An Evolutionary Approach towards Bean Conservation – from Wild Bean to its Genome to the Field

1. Applied Plant Breeding and Cultivar Development An Evolutionary Approach towards Bean Conservation – from Wild Bean to its Genome to the Field Paul Gepts Plant Sciences, UC Davis 6oCongresoBrasileiro de Melhoramento de Plantas 1 a 4 de agosto, 2011 – Búzios, RJ

2. Empiricism in plant breeding and genetic resources conservation • Boon or bane of the field? • Highly successful • Progress from selection • Different types of inheritance • Different degrees of environmental effects • Combination and correlation of traits • Adoption of new technologies • “Cannot get no respect” • “Basic information is lacking” • “Less precise” • Response? • Examples from germplasm conservation: Adoption of wide range of approaches: How to penetrate the “Black Box”?

3. Crop Biodiversity Conservation (I) • Ex situ: gene banks: • Largest: USA: 500,000 samples; China: 390, 000; Germany: 160,000; Brazil: 150,000 (EMBRAPA 2008 data) • CGIAR gene banks • Svalbard seed vault • Many other gene banks: 1,750 individual genebanks worldwide, about 130 of which hold more than 10,000 accessions each

4. Gene Banks around the World:> 10,000 accessions State of the World’s Plant Genetic Resources for Food and Agriculture (SOTW2), 2009

5. Crop Biodiversity Conservation (II) • In situ: • Natural vegetation • Farmers’ fields and backyards • Complements ex situ • Subject to evolutionary forces • Provides a bio-cultural context • More urgency • Global environmental change

6. Outline How to penetrate the “Black Box”? • A phylogenetic/genealogical approach to understanding genetic diversity of a crop species: • The diversification of common bean (Phaseolus vulgaris) • A GIS approach the discovery and use of genetic diversity in gene banks • Example of Brasilian bean landraces • A genomic approach to discovery and transfer of genetic diversity • Development of PhaseolusGenes: Bean breeder’s toolbox for marker discovery • Comparative genomics with model experimental systems

7. What are beans and why study them? San Agustín del Pulque, MEX (2004) • Complement cereals as a source of nitrogen during cultivation • Complement cereal and root crops as a source of protein • Among the 10 foods that pack the most anti-oxidants (USDA study, 2004): Small red, red kidney, pinto beans • CompositionPlant proteins • Minerals: iron and zinc (~ meats, poultry, and fish) • Dietary fiber • Vitamins: folate (low in diets of many Americans) • Reduces breast cancer (Thompson et al. 2008) Phaseolus beans

8. How to Penetrate the “Black Box”? A Phylogenetic/Genealogical Approach to Understanding Genetic Diversity of a Crop Species: The Diversification of Common Bean (Phaseolus vulgaris)

9. Phylogeny/Genealogy of Common Bean Domestication Dissemination M G D J Gene flow Domesticated outside Center of origin Domesticated landraces in Mesoamerica Wild Mesoamerican Feral or weedy Feral or weedy Wild ECD & N. PER C P NG Domesticated outside Center of origin Domesticated landraces in Andes Wild Andean OtherPhaseolus species Multiple Sources, Several Years

10. Applications to Bean Breeding

11. Domestications Mesoamerican Andean Two major geographic gene pools • Observation: Andean and Mesoamerican gene pools • Geographic differentiation prior to domesticationGepts & Bliss 1985; Gepts et al. 1986; Singh et al. 1991a,b,c,; Becerra-Velasquez & Gepts 1984; Debouck et al. 1993; Freyre et al. 1996; Papa & Gepts 2003; Kwak & Gepts 2009 • Consequence: • Bean breeding: • 2 breeding pools, Andean and Mesoamerican • 7 races • inter-racial crosses within gene pools • For inter-gene pool crosses: Adapt breeding method to account for wider genetic distance: e.g., 1 BC

12. Reduction in Levels of Genetic Diversity • Observation: Reduction in genetic diversity • Single domestication in each gene pool • Plant breedingGepts et al. 1986; Sonnante et al. 1994 • Consequence: • Use landraces and wild germplasm in breeding • Use other Phaseolus species M13-related RFLPs

