An Integrated European In Situ Management Workplan:Workplan:

Implementing Genetic  Reserve and On Farm Concepts

IV ECGPR BrassicaWorking Group meeting

AEGRO PROJECT

WP 7: Case study BrassicaWP  7:  Case study Brassica

Lead partner: 8 (DOFATA UNICT)

Linguaglossa,  1‐5  March  2010

Main objectivesMain objectives

• Objective 1: Collection of species and population distribution data existing in variousinformation systems.information systems.

• Objective 2: Priorisation of species and populations.

• Objective 3: Recommendation of sites suited to establish genetic reserves for Brassica inth EUthe EU.

• Objective 4: Development of species specific guidelines for genetic reserves design,management and monitoring.

• Objective 5: Establishment of a demographic and genetic baseline for a single Brassicagenetic reserve.

• Objective 6: Compilation of the national legal framework related to in situ management,annotation of the legal and organisational national framework and derivation of aannotation of the legal and organisational national framework and derivation of arecommendations for a national strategy for in situ management.

• Objective 7: Contribution to the establishment of an European integrated workplan for insitu management of crop wild relatives.

Research objectives to be d l d d i h ddeveloped during the second year

Objective 3: Recommendation of sites suited toObjective 3:  Recommendation of sites suited to establish genetic reserves  for Brassica in the EU.

Objective 4: Development of species specific guidelines for genetic reserves design, management and monitoring.

Objective 5: Establishment of a demographic j g pand genetic baseline for a single Brassicagenetic reserve.

Research objectives to be developed during the second year

It was considered necessary for to achieve the objectives(3, 4 and 5) to proceed with:

sowing of wild Brassica species identified during the first year and for which seed was available and to have plantyear and for which seed was available and to have plant for the characterisation

Research objectives to be d l d d i h ddeveloped during the second year

morphological, genetics

biologicalbiochemical

Research objectives to be d l d d i h ddeveloped during the second year

Characterize the population identified is veryimportant for planning their protection andconservation and avoid duplication

R h bj ti t bResearch objectives to be developed during the second year

The population were sown in September on

polystyrene containers with peat.

Among all Brassica macrocarpa B incana and BAmong all, Brassica macrocarpa, B. incana and B.

villosa were marked for high germination

Research objectives to be jdeveloped during the second year

General there has been a slow growth of seedlings in fact the transplant

i d t i ld h f d ft b t th th (thcarried out in a cold greenhouse was performed after about three months (the

22 of December 2008)

The II sowing date have been the 17 December 2009

Research objectives to be developed during the second yearp g y

growth rate of plants in different periods, during the

month of March has been achieved (in the same

greenhouse) a second transplantg ) p

for both fields the plant growth has been satisfactoryp g y

Accession used for the molecular and biomorphological characterizationp g

Code Plot Accession LocationCode Plot Accession LocationBY 5 1 Brassica incana Agnone Bagni

BW 2 Brassica incana Sortino

BN 3 B i i A B iBN 3 Brassica incana Agnone Bagni

DC 4 Brassica incana Lago Albano

DA 5 Brassica montana Monte Covero

BL 6 B i ill Pi l fBL 6 Brassica villosa Pizzo telegrafo

BX 7 Brassica villosa Marianopoli

BU 5 8 Brassica rupestris Roccella valdemone

BU 16 9 Brassica rupestris Bivongi

BU 9 10 Brassica rupestris Ragusa Ibla

BU 15 11 Brassica rupestris Stilo

BG 12 Brassica rupestris S. Vito Lo Capo

BB 13 Brassica macrocarpa Favignana

For some accessions there was a high adaptability to environmental conditions

B. macrocarpa

Brassica rupestris

The inflorescence has b d t t d l i Bbeen detected only in B. rupestris and B. incana

For each genotype were taken a few leaves from seedling plant by plantplant by plant.

DNA t ti f dDNA extraction was performed using the method (Xinrong Ye, March 2005))

The extracted DNA was then quantified by spectrophotometer(absorbance or optical density, OD) at wavelength λ = 260( p y, ) gnm.

The quantification and quality of DNA extracted (withoutsmearing) was performed by running electrophoresis ofsamples and vision to ultraviolet light transilluminatorsamples and vision to ultraviolet light transilluminator.

On the basis of quantifying the spectrophotometer wereOn the basis of quantifying the spectrophotometer werecompared to approximately 100 ng of DNA extracted with amolecular mass marker (DNA ladder).

Based on the literat re cons lted attention as placed onBased on the literature consulted attention was placed onmicrosatellite SSR, or non-coding repeated sequences ofDNA consisting of repeating units of very short (2-5 bp) used

l l k f l ias molecular markers of loci.

Primer adopted: BoAP1

Th lifi i f DNA i l d i h i i fThe amplification of DNA involved in the various portions ofprimers was carried out by PCR (Polymerase ChainReaction) and was performed with a thermocycler Only 96(Explera).

The program has providedfor amplifying the initialfor amplifying the initialDNA denaturation at 95C °for 15 ', followed by 35cycles of denaturation atcycles of denaturation at94C ° for 60'', phase forthe annealing of DNAt d t 55 ° C f 60' 'strands at 55 ° C for 60' ',

the phase of extension ofthe DNA 72C ° for60'' hi h f ll d b60''which was followed bya phase of extension at72C ° for 10 min.

An aliquot of 5 μl of amplifiedproduct was placed, along withDNA ladder on a 1% agarose gel toverify the quality of PCR.

• Two microliters of PCR were added to 15 microliters offormamide and 0.5 microliters of Genescan 350 ROX (AppliedBiosystems, USA), denatured at 96 ° C for 3 'and placed onice before analysis.

• Capillary electrophoresis was carried out in ABI PRISM 3130(Applied Biosystems, USA) using POP7 as polymer.

• The molecular weight of each allelic variant was determinedusing the software "Gene Mapper v.3.7 (Applied Biosystems,g pp ( pp y ,USA).

Allele frequency of different alleles observed for Bo AP1 SSR marker

B. incanaB B. macrocarpaB. rupestrisB. villosaAll 154All 154

All 155

All 156

All 164

All 168

All 176All 176

Brassica incanaBOAP1

BOAP1 Brassica macrocarpa

Brassica rupestrisBOAP1

BOAP1 Brassica villosa

Thanks for the attention Thanks for the attention ……