BIONET2017 – 2022

Regional Biodiversity network of agricultural and food interest

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Overview

➢ ‘’BIONET’’- Project:

a. Short overview of the project

a1. Short overview of the regional fruits production (apples and pears)

a2. Short overview of the method (phenotipyc and molecular biology approach)

Builds on the results of the previous project

b. What has been done?

c. Corrent results

➢ Applications

➢ Future Perspectives

➢ Acknowledgements

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

BIONET2017 – 2022

11 partners

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Program Priority of BIONET

Enrollment of genetic resources of Veneto, in the appropriate varietal registers

Conservation of genetic resources of Veneto region

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Alberto Storti

09 November 2017

Alberese - Tuscany (Italy)

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Bionet: begin of the project

Apple export in the world (2016):Millions of dollars

Reference: FreshPlaza su dati InsiderMonkey/Wolrd Top Exports

Fuji:

254.000 T

Red delicius:

630.000 T

Gala:

1.059.000 T

Golden delicius:

2.553.000 T

Total european apple production for different cultivar -Years 2008-2010

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Apple production of Italy (2016):2,5 millions of Tons

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a1. Short overview of the total regional production of pears and hectares cultivated

Cultivated surface: 3.130,00 haTotal production of pears: 72.000 tReference: Elisa Macchi – CSO Ferrara 2017

Rovigo: 1.030 ha-1,2%

Padova: 370,00 ha+5%

Verona: 1.360 ha+ 1,5%

Venezia: 210 ha-7,5%

Total European production of pears 2016

Alberto Storti, Maurizio Arduin:

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Alberto Storti, Maurizio Arduin:

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Commercial varieties and biodiversity

In the worldexisting

7.000 applevarieties

In the worldexisting, over2.000 peach

varieties In the worldexisting

2.000 strawberryvarieties

In the worldexisting over100 cherryvarieties

In the worldexisting

7.000 pearvarieties

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Are they same or not same varieties?

Alberto Storti, Maurizio Arduin:

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Traditional and modern approach for cultivar identification

Description of the

phenotypic traits of

apple

Genotyping ofdifferent cultivars of

apple (Molecularbiology approach)

The traditional approach is based on phenotypic character such as form of fruits, length of stem of fruit, form of calyx, basal color and

other characters.

The modern approach is based on genotypic analysis by using molecular markers (SSR, AFLP, RAPD, SNPs…)

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a2. Study and comparision between apple/pear varieties and pomological books

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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DNA and genetics information

Genetic dictionary (succession of 4 letters (A, C, G, T)

Complementary of basis

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Apple genomes:

1. 17 cromosom

2. 742,3 million of basis

DNA Mapping

(study of the position of the gene)

DNA, cromosome and genisReference: Bus et al. (2008) Tree Genetics & Genomes 4:233-236

Velasco et al., (The genome of the domesticated apple (Malus × domestica Borkh.) – Nature Genetic 2010

DNA of apple Succession of the basis of DNA

(Sequencying)

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Why is important the study of DNA?

CGACGGA

ACGGATTCGGAT GACGGAT

1. Position of different genes on the cromosomes (genes locus)

Gene of crunchiness Gene of disease resistence Gene of colour of pulp Gene of ripening time

2. Genes expression

DNA information is translated infunctional products (eg. Protein)

Cromosome

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Variety A

reference

?

Variety

unknow

Locus 1 Locus 2 Locus 3

176 182

154 182 198

198 222 244 258

244 258

176 182 198 222 244 258

Variety B

Variety B

reference

How the identification

by molecular markers works?