13. Breeding Strategies to Broaden the Genetic Diversity of Common Bean Kelly et al. 1998

14. Results of Gene Flow Studies in Mexico • Gene flow: • Introgression: 20-50% of wild individuals in sympatric populations • Asymmetric: Three- to four-fold higher in D  W than in W  D • Paradox: Self-pollinated species; yet, measurable effect of outcrossing • Displacement of alleles in W by alleles of D, except around genes for domestication in ~ 80 % of the genome • Implications: --In situ conservation? Complemented with ex situ conservation --Unwanted escape of genes but also strategy against escape: genetic footprint Photo: R. Papa Papa & Gepts 2003, 2004; Payró de la Cruz et al. 2004; Zizumbo-Villareal et al. 2005; Papa et al. 2007

15. Co-evolution between Common Bean and Pathogens • Observation: • Parallel geographic distribution of genetic diversity between beans and pathogens: angular leafspot, anthracnose, rust, BDMVGuzmán et al. 1995, Geffroy et al. 1999, 2000; Seo et al. 2004 • Consequence: • Facilitates breeding: • Identification of resistance • Broad representation of pathogen variation Geffroy et al. 1999

16. 1 The presumed domestication center of Phaseolus vulgaris in MesoamericaPhD thesis MyounghaiKwak (Korea) with Jim Kami

17. Phylogeny/Genealogy of Common Bean Domestication Dissemination M G D J Gene flow Domesticated outside Center of origin Domesticated landraces in Mesoamerica Wild Mesoamerican Feral or weedy Feral or weedy Wild ECD & N. PER C P NG Domesticated outside Center of origin Domesticated landraces in Andes Wild Andean OtherPhaseolus species 1 2

18. Relationship between Wild & Domesticated types in the Mesoamerican Gene Pool Also, close genetic relationship based on phaseolin protein electrophoresis (Gepts 1988) Kwak et al. 2009

19. The Suggested Domestication Center of Common Bean in Mexico M. Kwak, J. Kami & P. Gepts, Crop Sci., March 2009

20. Why the Lerma-Santiago Basin? • Climate: Cwa • Subtropical: t° coldest month: 5-18 °C • Subhumid: 4-6 months of humidity in summer • Semi-warm: average annual t°: 18-22 °C • Vegetation: • Dry deciduous forest to drier thorn forest

21. Westernmost putative domestication location, Mascota-Ameca Basin

22. Domestication Areas within Mesoamerica

23. How to Penetrate the “Black Box”? A GIS approach the discovery and use of genetic diversity in gene banks: Example of Brazilian bean landraces PhD thesis of Marilia Lobo Burle (EMBRAPA/CENARGEN) with help of M.J. del Peloso & L.C. Melo (EMBRAPA/CNPAF)

24. 2 Genetic Diversity in a Secondary Center of Origin: Brazil

25. Phylogeny/Genealogy of Common Bean Domestication Dissemination M G D J Gene flow Domesticated outside Center of origin Domesticated landraces in Mesoamerica Wild Mesoamerican Feral or weedy Feral or weedy Wild ECD & N. PER C P NG Domesticated outside Center of origin Domesticated landraces in Andes Wild Andean OtherPhaseolus species 1 2 2

26. General Approach • Maps (1:5,000,000): • Map of climate • Mean annual temperature • Mean annual precipitation • Biomes • Original vegetation • Pedology • CIAT: climatic database Latin America • Combined analysis of genetic diversity: • Molecular analysis: • Genetic relationships • Admixture • Phenotypic analysis: • Characterization: morphological and agronomic traits (UC Davis) • Agronomic traits: Yield, field resistance to CBB, rust (EMBRAPA) • Geographic information systems (GIS) • Climate • Biomes, etc.

