The Danish Apple Collection – Characterization and Utilization

07/12/2017 1

NORDAPP meeting on fruit and berry breeding September 4-5, 2017, Dobele and Jelgava, Latvia

The Danish Apple Collection – characterization and utilization

by Carsten Pedersen, with contribution from Bjarne Larsen, Maren Korsgaard, Marian Ørgaard, Mikael Agerlin Petersen and Torben Toldam-Andersen. University of Copenhagen, Denmark, Sean Myles and Kyle Gardner, Dalhausie University, Canada,

• The collection and some resistance characters • The Apple-oasis project • Genetic Diversity and Genome-Wide Association Mapping of Fruit Quality Traits – a PhD study

07/12/2017 2

The Danish Apple Collection: • Part of NordGen https://www.nordgen.org/nak/index.php?act=startsearch&letter=A • About 300 cultivars of mainly Danish origin

Characters recorded for: The tree: Shape Flowers: 6 Leaves and shoots: 6 Harvest and use: 4 Outer qualities of the fruit: Inner qualities of the fruit: Keeping qualities: 2 07/12/2017 3

Replica of the Danish Apple Collection at Gl. Estrup grown without spraying

212 of these have been evaluated from 2006 to 2009 for: • Apple scab on fruits and leaves (Venturia inaequalis) • Fruit tree canker (Neonectria ditissima) • Powdery mildew (Podosphaera leucotricha)

• Visual field-evaluation on a scale from 1-9 on tree level

• Rosy apple aphid (Dysaphis plantaginea) • Sooty blotch and flyspeck (SBFS) or apple summer disease Augustæble Augustæble, gult Bedstefars Æble Blankholm Bodil Neergård Bodils Æble Bredæble Brostrøm Guldreinette Brændekilde Brøndæble Bøghs Citronæble C. J. Hansen 603 C.J.H. 6-18 Dansk Rosenhæger Dauglykkeæble Degneæble Diana Drejæble Edle Wendelborg Ejby æble Else Lindstow Ferskenrødt Sommeræble Filippa 'Anka' Flaskehalser Flintinge Fynsk udvalg V Fynsk udvalg VI Glostrup Guldreinet Gravenfin Grænseæble Guldborg Guldæble Hans Mathiesen Henrik Jensen Æble Herschendsgave Augustæble 07/12/2017Hugonæble 4 Augustæble, gult Hyltofte Æble Bedstefars Æble Højbjerg Sommeræble Blankholm Hørningsholm Bodil Neergård Ildrød Eilstrup Bodils Æble Kronprins Frederik Bredæble Kundbyæble 07 Scab on fruits,Scab onaverage fruits 2006and leaves-9 Brostrøm Guldreinette Kyholm Reinette Brændekilde Ladbyæble/Fynsk udv I 07 06 Brøndæble Langeland/Fynsk udv IV Bøghs Citronæble Lise Legind æble 06 C. J. Hansen 603 Lunds Doucin 05 C.J.H. 6-18 Lyngby Æble Dansk Rosenhæger Maren Nis 05 Dauglykkeæble Marselisborg Sommeræble 04 Degneæble Mikkel Pedersæble 04 Diana Nonnetit Bastard Scab on fruits 03 Drejæble Ondrup Moseæble Scab on leaves Disease severity Disease 03 Edle Wendelborg Ormslevæble

Disease severity Disease Ejby æble Oudrupgaards Høstæble 02 02 Else Lindstow Pigeon fra Juellinge Ferskenrødt SommeræblePigeon fra Maribo Filippa 'Anka' Rifbjerg Gylling 01 Flaskehalser Ringkloster Kammerjunker Flintinge Rødby Æble

00 Fynsk udvalg V Sildig Sandholt Æble

1 8

15 85 22 29 36 43 50 57 64 71 78 92 99

29 78 15 22 36 43 50 57 64 71 85 92 99

134 183 106 113 120 127 141 148 155 162 169 176 190 197 204

127 176 113 120 134 141 148 155 162 169 183 190 197 204 106 Fynsk udvalg VI Skarridsø Æble CultivarsCultivars ordered ordered according according to to disease disease severity severity Glostrup Guldreinet Skovfoged Gravenfin Sorøæble Grænseæble Spiseæble Vejle Guldborg Søbjergæble Guldæble Sønderskov Hans Mathiesen Yduns æble Henrik Jensen Æble Aabyæble Herschendsgave Hans Wassard Hugonæble Hyltofte Æble Højbjerg Sommeræble Hørningsholm Ildrød Eilstrup Kronprins Frederik Kundbyæble Kyholm Reinette Ladbyæble/Fynsk udv I Langeland/Fynsk udv IV Lise Legind æble Lunds Doucin Lyngby Æble Maren Nis Marselisborg Sommeræble Mikkel Pedersæble Nonnetit Bastard Ondrup Moseæble Ormslevæble Oudrupgaards Høstæble Pigeon fra Juellinge Pigeon fra Maribo Rifbjerg Gylling Ringkloster Kammerjunker Rødby Æble Sildig Sandholt Æble Skarridsø Æble Skovfoged Sorøæble Spiseæble Vejle Søbjergæble Sønderskov Yduns æble Aabyæble Hans Wassard 07/12/2017 5