?Variety unknow

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Corrent Results

1. Phenotipic identification – pomological sheet

2. Statistical analysis of old apple varieties cultivated in the past of Veneto region

3. Network

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Current Results

1. Phenotipic identification – pomological sheet

Apple: 60

Pesco: 5

Ciliegio: 1

Pear: 40

106 Fruit accessions – pomological sheet

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Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Current Results

• 360 Apple accessions

• Over 100 Pear accessions

2. Statistical analysis of old apple varieties cultivated in the past of Veneto region

‘Rosa Gentile‘

‘Favortita di Claps‘

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3. NetworkMapping of genbanks and germplasm collections

Collezione di Veneto Agricoltura –Azienda si Sasse Rami (Ro)

Collezione dell`Università di Padova

Collezione del `giardino della Memoriafruttale` - Fregona (Tv)

Collezione dell`Associazione Opfel on Pira - Lusiana (Vi)

Collezione dell`Istituto Agrario –Della Lucia di Feltre (Bl)

Collezione dell`Istituto Agrario -Parolini di Bassano (Vi)

Collezione dell`Università di Padova

Collezione del `giardino della Memoriafruttale` - Fregona (Tv)

Collezione dell`Associazione Opfel on Pira - Lusiana (Vi)

Collezione dell`Istituto Agrario -Parolini di Bassano (Vi)

Collezione dell`Istituto Agrario –Della Lucia di Feltre (Bl)

Collezione di Veneto Agricoltura –Azienda si Sasse Rami (Ro)

Alberto Storti, Maurizio Arduin:

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Collezione dell`Istituto Agrario – Duca degli Abruzzi (Vr)

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Applications

1. Farmers cooperative (‘Winesap‘ for ‘Braeburn‘)

2. Apples from Cile (‘Cripps Red‘ for ‘Cripps Pink‘) both (‘Golden delicius‘ x ‘Lady william‘)

‘Cripps Pink‘

(Own Label Pink Lady)

REFERENCE

‘Cripps Red‘

(Own Label Sundwner)

REFERENCE

‘Unknow apple‘

(Sample from Cile)

Locus 14 Locus 1Locus 6

➢ Counterfeiting

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Applications

➢Genitor Analysis of breeding programm

‘Gala‘

(parental analysis

‘Liberty‘

parental analysis

‘Modì‘

(son)

Locus 6Locus 14 Locus 1

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Bionet Project: (achieving final results…)

2. Creatingthe contact

1. Search theGermoplasmcollections

3. Sampling of leaves

4. DNA extraction

from leaves

5. DNA analysis byPCR

6. SSR Analysis & Reporting

Final Results(Different Genetic

profiles)

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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3. Level ofPolymorphism

Database(VerifiedGeneticProfiles)

1. TaxonomicalRelationships in Malus

domestica

4. Breeding for

Improved Cultivars

5. DNA Markers Associated with Interest

Genes (MAS)

6. Database online

Color of peel/pulp

Harvest

Resistence Disease

2. GeneticMapping

Future perspectives

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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➢ Genetic resources

Modern apple cultivarsGenetic resources

Future perspectives

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Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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➢ True to type-Nursery

New apple cultivar

01 0 0 0 02 0 0 0 03 0 0 0 04 0 0 0 05 0 0 0 06 0 0 0 07 0 0 0 08 0 0 0 09 0 0 0 0

1 0 0 0 0 01 1 0 0 0 0

1 0 0 1 2 5 1 5 0 1 7 5 2 0 0 2 2 5 2 5 0 2 7 5 3 0 0

S h i n a n o G o l d _ O r i g i n a l B r e e d e r

S i z e ( n t )

Dye

Sig

nal

122

CH02 h11a

124

CH02 h11a

170

CH01 f02

184

CH01 f02 199

CH02 d12240

CH01 d08

250

CH01 d08

Future perspectives

23Alberto Storti, Maurizio Arduin:

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

➢ Creating food culture

➢ Creating orchards with old apple and old pear varieties with nutritional values.

➢ Micro - economy

➢ Functional foods

Future perspectives

Monitor Ortofrutta 2014

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Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Comunication ofresults

RESEARCH

PRACTISE INDUSTRY

FeedbackResults

Interationand

Innovation

Network

Future perspectives

25Alberto Storti, Maurizio Arduin:

Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

Thanks for the attentionFor contact and informations: alberto.storti73@gmail.com

Acknowledgements:Dr. Luigino Schiavon, Veneto Agricoltura,

Thanks to the organizers of meeting

The science is not just a discipline of reason, but also of fantasy and passion.

