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Grapes 101

 

Figure 1. Breeders at Cornell and elsewhere have identified several powdery mildew genes that they are incorporating in new varieties that will require fewer fungicide sprays.  DNA markers allow them to know which vines have which resistance genes by testing leaf tissue at the seedling stage.          Photo by Lance Cadle-Davidson

The Core Grape Genome and Cheap DNA Sequencing:  A New Roadmap for Grape Breeders

Grapes 101 is a series of brief articles highlighting the fundamentals of cool climate grape and wine production.

By Tim Martinson and Bruce Reisch

Those who are familiar with the Cornell grape breeding program realize that it has been very productive in releasing new varieties.  Since Bruce Reisch released “Horizon” in 1982 (a sibling of Cayuga White), there have been several successes,  including Traminette, Geneva Red (GR7), Corot noir, Noiret, Valvin Muscat, Aromella, and Arandell.  It’s fair to say that these were the ‘needles in the haystack’ that resulted from continuing cycling of new accessions (around 3,000 seedlings per year) through a 15-20 year process of evaluation. 

But understanding the underlying genetic traits that produce disease-resistant, high quality table and wine grapes has lagged.  Grapes are expensive to grow, and until recently, extracting the underlying genetic basis of desirable traits such as disease resistance and cold-hardiness was time-consuming and tedious.  Corn breeders have been able to produce thousands of seedlings each year and evaluate them at the end of a single growing season.  This (and a few other tricks like inbred breeding lines) allowed them to learn a lot about traits and their inheritance starting in the early 1920s.  Corn yields have quintupled (5x) since the 1930s as a result. 

Grape breeders can only screen around 2,000 seedlings annually, and have to continually winnow them down to a handful of ‘advanced selections’ they retain for several years of evaluation.  Low numbers, slow turnaround, and high cost to retain seedlings are three factors that handicapped grape geneticists and breeders in determining inheritance of traits and the genes responsible for them.

Cheap DNA Sequencing is a Game Changer

Inexpensive DNA sequencing and the “Polymerase Chain Reaction” or PCR reaction has changed the landscape.  Since 2007, according to the National Health Institute, the cost of sequencing a human genome has dropped from $10 Million to $1,000.  Its 10,000 times less expensive now than it was ten years ago.  To sequence one million DNA base pairs now costs around 1.2 cents – down from $500 in 2007.

This opened up the possibilities for grape breeders to map the grapevine genome and discover DNA markers that were related to traits such as disease resistance.  By 2010, European researchers published the first complete genome of an inbred Pinot noir line called “PN40024” – a major accomplishment, and  the result of a research investment of millions, and several years of effort. 

It worked well with Vitis vinifera genetics – but less well when it came to North American Vitis species that are the source of many disease resistance and cold-hardiness traits in breeding programs.  Simply put, markers from PN40024 were not informative for North American Vitis species used in many breeding programs. 

One of the reasons for this is the fact that European Vitis vinifera diverged from North American Vitis around 20 million years ago.  Twenty million years is a lot of time for the European species to diverge from the North American species.  The ~20 North American species and the European Vitis are all recognizable as grapevines and nearly all make fertile hybrids with V. vinifera – but their genomes have structurally rearranged themselves in the meantime. 

Core Genome and Transferable Markers

This led Cornell genetics researchers Qi Sun,Cheng Zou and postdoc Avi Karn, along with USDA researchers Lance Cadle-Davidson and Jason Londo to sequence nine North American Vitis genomes and try to align the ‘core genome’ (ie. the genes coding for what makes a grapevine a grapevine) that all of the species shared in common with the existing reference PN40024 genome. 

The result is that there was about 10% commonality among all species.  By using the ‘core genome’ sequences, the team was able to come up with DNA markers that spanned the 19 chromosomes (good coverage) and worked just as well with Vitis riparia and Vitis cinerea as they did with the Vitis vinifera PN40024 genome.  See (Grape Breeders Search for Reliable DNA Markers: Why the Pinot noir PN40024 Reference Genome is Not Enough).

Current situation

Now, breeders have access to a suite of 2,000 DNA markers, more or less evenly spaced throughout the 19 grape chromosomes, that work across the diverse Vitis genus.  This marker platform allows them to look at families of siblings called mapping populations and determine which combination of markers each vine has.  Like human brothers and sisters, these siblings of two different parents harbor different combinations of traits from each parent.  Researchers can then find out from these siblings where in the genome the genetic trait is located.  And do so at a reasonable cost of about $10 per vine.

Disease resistance markers and their use

Since 2000, several DNA markers for powdery mildew (10) and downy mildew (27) have been identified (see Figure 2).  These markers – designated by a three letter code involving the scientific name of the pathogen and a number (for example Ren2 = Resistance to Erysiphe necator 2 for powdery mildew) – are easily identified with simple DNA tests.  In practical terms, it allows the breeders to test each seedling they generate every year – and to learn which resistance genes the seedling contains. 

 

Figure 2.  Powdery and downy mildew resistance DNA markers discovered since 2000. These markers, designated with a three-letter abbreviation and a number, are now available to grape breeders for marker-assisted selection.

Instead of planting them out in the field and looking for natural powdery mildew infections, breeders can test tissue and discard those that don’t contain the genes they want.  This process, called Marker Assisted Selection is allowing breeding programs to stock the pipeline with vines that have known characteristics – and save time, space, and money.  As these new selections build up within the breeder’s plantings over years,  the investment in testing they are making now will pay increasing dividends in the future.

Figure 3.  This numbered selection called  4427075 is a new wine selection in the Cornell-Geneva grape breeding program. It has two genes for powdery mildew resistance (Run1 and Ren2), and one for downy mildew resistance (Rpv1). The cross was made in 2014, and though we have no wine results yet, the flavors of the fresh fruit reflect a lack of wild grape off-flavors, and presence of pleasing fruity flavors. Fruit ripen mid-season.

Bruce Reisch’s program already has several selections with known powdery mildew and downy mildew disease resistance in his program.  For example, (Figure 3), a new selection called 4427075 , with two powdery mildew (Run1 and Ren2) and one downy mildew (Rpv1) marker, produced in 2014, is currently being evaluated by the program.

Incorporating disease resistance genes into new varieties will pay dividends for grape growers worldwide.  Instead of needing to spray a variety like ‘Chardonnay’ 10 to 14 times, the disease-resistant varieties offer the prospect of reducing the number of spray applications down to two to three.  The economic, environmental, and social benefits of a potential 80% reduction in fungicide sprays as these new varieties are planted will be substantial.

The VitisGen2 project, led by Reisch and USDA scientist Lance Cadle-Davidson, is a nationwide project funded by the USDA's Specialty Crops Research Initiative involving researchers in California, Minnesota, Missouri, North Dakota, South Dakota, and New York.  Its aim is to discover DNA markers, and particularly those associated with powdery mildew resistance and fruit quality, that breeders can use for marker-assisted selection of new varieties.   To date, the project and its predecessor (Vitisgen1) have discovered over 70 DNA markers for desirable traits.  These DNA markers provide grape breeders with a roadmap for producing a new generation of more sustainable and resilient grapes of the future.

Tim Martinson is senior extension associate and Bruce Reisch is professor of grape breeding and genetics in the horticulture section of Cornell's School of Integrative Plant Sciences.  Both are based at Cornell AgriTech in Geneva, NY.