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Functionalizing the StrawberryGenome—A ReviewKevin M. Folta aa Horticultural Sciences Department and the Graduate Programin Plant Molecular and Cellular Biology, University of Florida,Gainesville, Florida, USA

To cite this article: Kevin M. Folta (2013): Functionalizing the Strawberry Genome—A Review,International Journal of Fruit Science, 13:1-2, 162-174

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International Journal of Fruit Science, 13:162–174, 2013Copyright © Taylor & Francis Group, LLCISSN: 1553-8362 print/1553-8621 onlineDOI: 10.1080/15538362.2012.698150

Functionalizing the StrawberryGenome—A Review

KEVIN M. FOLTAHorticultural Sciences Department and the Graduate Program in Plant Molecular

and Cellular Biology, University of Florida, Gainesville, Florida, USA

A draft sequence of the diploid strawberry was released to the publicearly in 2011. This milestone was achieved by an internationalteam of experts exploiting the newest advances in sequencing tech-nology and bioinformatics. While an excellent resource, the accom-plishment is simply a starting point for creating links between thegenes within and the traits they control. The central thrust of manyresearch programs is to now connect genes and traits using a vari-ety of genetic, physiological, and developmental tests. Ultimately,the derivation of the parts list for a strawberry should expand theuse of this organism as a system for studying questions important tothe industry, as well as expanding our understanding of the basicscience of plant physiology and development. This review is a sup-plement to the recent release of the genome sequence, focusing onthe rationale for sequencing a strawberry genome, how it will beput to work, and some of the attributes of strawberry that make itan attractive system to answer questions in plant biology.

KEYWORDS Fragaria, physical maps, sequencing, next-generation, Roche 454, F. vesca, F. × ananassa, molecularmarkers

INTRODUCTION

A Long Sequence of Events to a Short Sequence

In January of 2008, our grasp of the molecular underpinnings of straw-berry traits changed dramatically. Molecular-genetic research in strawberry

Address correspondence to Kevin M. Folta, Horticultural Sciences Department and theGraduate Program in Plant Molecular and Cellular Biology, 1301 Fifield Hall, P.O. Box 110690,University of Florida, Gainesville, FL 32611, USA. E-mail: kfolta@ufl.edu

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had been gaining momentum for a decade, owing to development of agiletransformation systems (Haymes and Davis, 1998; Mezzetti and Costantini,2006; Nehra et al., 1990; Oosumi et al., 2006; Slovin et al., 2009), gener-ation of increasingly complex genetic maps (Davis and Yu, 1997; Sargentet al., 2004, 2006; Spigler et al., 2010), and growing genomics-level resources(Aharoni et al., 2000; Davis et al., 2010; Folta et al., 2010; Pontaroli et al.,2009). Elegant studies examined molecular processes in strawberry fruits rel-evant to important traits, like flavor (Aharoni et al., 2000, 2004) and softening(Pombo et al., 2009; Rosli et al., 2004; Spolaore et al., 2003; Woolley et al.,2001). This experimental progress was achieved in the absence of wholegenome information, meaning that any molecular laboratory endeavor hadto start with procuring gene sequence. This primary characterization alonecould take weeks to months, and in some cases it was not possible at all. Thenew millennium brought with it new means to capture substantial genomesequence through methods that were relatively inexpensive, and sequencingthe strawberry genome shifted to high on the priority list for the strawberryresearch community. At the North American Strawberry Symposium in 2007,it was a lofty yet desperately desired objective.