27. Fradinho Boca Preta Brazilian Beans Macaçar pequeño Rosinha Mulatinho Bico de Ouro Jalo Carioca Bolinha Amarelo Preto Bolinha Vermelho Roxinho http://www.unifeijao.com.br/feijao_do_brasil/mapa.htm

28. Plant Materials & Molecular Markers • Plant sample: • 279 landraces • Collected by Jaime Fonseca • 1 per municipality • 2 control accessions: • BAT93 (Mesoamerican), Jalo EEP558 (Andean) • Marker sample: • 67 SSRs (Yu et al. 2000; Gaitan-Solis et al. 2002; Blair et al. 2003; Grisi et al. 2007) • 4 SCAR markers • 2 Seed proteins + 1 growth habit candidate gene

29. Molecular variation: STRUCTURE analysis Jalo EEP558: landrace; BAT93: (Veranic 2 x Tlalnepantla 64) x (Negro Jamapa x GN Tara)  

30. Molecular variation: NJ tree analysis K = 3

31. 2. Phenotypic diversity Field experiments: 281 varieties UC Davis Morphological traits: seed: pattern, color, brilliance, shape, weight leaflet: leaflet shape and length flower: color, days to flowering, … determinacy, growth habit EMBRAPA-CNPAF, Goiânia Agronomic traits: Yield Disease resistance: CBB, Rust

32. PCA of Morphological & Agronomic Traits Andean Mesoamerican Hybrid • First component: 39% • Flower color, seed weight, flower standard striping, and pod beak position • Second component: 13% • Growth habit, determinacy and number of days to flowering

33. 3. Eco-geographic variation • Biome: mainly semi-deciduous forest, pine forest • Only difference between A and M? • Altitude: ~ 100m • Yearly average T°: 23C • Average rainfall growing season: 549 mm

34. SUMMARY • Three-pronged approach to assessing genetic diversity: genetic, phenotypic, and environmental: • Reciprocal confirmation of findings • Generates hypotheses • Provides a model for large-scale characterization of germplasm collections • Availability of geo-referenced germplasm is a must • Most landraces of Mesoamerican origin; strong separation with Andean gene pool • Large “hybrid” group in Mesoamerican gene pool; may have superior adaptation to poor soil conditions? • Identification of markers potentially associated with tolerance to environmental conditions • Needs further corroboration before being adopted as a strategy for genetic diversity discovery

35. How to Penetrate the “Black Box”? A Genomic Approach to Marker & Gene Discovery and Transfer:Development of PhaseolusGenes, a breeder’s marker toolbox http://phaseolusgenes.bioinformatics.ucdavis.edu UCD Bioinformatics: Dawei Lin, Jose Boveda, Monica Britton, Joe Fass, Nikhil Joshi, Zhi-Wei Lu UCD Gepts group: James Kami, José Vicente Gomes dos Santos, Shelby Repinski Adriana Navarro Gómez, Paul Gepts

36. Overall design of PhaseolusGenes

37. URL: http://phaseolusgenes.bioinformatics.ucdavis.edu

38. Genome Browser

39. Early Applications of PhaseolusGenes: EXAMPLE TheorAppl Genet 122: 893–903 (2011)

40. Identifying additional markers linked to Co-14 and Phg-1 on PV01 • Previous information: • Phg-1 maps on PV01 & linked to SH13 • Co-14 linked to Phg-1 • Location of SH13 is ambiguous: Pv01 or Pv11 • Alternative markers on PV01? • Check Cmap • Run markers against Bulked DNA for R and S progenies

41. Two New Markers • Linkage distances: • CV542014: 0.7 cM • TGA1.1: 1.3 cM CV542014 TGA1.1

42. Summary • PhaseolusGenes: • Includes all known markers established so far • Used soybean whole-genome sequence as anchor because of macro- and micro-synteny • Facilitates marker discovery after initial mapping • Can also use synteny for candidate gene discovery • Further work: • Addition of three whole-genome sequences of bean • QTLs from beans

43. Conclusion • Adoption of different approaches: • Molecular Markers • GIS • Genomics • Facilitate use of germplasm and reduce the size of the “Black Box”: Black Box  Grey Box Crop Science 46:2278–2292 (2006)