Genetic improvement of ash ( Fraxinus excelsior ) – a European project

RAP : Realising Ash’s Potential

Dr. Gerry Douglas

Teagasc, Agriculture and Food Development AuthorityKinsealy Research Centre,

Malahide Rd. Dublin 17, Ireland

Utilisationstrategies

Propagation technology

Biodiversity assays

3 Workpackages

OVERALL OBJECTIVES:

1. To characterise the genetic diversity on regional & European scale

2. To accelerate the utilisation of scarce selections by vegetative propagation and induced flowering

3. To identify effective ways to exploit & use results & materials

Dr. Gerry Douglas Teagasc, IRELAND Coordinator …..Gerry.Douglas@teagasc.ie

Dr. Jean Dufour INRA ……………………………… Jean.Dufour@orleans.inra.fr

Dr. A. Meier Dinkel NFVA GERMANY............ ....nfv-abtc@t-online.de

Dr. D. Thompson Coillte Teo., IRELAND………. .david.thompson@coillte.ie

Dr. J. Hubert Forest Research, UK…………… jason.hubert@forestry.gsi.gov.uk

Dr. N.Frascaria-Lacoste , ENGREF, France……. .nathalie.frascaria@esv.u-psud.fr

Dr. B. Heinze FFRC, AUSTRIA,............................. Berthold.Heinze@bfw.gv.at

Dr J.F. Hausman CRP-GL, LUXEMBOURG.......... hausman@crpgl.lu

Mr. L. Somer Vitroform , Denmark…………………….Vitrom@mail.dk

Dr. M. Capuana IMGPF CNR , ITALY..................... maurizio.capuana@igv.cnr.it

Mr. B. De Cuyper INBO, BELGIUM.....................bart.decuyper@lin.vlaanderen.be

Ms. K. Russell EMR, UK …………………………...karen.russell@emr.ac.uk

Dr. P. Mertens MRW BELGIUM………………. P.Mertens@mrw.wallonie.be

Dr. Andreas Ottitsch EFI, FINLAND…………….. andreas.ottitsch@efi.fi

Contracting partners & contacts

WP 1: Biodiversity assays

Growth & adaptationanalyses

• Populations• Provenance trials• (10-15 yr old)• Families

• Molecular analyses• Genetic diversity• Gene flow• Hybridisation frequency

New International Ash

provenance trial

New International Ash

provenance trial

Evaluation of existing provenancetrials of ash Fraxinus excelsior

Biodiversity assays

EVALUATION OF 52 ASH PROVENANCES

(D Jacques, Gembloux B.)

Genotypic Heritability

Character

Significance

Site 1

Site 2

Height &

Circumference

**

0.48

0.76

Stem & Crown Form

**

0.68

0.52

Forking

NS

-

-

Frost Damage

**

0.60

0.72

Game Damage

NS

-

-

Global analysis of provenance trials ( 3 countries 50 provenances each in common France Belgium Germany)

Coefficient of variation as a % of the provenances, site and interaction effects( P. Mertens Gembloux)

26.928.544.1Residual19.924.329.0Interaction17.824.427.5Site6.43.75.7Provenance

Stemform

Stemheight

Stem girth

Coef. Varn.%

1. Site and Interaction effects > provenance effect.

2. Stem form has a relatively higher provenance effect relative to site and interaction effects.** Efficiency of provenance selection is not yet demonstrated.

3. Progeny selection is not more useful than Provenance selection

• Summary resultsHeritability values for ash:

Very high for----Susceptibility to frost-- bud flushing date

(At the level of country, provenance and progeny)

High for------ Stem straightness------- Height and circumference

Global analysis of existing trials

----Very strong site effects and interaction effects

--- stemform a good selection criterion

Much intra population variation– clonal selection

New European Provenance Trial

Biodiversity assays

• 600 Seedlots collected & germinated from 44 EU provenances

• Testing 30 provenances “ core collection” in 6 countries

-- Material already planted in France. Germany Belgium, Italy, Ireland& Denmark

Ash provenences in the new European trial

Nursery flushing of 40 provenances on seven European sites, (F, D, IRL, UK, IT, B)– first results

( B. De Cuyper)

EarlyLate

Lt(Kaisiadorys)

F (Sila Grande)

LT + F

IntermediateB, F, DCentral

Early (Stable)I, CZSouth + East

Late (Stable)IRL, UK, DK, LT

North and Western

Flushing Pattern

Countrysource

Provenance

Chloroplast DNA markers ( maternal inheritance) ( sequenced variable regions )

• atpB-rbc• Mat-K

Measuring genetic variation of ash

Molecular Nuclear DNA markers

Microsatellites ( five variable loci)• FEMSATL 11, 9, 4, 16, M2- 30

• AFLPs

• Analysis of provenances, populations, clonal material

Biodiversity assays

Genetic Diversity estimates• High – for intra population diversity• heterozygote deficiency

Gene flow estimates are • 42-56 % of pollen from outside 12ha stand• Gene flow ( seed & pollen flow within stands is restricted )

Biodiversity : Molecular analyses : Some results( N. Frascaria Lacoste, B. Heinze, J. Fernandez, G. Douglas)

Seed orchard trees

14 Provenances

Natural populations

Genetic variation of Ash in IrelandM. HarbourneT. HodkinsonG. Douglas

Allele number and overall gene diversity in Irish ash using nuclear microsatellites

0.881410.871800.82337

0.93280.90340.8874FEMSATL 19

0.66110.71150.6952FEMSATL 16

0.91240.89340.7860FEMSATL 11

0.95320.9380.8772FEMSATL 4

0.98460.94590.8979M2-30

Gene diversityHT

No. Alleles

Gene diversityHT

No. Alleles

Gene diversityHT

No. Alleles

Ms- Locus

Seed orchardNatural populations 3

n=50 / pop

Provenances14

n=6 / provenance

--intra population diversity --- high

--genetic differentiation between strands-- low.

