Bermudagrass DNA FingerprintingThis powerful tool can be used to distinguish genetic differencesthat are important in protecting plant patents.

BY MICHAEL P. ANDERSON AND YANQI WU

The fingerprinting of plant,animal, and human DNA(deoxyribonucleic acid) has

been practiced among researchersand forensic scientists for many years,especially garnering widespread atten-tion from notorious criminal cases in-volving DNA evidence. DNA finger-print analysis is powerful and capableof distinguishing one individual fromanother. Each of us has a unique DNApattern, as do plant species and plantvarieties.

DNA DIFFERENCESAll organisms, including grasses, haveidentifiable characteristics. These char-acteristics make an organism uniquefrom all others. Physical characteristicsin bermudagrass, such as leaf textureor leaf color, are obvious and readilydiscernable. However, some character-istics require detailed measurements,while others are more qualitative innature. Some distinguishing featurescan be observed with little or no train-ing, while others need close inspectionby trained and experienced personnel.Many subtle differences among closelyrelated bermudagrasses cannot bereadily distinguished visually. Anothermethod is necessary to differentiatethese bermudagrasses: DNA finger-printing.

Differences among organisms arecoded by their DNA, which is a verylong molecule made up of a specificsequence, in linear order, of fourdistinct chemicals called nucleotides.

14 GREEN SECTION RECORD

If human DNA were represented bysingle letters standing for each distinctnucleotide (adenine, cytosine, guanine,and thymine) on a blank page, thelength of the alphabetic sequencewould run at least to one million pages,enough to fill 1,000 large volumes.

The DNA sequence dictates the lookof an organism and how it responds tothe immediate environment, and it isdifferent for every organism. Conse-quently, the DNA sequence can beused to distinguish one organism fromanother. DNA fingerprinting is nothingmore than a sophisticated technique tosample an organism's DNA sequence,projecting the differences as a kind ofbar code for ready identification andcompanson.

Most DNA fingerprinting dependson a technique known as PCR orpolymerase chain reaction. PCR wasdeveloped in the mid-'80s to efficientlyamplify specific segments of DNAmany-fold. The PCR technique usesshort DNA segments composed ofanywhere from 6 to 20 nucleotidesknown as primers, which are comple-mentary to segments of the targetDNA. The primers figuratively scanfor matches in the target DNAsequences. Once a match is found,then amplification of that segmentbegins. If there are many matches,many segments will be amplified.

This mixture of amplified segments,known as amplicons, can be separatedon an electrophoretic gel system, whicheffectively sieves amplicons based on

size. The gel is stained with fluorescentdyes to reveal what looks like a band-ing pattern or a bar code. Multipleprimers can be used to scan differentportions or the total genomic DNA,revealing additional bar coding. Finger-printing with many primers is capableof differentiating even the most closelyrelated of all organisms. Thus, whiletwo bermudagrasses may be physicallyindistinguishable from each other, theDNA fingerprinting can highlight theintrinsic differences in their DNA byusing PCR-based techniques.

All organisms can be fingerprintedand their DNA patterns stored andanalyzed. Analysis of the banding pat-tern is performed using a variety ofstatistical techniques known as clusteranalyses. The data are inputted in theform of presence or absence of a par-ticular PCR amplicon or electrophoreticband and cluster analysis analyzes thedata and connects those organisms thatshow similar patterns. However, to beeffective, there must be enough simi-larities, as well as differences, in thepattern to reveal relationships amongall tested organisms.

A number of fingerprinting tech-niques exist. These techniques differ inthe ability to differentiate organisms,the amount oflabor required, theextent of automation available, theexpense of use, and the nature of thespecific targeted DNA segments. AFLP(Amplified Fragment Length Poly-morphism), DAF (DNA AmplificationFingerprinting), SSR (Simple Sequence

Drs. Mark Gatschett (left) and Mike Anderson use advanced biotechnological and molecular genetictools to understand the genetics of Oklahoma State University's bermudagrasses.

Repeats), and RAPD (Random Ampli-fication of Polymorphic DNA) are afew of the more commonly used tech-niques used to fingerprint DNA. All ofthese utilize PCR to amplify segmentsof DNA based on the DNA sequence.Sophisticated and expensive commer-cial packages and instrumentation existto automate and increase the resolutionof the fingerprinting procedure.

