The apple genome: origin of
the species and perspectives
Riccardo Velasco, FEM-IASMA,
San Michele all’Adige, Trento
Bolzano, 6 novembre 2010
Apple genome sequencing
Cultivar ‘Golden delicious’ (most important world
and Trentino cultivar)
Diploid (2n = 34)
Genome size: ca. 750 Mb
Sequenced 17x (Roche ca. 13x + Sanger ca. 4x)
Ca. 70% of genome anchored on linkage map,
including ca. 90% of genes.
Estimated ca. 2 millions molecular markers (SNPs
single nucleotide polymorphisms)
Genome sequencing: neverending puzzle
sequencing 2 kb Clones
Contigs
Metacontigs da
2-15 kb Clones
BACs e Fosmids
Metacontigs da
BACs and
Fosmids
Molecular markers
Chromosomes
Details on the Golden genome
Third round of assembly (15.4x Golden Delicious + 1.5x DH)
122,146 contigs assembled into 20,738 clusters after corrections
for overlaps of which 19,084 singletons and 1,654 metacontigs
(average lenght ca. 7 kb), for a total length of 658,7 Mb
repeat estimation length 80 Mb
1,654 metacontigs are assembled into 605 Mb, out of them 554
cover 535 Mb anchored (max length 9.1
70000
Mb - from 0 to 31.1 cM of LG15)
60000
-
28,400 TC from 255,000 ESTs
→ 23,058 TC aligned to the genome with
90% of identity and 80% of length
- approx. 57,000 genes predicted.
-
# c o n ti g
50000
40000
30000
20000
10000
0
N100
N90
Ca. 93% of predicted genes are positioned in anchored
metacontigs
N80
N70
N60
N50
2
1
negative
control
3
The apple genome: milestones in plant evolution
4
Apple applied genetics and genomics
Germplasm collection at FEM IASMA
Genetic distance between Malus species and the origin of the cultivated apple
23 genes re-sequenced
74 Malus accessions
• 12 M. x domestica
• 10 M. sieversii
• 21 M. sylvestris
Splits-Tree (Hamming distance)
Apple applied genetics and genomics
Cultivar fingerprinting
MDFiesta
MDFreedom
MDKent
MDCoxOrange
MDIngridMarie
MDDelcoros
MDJamesGrieve
MDLordLamboune
MDAmorosa
MDClivia
MDPilot
MDReanda
MDRewenda
MDBenDavis
MDLaxtonSuperba
MDMcIntosh
MDWagener
MDJanamac
MDAkane
MDWorcesterPearmain
MDny5822McIntosh
MDBmypol
MDEnterprise
MDSpartan
MDRenettaBianca
MDRenettaGrigiaGranFaje
MDCalvilla
MDBoskoop
MDJonafree
MDJonathan
MDIdared
MDRedRome
MDRomeBeauty
MDDiwa faw
MDEarlychief
MDHapke
MDJeromine
MDOregonSpur
MDStarkingDelicious
MDCatarina
MDFuiji
MDCarlaOpaca
MDStayman
MDBraeburn
MDGloster
MDCrimson Snow
MDFlorina
MDPriscilla
MDAmbrosia
MDGotha
MDGrannySmith
MDForlady
MDBoujade
MDDalinred
MDAntonovka
MDGravenstein
MDDiscovery
MDSanLugano
MDPiros
MDRenettaAnanas
MDRosmarinaBianca
MDSpitzlederer
MDNapoleone
MDDurelloDiForli
MDRosaDiCaldaro
MDSila
MDGenevaCrab
MDGoldenHornet
MDRedField
MDAnnurca
MDPioneerScarlet
MDRadiant
MDWinterGold
MDRedJade
MDVanEsentine
MDGoldenGem
MDJohnDownie
MDPearmainDorata
MDEvereste
MDPlatycarpa132478
MDDolgo
MDMelaDellaSerla
MDResista
MDSunkrisp
MDRedEarly
MDEarlyRedGold
MDGala
MDRubicon
MDRubyGala
MDInitialPajam2
MDSonya
MDSansa
MDModì
MDRubin
MDTopaz
MDRosana
MDGoldenOrange
MDElstar
MDRubens
MDElan
MDGoldenDel
MDMutsu
MDPinkGold
MDDelblush
MDGoldrush
MDRubinette
MDDoriane
MDPinkLady
MDLygol
MDPinova
MDGingerGold
MDRebella
MDPrima
MDRubinola
MDRetina
MDBrina
MDRenora
MDSummerfree
MDPri14126
MDStettino
MDBelfioreGiallo
MDSaturn
MDCoop36
MDDelgrared
MDCameo
MDPrimiera
MDAriwa
MDDelorina
MDAmbassy
MDTunda
MDEarlyGold
MDMonroe
• Internally validated
system on:
• 132 apple cv
• Ca. 200 SNP on the 17
chromosomes
• 100% of distinguibility
between cultivars
Micheletti et al. unpublished
Least difference
between cvs
Red Del
clones
Gala clones
cv Initial (Gala
x Redfree)
GD clones
Difference between
apple clones ~
experimental error
Golden Delicious × Scarlet
IASMA reference map
×
Golden
delicious
Scarlet
spur
• 270 individuals
• 1,571 markers
• 1,413 SNP
• 158 SSR
• Map length: 1,159.7 cM
• Average marker interval:
0.7 cM
Troggio et al. submitted
Linkage based-QTL mapping projects at
FEM-IASMA
Mapping populations for genome anchoring and generation of consensus map
