Structural genomics in Fragaria x ananassa Denise Cristina Manfrim Tombolato

1. Structural genomics in Fragaria x ananassaDenise Cristina Manfrim Tombolato

2. Challenges during strawberry production • Diseases - Florida: anthracnose • Pests

3. Challenges for Florida strawberry production • Diseases • Pests • Phase-out of methylbromide • Market competition/consolidation

4. One Remedy = Plant improvement • Traditional strawberry breeding • Desirable fruit quality (appearance, size, firmness, flavor) • Open-plant habit (for efficient harvesting and pesticide application) • Disease/pest resistance • Precocious, prolific cultivars • Molecular approaches • Structural genomics • Marker-assisted selection • Functional genomics • Analysis of gene function • Transgenic plants

5. One Remedy = Plant improvement • Traditional strawberry breeding • Desirable fruit quality (appearance, size, firmness, flavor) • Open-plant habit (for efficient harvesting and pesticide application) • Disease/pest resistance • Precocious, prolific cultivars • Molecular approaches • Structural genomics MOLECULAR MARKERS • Marker-assisted selection • Functional genomics • Analysis of gene function • Transgenic plants

6. Uses of Molecular Markers • Genetic characterization of plants and animals • Conservation of germplasm • Insights on evolution • Proprietary issues • Marker-assisted selection • Linkage maps • Map-based positional cloning • Comparative mapping • Detection of chromosome rearrangements between related taxa

7. Molecular Markers in Strawberry • Isozymes • Cultivar identification • Arulsekar et al. 1981 • Bringhurst et al. 1981 • Nehra et al. 1991 • Bell and Simpson, 1994 • Linkage mapping • fruit color & shikimate dehydrogenase isozyme locusWilliamson et al. 1995 • runnering & Pgi-2 isozyme locusYu & Davis1995 • Intron length polymorphism • Primers specific for genes in the anthocyanin biosynthesis pathwayDeng and Davis 2001

8. Molecular Markers in Strawberry • Randomly Amplified Polymorphim DNA (RAPD) • Cultivar identification • Gidoni et al. 1994 • Hancock et al. 1994 • Levi et al. 1994 • Parent & Page, 1995 • Graham et al. 1996 • Landry et al., 1997 • Harrison et al. 1997 • Degani et al. 1998 • Degani et al. 2001 • Linkage mapping in the diploid F. vesca • Davis and Yu, 1997 • Marker-assisted selection • Rpf1 = resistance gene to race 1 of Phytophthora fragariae Haymes et al. 1997 • Rpf2 = resistance gene to race 2, Van de Weg 1997

9. Molecular Markers in Strawberry • Inter Simple Sequence Repeat (ISSR) • Cultivar identification Arnau et al. 2003 • Marker-assisted selection to seasonal flowering • SCAR markers, derived from ISSR Albani et al. 2004 • Sequence-Characterized Amplified Region (SCAR) • Davis et al. 1995 • Haymes et al. 2000 • Albani et al. 2004 • AFLP • Cultivar identification Degani et al. 2001 • Linkage mapping in F. x ananassaLerceteau-Kohler et al. 2003

10. Molecular Markers in Strawberry • Simple Sequence repeat (SSR) • Diploids • F. viridis Sargent et al. 2003 • F. vesca James et al. 2003 • F. vesca Hadonou et al. 2004 • F. vesca x F. nubicola map Sargent et al. 2004 • F. vesca , transferable to other Fragaria Hadonou et al. 2004 • F. vesca + 5 other diploids Monfort et al. 2005 • Octoploids • F. x ananassa Nourse et al. 2002 • F. virginiana Ashley et al. 2003 • F. x ananassa Monfort 2005

11. Published Strawberry Maps • Diploid F. vesca • RAPD Davis and Yu, 1997 • SSR Sargent et al. 2004 • Octoploid • AFLP Lerceteau-Köhler et al., 2003

12. Published Strawberry Maps • Diploid F. vesca • RAPD Davis and Yu, 1997 • SSR Sargent et al. 2004 • Octoploid • AFLP Lerceteau-Köhler et al., 2003 Need for a robust, transferable marker for the octoploid, cultivated strawberry!