HOW IS DNAFINGERPRINTING USED?We have used DNA fingerprinting tolook at the genetic relationship amonga wide range of bermuda grasses. Someof the first work highlighted the differ-ences among high-quality commercialcultivars and select bermudagrassesfound in germplasm collections.Caetano Anolles et aF surveyed 13bermudagrass cultivars, includingAfrican, common bermudagrass, andseveral interspecific hybrids for geneticrelatedness using DAF. Results showedthat DNA fingerprints were easily dis-tinguishable, and the analysis showedclear genetic relationships among allbermudagrass varieties.

To probe the limits of the ability todistinguish bermudagrasses, we finger-printed Tifway and its irradiation-induced mutant Tifway II, whichpresumably differed in one or a fewnucleotide changes in the DNAsequence. In order to differentiatethese very closely related varieties, wefound it necessary to use 81 distinctprimer combinations to find a one-band difference among all 81 finger-prints.2 From this early work, it wasclear that investigators can differentiateand draw genetic relationships evenamong the most closely relatedbermudagrasses.

Breeders often collect from aroundthe world a wide range of plant intro-ductions in the hope of finding specificgenetic traits that may be put to pro-ductive use. The genus Cynodon (ber-mudagrasses) is comprised of9 species.4

Oklahoma State University is home toa worldwide collection of bermuda-

grass varieties and plant introductionsthat was initiated by the geneticist JackHarlan. Charles Taliaferro, and morerecently Yanqi Wu, two bermudagrassbreeders at OSU, have added signifi-cantly to this collection, making it oneof the most comprehensive collectionsof Cynodon germplasm in the world.Understanding the genetic relatednessamong Cynodon spp. and varieties givesus a better understanding of the geneticmakeup of the Cynodon genus.

At times, doubts about the geneticidentity of a particular variety surface.In previous work, our laboratoryresponded to the need to evaluate thewidely used variety U3 for geneticfidelity.1 U3 was an early success madeup of bermuda grasses collected fromgolf courses in the southern U.S. inthe 1930s. U3 showed moderate coldtolerance and fine-textured leaves andwas a general improvement whencompared to previous cultivars.

DNA fingerprinting was employedto distinguish the current labeled U3from presumably authentic U3 collec-tions assembled from around thecountry. Results showed that the cur-rently labeled U3 varieties differedsubstantially from the presumablyauthentic U3 varieties. How these dif-

ferences came about could not beaddressed by the fingerprinting tech-nique, but the research underscoredthe need for evaluating current varietiesfor genetic stability and purity. Inaddition, our research, as well as thatof others,9 has discovered a few otherdiscrepancies between the historicalpedigree claims of several varieties andtheir actual genetic relationships usingfingerprinting techniques.

GAINING BERMUDAGRASSDIVERSITY WORLDWIDENew bermudagrass germplasm hasbeen and is now being collected andassembled into worldwide collectionsfrom many sources. There are areaswhere collections have only recentlybeen assembled from specific locationssuch as southern and southeastern Asia.Recently, a number of bermuda grassesfrom China were added to the OSUgermplasm collection. DNA finger-printing using the AFLP techniquewas used to evaluate the diversitywithin this germplasm.

The Chinese collection seemedsurprisingly diverse7 and distinct fromother bermudagrasses from otherlocations around the world.6 Furtherwork in our laboratory easily separated

SEPTEMBER-OCTOBER 2007 IS

Oklahoma State University is home to a worldwide collection of bermudagrass varieties, much tothe credit of Dr. Charles Taliaferro (pictured). Dr. Mike Anderson and his colleagues are using DNAfingerprinting techniques to understand the genetic relatedness and gene function of this importantturfgrass.

the Chinese collection from all U.S.varieties tested. Overall, the work indi-cated a source of significant variationin the new Chinese collection, whichmay contain valuable genes for ber-mudagrass development. Additionaldiversity assessments need to be doneon collections from India and otherareas not previously surveyed.

The same techniques used forDNA fingerprinting are also used for

molecular genetic analysis of specifictraits. The goal here is to locate specificgenetic elements or genes that contrib-ute substantially to those traits. This isperformed by first constructing popu-lations with significant variation in aparticular trait of interest, and then per-forming the DNA fingerprinting tech-nique on members of the populationto identify specific genetic elementsthat correlate with the expression ofthat trait. These genetic elements arevisualized as unique bands on electro-phoretic gels that appear to correlatewith traits of interest. The bands arevaluable in that they can serve asgenetic markers, markers that are based

16 GREEN SECTION RECORD

on the DNA sequence rather than somephysical characteristic of the plant.