Stephen Hawking

Maurizio ArduinSettore Ricerca AgrariaStaff Progetti SpecialiVeneto Agricoltura* Viale dell’Università, 14 – 35020 Legnaro (Pd)( 049 8293915( mobile : 338 9129651@ maurizio.arduin@venetoagricoltura.orgwebsite: www.venetoagricoltura.org

For contacts

………….....

➢ Structure

= Repeat motif e.g.

(GATA)n, (GCC)n, (CA)n, (ATT)n

…………..…………..………………………………

Unique flanking regions

Double strands of DNA

ATTATTATTATTATTATT

TAATAATAATAATAATAA

2-5 bp

………….. n

Microsatellites (Simple Sequence Repeats-SSR)

…………........

➢ The number of repeats is highly variable among apple varieties

…………........ ..……………

………….. …………………….. …………

..………………………............. ………….....………….............

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

.…………

.……

➢Design primers ( ) complementary to conserved flanking regions

………….. …………………………………………..

➢Amplify repeat regions by PCR

➢Analyse PCR products by DNA sequencer

Locus 14 Locus 1Locus 6

104 bp

94 bp

……..

…………

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region

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Bionet Project with SSR

Liebhard et al., 2002 140 Microsatellites in appleLB 14 Microsatellites for different position on the chromosomeand perfect Repeat Type

SSR LocusLinkage

GroupPrimers N. Dye-Fluorophore Locus Range Multiplex Fw primer RV primer References

CH01c06 8 1 Bleu_ D4 144190 1 TTCCCCATCATCGATCTCTC AAACTGAAGCCATGAGGGC Liebhard et al . 2002

CH02b10 2 6 Black_ D2 107-159 CAAGGAAATCATCAAAGATTCAAG CAAGTGGCTTCGGATAGTTG Liebhard et al . 2002

CH03a04 5 14 Bleu_ D4 90-132 GACGCATAACTTCTCTTCCACC TCAAGGTGTGCTAGACAAGGAG Liebhard et al . 2002

CH01d08 15 2 Bleu_ D4 204-294 2 CTCCGCCGCTATAACACTTC TACTCTGGAGGGTATGTCAAAG Liebhard et al . 2002

CH01f02 12 3 Bleu_ D4 162-224 ACCACATTAGAGCAGTTGAGG CTGGTTTGTTTTCCTCCAGC Liebhard et al . 2002

CH02d12 11 12 Black_ D2 177-219 AACCAGATTTGCTTGCCATC GCTGGTGGTAAACGTGGTG Liebhard et al . 2002

CH02h11a 4 13 Green_ D3 98-144 CGTGGCATGCCTATCATTTG CTGTTTGAACCGCTTCCTTC Liebhard et al . 2002

CH02c02a 2 7 Green_ D3 121-199 3 CTTCAAGTTCAGCATCAAGACAA TAGGGCACACTTGCTGGTC Liebhard et al . 2002

CH02c09 15 9 Green_ D3 233-269 TTATGTACCAACTTTGCTAACCTC AGAAGCAGCAGAGGAGGATG Liebhard et al . 2002

CH02c11 10 10 Bleu_ D4 206-268 TGAAGGCAATCACTCTGTGC TTCCGAGAATCCTCTTCGAC Liebhard et al . 2002

CH01h01 17 16 Bleu_Cy5 90-146 GAAAGACTTGCAGTGGGAGC GGAGTGGGTTTGAGAAGGTT Liebhard et al . 2002

CH01f07a 10 4 Green_ D3 173-205 4 CCCTACACAGTTTCTCAACCC CGTTTTTGGAGCGTAGGAAC Liebhard et al . 2002

CH02d08 11 11 Black_ D2 208-262 TCCAAAATGGCGTACCTCTC GCAGACACTCACTCACTATCTCTC Liebhard et al . 2002

COL 10 15 Bleu_ D4 203-243 AGGAGAAAGGCGTTTACCTG GACTCATTCTTCGTCGTCACTG Liebhard et al . 2002

Reference: Development and characterisation of 140 new Microsatellites in apple (Malus x domestica Borkh.) -(Liebhard et al., 2002)

Alberto Storti, Maurizio Arduin: Phenotypic study and molecular approach for a correct assignment of apple (Malus domestica x Borkh) and pear (Pirus L.) genetics resources in Veneto (Italy) region