In the era of new high-throughput sequencing, application of these tech-nologies to generate strawberry genome sequence information made perfectsense. Strawberry is among the smallest plant genomes, certainly amongplants directly related to valuable crops. The diploid strawberry genome iscomposed of approximately 240 million base pairs of DNA (Mb), quite smallwhen compared to other crops, such as rice (∼430 Mb), tomato (950 Mb),onion (∼15,000 Mb), or pine (21,658 Mb). A small genome means lessgenetic real estate to decipher, so in this sense the strawberry genome wasan extremely attractive technology target. However, strawberry lacked funda-mental resources that would enable a successful genome sequencing effort,such as dense linkage maps, physical maps, and large-sequenced genomicfragments from BAC libraries. The only resource approaching a hard genomicsequence starting point was the report of 1% of the Fragaria vesca genome(a diploid species), from 30–50 kb pieces from Sanger sequencing (Daviset al., 2010; Pontaroli et al., 2009). The sequences were present in fosmids,bacterial plasmids engineered to contain large pieces of strawberry DNA.The fosmids sequenced were selected randomly in about half the cases(Pontaroli et al., 2009), while the other half was comprised of fosmids bearingsequences likely to influence flowering time, disease resistance, fruit devel-opment, or other traits important to horticulture (Davis et al., 2010). Whilehaving 1% of the genome in hand was an important step in understand-ing the fundamentals of strawberry genome structure and gene arrangement,the lack of hard physical maps made a larger effort less promising. On theother hand, peach and apple, strawberry relatives in the family Rosaceae,possessed well developed maps that would allow assembly and orientation

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of next-gen sequencing scaffolds. These resources made the likelihood ofsuccess much higher in these species, so sequencing resources and inter-est were diverted away from strawberry and over to its valuable tree croprelatives.

Time, technology, and a bold proposal would ultimately conspireto raise interest in strawberry genome sequencing. At the 2008 Plant-Animal Genome meeting in San Diego, California, a group of rosaceouscrop researchers met for an annual meeting. Those in attendance heardupdates on the status of apple and peach sequencing. Late in the session,Drs. Vladimir Shulaev and Richard Veilleux of Virginia Tech proposed a chal-lenge to those in the room. Shulaev and Veilleux had forged an arrangementwith Virginia Tech Bioinformatics Institute to sequence the diploid strawberry(Fragaria vesca) using a newly-acquired piece of equipment, the Roche-454 Sequencing Platform (Margulies et al., 2005). According to the plan,VBI would provide the service at a discounted rate in conjunction withRoche-454, the company supplying the reagents. For Roche-454 it was anopportunity to test the mettle of their technology, showing that their short-read sequencing platform and software could generate large assemblies ofcontiguous sequence from plant genomes. Shulaev and Veilleux were look-ing for buy-in from a hungry strawberry genomics community. Stopping justshort of passing a hat around the table for contributions, the quest for astrawberry sequence was set in motion. A formal attempt to sequence thestrawberry genome would be made with a core of informatics expertise, anew technology, a community of diverse talents—and absolutely no singlesource of substantial funding.

Financial support and intellectual contributions eventually trickled infrom around the globe and the amount of raw sequence grew exponentially.Industry, government, and university labs brought funds to the effort. Manyresearchers joined from other plant systems, seeing the utility of a strawberrygenome in their own studies. The project gained the attention of recognizedcomputational experts that looked at strawberry as a new frontier to applytheir expertise. Other scientists sought to guide writing and submission forpublication. Over the next 2 years and countless conference calls, a draftsequence of the strawberry genome was submitted for publication, findingfavor at Nature Genetics (Shulaev et al., 2011).

It was interesting to observe the spectrum of responses heard upon finalword of manuscript acceptance. While computational biologists exclaimed,“Cheers! Now we’re done!” the biologists in the consortium ignited with,“Cheers! Now we can begin!” The varied response represents two uniqueperspectives on a published genome. It is analogous to the disparity inattitude between the workers that plan and build the playground and thechildren that want to play in it. Many of us took off our work hats—now itwas our time to play.

