STRUCTURAL GENOMICS OF Fragaria—WILD AND CULTIVATED .

STRUCTURAL GENOMICS OF Fragaria—WILD AND CULTIVATED STRAWBERRIESByDENISE CRISTINA MANFRIM TOMBOLATOA DISSERTATION PRESENTED TO THE GRADUATE SCHOOLOF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENTOF THE REQUIREMENTS FOR THE DEGREE OFDOCTOR OF PHILOSOPHYUNIVERSITY OF FLORIDA20071

2007 by Denise Cristina Manfrim Tombolato2

To:- my father Vadir Tombolato, who has taught me the importance of moral integrity;- my mother, Marlene Tombolato, who has, by example, taught me persistence;- my professor, Kevin Folta, who permitted and encouraged me to exercise those virtues.‘Yes,’ you will say, ‘but the plank is very long.’ That is true, and so if you do not have a surefoot and a steady eye, and are afraid of stumbling, do not venture down the path.Jean de Léry, in "History of a Voyage to the Land of Brazil, Otherwise Called America", 15783

ACKNOWLEDGMENTSI thank my parents Vadir and Marlene, and my brothers Eduardo and Ricardo, for theirteachings, advice, support, and, above all, for their unconditional love. Though not content withmy departure from Brazil, my family always supported my decisions. I appreciate theirconfidence in my choices and me, for it reaffirmed my personal mission in moments of doubt.I am grateful to my professor Dr. Kevin M. Folta, who accepted me as his student in analtruistic gesture, and who has been a lato sensu adviser since. I thank the members of mycommittee for the enjoyable discussions about my project and about science in general: drs. A.Mark Settles, Natália A. R. Peres, and Craig K. Chandler. I also wish to thank my laboratorycolleagues and friends drs. Philip J. Stewart and Amit Dhingra, Thelma F. Madzima, Stefanie A.Maruhnich, Jeremy Ramdial, Dawn Bies, and Maureen Clancy, as well as project collaboratorsdrs. Thomas M. Davis and Daniel J. Sargent, for DNA sequences and plant material from thegenetic linkage mapping population.Many people made special the almost-9 years I spent in Gainesville, while I pursued partof my undergraduate training and two advanced degrees. I convey my gratitude to all those whofacilitated not only my adaptation to a new country and language, but also the discovery of who Iam and of matters I learned to be truly meaningful. I recognize Welch McNair Bostick III(“McNair”), whose short life was vastly fruitful. McNair caused positive impact into the lives ofwhomever surrounded him: his wife and my friend Carmen Valero, his neighbors (includingmyself), and his colleagues. I thank him for having shown to me the importance of treasuring thetime shared with loved ones, expressing honest opinions and making a difference in society.I express my appreciation for the time and assistance granted to me by professors andtechnicians with whom I worked since my arrival to the University of Florida: Richard D.4

Berger, Terry A. Davoli, D. Pete Weingartner, Jeffrey A. Rollins, Ulla Benny, Valerie Jones,Jeffrey B. Jones, and Jerry Minsavage.I thank these individuals for the attention they have dedicated to me: Balša Terzic', SylviaMorais de Sousa, Gisele, Jens, and Gabriel Schöene, Mark D. Skowronski, Luciana C. B.Manfrim Bchir, Gustavo Ramirez, Juliana and Gustavo Astua, Aaron Hert, Botond Balogh,Abby Guerra, Ahu Demir, Petrônio Pinheiro, Ilka V. Araújo, Maggie Kellogg, Maria BeatrizPádua, Melissa Webb, Bruno Maciel, Camila A. Brito C. Paula, Luiz Augusto de Castro e Paula,Hazar Dib, Marlise Klein, Marcus Martin, Michelle Bolton, Sonja I. Parisek, Penny E. Robinson,Anne Visscher, Ricardo da Costa Mattos, Claudia Riegel, Valerie Rodriguez-Garcia Schweigert,Lisa Olsen, Jared Greenberg, Wendy Gonzalez, and David Adato. Every one of them made mylife in Gainesville a more enjoyable experience.5