13. History of strawberry 1759 “F. ananassa” 1765 Duchesne F. ananassa’s parents 1714

14. Objetives • Characterize 35 gene-pair haplotype loci • Sequence polymorphic alleles • Test feasibility to establish relationships between diploid versus octoploid alleles • Develop a fingerprint strategy for strawberry cultivars • Establish a DNA extraction protocol for strawberry • Research phylogenetic relationships between strawberry cultivars by plastid markers

15. 8-ploid strawberries 1600’s chiloensis virginiana iturupensis

16. Octoploid strawberries 1759 “F. ananassa” 1765 F. ananassa’s parentage proposed chiloensis virginiana 1714

17. gracilis daltoniana vesca nilgerrensis nubicola viridis iinumae mandshurica yezoensis moupinensis 4x nipponica orientalis 4x pentaphylla moschata 2x,4x, 6x strawberries 2x 6x 4x

18. vesca nilgerrensis nubicola viridis iinumae mandshurica Diploids used in this study 2x

19. The cultivated strawberry, Fragaria x ananassa, is an octoploid (2n=8x=56) Genomic complexity: AAA’A’BBB’B’ (Bringhurst, 1990) Small basic (x=7) genome: C < 200 Mb

20. Structural genomics in Fragaria x ananassaDenise Cristina Manfrim Tombolato • DNA extraction from a recalcitrant plant species • Gene-Pair Haplotypes: novel, complex markers for development of linkage association in a polyploid • Development of a fingerprinting strategy for cultivated strawberry • Phylogenetic relationships between strawberry cultivars revealed by hypervariable plastid markers

21. Possible Remedy = Cultivar improvement • Traditional strawberry breeding • Linkage mapping in the octoploid background is difficult • Molecular approaches Structural genomics Molecular markers for MAS

22. Strawberry Genomics at UF Functional genomics Structural genomics Molecular markers for MAS Gene discovery and analysis of expression Assessment of gene function in transgenic strawberry

23. Strawberry Genomics at UF Functional genomics Structural genomics Molecular markers for MAS Gene discovery and analysis of expression Assessment of gene function in transgenic strawberry

24. DNA extraction from a recalcitrant plant species Plants reported as recalcitrant to DNA extraction • Cotton • Sugarcane • Tomato • Grapevine • Conifers • Chestnut rose • Strawberry

25. DNA extraction protocols • 1953: Jones observed differential precipitation of CTAB • high ionic strength solutions (>0.7M NaCl), CTAB complexes with proteins and basic and neutral polysaccharides • low ionic strength, CTAB precipitates nucleic acids and acidic polysaccharides, leaving proteins and neutral sugars in solution • 1980: Murray and Thompson • lyophilized plant material • 1987: Doyle and Doyle • fresh plant material • more concentrated buffer to compensate for water in tissue

26. My 30 attempts to extract DNA • Fresh and lyophilized tissue • Tissue-to-buffer ratio: • from 1.5mg/ml to 500mg/ml

27. My 30 attempts to extract DNA • Murray and Thompson, with no CsCl gradient • Precipitation by • differential ionic strength • addition of alcohol at high ionic strength • [CTAB]: 1-6% • Pre-extraction buffers containing sorbitol and/or PEG • Guanidine thiocyanate, with and without CTAB • DNAzol kit • Urea • Differential precipitation by butoxyethanol • Isolation of nuclei prior to DNA extraction

28. Nuclei isolation

29. DNA extraction following nuclei isolation • SDS • 1% • 2% • 5%, with TIPS and PAS • CTAB • 2% • Qiagen Kit • Guanidine thiocyanate

30. Deproteination of DNA • Phenol:chloroform • Kirby, 1957 • Sodium perchlorate • Wilcockson, 1973

31. Results • Mostly low to moderate yields, including post-nuclei isolation methods • High DNA yields = non-digestable DNA • Guanidine thiocyanate protocols • Butoxyethanol

32. Results

33. Results

34. Results a undigested and supposedly digested samples had smeary aspect, though with most DNA as high molecular weight, in 0.8% agarose gel b no amplicons observed after PCR with primers for 18S ribosomal DNA c undigested and allegedly digested samples showed a single high molecular band in 0.8% agarose gel d determined by spectrophotometer reading of absorption at 260nm; values are a linear extrapolation of milligrams of tissue in fact used for extraction e tissue-to-buffer ratios were 15mg/ml and 1.5mg/ml f newly expanded and non-expanded leaflets, respectively g quantification in agarose gel did not reflect spectrophotometer reading h loading dye ran to negative pole

35. Results

36. Results Precipitation by differential ionic strength versus ethanol

37. Future attempts - Manning protocol • Extraction buffer • Boric acid (BH3O3) to adjust pH of Tris buffer • At pH 7.6, boric acid complexes with polyphenols and carbohydrates • DNA Precipitation by butoxyethanol • 40% volume butoxyEtOH precipitates sugars • Equal volume butoxyEtOH precipitates DNA & RNA • [Na] = 80mM

38. Future attempts - Manning/CTAB/Guanidine • Murray and Thompson and guanidine protocols with precipitation by butoxyethanol? • Test different Na concentrations?