Sophisticated computer softwareanalysis can gauge the contribution ofthe DNA element associated with themarker to the genetic makeup of thephenotype. These markers can be usedto increase the efficiency of selectionin a process known as marker-assistedselection. Marker-assisted selection hasbeen shown to be very effective in

enhancing germplasm improvementin a variety of cropping systems.3•5•8

Constructions and evaluation of map-ping populations, and utilization ofmolecular genetic analysis, are majorgoals of the OSU bermudagrass team.

DNA fingerprinting of individualswithin a population provides informa-tion concerning the genetic makeup ofa population. The individual makeupof the population may change withtime, depending on natural selectionand genetic inflow from neighboringbermudagrasses. To observe theseshifts, DNA fingerprinting can be usedto document and track alterations inpopulation makeup of seeded bermuda-

grasses under a variety of environ-mental conditions over time.

PLANT PATENTINGDNA fingerprinting can have animpact in the area of patent protection.Many years of effort are expended todevelop commercial varieties. Institu-tions have a substantial investment indevelopmental costs and are increas-ingly desirous of recovering some ofthose costs through plant variety pro-tection and the collection of royaltiesfrom consumers. To support the patentapplication process, differences inmorphology, cultural characteristics,and pedigree need to be presented inorder to distinguish the proposedvariety from those that are currentlyavailable. DNA fingerprinting is cur-rently being used on a limited basis todocument the genetic differences ofnew varieties in the patent process.Any infringement on the patent wouldhave to use the DNA fingerprints andother characteristics to justify a patentinfringement lawsuit. The process maybe costly and subject to interpretationby experts, but it may be worth theeffort when the stakes are large.

In summary, DNA fingerprintingis a valuable technology that is beingused to assist producers, breeders,geneticists, and researchers in evaluat-ing bermudagrass populations andgermplasm for genetic diversity andbackground. Information from DNAfingerprinting techniques allowsresearchers to make informed decisionsconcerning progress in developinghigh-quality bermudagrass lines. DNAfingerprinting technology remains apowerful technique to assess thegenetic diversity of bermuda grassesworldwide and to protect plant varietiesfrom infringement. At OSU, ourprojects have been involved in usingDNA fingerprinting to furtherbermudagrass improvement.

ACKNOWLEDGEMENTThe authors acknowledge the contribu-tions of Dr. Charles Taliaferro, Praveen

An interview with DRS. MICHAEL ANDERSON and YANQI Wu regardingDNA fingerprinting.

Q: As you note, most of us are aware of DNA fingerprinting fromcriminal cases, but from a research perspective, how long have DNAfingerprinting techniques been available?A: Fingerprinting has been around for quite some time. Inplants, some of the earliest DNA fingerprinting involved atechnique known as RAPD, which was developed in the late1980s. Bermudagrass fingerprinting did not take place until aboutthe early 1990s. Advancement in fingerprinting mainly comes fromthe use of high-resolution instrumentation that greatly increasesthe accuracy and resolution of the technique, but at a cost.Instruments typically cost from $70,000 to $500,000, and the kitsfor doing the fingerprinting are expensive as well.

Q: Do you think that at some time in the future, in order to receive aplant patent for a new cultivar, breeders will have to submit DNAfingerprint evidence that establishes this new cultivar as geneticallyunique from existing cultivars?A: Currently this is not a requirement, but it may be advisable.A patent contains morphological descriptions that distinguish thenew cultivar from those already released. Whether it becomes arequirement depends on the decisions of the courts. Patents aregranted for inventions (including new varieties) that are useful,new, and non-obvious. The DNA fingerprint establishes whethera new variety is new genetically, but it does not indicate utility.The utility factors must also be documented to distinguish thenew variety.

Q: You mentioned the use of primers to characterize specific geno-types. Is there a ballpark number of primers that are necessary toadequately characterize a genotype, or does it depend completely onthe relatedness of the genotypes?A: It depends on how closely related your cultivars are and whattechnique you are using. When using AFLP, you may need from 8to 14 primer pairs to differentiate bermudagrasses adequately.With DAF you need anywhere from 4 to 12 primer pairs. If thebermudagrasses are very divergent, 4 primers give satisfactoryresults. There is an additional technique known as mini-hairpinDAF or MHP-DAF, which scans the amplicons created in the first

DAF reaction for additional differences. With this technique it ispossible to distinguish even very closely related bermudagrasseswith no more than 4 MHP-DAF primers.

