Using Molecular Biology for Management and Genetic ...

CHAPTER 31: Using Molecular Biology for Management and Genetic Enhancements 303

The purpose of this chapter is to describe how molecular biology, molecular enhancements, and integrated crop production research can lead to genetic improvements and the development of management systems that fully utilize the genetic capacity of wheat varieties.

Introduction Researchers are often asked how our experiments with genes and DNA in the laboratory can possibly benefit farmers and their crops out in the field. Sometimes, research scientists seem just too far removed from the reality of what happens on the land. Can these molecular approaches really lead to benefits for growers and producers? The short answer to that is yes. Information produced by molecular biology can be used to:

• Improveourunderstandingonhowwheatgrowsanddevelops.• DevelopinformationthatcanleadtoimprovedBestManagementPrograms.• Speedupandincreasetheefficiencyofwheatbreedingprograms.

Using Molecular Biology for Management and Genetic Enhancements

C H A P T E R T H I R T Y- O N E

Paul Rushton ([email protected]) Wanlong Li ([email protected])

Stephanie Hansen ([email protected])

Rules of Thumb for Using Molecular Biology to Increase Profitability

• Molecularbiologyprovidesinformationthatspeedsupcropbreedingbyapproximately50%.

• Unlikecorn,soybean,andrice,thesequencingofthewheatgenomecontinues.Whenthewheat genomeissequenced(estimated5years),theabilitytoenhancebothgeneticsandmanagement practicesshouldbeimproved.

• Molecularbiologyprovidesinformationthatcanbeusedtobetterunderstandhowgenes,climate, andmanagementinteract.

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Ourancestorsstartedmanipulatinggenesinwheataround12,000yearsago.Theseearlyfarmers had the same goals as we do, and in many ways used similar approaches to improve their crops. The wild relatives of our crop plants had many undesirable qualities that made earlyfarmingmuchharderthanitistoday.In1884,AlfonseDeCandollewroteinOrigin of Cultivated Plants that:

a cultivated species varies chiefly in those parts for which it is cultivated. . . . We

may expect, therefore, to find the fruit of a wild fruit tree small and of a doubtfully

agreeable flavour, the grain of a cereal in its wild state small, the tubercles of a wild

potatosmall,theleavesofindigenoustobacconarrow...(13-14)

The transformation from wild to domesticated varieties is called the Domestication Syndrome. In this process, spontaneous mutations occur in wild populations and these mutant individuals are selected for use by humans for their more desirable traits. Interestingly, the traits selected for “under” domestication would often be detrimental to the crop in the wild. As a consequence, fully domesticated crops may not survive in the wild without human intervention.

Wheat provides an excellent example of this. The ears of wheat are separated from the stem that bears them by a structure called the rachis. Wild forms of wheat need to disperse seeds effectively, so they have easily shattered ears with brittle rachises. When the wheat seeds mature, the rachis shatters and the seeds penetrate surface litter embedding into ground cracks. This is an important mechanism for effective seed dispersal. The problem with this is that when the seeds fall they also become difficult for humans to gather. Wild forms of wheat, such as Wild Emmer,haveabrittlerachis,thereforemakingharvestingtimeconsumingandinefficient. During early wheat domestication, farmers selected for a rare single gene mutation (br –brittlerachis)thatpreventsshattering(DubcovskyandDvorak2007).Thismutationislethalinthewild(becausetheseedsfailtodrop),butconvenientlyconcentratestheseedsforhuman gatherers. All domesticated forms of wheat have this mutation. Wild wheat also had tough glumes, making threshing difficult. A genetic mutation convertinghulledwheatintofree-threshingwheatwasselectedfor,andispresentinDuramandBreadWheat,butnotEmmer. Themaingenethatisresponsibleforthisfree-threshinghabitiscalledTg (tenacious glume).Anothergenethatalsoproducesfree-threshingwheatissimplycalledQ. Q is a transcription factor. Transcription factors are proteins that turn other genes on or off. Molecularbiologyhasshownthatmostofthekeydomesticationgenesinwheatandothercerealspeciesaretranscriptionfactors(Table31.1).Understandingtranscriptionfactorgenesis important because mutation in a single factor can turn a whole process on or off.

Table 31.1. Selected genes and their roles in cereals.