Golden Delicious x Scarlet (reference cross, 270 ind.) (quality)
Golden Delicious x Braeburn (96 ind.) (quality)
Golden Delicious x Freedom (300 ind.) (mapping Va -scab resistance)
Mapping populations – Projects for gene/QTL mapping (Internal +
collaborations)
Fuji x Pink Lady (96, quality, internal project)
Fuji x Delearly (96, quality, internal project)
Golden Delicious x M. hupehensis (Erwinia, internal project)
Golden Delicious x M. fusca (Erwinia, flowering biology, internal project)
Golden Delicious x M. prunifolia (Erwinia, internal project)
GoldRush x McIntosh Wicjik (Habitus, internal project)
Golden Delicious x McIntosh Wicjik (103, Habitus, internal project)
Gala x X-3191 (self-thinning, coll INRA)
Pink Lady x Idared (192, self-thinning, internal project)
Gala x Rubinette (96, resistance, quality, internal project)
Mapping populations – collaborations
Royal Gala x Braeburn (IRTA-HortResearch-INRA)
TN x Discovery (INRA)
Gala x Elstar (INRA)
Pyrus communis Max Red Burtlett x P. pyrifolia (HortResearch)
LOD
×
CH01h02
GD_SNP01466
HI01d01
GD_SNP00509
GD_SNP02441
GD_SNP02100
GD_SNP02014
Scarlet
spur
Apple applied genetics and genomics
3.0
2.5
2.0
0.5
0.0
Genotypic mean value
•270 F1 plants
GD_SNP01891
•2-year phenotype
LOD
GD_SNP00169
GD_SNP02564
GD_SNP01646
Golden1/Scarlet(+)
GD_SNP01948
GD_SNP01771
Golden1/Scarlet(-)
GD_SNP01911
Golden2/Scarlet(+)
Golden2/Scarlet(-)
GD_SNP00514
•705 markers
GD_SNP02031
GD_SNP00400
CH01f03b
GD_SNP01529
GD_SNP00337
GD_SNP01700
GD_SNP00893
GD_SNP01367
GD_SNP01706
GD_SNP00280
•QTL analysis
(MapQTL6, CP, IM)
Chromosome 9
Chromosome
9
GD_SNP00331
GD_SNP02046
GD_SNP00109
HI23d06
GD_SNP01959
CH05c07
GD_SNP01880
GD_SNP00947
QTL analysis for skin color
Golden
delicious
120
1.5
100
60
1.0
80
40
20
0
Rf
GD_SNP00332
GD_SNP01200
GD_SNP00352
GD_SNP00886
GD_SNP00766
GD_SNP00205
GD_SNP01817
GD_SNP01416
CN444542GD_SNP00659
GD_SNP01066
GD_SNP00206
GD_SNP02482
GD_SNP01596
Apple applied genetics and genomics
QTL analysis for self-thinning
• Apple overproduction is currently controlled by chemical thinning (to be strongly
restricted for health and environmental risks and cost concerns)
• Sources of genetic variability is available
• QTL analyses on crosses:
R. Gala x INRA X-3191
LOD
Pink Lady x Idared
Zini et al. unpublished
Chrom. 3 linkage map
Apple applied genetics and genomics
Cloning of the Co gene
• Golden Delicious x McIntosh Wijick
• 1,298 plants screened (markers and phenotype)
• Ca. 1,800 new seeds in 2009, now in pots
• Locus lower to < 1 cM, ca. 300 kb, 3-4 candidate
genes.
Chr 10
600,000 bp interval
Breeding by design
Example of gene/QTL pyramiding
Molecular markers assisted selection
n
GD
traditionsl
x
Breeding by design
assisted
M. floribunda
Source of Vf
gene, resitance to
apple scab
Acknowledgements
FEM - Istituto Agrario di San Michele all’ Adige
Sequencing/Mapping Silvio Salvi, Michela Troggio,
Massimo Pindo, Diego Micheletti, Giuseppina Coppola,
Fabrizio Costa, Paolo Baldi, Mickael Malnoy, Francesco
Salamini, Roberto Viola
Bioinformatics Paolo Fontana, Alessandro Cestaro, Marco Moretto,
Stefano Toppo
Annotation Claudio Moser, Giulia Malacarne, Azeddine Si-Ammour
Breeding Pierluigi Magnago, Marco Fontanari
Dep. of Plant System Biology, Gent University, Belgium
Yves Van de Peer, Jeffrey Fawcett, Lieven Sterck, Klaas Vandepoele
Roche/454
Egholm Michael, Jason Affourtit, Tim Harkins, Kodira Chinnappa
Amplicon Express
Amy Mraz, Keith Stormo, Quanzhou Tao, Robert Bogden
Myriad Genetics
Andrey Zharkikh, Alexander Gutin, Dmitry Pruss, Satish Bhatnagar, Natalia Gutin, Glenn
Eldredge, Jerry Lanchbury, Mark Skolnick, Dustin Cartwright
Washington State University – Amit Dhingra
INRA - Charles-Eric Durel, Yves Lespinasse, Pauline Lasserre
University of Western Cape, South Africa – Jasper Rees
HortResearch – Sue Gardiner, Andrew Allan, Roger Hellens, Vincent Bus, David Chagne