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Apple cDNA libraries.

 
Library code Source Library strategy Apple cultivar
Mdas Leaf tissue challenged with Venturia inaequalis Primary M. × domestica cv. GoldRush
Mdbd Mixed-bud stages Normalized M. × domestica cv. GoldRush
Mdfb Leaf tissue challenged with Erwinia amylovora Primary M. × domestica cv. Red Delicious
Mdfr Fruit – 9 DAP Primary M. × domestica cv. GoldRush
Mdfbg Leaf tissue challenged with Erwinia amylovora Primary Apple rootstock Geneva 3041
Mdfrb Fruit – 36 DAP Primary M. × domestica cv. Braeburn
Mdfrf Fruit – 36 DAP Primary M. × domestica cv. Fuji
Mdfrg Fruit – 36 DAP Primary M. × domestica cv. Granny Smith
Mdfrj Fruit – 36 DAP Primary M. × domestica cv. Jonagold
Mdfrs Fruit – 36 DAP Primary M. × domestica cv. Suncrisp
Mdfrt Mixed-fruit stages Normalized M. × domestica cv. GoldRush
Mdfw Mixed-floral stages Normalized M. × domestica cv. GoldRush
Mdfwb Flower balloon stage Primary M. × domestica cv. Braeburn
Mdfwf Flower balloon stage Primary M. × domestica cv. Fuji
Mdfwg Flower balloon stage Primary M. × domestica cv. Granny Smith
Mdfwj Flower balloon stage Primary M. × domesticac cv. Jonagold
Mdfws Flower balloon stage Primary M. × domestica cv. Suncrisp
Mdlr Leaf challenged with leaf roller insect Primary M. × domestica cv. GoldRush
Mdltb Bud tissue exposed to low temperature Primary M. × domestica cv. Royal Gala
Mdltl Leaf tissue exposed to low temperature Primary M. × domestica cv. Royal Gala
Mdltx Xylem exposed to low temperature Primary M. × domestica cv. Royal Gala
Mdlv Leaf – Stage I Primary M. × domestica cv. GoldRush
Mdlv2 Leaf – Stage II Primary M. × domestica cv. GoldRush
Mdlv3 Leaf – Stage III Primary M. × domestica cv. GoldRush
Mdlv4 Leaf – Stage IV Primary M. × domestica cv. GoldRush
Mdrta Root tissue Primary Apple rootstock M.9.
Mdrtb Root tissue Primary Apple rootstock M.111
Mdrtc Root tissue Primary Apple rootstock Geneva 3041
Mdrtp Root tissue challenged with Phytophtora cactorum Primary M. sieversii × Geneva 3041
Mdst Mixed-shoot stages Normalized M. × domestica cv. GoldRush
Mdstw Actively growing shoot Primary M. × domestica cv. Wijcik
Mdwdb Bud tissue exposed to water deficit Primary M. × domestica cv. Royal Gala
Mdwdl Leaf tissue exposed to water deficit Primary M. × domestica cv. Royal Gala
Mdwdr Root tissue exposed to water deficit Primary M. × domestica cv. Royal Gala
Primary library construction using Approach 2—see Materials and Methods.
Normalized cDNA libraries were constructed from several developmental stages: bud—three stages (dormant terminal and lateral, and active lateral); flower—four stages (bud—pink stage; balloon—full pink stage; full bloom and petal fall—after pollination); fruit—six stages (young fruitlets 9, 16, and 44 d after pollination [DAP]; maturing fruit 104 and 145 DAP; and ripe fruit 166 DAP); shoot—three stages (dormant, active, and actively growing).
§Days after pollination.
Primary library construction using Approach 1—see Materials and Methods.



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Modified oligo18(dT) primers with identifying tag sequence.

 
Tag sequence Tag identification from 5′ end Tag identification from 3′ end
A Insert 18(A)TCGTG CACGA18(T) insert
B Insert 18(A)TGCTG CAGCA18(T) insert
I Insert 18(A)TCGGT ACCGA18(T) insert
J Insert 18(A)TGCGA TCGCA18(T) insert
K Insert 18(A)TCGGA TCCGA18(T) insert
H Insert 18(A)TGCGT ACGCA18(T) insert



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Sequence assembly results.