Q: You mentioned that the use of DNA fingerprinting can be used toprotect plant patents. Have there been cases where DNA fingerprintinghas been used and either found patent infringement or a situationwhere the plant cultivar was not what it was supposed to be?A: I am not aware of any at this time. Patent lawyers whospecialize in plant variety protection would be aware of the legalhistory behind this particular question. In answer to your secondquestion, yes, there are cultivars out there that claim a certainpedigree, but in reality they are not closely related to thedescribed variety. I know of three such cases. The most obviousone is the U3 variety referred to in the article. It seems to methat if a company is selling a variety labeled as a protected varietyand if the actual variety does not conform to the legal patentdescription, then that company's variety is open to legal challengeas far as ownership is concerned.

Q: How important are the Chinese bermudagrass germplasm additionsto the bermudagrass breeding effort at OSU? Are there specific traits inthe Chinese bermudagrasses that have a high priority for introductioninto new bermudagrass cultivars here in the U.S.?A: Currently there is great interest in screening this collection forproductive traits. Some of the germplasm have desirable seedyield, seed quality, genetic color, and/or some other traits relatedto turf performance. Our best guess is that some of thecollections will be incorporated into our existing breedingprogram and contribute substantially to future OSU releases.

Q: Toyour knowledge, are most breeding programs using marker-assisted selection (MAS) as an integral part of cultivar development?A: Most breeding programs are not using marker-assistedselection for their variety development. Part of the impediment tousing the molecular techniques is due to lack of training andexpertise. However, experience in molecular aspects of breedingis becoming very common for the breeders coming out ofgraduate school, so I expect the trend towards the acceptance ofmolecular approaches to continue with a newer crop of breeders.

JEFF Nus, PH.D., manager, Green Section research.

Yerramsetty, and Carole Anderson,and appreciate the funding from theUSGA Turfgrass and EnvironmentalResearch Program and the OklahomaState Agricultural Experiment Station.

LITERATURE CITED1. Anderson, M. P., C. M. Taliaferro, D. L.Martin, and C. S. Anderson. 2001. Compara-tive DNA profiling ofU3 turfbermudagrassstrains. Crop Science 41:1184-1189.

2. Caetano-Anolles, G., L. M. Callahan, P. E.Williams, K. R. Weaver, and P. M. Gresshoff1995. DNA amplification fingerprintinganalysis of bermuda grass (Cynodon) - geneticrelationships between species and interspecificcrosses. Theoretical and Applied Genetics 91:228-235.

3. Mackay, 1., and W. Powell. 2007. Methodsfor linkage disequilibrium mapping in crops.Trends in Plant Science 12:57-63.

4. Taliaferro, C. M. 1995. Diversity andvulnerability of bermuda turf grass species.Crop Science 35:327-332.5. Tuberosa, R., and S. Salvi. 2006. Genomics-based approaches to improve drought toleranceof crops. Trends in Plant Science 11:405-412.6. Wu, Y. Q., C. M. Taliaferro, G. H. Bai,and M. P. Anderson. 2004. AFLP analysis ofCynodon dactylon (L.) Pers. var. dactylon geneticvariation. Genome 47:689-696.7. Wu, Y. Q., C. M. Taliaferro, G. H. Bai,D. L. Martin, J. A. Anderson, M. P. Anderson,and R. M. Edwards. 2006. Genetic analyses ofChinese Cynodon accessions by flow cytometryand AFLP markers. Crop Science 46:917-926.

8. Yamaguchi, T., and E. Blumwald. 2005.Developing salt-tolerant crop plants: challenges

and opportunities. Trends in Plant Science10:615-620.

9. Zhang, L. H., P. Ozias-Akins, G. Kochert,S. Kresovich, R. Dean, and W. Hanna. 1999.Differentiation of bermuda grass (Cynodon spp.)genotypes by AFLP analyses. Theoretical andApplied Genetics 98:895-902.

EDITORS NOTE: This complete papercan befound at the USGA's Tuifgrassand Environmental Research Online(http://usgatero.msu.edu).

MICHAEL P. ANDERSON, PH.D.,

Associate Prcifessor,and YANQI Wu,PH.D., Assistant Prcifessor,Department ojPlant and Soil Sciences, Oklahoma StateUniversity, Stillwater, Okla.

SEPTEMBER-OCTOBER 2007 17