Thursday, October 6, 2011

What Secrets Lurk in Peony DNA?

Last year we had the opportunity to start a project conserving peony (Paeonia L.) genetic resources in the National Plant Germplasm System.  We received approval from the the US Department of Agriculture, Agricultural Research Service, National Program Leaders, to begin obtaining and managing peonies at the Arctic and Subarctic Plant Gene Bank in Palmer, Alaska and the National Clonal Germplasm Repository in Corvallils, Oregon.

This project is in its infancy and its financial continuation is subject to the federal budget.  Despite these difficult times, collections of peonies were begun at both locations.

In concert with the establishment of the US peony gene back, a group of about 55 scientists, from across the United States, banded together and submitted a proposal for US Specialty Crop Research  initiative 2011 funding to research Peony as a Model for Cut-Flower Production.  This proposal was quite extensive, requesting $6.7 million, to study everything from tunnel production, plant nutrition, and clean stock program development, to genetic analysis.  Unfortunately, our proposal was not chosen to be funded, but the reviewers encouraged us to revise and resubmit in 2012.  We scientists suppressed our dismay and retreated to our drawing boards to consider resubmission.

One of the projects within the proposed research plan included examining peony DNA for identity and diversity determination.  We couldn't just let this project sit idly by, so with the limited resources we had on hand, we began this study.

Have you ever picked up a dormant peony root with a few  buds on it and wondered, "What is this cultivar?"  Have you planted some area with peonies, had them bloom a year or two later and realized that they were not the cultivar that you thought you ordered?  Have you ever received a hybrid peony and wondered "What cross is this?  Who's your daddy?"  The DNA analysis protocols that we are developing could answer these types of questions.

The kinds of techniques we are using are similar to laboratory forensics used in police work or paternity testing. First we extract DNA from the peony plant.  The standard way to get the DNA from plants is to grind up young, rapidly expanding leaves.  But because it was winter, all we had were dormant roots and no leaves.

We were very happy to find that we could easily extract large quantities of DNA from dormant peony buds.

We thought that this would be of great use to nursery folks who wanted to identify dormant material when asking for roots prior to shipping.  We also tried dormant buds from 11 other plants (pear, quince, blueberry, service berry and others).  Peony yielded the highest amounts of excellent quality DNA, more than any dormant buds of the 11 other plants.  We are in the process of publishing this information.  If anyone is interested in knowing more about our protocols we would be happy to discuss our approach with them.

We began our analyses by looking up publications from laboratories who had been working on DNA analysis in peonies.  We found two laboratories in China had begun using DNA from leaves of tree peonies.  These reports were moderately successful using what is called simple sequence repeats (SSR).  This is the kind of analysis that we also wanted to do.  But we realized that we would need more SSR options than what the folks in China were using, so we could examine DNA form each of the different peony species.

In April 2011, the Center for Genome Research and Bio-computing at Oregon State University requested samples to test using their new DNA sequencing equipment.  They offered to sequence our peonies' DNA for  a much lower than usual rate to test their instrument operation.  Of course, we accepted.  We had them analyse DNA from herbaceous peonies, tree peonies, hybrid peonies, and P. brownii.  As a result we were provided with a wealth of DNA sequencing information.  We can now design 8,000 SSRs (way more than we need!), which will help us find additional SSR markers.

In late May 2011, we obtained some plant material of diverse peonies generously provided by Jim and Carol Adelman, of Adelman Peony Gardens, Brooks, Oregon.  We visited their peony fields and collected young leaf samples from 94 cultivars (herbaceous, intersectional, and tree) and two breeding lines.  These two lines were from Dr. Don Smith's (West Newton, Massachusetts) breeding program.  Dr. Smith was very interested in our work and had some specific questions on parentage that he was hoping we would be able to resolve.  So we took the young leaves, extracted the DNA and started screening our SSRs with this material.

So far we have screened 16 peonies with 250 SSRs.  This will give us preliminary data to choose which markers will be most successful for genetic fingerprinting.  We will also identify SSRs that are specific for different species.  That will present allow us to distinguish species present in a hybrid plant's genetic background.  Another of our goals is to develop a phylogenic map (like an evolutionary family history) that would show the ancestry and relatedness of peony species.

Our research is in the early phase. We anticipate that these molecular markers will be as useful to peony nurseries and growers for identification of cultivars as they are to those working with berry and fruit crops.

We are very happy working with the peony community in continuing to research this royal plant, the queen of the garden.  We greatly appreciate the generosity of information and plant material from the American Peony Society, the Wisonsin Peony Society, Minnesota Peony Society, the Pacific Northwest Peony Society, the Alaska Peony Growers Association, Dr. Patricia Holloway of the Georgeson Botanical Garden, Hidden Springs Nursery, Kennicott Brothers Company, Adelman Peony Gardens, and Oregon Perennial Company.

Who knows what secrets lay within peony DNA? Hopefully within a year we will have some answers.

Barbara Gilmore, USDA ARS Arctic and Subartic Plant Gene Bank, Alaska., 
Nahla Bassil, and Kim Hummer, USDA ARS National Clonal Germplasm Repository,

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