Gene Crop species Type of gene Role

Q Wheat AP2Transcriptionfactor Free-threshingRht-B1 Wheat GRASTranscriptionfactor Semi-dwarfplantTb1 Maize TCPTranscriptionfactor LateralbranchesTGA Maize SBPTranscriptionfactor GlumesizeSH4 Rice MYBTranscriptionfactor GrainshatteringqSH1 Rice BELLHDTranscriptionfactor Grainshattering

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The family history of wheat Molecularbiologycanspeedupbreedingprograms.Tounderstandhowthesenewmolecular methods can do this, fi rst we need to consider wheat itself. First, wheat is different inseveralimportantrespectsfromcorn,riceandbarley.Breadwheatistheresultofmultiplecrosses between goat grass (the “grandmother” of wheat—gene set AA)andwildwheat(the“grandfather” of wheat—gene set BB).Theprogenyofthesecrossesenabledasecondtypeofcross between durum (called the “mother” of wheat—gene set AABB)andanothergoatgrass(called the “father” of wheat—gene set DD)(Fig.31.1). TheresultisthatBreadWheathasthreegenomes:theA,B,andDgenomes.Thiscross(gene set AABBDD)wasprobablymadebyancientfarmerslivinginwhatwenowcallIraq.The resultant cross demonstrated “hybrid vigor,” and outperformed its wild ancestors in yield and environmental adaptation, leading to further cultivation and hybrid improvement. The initial crosses, which were a boon to ancient farmers, also had the unfortunate side effect of creatinganextremelycomplexgeneticcodefortwenty-firstcenturyscientists.

Thecompletesetofgenes(ageneisasectionofDNAthatisresponsiblefortraits)thatmakes up any living organism is called its genome. This can be compared to a large book of blueprints, for instance, or the code that makes up a computer program. Wheat has a huge genome that is forty times larger than that of rice, and fi fteen times the size of soybean. Becauseofitsgeneticbackgroundwedescribedpreviously,wheatpotentiallyhasthreegenesfor each trait. This complicates matters considerably. For example, if we want to produce an improved wheat variety by eliminating a gene with negative properties, we are faced with the possibility that we will have to actually eliminate three genes because each of the three genomeshasacopyofthisgene.Eliminatingonlyonegeneislikelytohavenoeffectbecausethere will still be two of these genes left that can carry out the job that this gene performs.

Figure 31.1. A diagram showing the genetic source of bread wheat. (Source:PaulRushton)

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The sequence of the wheat genome To effectively use molecular biology for genetic improvement, we need the genetic code. Given that wheat contains three genomes, making sense of the genetic code is like trying to assemble three jigsaw puzzles that have been mixed in the same box. It was therefore a surprise to wheat scientists when it was recently announced that the wheat genome had been sequenced. This accomplishment, although immensely important, was not quite what it seemed.Unfortunately,whatthegroupofscientistshadproducedwasnotagenomesequencein a form that scientists and breeders can easily use. A simple jigsaw puzzle example again explains what happened. The scientists had chopped the wheat genome up into small chunks and sequenced all of the chunks—just like making all of the pieces of a jigsaw puzzle, without putting the puzzle together. Without putting the pieces ofthejigsawpuzzletogether,youcannotseethecompletepicture.ExactlythesameistrueofthesequencethatwasgeneratedfromwheatintheUK.ThesmallpiecesofDNAsequencenowneedtobeputtogether(assembled)beforewecanusethem. TheInternationalWheatGenomeSequenceConsortium,amorethan200-memberorganizationofgrowers,breedersandscientistsnotaffiliatedwiththeU.K.project,issuedapressreleaseinWashington,D.C.onAugust30,2010http://www.wheatgenome.org/News-and-Reports/News/Significant-Work-Still-Needed-to-Really-Crack-Wheat-s-Genetic-Code. They echoedwhattheUKfundingbodyhadsaid,namelythat“significantworkremainstobedoneto achieve a complete genome sequence” http://www.bbsrc.ac.uk/news/archive/2010/2010-archive-index.aspx. In contrast, the International Wheat Genome Sequence Consortium is attempting to produce the complete assembled wheat genome “in the next five years.” This seems a reasonable target. Some believe that the develop of the wheat genome will be “the most significantbreakthroughinwheatproductionin10,000years.”Currentwheatgrowersliveinexciting times.

Why will the wheat genome sequence be such a big breakthrough? Ourforefathersimprovedwheatinanuntargetedway.Theyobservedmutantsandifthisresulted in an improvement, they selected from those plants. It was completely dependent on theoccurrenceofnaturalmutations,whichisaslowprocess.Bycontrast,modernmolecularapproaches are rapid and highly targeted. We take a specific gene or genes and alter it. We then monitor the effect to see if there is an improvement in the wheat cultivar in some important trait such as yield or resistance to disease. To do this effectively, however, we need all of the genes in wheat so that we know what to manipulate. We can’t modify something if we don’t know of its existence. The genome sequence provides the blue print for this approach. When a plant is affected by drought or water stress, are there management practices producerscanimplementtohelpreducetheyieldloss?Probably.Researchoncornindicatesthatcornplantsunderwater-deficitstressinsummitlandscapepositionsaremoresusceptibletodiseaseandnutrientstressthannon-water-deficitcorninfootslopepositions(unpublisheddata,Clayetal.).Thiscanbecomparedtoahuman’simmunesystem;ifyoustressapersonbywithholding water, nutrients, or sleep, the person’s immune system will be lowered. It seems to be the same with plants.

