Coffee Breeding UpdateCoffee Breeding Updatewith new toolswith new tools

HCA Conference 2003Chifumi Nagai

Hawaii Agriculture Research Center

New coffee varieties with high yielding and superior quality

with rust and nematode resistance having cupping quality

Coffee Breeding and SelectionCoffee Breeding and Selection

Quality evaluation (Cupping)

Flavor chemistry

New tools

DNA markers

Genetic Engineering

Traditional tools

Existing varieties

Foreign germplasm

Hybridization

Visual selection

Data collection

Backcross

HARC Coffee Program • Breeding and Selection

HCGA/State,DOA 1997-

• Molecular Markers

USDA grant (CMBRU)

Ray Ming, Heather Pearl, C. Nagai

• Transgenic coffee for nematode resistance

USDA grant ( T-STAR )

Brent Sipes, D. Schmit. H. Atkinson, Roxana Cabos, C.Nagai

• Green Bean Chemistry

USDA grant (CMBRU) Mel Jackson, Cathy Cavaletto, Shawn Steiner, C. Nagai

Coffee Breeding and Selection

HARC- teamC. Nagai, B. Osgood, R. Ming, M. Jackson, L. Poland, K. Wiever, J. Buenafe, J. Clayton

UHC. Cavaletto, N.Nagai and S. Bittenbender,

Hawaii Coffee Growers’ Association (HCGA)

Kauaiyellow Catuaired Catuai

Oahumokka hyb.Promecafe

Molokaired Catuaired Caturra

Kona‘Guatemalan’ typica‘Old Hawaiian’Hawaiian

Mauimokkared Catuai

Common FieldKunia, HARCplanted April,1998

Crosses (spring 1999)Group # of crosses

mokka and other arabica varieties 66 with Promecafe (Catimor) 53with Icatu 8mokka and other selfs 40

Total 167

H99 - series Progeny

New New CultivarCultivar Development Development using using MokkaMokka HybridsHybrids

•Mokka was introduced to the UH coffee germplasm collection in the mid 1950s

•Commercially cultivated on Maui (Kaanapali Coffee Co). The accession is now considered to be a Mokka hybrid.

•The cupping quality of Mokka hybrid is considered excellent but bean size is very small ( about 30-40% of Red Catuai beans).

•In 1999, we made crosses focused on increasing mokka seed size while maintaining its excellent cupping quality.

Goal:The introgression of Mokka flavor traits into high yielding and larger bean size cultivars

• Tree heightRed Catuai x Mokka hyb Red catuai dwarf x mokka

hybRed Catuai x typicaYel Caturra x mokka

• Cupping Quality• Increase bean Size

Yellow catuai x MargogipeMokka hyb. SelfMokka x Red CatuaiRed Catuai x SL28Mokka hyb selfRed Catuai x 6661

Example of Progenies from 1999 CrossExample of Progenies from 1999 Cross

Cherry and green bean) size Cherry and green bean) size of families with of families with mokkamokka andand

MargogipeMargogipe crosses crosses Fall 2002Fall 2002

Cross# Parentage n 100 fruit wt Green beangram gram

H99-34 KA17-5 x Margog 3 241 ± 3 41.7 ± 0.2(Y. catuai)

H99-36 MA1-12 x Margog 5 258 ± 17 42.9 ± 4.3(mokka)

H99-22A MA1-12 self 5 179± 9 29.2 ± 1.4(mokka select )

H99-101 KA16-4 self 6 148 ± 1 25.5 ± 1.7(Y. catuai)

H99-36:MA1-12 x Margogipe(mokka)

Potential New cultivarPotential New cultivar

Cupping of 3 genotypes of coffee Cupping of 3 genotypes of coffee harvested at harvested at KuniaKunia in 2002in 2002--03 Season03 Season

By Cathy Cavaletto’s Panel ( April 2003)

Cupping Panel: 7 trained cuppers (Judge)

Genotypes ( 2 hybrids + 1 parent)H97-1700: Yellow Catuai (KA17C)H99-34: KA17 x MargogipeH99-36: MA1-12(mokka) x Margogipe

Replication: 3 days

Cupping ResultsCupping Results

_________________________________________________________Source df Dry Wet Acidity Flavor Body

aroma________________________________________________Judge 6 ** ns *** ns **Genotype 2 ns ns ns ns nsRep 2 ns ns ns ** nsResidual 43_________________________________________