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The F. vesca Sequencing Strategy

The cultivated strawberry is known as Fragaria × ananassa. The “×”reminds us that the modern commercial strawberry is a hybrid of two wildspecies, one from North America and one from Chile in South America. Thesetwo species first crossed in the royal botanical gardens in Versailles, Francein the mid-1700s, making the dessert strawberry a relatively recent species(Darrow, 1966). F. × ananassa does not contain a simple genome (Bringhurstet al., 1966; Rousseau-Gueutin et al., 2008; Senanayake and Bringhurst, 1967).It is the fortunate victim of a chance trick of the plant world—polyploidy (forreview: Wendel, 2000). Whereas the cells of diploid organisms contain twocopies of every gene (typically one maternal and one paternal), polyploidscontain additional copies, residing on an entirely separate set of chromo-somes. These may occur from doubling of genetic materials or possibly fromabnormal production of gametes resulting in atypical doses of genetic mate-rial passed from pollen or ovule (Islam, 1960). F. × ananassa contains acomplex genome (reviewed in Folta and Davis, 2006), composed of foursubgenomes (one subgenome would be diploid, four subgenomes result inan octoploid). This arrangement makes sequencing and assembly of the cul-tivated strawberry almost impossible (at least at this point), due to constraintsof the sequencing and assembly strategy (next section). Instead, a simplerrelative of the octoploid strawberry, F. vesca, was chosen. F. vesca containsa simple, diploid genome, but more importantly it shares a close commonancestor with at least one of the four subgenomes of octoploid strawberry.In this way, F. vesca makes an appropriate selection for sequencing as manysequences will be identical, while the differences will tell us something aboutstrawberry history and evolution.

The diploid strawberry was sequenced using “next generation” sequenc-ing technology. The concept is simple. Using new technology it is easy andinexpensive to obtain short runs of DNA sequence, on the order of 30 to500 bases. These short runs of sequence (reads) are gathered by the millions.The reads are then compared to each other computationally, aligning themand stacking them by shared sequence to derive a consensus sequence.An analogy of this process is shown in Figure 1. Imagine that someonewanted to resolve the sequence of the letters of the alphabet—but it wasunknown. A next-generation sequencing approach would produce a few let-ters at a time in the correct order (Figure 1A; ABCD, CDEF, TUVW, etc). Whenmany of the short sequences are compared and aligned against each other(Figure 1B) it becomes possible to determine an entire consensus sequencefrom end-to-end (Figure 1C) with great confidence.

This method is quite suitable for use on F. vesca, a plant with a smalland simple genome. Whereas many genomes are cluttered with repeatedinformation, strawberry parks its valuable genetic information into discreteparcels that could be sequenced and assembled. The median scaffold (large

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FIGURE 1 The theory of next-generation, short-read sequencing, and assembly as appliedto the alphabet: Relatively short runs of letters are obtained where the letters appear in thecorrect order (A). The letters are computationally aligned based on common features (B).A consensus sequence is derived (C). The same process was used to sequence the strawberrygenome, only using millions of fragments that were between 76 and ∼400 bp in length.

contiguous genomic sequence) size was over 1.3 million bases long, demon-strating that the small runs produced via short read sequencing couldbe constructed into substantial assemblies of contiguous strawberry genes.Innovative methods were then used to order and orient the scaffolds usingthe Fragaria diploid linkage map (Sargent et al., 2011). This feat was a sub-stantial accomplishment as it allowed the long scaffolds to be ordered in amanner that reflects their true physical position relative to one another, inessence, assembling them into their actual positions on the chromosomes.

The Parts of the Strawberry-Making Machine

Any sequenced genome is simply a parts list. It is a comprehensive account-ing of the components that make up the genetic basis of the organism andthe elements that control their expression and activity. Strawberry is no dif-ferent. Successful sequencing and assembly of the strawberry genome nowprovides researchers with a solid starting point to begin functional inquiry asto how the various genetic elements influence each other and work togetherto affect the biology of seeds, plants, and ultimately the fruit product in theclamshell that entices the consumer’s senses.