 
Sequence assembly No. of sequences Avg. length
bp
Total sequences 190,425
Total high-quality sequences 182,241
ESTs in contigs 172,398 ND
Total no. of contigs 23,442 865
Singleton ESTs 9,843 441
Total no. of apple unique sequences (unigenes) 33,285
Number of assembled sequences matching known genes 26,333 ND
Number of sequences specific to apple 6,952 ND
Avg. insert size 1500
Avg. sequence size 465
Clean length.
ESTs, expressed sequence tags.
§Not determined.



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Munich Information Center for Protein Sequences (http://mips.gsf.de) Functional Catalogue analysis of subset of apple unique sequences.

 
No. Functional category Apple unique sequences
%
01 Metabolism 7.61
02 Energy 0.13
10 Cell cycle and DNA processing 1.34
11 Transcription 2.64
12 Protein synthesis 1.95
14 Protein fate 5.02
16 Protein with binding function or cofactor requirement 2.77
18 Protein activity regulation 0.22
20 Cellular transport 4.76
30 Cellular communication/signal transduction mechanism 4.11
32 Cell rescue, defense, and virulence 2.77
34 Interaction with the cellular environment 0.43
36 Interaction with the environment 0.82
40 Cell fate 1.86
41 Development 1.90
42 Biogenesis of cellular components 4.15
70 Subcellular localization 6.53
73 Cell type localization 0.09
75 Tissue localization 0.13
77 Organ localization 0.30
98 Classification not yet clear-cut 5.71
99 Unclassified proteins 44.79



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Fifty most common InterPro families represented within the apple unique sequences.

 
InterPro no. Description Frequency
IPR000719 Protein kinase 1040
IPR001611 Leucine-rich repeat 380
IPR001245 Tyr protein kinase 297
IPR002290 Ser-Thr protein kinase 257
IPR000504 RNA recognition motif 250
IPR008271 Ser-Thr protein kinase, active site 227
IPR001680 G-protein β WD-40 repeat 196
IPR000504 RNA-binding region RNP-1 (RNA recognition motif) 186
IPR001841 Zinc finger, RING 173
IPR001128 Cytochrome P450 167
IPR002048 Calcium-binding EF-hand 132
IPR002885 PPR repeat 119
IPR014778 MYB DNA-binding domain 104
IPR000608 Ubiquitin-conjugating enzymes 92
IPR001471 Pathogenesis-related transcriptional factor and ERF 87
IPR001344 Chlorophyll a/b-binding protein 86
IPR013753 Ras GTPase superfamily 86
IPR001440 TPR repeat 85
IPR000571 Zinc finger, C-x8-C-x5-C-x3-H type 80
IPR005123 2OG-Fe(II) oxygenase 80
IPR002110 Ankyrin 77
IPR001810 Cyclin-like F-box 76
IPR001993 Mitochondrial substrate carrier 74
IPR001471 Epimearase–NAD-dependent epimerase/dehydratase 70
IPR001878 Zinc finger, CCHC type 70
IPR013766 Thioredoxin-type domain 66
IPR007125 Histone core 62
IPR014045 Protein phosphatase 2C,N-terminal 62
IPR001087 Lipolytic enzyme, G-D-S-L 59
IPR002016 Haem peroxidase, plant/fungal/bacterial 59
IPR001092 Basic helix-loop-helix (bHLH) dimerization domain bHLH 57
IPR001623 Heat-shock protein DnaJ, N terminus 57
IPR000008 C2 domain 56
IPR006121 Heavy metal transport/detoxification protein 56
IPR002198 Short-chain dehydrogenase/reductase 55
IPR003439 ABC transporter 53
IPR013057 Amino acid transporter, transmembrane 53
IPR000626 Ubiquitin 52
IPR007087 Zinc finger, C2H2 type 49
IPR010847 Harpin-induced I 49
IPR000916 Bet v I allergen 46
IPR001356 Homeobox 44
IPR013126 Heat-shock protein 70 40
IPR002182 NB-ARC 39
IPR000425 Major intrinsic protein 38
IPR001395 Aldo/keto reductase 37
IPR002130 Peptidyl-prolyl cis-trans isomerase, cyclophilin type 33
IPR000157 TIR 30
IPR007493 Protein of unknown function DUF538 28
IPR001938 Thaumatin, pathogenesis related 24
2OG-Fe(II), 2-oxoglutarate and Fe (II)-dependent oxygenase; ABC, adenosine triphosphate–binding cassette; CCHC, CysCysHisCys; ERF, ethylene-responsive-element-binding factor; NAD, nicotinamide adenine dinucleotide; NB-ARC, nucleotide binding domain shared by Apaf-1, certain R (resistance) gene products, and CED-4; PPR, pentatricopeptide repeat; TIR, Toll/interleukin-1 receptor; TPR, tetratrico peptide repeat.