TABLE OF CONTENTSpageACKNOWLEDGMENTS .4LIST OF TABLES.8LIST OF FIGURES .9ABSTRACT.11CHAPTER1STRAWBERRY AND THE GENOMICS ERA.12Introduction.12Molecular Markers for Strawberry .13The Genomics Era .142DNA EXTRACTION FROM RECALCITRANT SPECIES.16Introduction.16The DNA Extraction Procedure .16DNA Extraction from Plants .19Material and Methods .21Results.24Components of the “Strawberry Protocol”.26Optimization of the CTAB Protocol.27Leaf tissue state .27Incubation temperature and duration.28Tissue-to-buffer ratio.28Tissue maceration method.30Discussion.313PRIMARY ANALYSES OF Fragaria GENE distribution .42Introduction.42Materials and Methods .45Results.48Expressed Sequence Tags (ESTs) .49Simple Sequence Repeats (SSRs) .49Discussion.494GENE-PAIR HAPLOTYPES: NOVEL MOLECULAR MARKERS FORINVESTIGATION OF THE Fragaria ananassa OCTOPLOID GENOME.55Introduction.556

Materials and Methods .58Results.62GPH5 .63GPH23 .64GPH10 .6472E18.65Discussion.665GENE-PAIR HAPLOTYPES: FUNCTIONAL AND TRANSFERABLE MARKERSAS NOVEL ADDITIONS TO THE DIPLOID Fragaria GENETIC LINKAGEREFERENCE MAP.82Introduction.82Materials and Methods NA EXTRACTION PROTOCOLS.98DNA Extraction from Leaves.98DNA Extraction from Isolated Nuclei.101Modifications of Murray and Thompson DNA Isolation Protocol .102BIn silico ANNOTATION AND DISTRIBUTION OF Fragaria vesca GENES.106CPCR PRIMERS USED TO AMPLIFY AND SEQUENCE GENE-PAIRHAPLOTYPES.115DSEQUENCES GENERATED DURING CHARACTERIZATION OF “GENENPAIRHAPLOTYPES” .117EGENE-PAIR HAPLOTYPE INDIVIDUAL LOCI ALIGNMENTS.153Gene Pairs Detected by Microcolinearity.153Gene Pairs Detected Through Prediction from Genomic Sequence.166LIST OF REFERENCES.205BIOGRAPHICAL SKETCH .2207

LIST OF TABLESTablepage2-1Nucleic acid yields from isolation protocols. .342-2Ranking of 4 best nucleic acid extraction protocols .362-3DNA yields (µg DNA) from ten strawberry genotypes.382-4Impact of interactions between maceration methods and incubation temperatures onDNA yield and purity.383-1Number of simple sequence repeats (with a minimum of 5 repeats) observed inFragaria vesca genomic sequence.543-2Different types of dinucleotide and trinucleotide repeats observed in Fragaria vescagenomic sequence .544-1PCR primers designed for amplification of micro-colinearity-inferred putativeintergenic fragments.724-2PCR primers that allowed amplicon generation. .734-3Overview of insertions and deletions detected through alignment of all sequencedclones. .805-1PCR primer pairs and amplification conditions used in this study.945-2Fragment sizes of parental amplicons digested with restriction enzymes .958

LIST OF FIGURESFigurepage2-1Design of incubation temperatures and durations experiment.332-2Effect of incubation temperature and time on DNA yields. .372-3Effect of tissue-to-buffer ratios on DNA yields.372-4Relationships between DNA yield, tissue-to-buffer ratios, and sample amenability toamplification by PCR.392-5DNA contamination by carbohydrate (estimated by the ratio between absorbance at260nm and 230nm) and its influence on PCR outcome. .402-6Effect of interactions between maceration method and incubation temperature in theabsorbance at 220-340nm. .412-7The effect of Polytron homogenization on nucleic acid recovery. .413-1Flowchart of genomic DNA sequence annotation scheme. .523-2Diagram of two fosmid inserts of variable length, with their putative proteins andSimple Sequence Repeats (SSRs).533-3EST classes identified by homology searches between large genomic F. vescasequence and Rosaceae ESTs. .544-1An idealized GPH locus.704-2Fragaria species and their geographical locations .704-3GPH design upon comparison between strawberry ESTs and Arabidopsis database. .714-4Subset of the alignment of GPH5 octoploid and diploid clones.764-5Diagrammatic representation of alignment of full GPH23 clones.764-6EcoRI Restriction patterns observed for GPH10 clones from the octoploid‘Strawberry Festival’, indicating four different allele classes .774-7GPH10 clones, 4 alleles from the octoploid Fragaria ananassa. .774-8Subset of GPH72E18 alignment displaying SSR polymorphisms. .784-9Cladograms of F. ananassa and diploid alleles for six independent GPH loci.799