The strawberry plant may be thought of as a factory, a factory thattakes water, sunlight, carbon dioxide, and a pinch of minerals to assemblea desirable product. If you want to understand the product and how tomake it better, cheaper, or faster, you need to understand the mechanicsof the factory at a nuts-and-bolts level. This level of understanding comesquite quickly if you have the blueprints. Blueprints show you how partsare assembled and interact. Unfortunately, blueprint-level resolution of the

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strawberry is still decades in the future. But we do have that parts list. Theparts list contains all of the raw information about the plant; it is the researchcommunity’s job to experimentally link the parts together into pathways,networks, and webs that ultimately will build that needed blueprint.

The Visible Genome

As presented, the diploid strawberry genome is not just a long string ofletters that define the genes of strawberry. Instead, it is viewable in an anno-tated and dynamic format that is freely available to any interested user on theinternet, at www.strawberrygenome.org. At this website, one can explore thegenome, either on substantial pieces of chromosomes or one gene at a time.Most importantly, the genome browser hosted by Plant and Food Researchin New Zealand provides a means to compare strawberry gene sequences tothose of grape, tomato, or Arabidopsis. It shows where genes are predicted tooccur, even if there is no previous evidence of such a gene’s function, provid-ing new areas for research into strawberry-specific sequences. The genomebrowser also allows a user to visualize if a gene is expressed, as evidencedby the existing RNA transcripts that correspond to the gene sequence. Manyother features of the genome sequence may be observed, such as the pres-ence of potential molecular markers and comparisons to other strawberryspecies. These tools permit anyone to explore strawberry gene space, andcompare the parts of strawberry to those of other important species.

Major Categories of Important Parts

The strawberry genome contains genes that are similar to genes identi-fied in other plants, such as Arabidopsis or rice, that are known to beinvolved in processes interesting to the farmer and/or the biologist. Theseare enumerated in detail in the Supplemental Materials presented by Shulaevet al. (2011). Of the major classes of genes uncovered are those relatedto flavors and aromas (Aharoni et al., 2000, 2004; Lunkenbein et al., 2006;Raab et al., 2006). Many of these have been previously characterized,such as quinone oxidoreductase, alcohol dehydogenases, alcohol acyl trans-ferases, O-methyltransferases, and various terpene synthases. While previousresearch has implicated these genes in the production of specific flavor com-pounds, examination of genome sequence reveals multiple family members.These various gene versions may catalyze production of other flavor com-pounds or possibly are expressed in only particular parts of the fruit orplant.

Many genes associated with softening and cellulose degradation (Dottoet al., 2006; Jimenez-Bermudez et al., 2002; Rosli et al., 2004, 2009;Woolley et al., 2001), such as pectate lyases, β-galactosidases, chitinases,

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and β-glucanases, are among those found in the genome. Most impor-tantly, these genes also appear to be found in families of closely-relatedmembers, suggesting that various members may have discrete roles thatmay change throughout development. Other genes affecting fruit qualitylikely play a role in fruit expansion in response to auxin secreted from theachenes (Manning, 1994; Nam et al., 1999). Still, others likely control pig-mentation and the development of secondary compounds that make fruitsattractive to the eye and possibly provide benefits to human health (Seeram,2008).

Another class of genes is known to encode proteins with roles in plantdefense to pests or pathogens (Dong, 2004; Tor et al., 2009). The familiargene sequences, such as Npr1, the Pathogenesis Related gene family, var-ious WRKY-motif factors, and NB-LRR proteins, are often found in familieswhere individuals may direct resistance to various strawberry diseases. Genesplaying a role in hormone synthesis and sensing (GID1, TIR1), as well asthose involved in light sensing (cryptochromes, phytochromes) and flower-ing (Constans, Flowering Locus T ) are present, showing parallels with otherplant systems. Functional tests in plants will permit description of the rolesof these genes in strawberry and allow scientists to compare and contrastthese genes with those described in other systems.