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The 10 most common transcription factor (TF) families in apple identified by searches of automated predictions using InterPro.

 
Top 10 TF family descriptions No. apple unigene sequences InterPro accession nos. TF family rank
Apple Arabidopsis Rice
MYB 228 IPR014778 1 (1) 1, 11, 14 1, 9
IPR001005
IPR006447
Pathogenesis related 87 IPR001471 2 (2) 2 2
C2H2 Zn finger 52 IPR007087 3 (3) 6 7, 8, 10
IPR003656
Homeobox 65 IPR001356 4 (4) 7 ND
IPR003106
C2C2 Zn finger 62 IPR000315 5 (5) 5 3
IPR000679
IPR003851
IPR006780
Basic helix-loop-helix 61 IPR001092 6 (7) 3 ND
C3H-type 1 Zn finger 43 IPR000571 7 (8) 18 ND
NAC 39 IPR008917 8 (6) 4 ND
IPR003441
WRKY 38 IPR003657 9 (9) 10 4
bZip 35 IPR004827 10 (10) 9 5
Total no. TFs 1091 1, 470 1306
TF family rank based on data from Newcomb et al. (2006).
Based on data from Riechmann et al. (2000).
§Based on data from Goff et al. (2002).
Family not determined by Goff et al. (2002).



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Comparisons of apple unique expressed sequence tags (ESTs) (33,285) with unigenes or proteomes from other plant species.

 
Species Database No. of hits Similarity
%
Arabidopsis Proteome 24,848 75
Citrus UniGene 13,521 40
Grape UniGene 18,691 56
Pine UniGene 14,693 44
Poplar UniGene 11,061 33
Poplar Proteome 25,817 77
Rice Proteome 23,768 71
Soybean UniGene 18,690 56
Tomato UniGene 16,871 50
Nonredundant Proteome 26,333 79
Total number of apple uniqe ESTs matching sequences in a given database.
Similarity was calculated as no. of hits/no. of apple unique ESTs.
§Populus trichocarpa v1.1 protein database.



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Putative functions of Malus-specific genes not conserved between apple and Arabidopsis.

 
Match description, species (GenBank no.) E-value Length query (amino acids) No. of isoforms
Mal d 1-like, Malus × domestica (AAS00042–AAD00053) 1.0 × 10−160 101–163 9
Polyphenol oxidase 2 precursor, Malus × domestica (AAK56323) 1.0 × 10−194 191–587 3
Polyphenol oxidase precursor, Prunus armeniaca (AAC28935) 1.0 × 10−108 99–245 2
Polyphenol oxidase, Prunus salicina var. cordata (AAW58109) 1.0 × 10−184 385 2
Transcription factor AHAP2, Malus × domestica (AAL57045) 1.0 × 10−70 180 1
Dehydrin, Malus × domestica 1.0 × 10−59 172 1
MADS box protein, Malus × domestica (CAC86183) 1.0 × 10−41 84 1
Fruit acidity–related protein, Malus × domestica (Mal-DDNA–DQ417661) 1.0 × 10−37 112 1