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Becausenotallfieldshaveconsistentnutrientorwateravailability,applyingafield-widefungicidecanbewasteful,assomeareasjustdon’tneedit.Molecularbiologycanleadtoin-field tests that will allow producers to assess the activity of specific genes. This information can be used to improve management decisions. At the present time, producers and consultants can make assumptions regarding what management options are needed, but until weunderstandwhatisreallyhappeninginsidetheplant,theyarejust“best-guesses.”

What are “molecular approaches?” Moleculartechniquescanbeintegratedintotraditionalbreedingandagronomicproduction approaches. When linked with breeding, molecular biology techniques speed up thecultivarselectionprocess.Molecularapproachescanalsoprovidecriticalfieldproductioninformation needed to take full advantage of the genetic potential of crops. For example, by using molecular biology, the impacts of seeding density, fertilizer rates, and heat stress ontheup-and-downregulationofspecificgenescanbeassessed.Thebottomlineisthatmolecularbiologyenhancestraditionaltestingapproaches.Moleculartoolsthatareroutinelyusedinbreedingandcropproductionresearcharemolecularmarkersandmicro-arrays.Thedevelopment of transgenic corn and soybeans relied on molecular approaches.

Molecular markers Abreedercrossesonewheatvarietywithanother,getting500seedsthatpotentiallycontainthetraitbeingbreedfor.BeforeMarkerAssistedSelection(MAS),thebreederwouldhavetogrowoutall500seedsandassaythemforthedesiredtrait,sometimestofullmaturity,depending upon which trait was being sought. This method uses valuable greenhouse or field space,labor,andresources.UsingMAS,breederscangerminatetheseeds,takeasmalltissuesample, and save the seedlings that have the marker for the desired trait. Perhapsonlytenofthe500seedscontainthetrait,butbreederswillnowonlyhavetogrowoutthosetenplantsknowingtheycontainthedesiredtrait.Markersareunique,shortstrings of DNA located near a gene of interest. Small genetic differences in the DNA sequence of traits can be responsible for one plant being resistant to a disease and another not being resistant.UsingMAS,thetimerequiredtobringanewtraittothepublicisreducedby50%.Inwheat,approximately6,000molecularmarkershavebeendiscovered.Thesemarkersfunctionas an additional set of “index tabs” in the wheat set of blueprints.

Microarray technology assesses plant responses to stress Plantsrespondtosoil,climate,andpeststressbychangingthegenesthatareexpressed.Microarray(orchip)technologyallowsustopinpointwhichgeneshavebeenaffectedbystresstreatments by comparing the gene expression of a control plant to the gene expression of a test (ortreated)plant.Wheatchipshavebeenusedtoexploregeneexpressionduringpathogeninfection, environmental stress, and plant development. In corn we have used microarray analysis to assess the influence of plant density and weed competition on gene expression. Understandingwhatishappeningintheplantunderstressfulconditionswillleadtobetterdecision making regarding planting populations, choice of variety, fungicide and fertilizer applications, and other management decisions.

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Some routes to improved wheat varieties Traits that wheat breeders are specifi cally interested in include: vernalization and photoperiod response, plant architecture, grain quality, pest resistance, and tolerance to abiotic stresses. Vernalization and photoperiod responses are of interest because they infl uence the wheat fl owering time. Increasing the length of time of grain fi lling may lead to higher yields. Plant architecture is important because it impacts the ability of the plant to withstand lodging. As stand density and use of fertilizer increased, lodging became a critical problem. Approaches to solve this problem are breeding shorter plants and delaying N fertilizer applications(Chapter11).Oneofmostsignificantcontributionofthe“greenrevolution”isthereduction of wheat plant height. Two reduced height (Rht)(Table31.1)genesarenowfoundinmostmodernsemi-dwarfwheatcultivars.Themanipulationofthesegenessignificantlyreducedwheatplantheightsto80to90cmandimprovedwheatresistancetolodging.