While the function of many of the genes predicted may be easily inferredfrom what is known in other plant systems, some predicted strawberrygenes defy intuitive functional annotation. When compared against otherplant genes, these sequences report back as “unknown,” “hypothetical,” or“predicted” with no described function. Others are unique to strawberry; thegenes that make strawberry a strawberry. Now that they have been identifiedthey can be tested using transgenic technologies, defining their specific rolesin strawberry biology.

How Will the Sequence Impact Consumers?

There is an undeniable demand for perfect, large, fresh, strawberry fruitsavailable throughout the year. The first gains that will come from a sequencedgenome will be from identification of molecular markers, easily traceableDNA patterns that associate with a trait of interest, complementing the cur-rent set available in strawberry (Whitaker, 2011). For example, it is possible toamplify a short piece of DNA in plants that tend to be resistant to anthracnosefruit rot, caused by Colletotrichum acutatum (Lerceteau-Kohler et al., 2005).Plants containing this fragment are resistant to the disease, whereas suscep-tible plants fail to permit amplification. In other words, this small stretch ofDNA is likely to be physically proximal to a gene or a chromosome segmentthat confers resistance. The distillation of the genome sequence providesresearchers with a starting point to identify genes potentially linked to animportant horticultural process. The same genes may be examined for other

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germplasm for variation that can be used and easily traced in breeding.In the future, such markers will be developed for resistance to other diseaseslike Verticillium wilt, photoperiodic flowering habits, or flavor compoundcontent.

The advantage to using markers is that seedlings may be screened to testfor candidates likely to possess a series of markers consistent with favorablehorticultural qualities. Right now a strawberry breeder may screen tens ofthousands of plants to find just one that has a favorable combination ofhigh yield, large fruits, resistance to disease and pests, insensitivity to abioticstress, and, to some extent, acceptable flavor. Such searches require acres ofmanicured fields, complete with mechanisms to fertilize, water, and protectplants. Finding a new variety is labor intensive and expensive—in dollarsand time.

Imagine if a breeder could make a cross between two elite strawberryplants and then screen the offspring at the seedling stage. A single snippetfrom a juvenile leaf would provide enough DNA to screen for hundreds ofmarkers associated with favorable traits. Such technologies might assist abreeder to narrow 10,000 seedlings down to the 100 most likely to exhibitimproved traits. Not only would this save tremendous resources in labor,fuel, water, land, and time, it also would permit a breeder to test largernumbers of candidates known to possess a higher likelihood of becominga new elite variety. This process of identifying the genetic variants that maybe elevated to useful molecular markers is greatly facilitated by capture of awhole genome sequence.

So Now What?

The derivation of a strawberry genome sequence enables accelerated studyon two levels—findings that will benefit farmers and consumers, and findingsthat will advance our understanding of processes that control plant growthand development. Whether it is fruit size, disease resistance/susceptibility,fertilizer requirements, stress tolerance, or fruit flavor, every trait in a straw-berry is shaped by contributions from at least one gene, but usually manygenes. The challenge before the strawberry research community is to nowconnect the 34,809 genes in the new genome sequence with the traits thatthey support. While obtaining and describing the strawberry genome tookover 70 scientists a few years, even a cursory characterization of strawberry’smany genes will easily consume the next decade. This ambitious timeline ispredicated on increasing interest from a wider plant-science community, asexpertise from physiology, biochemistry, and molecular biology will need tobe pooled to exploit this new resource.

It should not be a problem to generate substantial interest in the straw-berry system. As funding agencies focus on translational systems to advancefindings from models to plants of agricultural import, diploid strawberry

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plugs the chasm between the model plant Arabidopsis thaliana and large-format rosaceous tree crops. The diploid strawberry has many attractiveadvantages over other experimental plant systems:

● It may be grown, seed to seed in 10-cm pots.● It may be propagated sexually as well as by branch crowns or runners.● There is substantial transcriptome coverage.● It is a perennial, unlike other models.● Strawberry cycles from seed to seed in 12–16 weeks.● Construction of transgenic lines is possible.● It is possible to suppress transcripts or overexpress them, allowing for loss-

and gain-of-function studies.● Hundreds of plants may be grown on a single greenhouse bench.● It has a tiny, sequenced genome!

