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

the Field

Paul GeptsPlant Sciences, UC Davis

6o Congreso Brasileiro de Melhoramento de Plantas1 a 4 de agosto, 2011 – Búzios, RJ

Applied Plant Breeding and Cultivar Development

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”?

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

Gene Banks around the World:> 10,000 accessions

State of the World’s Plant Genetic Resources for Food and Agriculture (SOTW2), 2009

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

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?

Phaseolus beans

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 proteinsMinerals: iron and zinc (~ meats, poultry, and fish)Dietary fiberVitamins: folate (low in diets of many Americans)

Reduces breast cancer (Thompson et al. 2008)

San Agustín del Pulque, MEX (2004)

A Phylogenetic/Genealogical Approach to Understanding Genetic Diversity of a Crop Species: The Diversification of Common Bean (Phaseolus vulgaris)

How to Penetrate the “Black Box”?

9

Gene flow

Feral or

weedy

Feral or weedy

Phylogeny/Genealogy of Common Bean

Domestication

Wild Mesoamerican

Domesticated outsideCenter of origin

Dissemination

Wild Andean

Domesticated outsideCenter of originDomesticated landraces

in Andes

P NG

C

WildECD &N. PER

Domesticated landracesin Mesoamerica

J

M

D

G

Other Phaseolus species

Multiple Sources, Several Years

• Applications to Bean Breeding

10

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 racesinter-racial crosses within

gene pools• For inter-gene pool crosses:

Adapt breeding method to account for wider genetic distance: e.g., 1 BC

11

Mesoamerican

Andean

Domestications

Reduction in Levels of Genetic Diversity

• Observation: Reduction in genetic diversity– Single domestication

in each gene pool– Plant breeding

Gepts et al. 1986; Sonnante et al. 1994

• Consequence:– Use landraces and

wild germplasm in breeding

– Use other Phaseolus species

Wild Landraces US Cultivars0

0.05

0.1

0.15

0.2

0.25

MesoAndean

12

M13-related RFLPs

13

Breeding Strategies to Broaden the Genetic Diversity of Common Bean

Kelly et al. 1998

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 footprintPapa & Gepts 2003, 2004; Payró de la Cruz et al. 2004; Zizumbo-Villareal et al. 2005; Papa et al. 2007

Photo: R. Papa

Co-evolution between Common Bean and Pathogens

Colletotrichum lindemuthianumInteractions

MEXICO ECUADOR ARGENTINA

MEXICO

ECUADOR

ARGENTINA

Phaseolus

vulgaris

• 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

15Geffroy et al. 1999

• The presumed domestication center of Phaseolus vulgaris in Mesoamerica

PhD thesis Myounghai Kwak (Korea) with Jim Kami

16

1

17

Gene flow

Feral or

weedy

Feral or weedy

Phylogeny/Genealogy of Common Bean

Domestication

Wild Mesoamerican

Domesticated outsideCenter of origin

Dissemination

Wild Andean

Domesticated outsideCenter of originDomesticated landraces

in Andes

P NG

C

WildECD &N. PER

Domesticated landracesin Mesoamerica

J

M

D

G

12

Other Phaseolus species

Relationship between Wild & Domesticated types in the Mesoamerican Gene Pool

18

Also, close genetic relationship based on phaseolin protein electrophoresis (Gepts 1988) Kwak et al. 2009

The Suggested Domestication Center of Common Bean in Mexico

M. Kwak, J. Kami & P. Gepts, Crop Sci., March 200919

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

Westernmost putative domestication location, Mascota-Ameca Basin

21

Domestication Areas within Mesoamerica

22

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)

How to Penetrate the “Black Box”?

• Genetic Diversity in a Secondary Center of Origin: Brazil

24

2

25

Gene flow

Feral or

weedy

Feral or weedy

Phylogeny/Genealogy of Common Bean

Domestication

Wild Mesoamerican

Domesticated outsideCenter of origin

Dissemination

Wild Andean

Domesticated outsideCenter of originDomesticated landraces

in Andes

P NG

C

WildECD &N. PER

Domesticated landracesin Mesoamerica

J

M

D

G

12

Other Phaseolus species

2

26

General Approach• 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.

Maps (1:5,000,000): Map of climate Mean annual

temperature Mean annual

precipitation Biomes Original vegetation Pedology

CIAT: climatic database Latin America

27

Brazilian Beans

http://www.unifeijao.com.br/feijao_do_brasil/mapa.htm

Macaçar pequeño

Rosinha

Fradinho Boca Preta

Mulatinho

Jalo

Bolinha Amarelo

Bico de Ouro

Carioca

Preto

Roxinho Bolinha Vermelho

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

29Jalo EEP558: landrace; BAT93: (Veranic 2 x Tlalnepantla 64) x (Negro Jamapa x GN Tara)

Molecular variation: STRUCTURE analysis

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• 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

Andean Mesoamerican Hybrid

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

A Genomic Approach to Marker & Gene Discovery and Transfer:Development of PhaseolusGenes, a breeder’s marker toolbox

How to Penetrate the “Black Box”?

http://phaseolusgenes.bioinformatics.ucdavis.edu

UCD Bioinformatics: Dawei Lin, Jose Boveda, Monica Britton, Joe Fass, Nikhil Joshi, Zhi-Wei LuUCD Gepts group: James Kami, José Vicente Gomes dos Santos, Shelby RepinskiAdriana Navarro Gómez, Paul Gepts

Overall design of PhaseolusGenes

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

Genome Browser

Early Applications of PhaseolusGenes: EXAMPLE

Theor Appl Genet 122: 893–903 (2011)

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

Two New Markers

• Linkage distances:– CV542014:

0.7 cM– TGA1.1: 1.3

cM

CV542014

TGA1.1

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

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)