resistant wild apple and a Royal Gala.

Then scientists compared the DNA of all the offspring to find the DNA associated with the resistance trait. In the case of blue mold, there was one clear spot on one of the apple’s 17 chromosomes associated with resistance.

That key finding enabled the research to move forward much more quickly and is much easier to work with than a trait that appears to be associated with multiple genes, Norelli said.

Once that locus — a location on a chromosome — was identified, WSU researchers built a DNA test to assess which seedlings inherited the resistance gene.

The growing library of DNA tests means that Norelli’s seedlings can also be screened for about 10 other desirable traits, such as acidity and skin color, Peace said.

In traditional breeding efforts combining two already high-quality cultivars, fewer DNA tests are usually needed, but “there’s a lot more bad genetics in this material from the wild apple,” Peace said. Using all the tests on each generation of seedlings helps to weed out those unwanted wild genes faster.

Before DNA tests, breeders measured disease resistance by purposely infecting new trees. But having access to the genetic markers is a huge advantage, especially for a disease like blue mold, which causes fruit decay during storage rather than damages the tree itself.

“For a lot of diseases like scab and fire blight, we can screen seedlings directly with the pathogens, but DNA tests are better. And one of the big advantages of DNA markers for fruit traits is it saves us years versus waiting for apples,” Norelli said.

To speed up the breeding process even further, Norelli is using a genetically modified apple that carries a gene from a birch tree that initiates early flowering. By using it as a parent, his trees are blooming and ready to breed at just over a year old, instead of having to wait three years for each generation to flower.

That transgene for early flowering was introduced into the Pinata cultivar by German researchers, who used it in a similar way to breed fire blight resistance into modern cultivars as part of the FruitBreedomics project.

The early flowering trait comes from a single, dominant gene, which means that every generation, half the seedlings produced are early flowering; the other half flower normally because they did not inherit the chromosome with the transgene.

To accelerate this breeding process, Norelli crossed the parents — the offspring of the wild apple and the Royal Gala and the Pinata cultivar containing the birch tree gene — in the conventional manner and selected offspring with both the resistance gene and the early flowering gene. Now, he is continuing to cross those offspring for several more generations to weed out unwanted wild apple genes.

Once that breeding process creates high-quality, blue mold resistant cultivars, Norelli will no longer need the early flowering gene. So in the final round of the breeding process, he will select offspring that don’t carry the transgenic gene from the birch tree — and thus are not considered genetically modified — to grow into normal apple trees.

Testing the test

The second generation of blue-mold resistant, early flowering apple trees are growing in the U.S. Department of Agriculture greenhouse in West Virginia. Some of the spindly trees are already fruiting. But even high-tech tools need to be proven in the nursery. So, Norelli is preparing to test the DNA-based breeding by exposing the first crop of fruit to the blue mold fungus, so he can evaluate how susceptible they really are.

“We are validating whether that test actually predicts resistance. That’s really important because before we at RosBREED release a tool, we test it so breeders can use it with confidence,” Norelli said.

If the test proves itself, as scientists suspect, the cultivar should be ready for breeders by 2019. There are a few more crosses to go, Norelli said, to maximize the domestic apple genes and minimize the wild genes. To meet that deadline, the team is employing one more novel genetic tool that helps to select seedlings with the least wild DNA.

Every new generation has a mix of its parents’ traits — typically 50-50 — but due to some genetic rearranging that happens as chromosomes are passed on, there’s always a little variation.

“By the third generation, about 25 percent of the genetics should be M. seversii, but because of crossing over of the chromosomes, some have less and some have more,” Norelli said. “We’re using a DNA test to track how much wild DNA is left.”

FruitBreedomics developed the test to track apples’ lineage by looking at about 20,000 loci. That’s far from sequencing the entire genome, but it provides a significant snapshot of an apple’s 17 chromosomes.

After the test is run on each parent — the wild apple, Royal Gala, and Pinata with the early flowering gene — the offspring can be compared to see how much they still resemble the wild apple. With that insight, Norelli can beat the 50-50 odds slightly with each cross and select those seedlings with both the key traits and the least wild DNA to give rise to his next generation.

“With the accelerated system, that should be a one- to two-year window to get that next generation,” he said. “Our first objective is to produce elite breeding parents with resistance alleles and then trying to incorporate other resistance alleles for fire blight and scab.” •

– by Kate Prengaman