Research has been conducted to understand how genetics, management, and climate interact to impact grain quality(proteincomposition,baking,andmixingcharacteristics).Progresshasbeenmadeinunderstandingthegeneticcomponentsofwheatgrainqualityin two aspects: grain hardness and grain protein content. Genes controlling hardness of wheathavebeendiscovered(PinAandPinBgenes)(Hoggetal.2004).Varietieswithspecificmutations(sequences)inthesegenesarehardtextured,whileothervarietieshavesequencescontributing to the soft wheat type. Genescontrollingtheproteincontentofwheathaveadirecteffectonthebread-makingquality of the grain produced. Several of these genes that effect protein content have beendiscoveredinwheatandhavebeenmanipulatedinmodern-daywheat,withmoreimprovements to come. http://deltafarmpress.com/promise-better-wheat-varieties Disease resistance research has focused on rotational effects as well as better understandingseedlingandadultplantresistance.Seedlingresistanceisrace-specific,whereasadultplantresistanceisbroad-spectrum.Adultplantresistanceismoredurable,althoughatlowerlevelscomparedtoseedlingresistance.Morerecently,progresshasalsomadeinunderstanding wheat plant susceptibility to rusts, powdery mildew, tan spot, and plant resistance to Hessian fl y and greenbugs. Improving disease resistance is one of the areas where molecular biology holds most promise for breeders and growers. Wheat production is facing numerous challenges from drought, excess water, heat, salinity, andothersoil-derivedtoxicities.Complextraitssuchasdroughtandheat-stresstolerancehave started to reveal themselves through the use of molecular tools. It will take hard work and

Figure 31.2. Lodging in wheat has been greatly improved by selective breeding centered on the two Rht genes. (Photocourtesyofhttp://faculty.uca.edu/johnc/greenrev3390.htm)

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timetounderstandtraitssuchasthesesothatwemayusethemtoouradvantage.Bybetterunderstanding how the plant responds to stress, we can develop more effective management practices. In the future by linking our breeding, crop production, and molecular biology programs, we will be more effective at producing resilient tools that can respond to climate variability. Compared to rice, maize and soybean, a bottleneck limiting wheat research is the lack of a complete set of “blueprints.” Currently,labsin16countrieswithintheInternationalWheatGenomeSequencingConsortium(IWGSC)areengagedindecodingthisimmensebookofblueprints.Thisknowledge will increase our capacity, effi ciency, and dimensions in dealing with complex traits, such as drought and heat tolerance. Successful decoding of these blueprints will unlock the bank and pave the way to realize the potential of the sleeping wheat germplasm. As mentionedabove,wheatgrowersinthetwenty-firstcenturyliveinexcitingtimes.

Additional information and referencesBennett, David. The Promise of Better Wheat Varieties. Delta Farm Press, December 28, 2006. Available at http://deltafarmpress.com/promise-better-wheat-varieties

Biotechnology and Biological Sciences Research Council. UK researchers release draft sequence coverage of wheat genome, news release, August 27, 2010. Available at http://www.bbsrc.ac.uk/news/archive/2010/2010-archive-index.aspx

de Candolle, Alphonse. 1884. Origin of Cultivated Plants. Edited by Paul C. Kegan, Trench & Co., London.

Dubcovsky, J., and J. Dvorak. 2007. Genome plasticity: a key factor in the success of polypolid wheat under domestication. Science 316:1862-1866.

Hogg, A.C., T. Sripo, B. Beecher, J.M. Martin, and M.J. Giroux. 2004. Wheat puroindolines interact to form friabilin and control wheat grain hardness. Theor Appl Genet 108(6):1089-97.

International Wheat Genome Sequencing Consortium (IWGSC). Available at http://www.wheatgenome.org

International Wheat Genome Sequencing Consortium. Signifi cant work still needed to really crack wheat’s genetic code, news release, September 9, 2010. Available at www.wheatgenome.org/News-and-Reports/News/Signifi cant-Work-Still-Needed-to-Really-Crack-Wheat-s-Genetic-Code

AcknowledgementsSupport was provided by SDSU, South Dakota 2010 Initiative through the SD Drought Tolerance Center, USDA-AFRI, and the SD Agricultural Experiment Station.

Rushton, P, W. Li, and S. Hansen. 2012. Using molecular biology for management and genetic enhancements. In Clay, D.E., C.G. Carlson, and K. Dalsted (eds). iGrow Wheat: Best Management Practices for Wheat Production. South Dakota State University, SDSU Extension, Brookings, SD.

Figure 31.3. Fusarium head blight and rust are two diseases that researchers are battling through molecular means. (PhotocourtesyofMaryBurrows,MontanaStateUniversity,Bugwood.org)