5-1Fosmid 40M11 with primers designed on exons of FGENESH-predicted genicregions.945-2Amplicon restriction patterns for GPHs 34D20 and 72E18. .965-3Gene-Pair Haplotypes assigned to linkage groups of the reference Fragaria map.9710

Abstract of Dissertation Presented to the Graduate Schoolof the University of Florida in Partial Fulfillment of theRequirements for the Degree of Doctor of PhilosophySTRUCTURAL GENOMICS OF Fragaria—WILD AND CULTIVATED STRAWBERRIESByDenise Cristina Manfrim TombolatoAugust 2007Chair: Kevin M. FoltaMajor: Horticultural ScienceThe extensive phenotypic variability and complex genetic makeup of the cultivatedstrawberry Fragaria ananassa permits advances in plant improvement, a factor breeders haveexploited to great benefit. However, the introgression of specific characters is complicated due tothe cumbersome genetics and limited knowledge of genome structure and function of genesrelevant to traits of interest. The present study represents the first genomics-level insight intostrawberry genome structure and explores the hypothesis that a new type of molecular marker,the Gene-Pair Haplotype represents a transferable marker that may hasten linkage mapping in thediploid and octoploid strawberry.My research presents the findings of four related research activities. First, an efficient andunified method for genomic DNA isolation was derived from over 100 experimental tests andconditions. Next, 1% of the Fragaria genome was sequenced and functionally annotated, using abioinformatics approach and computational tools. Over 120 kb of intergenic regions weresequenced using the Gene-Pair-Haplotype approach, allowing for some initial relationships to beformulated concerning the diploid subgenome contribution to octoploid strawberry. Finally,Gene-Pair Haplotypes were used to add a suite of alleles to the growing Fragaria linkage map.These findings provide a starting point for further analyses of the strawberry genome.11

CHAPTER 1STRAWBERRY AND THE GENOMICS ERAIntroductionThe cultivated strawberry, Fragaria ananassa Duch, belongs to the family Rosaceae asdo the also economically important crops rose, apple, pear, peach, cherry, plum, raspberry, andalmond. Linnaeus named the genus Fragaria due to its fragrant properties, whereas the odor,taste and berry shape was thought to be similar to pineapple, or “ananas”, in Latin (Darrow,1966). In 1765, the F. ananassa parentage was proposed by Antoine Nicolas Duchesne, whosefather worked at the Court of Louis XV (Darrow, 1966). F. ananassa was first observed inseveral countries in Europe since the 1750’s and it originated from a spontaneous hybridizationbetween F. virginiana and F. chiloensis, both from the American continent. F. virginiana isthought to have been imported to Europe by two routes (Wilhelm and Sagen, 1974): to France bythe explorer Jacques Cartier during his first expedition to the Quebec Canadian Province in 1534;and to England, by Thomas Hariot, who visited the “New Found Land of Virginia” in 1588.Later, in 1714, F. chiloensis was taken to France by the engineer Amédée François Frézier.During his mission to study the defense fortifications of Chile and Peru, Frézier noticed thelarge-fruited berries at Concepción, Chile, and collected several plants to take back to his country(Darrow, 1966). The result of the accidental cross between the two Fragaria species was thebasis for the creation of the fruit cultivated and appreciated throughout the world today.Profitable strawberry production is challenged by several factors: diseases, pests, marketcompetition, and, arguably most importantly, by the phase-out of methyl bromide. This fumigantis considered essential for the production of many crops, including strawberry (Rosskopf et al.,2005), but because methyl bromide has great stratospheric ozone depletion ability, the MontrealProtocol mandates that its use be reduced (Anonymous, 1998). Although traditional plant12