-- significant heterozygote deficiency

Gene diversity

France : 0.52-0.81Bulgaria 0.57- 0.92

Allele number in chloroplast microsatellite regions (haplotypes)

in Fraxinus excelsior from Ireland & Europe.

N = number of alleles / haplotypes detectedHT = is the gene diversity (Nei, 1973). Most variation within provenances ( 79- 63%)14 Irish provenances;Europe from B, Cz. Rep., Dk,UK (S), UK, F, D, L., Poland, Spain.

0.81260.7114Multilocus Haplotype

0.51120.6812352-370T4ACTATCT9-11

&GCGC3-4A5-6

CPFRAX6

0.6950.324252-257T4ACTATCT9-11CPFRAX5

0.6850.093152-161T9-10AC2T6CPFRAX2

HTNHTNSize rangeSSr - typeCode

EuropeIreland

Distribution map of most

common haplotypes

of Irish ash

from provenancetrial

with new cpDNA markers

Analysis of provenance relationships

Neighbour – joining tree of European

ashfrom AFLP data

Distribution map of Common haplotypes in European ash

Sources of material Selected trees

Propagation technology

Micropropagation

Somatic embryogenesis

Cryopreservation

Cutting propagation

Medium: (mg/L) M9 = MS : BA 5.5; TDZ 0.55, IBA 0.2 QRC = WPM*with 3.0 g/L charcoal.

Culture initiation : 2003• Buds from 55 selected clones• 18 clones viable

(@ 4 subcultures)

Rooting spontaneouslyor induced with auxin

Micropropagation

24 14 35 25 31clone

0

1

2

3

4

5multiplication rate

14/6314/6814/66

Influence of TDZ on the initiation of shoocultures WPM with:14/63: 4mg/l BAP + 0.15mg/l IBA, 14/68: 4mg/l BAP + 0.15mg/l IBA + 0.2mg/l TDZ,14/66: 0.15mg/l IBA + 1mg/l TDZ

Culture establishment is difficult

0

0,5

1

1,5

2

2,5

3

3,5

4

1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19

Subcultures

Prop

agat

ion

rate

JK49

T70

F5

Micropropagation over time

Somatic embryogenesis ( M. Capuana )

MS medium containing 2,4-D (0.1 mg l-1 ) & BA (1.0 mg l-1 )

30-35 % germination

10- 20 % conversion

Cryopreservation of clones of Fraxinus excelsior

5780519-21

808077-8

758077-3

758077-27

508077-23

536077-7

08077-24

786877-5

717077-2

adult

8070126.103.1

70607-3.3

607016.1.1.

507047.1.1

8070129.6.2

juvenile

regeneration rate [%]

no. of cryopreservedshoot tips

clone

1- week( 1-2 mm)

3- weeks

8- weeks

(A. Meier Dinkel)

Types of cuttings

Apical = Apex + 2 nodes Subapical= 2 nodes

Rooting cuttings of Ash cuttings from seedlings and micropropagated plants:

Cutting propagation ( G. Douglas )

Rooting in cuttings from micropropagated ash

2 wks.

3 months

Rooting: warmbench with plastic

Hedges of stock plants Cuttings

Cuttings collected from the same hedge

April ( dormant )MayJuneJuly

0

20

40

60

80

100

120

72 F5 49 47

Clone

Roo

ting

%May June July

Rooting in cuttings from micropropagatedhedged plants in May June & July

400 rooted / m2

: 4 cutting crops / yr = 1600 rooted / yr / m2

glasshouse of 200 m 2 = 320, 000 rooted ash plants / yr.

1-2 yr growthField planting

Hedges from micropropagation .

Four crops of cuttings per year

Rooted cuttings (85-100%)

Planting broadleaves:

• Farmers survey• Focus group discussions

Utilisationstrategies

Improved broadleaves Field User

Factors of influence ??

(A. Ottitsch, J. Frawley)

Flow of information & materials from research institutesto forestry entrepreneurs – model

Afforestation environment in Ireland• 14 000 ha / yr.• Broadleaf 15-22 %-- target 30%

Grants and Premia

4674951Other 4996729Beech4996348Oak

Euro p.a. /ha Premium (20 yrs)

Total / ha grantEuro

Species

7Other2Beech4Sycamore20Alder21Oak46Ash

%

Species

10060Total1592017Other

106715Environmental effects

1381312Land suitability34203032Aesthetic/amenity28173024Higher subsidies

All %No*

%%

Broadleaf + conifersowners

Broadleafowners

Main influence

Farmer survey : Factors which Influence the

planting of broadleaf species

Landholders survey views: planting improved material ( when available ).

100All

16Not sure

2No, definitely

5No, advantage probably

27Yes, probably

50Advantage definitely

%View on advantages

100100100100100All

3326224253Third level

4551384838Secondary level

222340109Primary only

%%%%%

AllNoneConiferowner

Broadleaf &

conifer owner

Broadleafowner

Highest education

level obtained

Education level of landowners by plantation type.

Revised model -- Strong role for opinion leaders

Thanks to the RAP team&

Thank you for your attentio