New genetic and genomic tools and resources are being devised and willgreatly enhance the resources available for scientific inquiry. Such resourcesinclude collections of mutants:

● Production of T-DNA insertion lines: Almost random disruptions in genescan be identified by a few simple steps to help researchers identify plantslacking function in genes of interest (Oosumi et al., 2010; Ruiz-Rojas et al.2011).

A generation of activation-tagged lines or plants containing transgenes thatactivate expression from proximal genes will allow researchers to identifygenes associated with an important process by turning on their expressioninappropriately, possibly beaconing their activity.

● EMS populations: Several are in development. These plants feature randomchemically-induced mutations throughout the genome, producing plantsthat feature abnormalities in important traits. These traits may be dissectedback to the gene using genetics and/or sequencing techniques.

Lines that are homozygous for most genes:

● Recombinant-inbred lines for notable crosses: These lines will help toidentify genes associated with traits of interest that are segregating inestablished populations.

● Inbred lines for transgenic use: These lines allow the production of trans-genic plants in backgrounds that are genetically homogenous and stable,allowing more reliable and consistent presentation of phenotypes (e.g.,Slovin et al., 2009).

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These are just a few of the important new plant genetic resources developedin the past few years. All of these inherent qualities and enabling resourcestruly render strawberry as a superior system to analyze processes that mayfurther illuminate our understanding of plant biology. More importantly, thestrawberry is a transitional system into all species within the Rosaceae family.Its rapid growth, large seed set, transformability, and small stature are alltraits shared between strawberry and Arabidopsis, the white lab mouse of theplant world. Studies in strawberry may rapidly advance the understanding ofprocesses in apples, pears, roses, cherries, peaches, and other valuable cropsthat require more time and space to make research gains. Strawberry shouldserve as an important intermediate in many research schemes, and may makelofty research ideas a bit more tractable and less risky than working solely intree crops or brambles.

Strawberry also adds to the understanding of how fruits grow anddevelop. Strawberry is an unusual fruit. In fact, botanically speaking it is nota fruit at all, but rather an accessory fruit. The valuable red “berry” is reallyan enlarged, fleshy receptacle, dotted with achenes that are the true fruits.How is this “berry” different developmentally from tomatoes, grapes, kiwis,or other important berries? How do the genes and gene expression patternsdiffer between raspberries, roses, peaches, and apples? The successful addi-tion of a strawberry sequence to the body of sequenced genomes providesanother important node of comparisons from an unusual plant that bears anunusual fruit.

CONCLUSIONS AND PERSPECTIVES

From the perspective of a scientist that was intimately intertwined in thestrawberry genome process from the day that Drs. Veilleux and Shulaev pre-sented their proposal to the day that the final proofs magically materializedon the computer monitor, the whole genome sequence represents a startingpoint much more than an end. This sentiment is shared across the strawberryresearch community as well as the industry it serves. The draft sequence isa significant step that will enable researchers from other plant systems tolend their expertise to answering questions in strawberry. With this resource,there will be increased interest in the strawberry as a crop as well as anexperimental system to study plant biology. Together, the enabling technol-ogy of a genome sequence will put a better product on the grocery storeshelf and expand the scope of plant biology textbooks.

ACKNOWLEDGMENTS

This work represents the extensive contributions made by members of theInternational Strawberry Sequencing Consortium—financial, intellectual, and

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experimental. Whole-genome sequencing of strawberry would not havebeen possible without the primary vision and actions of Vladimir Shulaevand Richard Veilleux, in conjunction with Virginia Bioinformatics Institute atVirginia Tech.

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