08 Mildew

04 2006-8

2006-9 Disease severity Disease

00 Cultivars 07/12/2017 6

07 Canker

2006-8 2006-9 Disease severity Disease 03

1 6

11 56 16 21 26 31 36 41 46 51 61 66 71 76 81 86 91 96

101 111 156 201 211 106 116 121 126 131 136 141 146 151 161 166 171 176 181 186 191 196 206 Cultivars 07/12/2017 7

General disease index: Hans Wassard 1.0 Augustæble 1.3 Bedstefars Æble 1.1 Brostrøm Guldreinette 1.3 2006-9 average of: Brøndæble 1.1 Farum Æble 1.3 • Scab on leaves Langeland/Fynsk udv IV 1.1 Fynsk udvalg VI 1.3 • Scab on fruits Lise Legind æble 1.1 Kundbyæble 1.3 • Mildew Sildig Sandholt Æble 1.1 Ludvigsens Æble 1.3 Antonius 1.1 Ringkloster Kammerjunker 1.3 • Canker Hans Mathiesen 1.1 Sukkeræble 1.3 Oudrupgaards Høstæble 1.1 Søde Æbler fra Aalsrode 1.3 Filippa 1.2 Augustæble, gult 1.3 Dansk Rosenhæger 1.2 Aunhusæble 1.3 Flintinge 1.2 Bodils Æble 1.3 Guldæble 1.2 C. J. Hansen 603 1.3 Hugonæble 1.2 Edle Wendelborg 1.3 Maren Nis 1.2 Fynsk udvalg V 1.3 Skenkelsø Æble 1.2 Lundbytorp 1.3 Lyngby Æble 1.3 Conclusion: Ormslevæble 1.3 A number of the cultivars are showing high Pigeon Rød Vinter 1.3 resistances towards scab, mildew and canker. Sorøæble 1.3 Søbjergæble 1.3 Aabyæble 1.3 Kathrineæble 1.3 Rødhætte Æble 1.3 07/12/2017 8

The Apple-oasis project:

“Crowd breeding”

• Seeds from ca. 50 selected cultivars • distributed to “apple-enthusiastic people” in autumn 2013

• The cultivars were selected to be robust and tasty

• The progenies are from random pollination (more than 1000 possible fathers)

• Totally about 10000 apple seeds were distributed and seedlings from many of these are now growing up.

Indicate cultivars found to have very good disease resistance under unsprayed conditions at Gl. Estrup. Many of the others may have as well. 07/12/2017 9 07/12/2017 10

Breeding Potential in Danish Apple Cultivars: Genetic Diversity and Genome-Wide Association Mapping of Fruit Quality Traits SUPPORT: A PHD STUDY BY BJARNE LARSEN DEPARTMENT OF PLANT AND ENVIRONMENTAL SCIENCES SUPERVISIORS: TORBEN TOLDAM-ANDERSEN (POMETUM) MARIAN ØRGAARD (BOTANY) CARSTEN PEDERSEN (MOLECULAR PLANT BREEDING)

OTHER PEOPLE INVOLVED: MIKAEL AGERLIN PETERSEN (FOOD SCIENCE) SEAN MYLES AND KYLE GARDNER (DALHAUSIE UNIVERSITY, CANADA) 07/12/2017 11

Materials:

• The Pometum collection: 287 Danish cultivars • A set of reference cultivars primarily from other European countries: 86 • The Assens collection: 76 • A selection of M. sieversii, M. sylvestris and small-fruited, ornamental cultivars: 36

Methods: Extended with ca. 400 more cultivars!

• Genotyping with 15 SSR-markers (485 acc.) : Diversity, parentages, duplicates • Genotyping S-RNase alleles • Determine ploidy levels by flow cytometry • Genotyping by sequencing (GBS) (380 acc.): ca. 30.000 SNP-markers : Diversity, parentages, duplicates • Phenotypic traits for GWAS: - “Apple-key traits” - Sugars and acids - Apple juice aroma volatiles (YDUN-project) • GWAS using TASSEL 07/12/2017 12 Parent – offspring relationships: 07/12/2017 13 07/12/2017 14 07/12/2017 15 Duplicates: 07/12/2017 16

Genotyping by sequencing (GBS):

• Relative cheap (380 cultivars for 90.000 dkr)

Ca. 6 dkr/marker or 237 dkr/genotype (apple cultivar)

• Produces many SNP-markers

• Mostly random locations 07/12/2017 17 07/12/2017 18

Conclusions:

• High diversity in The Pometum Apple collection • Parent-offspring relationships and duplicates can be determined by both SSR-markers and SNP-markers • Ploidy-levels can also be determined by GBS (based on level of heterozygosity) • High-throughput S-RNase genotyping protocol established. • GWAS: major genes can easily and precisely be mapped • GWAS: minor genes much more challenging – requires many accessions and accurate phenotyping • SNP-markers provided for MAS of important traits • High-quality phenotyping is important for future GWAS • Verifications of candidate genes is another future goal 07/12/2017 19

Suggestions for topics for Nordic collaborations:

1. Study alleles of some of the candidate genes in Nordic cultivars/breeding material: Harvest time would be interesting. The aim would be to validate the significance of the gene and develop a marker for MAS 2. Phenotype the SNP-genotyped 380 cultivars for canker and storage rot susceptibility in order to map involved genes and develop markers for MAS 3. Collect trees (grafting) from the “Apple-oasis” project consisting of half-sip families from (50) selected cultivars for pre-breeding e.g. with markers from 1 and 2.