39. Gene-Pair Haplotypes: novel, complex markers for development of linkage association in a polyploid

40. Uses of Molecular Markers • Genetic characterization of plants and animals • Conservation of germplasm • Insights on evolution • Proprietary issues • Marker-assisted selection • Linkage maps • Map-based positional cloning • Comparative mapping • Detection of chromosome rearrangements between related taxa

41. Mapping in a polyploid background Autoploids Banana 3n=33 Alfalfa 2n=4x=32 Corn 2n=4x=20 Sowerbaea In an alloploid plant, the number of possible genotypes for one locus with eight different alleles is

42. Mapping in a polyploid background Autoploids Alloploids Banana 3n=33 Alfalfa 2n=4x=32 Corn 2n=4x=20 Sowerbaea Wheat 6x(Kam-Morgan et al. 1989) Oat 6x(O’Donoughue et al., 1992) Sugarcane 2n=100 to 130(Arruda, 2001) In an alloploid plant, the number of possible genotypes for one locus with eight different alleles is

43. Mapping of strawberry • Diploid Fragaria vesca Davis and Yu, 1997 • SSR markers added to map Sargent et al., 2004 • Octoploid, AFLP markers Lerceteau-Köhler et al., 2003

44. Markers/Maps in strawberry • Linkage: fruit color & Sdh isozyme locus Williamson et al. 1995 • F. vesca ‘Alpine’ varieties • Yellow Wonder x Baron Solemacher • Linkage: runnering & Pgi-2 isozyme locus Yu & Davis1995

45. Rpf1 = resistance gene Haymes et al. 1997 • RAPD, bulked segregant analysis • Gene-specific primers for proteins involved in the anthocyanin biosynthesis pathway and linkage to ‘c’ locus Deng and Davis 2001 • Northern California F. vesca x F. vesca ‘Alpine’ YW • F. vesca ‘Alpine’ YW x F. nubicola • PCR-amplified intron length polymorphism of candidate genes • SSR octoploid Nourse et al. 2002 • SSR F. virginiana Ashley et al. 2003 • SSR F. vesca James et al. 2003 • SSR F. viridis Sargent et al. 2003 • SSR from vesca to 8 vescas, ananassa, nubicola, mandschurica, viridis, iinumae, nilgerrensis Monfort 2005 • 2003 Arnau et al. Inter Simple Sequence Repeat (ISSR) to ID cultivars • ISSR-derived SCAR markers Albani et al. 2004 • SSR in Fragaria Hadonou et al. 2004 • SSR F. vesca Hadonou et al. 2004 • SSR Lewers et al. 2005 • SCAR Haymes et al. 2000 • SCAR Albani et al. 2004 • SCAR Davis et al. 1995

46. Markers in strawberry • For cultivar identification • Isozymes • Arulsekar et al. 1981 • Bringhurst et al. 1981 • Nehra et al. 1991 • Bell and Simpson, 1994 • RAPD • Gidoni et al. 1994 • Hancock et al. 1994 • Levi et al. 1994 • Parent & Page, 1995 • Graham et al. 1996 • Landry et al., 1997 • Harrison et al. 1997 • Degani et al. 1998 • Degani et al. 2001 • AFLP • Degani et al. 2001

47. Strawberry Linkage Maps • First strawberry linkage map Davis and Yu 1997 • RAPD markers, including codominant ones • F. vesca ‘Alpine’ Baron Solemacher x WC6 (wild, NH) • Gene-specific primers for proteins involved in the anthocyanin biosynthesis pathway Deng and Davis 2001 • AFLP Lerceteau-Kohler et al. 2003 • Microsatellite Sargent et al. 2004 • F. vesca f. semperflores x F. nubicola • SSR markers from a gDNA library enriched for SSR • Library enrichment Edwards, 1996: • Hybridize synthetic SSRs to a membrane • Restriction-digest gDNA • Attach adaptors to DNA cut ends • Hybridize • Elute bound DNA • PCR-enrich DNA fragments by using primers for the adaptors

48. Phytophthora fragariae red stele root rot • 5 race-specific plant resistance genes • 5 avirulent genes • Interact in gene-for-gene system • Resistance gene Rpf1 shown to segregate monogenically by use of RAPD markers linked to Rpf1 Haymes 1997

49. F. vesca linkage map

50. AFLP linkage map of the female parent AFLP linkage map of the male parent