**: significant at P = 0.01***: significant at P= = 0.001ns= not significant

ANOVA

Propagation of Selected Hybrids by cloning

Preparation of treesInduction of vertical shoots

Kunia H99- fields

Cloning1. Shoot tip culture

2. Root induction of cuttingsUsing misting/ bottom heat

3. Bioreactor of somatic embryos(root stock production)

Summary and plan ‘03-’04• Field Trial at Kauai Coffee- “FT7” design

Germinated Seeds from selected hybrids at MWüShip seedlings to Kauai (9/03)Plant seedlings at 2 fields, Kauai Coffee(12/03)Data collection (5/04-)

• Field planting of 2002 hybrids ( 7/03) ü

• Harvest and data collection of H99- series at Kunia (10-12/03)

• Crosses using new germplasm and selected genotypes (12/03-3/04)

• Send selected individuals for rust resistance tests.• Make new crosses of selected F1 plants

DNA Marker Study 2002-2003

Ray Ming, Heather Pearl and C. Nagai

• Genetic evaluation of Fukunaga and newly introduced coffee

• DNA analyses of green beans

• Development of coffee populations for QTL mapping

• Construction of a genetic map of Arabica coffee

DNACoffee leaves

Genetic Similarity (Cophenetic coefficient = .99)0.41 0.56 0.71 0.85 1.00

Bourbon1Bourbon3Typica-H1Typica-H2Typica-K6Typica-H3Typica-K2Typica-H4Typica-H5Typica-K10Typica-K7Typica-K9Typica-K3Typica-H8Typica-H9Typica-H7Typica-K1Typica-K11Typica-K32Typica-K33Typica-K8Catuai8Typica-K4Typica-K34MH3MH2MH5 MH6MH7Catuai1Catuai2Catuai5Caturra1Caturra2Catuai3 Catuai4Typica-K12Typica-K13MH8Catuai6Typica-K5Catimor4Catimor5Catimor7 Catimor8Catuai9MH4Catuai10Bourbon4Catimor6Bourbon2MH1Typica-H6Catuai7Catimor1Catimor3Catimor2C-canephoraC-libericaDeweveri

Genetic Relationships Within and Genetic Relationships Within and Between Between CoffeaCoffea SpeciesSpecies

Brown- Bourbon

Green- Typica

Blue- Catuai/Caturra

Lt. Blue- Mokka hybrid

Pink- CatimorBlack- Diploid Species

Application of Fingerprinting:

– Determine the genetic relationship of Fukunaga (Coffea liberica, deweveri) trees from various sources in Konaand other trees showing liberica traits

– Examine the differences between newly introduced cultivars and long established Hawaiian cultivars such as Kona typica

SamplesArabica (2n=44) 30 smples– tetraploid and self fertile– Blue Mountain(Jamaica), Blue Mountain (HARC)

Kona Typica, Mokka, Laurina, tall Mokka Hybrid, Bourbon, Mundo Nova

New Brazilian cultivars : Icatu, Obata, and Tupi

Liberica “Fukunaga “ (2n=22) 20 samples– diploid and self-sterile – torlelet nematodes , used as root stock for Kona typica

Canephora (robusta) 2 samples– diploid and self-sterile

List of List of Fukunaga Fukunaga samples from samples from KonaKona(collected by Mario (collected by Mario SarracinSarracin 11/20/01)11/20/01)

Sample Name Source Additional InformationKES1 UH Kainaliu H-Field, Marc Meinsner's selec. From P.Ito's original treesKES2 UH Kainaliu H-Field, Marc Meinsner's selec. From P.Ito's original trees

KES (Original) UH KainaliuKES 16 KraigLeeKES 6658 See MareM., Next to Arnold Diama 6658CTS 2F TwigSmith Farm Flower PlantedCTS 2S TwigSmith Farm Seed PlantedCTS 3 TwigSmith FarmCTS 4 TwigSmith FarmCTS 5 TwigSmith FarmCTS 6 TwigSmith FarmCTS 7 TwigSmith FarmCTS 9 TwigSmith FarmCTS 10 TwigSmith FarmCTS 11 TwigSmith FarmCTS 12 TwigSmith FarmCTS 13 TwigSmith Farm Reddish leaves, pointed, fruit not clustered, multiple verticalsCTS 14 TwigSmith FarmCTS 15 TwigSmith Farm Seeds Taken

C. liberica dewevrei (Fukunaga)Kraig Lee C. liberica dewevrei (Fukunaga)

Looks like dewevrei type, not fruiting

Genetic Similarity Tree

Genetic Similarity Coefficient0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90 1.00

MWBlueMountain1

BlueMountain3 KonaTypica4 KonaTypica3 KonaTypica2 MokkaHybrid4 Mokka3 Luurina4 Luurina2 Luurina3 KonaTypica1 MundoNova1 MO2-7-2 MundoNova2 Bourbon1 Bourbon2 MokkaHybrid3 Tupi1 Mokka1 BlueMountain6 MO2-7-1 YellowBourbon1 BlueMountain4 BlueMountain5 5175-1 5175-2 MWBlueMountain1 Icatu2 Obata1 Obata2 Tupi2 Canephora1 Canephora2 KESLiberica1 KESLiberica2 KESlibericaOrig KESLiberica16 KESLiberica6658 CTSLiberica12 CTSLiberica2S CTSLiberica2F CTSLiberica4 CTSLiberica13 CTSLiberica3 CTSLiberica8 CTSLiberica11 CTSLiberica14 CTSLiberica7 CTSLiberica6 CTSLiberica15 CTSLiberica5 CTSLiberica9 CTSLiberica10

Easily distinguishable groups were seen between the three species:

Arabica

Canephora

Liberica (Fukunaga)

Species Specific Marker Bands:ArabicaMarkerBand

LibericaMarkerBand

Genomic DNA analyses of green beans

• Isolated DNA from commercially processed green beans

• Tested DNA quality -- partially degraded

Polymorphic DNA Markers

Linkage Groups

Mapping of arabica coffee Development of a mapping population

P1: Promecafe1(C): X P2: Mokka (M) (1999)T5175-1 MA2-7

large bean size, small bean sizelow cupping quality, high cupping qualityRust resistance rust susceptible

F1(Pseudo F2)130 progeny

F2300 progeny(will be planted 9/03)

QTL (Quantitative Trait Locus) for marker assisted selection

Find correlation between DNA markers and coffee traits such as Tree Characters

leaf shapestree heightbranching angles

Bean Charactersbean sizeyieldcupping quality

• Developed a true F2 mapping population with 323 progeny

• Constructed the first Linkage map of Arabica coffee( Pearl et al. 2003 submitted)

• Mapped QTLs for five traits• Evaluated the possibility of applying DNA

markers on coffee green beans• Fingerprinted a new set of coffee

germplasm including Fukunaga

Summary 2002-2003

Genetic Transformation of CoffeeGenetic Transformation of Coffeefor Nematode Resistancefor Nematode Resistance

Team: Roxana Cabos(UH), Brent Sipes(UH), Don Schmitt(UH), Howard Atkinson(Leeds U)and C. Nagai

Damage to M. konaensis Infested Coffee Trees

Galls

Serracin et al ,2001

Cystatin

• From Rice Flower

• Inhibitor for Cysteine Proteinases

• Reduces Nematode Growth & Fecundity

• Tested for Safety

• Field testing in potato,rice, banana by Atkinson’s group

Atkinson et al, 2001

Coffee Tissue CultureCoffee Tissue Culture

Germination

Plants with roots Nursery

Somatic embryosCallus from young leaves

Production of Nematode Resistant Coffee Plants by Genetic Engineering

Callus Induction of Coffee Leaves

Somatic Embryos

Selection & Regeneration

Insertion of Cystatin GeneCystatin OcID86

Gene bombardment

Agrobacterium-mediated transformation

Selection of Transgenic Somatic Embryos and production of transgenic plants

Coffee Plantlets with cystatin gene

435 Plantlets Transformed w/Cystatin Constructs

Acknowledgements

hHawaii Coffee Growers Association

hUniversity of Hawaii, Kainaliu Exp. Station

hDr. Loren Gautz, University of Hawaii

hDr. Mario Serracin

hDr. H. Medina-Filho, Instituto Agronomico de Campinas

hJamaica Coffee Industry Board

hSusan Ancheta

hLaetitia Fournier, E.S.I.P.A