Supplementary Figure S1. Taxonomic tree of major sequenced eudicots. The tree was constructed using Interactive Tree Of Life (itol)

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Russell Beasley
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de/) based on the NCBI taxonomy database (http://www.ncbi.nlm.nih.gov/taxonomy).

1 Supplementary Figure S1. Taxonomic tree of major sequenced eudicots. The tree was constructed using Interactive Tree Of Life (itol) ( based on the NCBI taxonomy database ( The number on the branches indicates the time of divergence (million years ago) among different organisms, which was obtained from the TimeTree website ( 1

2 Supplementary Figure S2. 17-mer depth distribution of the kiwifruit genome sequencing reads. The frequency of each 17-mer was calculated based on the filtered paired-end reads from libraries with short inserts ( 500bp). Two peaks were observed (at 78x and 157x, respectively) indicating heterozygosity in Hongyang. 2

3 Supplementary Figure S3. Kiwifruit tissues used for RNA-seq analysis. Mature leaves (a), immature fruits [20 days after pollination (DAP)] (b), mature green fruits (120 DAP) (c) and ripe fruits (127 DAP) (d) were collected from a five-year-old Hongyang tree. Cross-sections of immature fruits (e), mature green fruits (f), and ripe fruits (g) are also shown. Scale bar = 1 cm 3

4 Supplementary Figure S4. Distribution of synonymous nucleotide substitution (Ks) rates. (a) Ks distribution between syntenic gene pairs within kiwifruit (orange), grape (red) and between kiwifruit and grape (blue). (b) Ks distribution between syntenic gene pairs within kiwifruit (orange), tomato (green), potato (cyan), and between kiwifruit and tomato (blue) and kiwifruit and potato (pink). (c) Distribution of the mean Ks value of each syntenic block in kiwifruit (orange) and tomato (green). (d) Kiwifruit synteny blocks can be grouped into three different age classes by their mean Ks values. 4

5 Supplementary Figure S5. Ascorbic acid (vitamin C) biosynthesis and recycling pathway in kiwifruit. Gene families with expanded members in kiwifruit are shown in red. Alase, aldonolactonase; AO, L- ascorbate oxidase; APX, L-ascorbate peroxidase; DHAR, dehydroascorbate reductase; GalLDH, L- galactono-1,4-lactone dehydrogenase; GalUR, D-galacturonic acid reductase; GDH, L-galactose dehydrogenase; GGP, GDP-L-galactose phosphorylase; GLOase, L-galactono-1,4-lactone dehydrogenase; GME, GDP-D-mannose-3,5-epimerase; GMP, GDP-D-mannose pyrophosphorylase; GPP, L-galactose-1- phosphate phosphatase; IPS, inositol-3-phosphate synthase; MDHAR, monodehydroascorbate reductase; MIOX, myo-inositol oxygenase; PG, polygalacturonase; PGI, glucose-6-phosphate isomerase; PME, pectinesterase; PMI, mannose-6-phosphate isomerase; PMM, phosphomannomutase. 5

6 Supplementary Figure S6. Phylogenetic analysis of APX (L-ascorbate peroxidase) genes in kiwifruit (green), Arabidopsis (cyan), tomato (red), grape (purple), and sweet orange (yellow). Heatmaps representing expression levels of kiwifruit APX genes in leaf, immature fruit, mature green fruit and ripe fruit (from left to right) are shown on the right of the tree. Log2-transformed gene expression values were used to generate heatmaps. 6

7 0 20 Supplementary Figure S7. Phylegenetic analysis of MIOX (myo-inositol oxygenase) genes in kiwifruit (green), Arabidopsis (cyan), tomato (red), grape (purple), and sweet orange (yellow). Heatmaps representing expression levels of kiwifruit MIOX genes in leaf, immature fruit, mature green fruit and ripe fruit (from left to right) are shown on the right of the tree. Normalized gene expression values were used to generate heatmaps. 7

8 Supplementary Figure S8. Phylegenetic analysis of Alase (aldonolactonase) genes in kiwifruit (green), Arabidopsis (cyan), tomato (red), grape (pink), and sweet orange (yellow). Heatmaps representing expression levels of kiwifruit Alase genes in leaf, immature fruit, mature green fruit and ripe fruit (from left to right) are shown on the right of the tree. Normalized gene expression values were used to generate heatmaps. 8

9 Supplementary Figure S9. Carotenoid biosynthesis pathway in kiwifruit. (a) Simplified carotenoid biosynthesis pathway in kiwifruit. CCD, carotenoid cleavage dioxygenases; CHY, non-heme hydroxylases; CrtISO, 7,9,7',9'-tetra-cis-lycopene isomerase; CYCB, lycopene beta-cyclase; CYP, P450 hydroxylase; LCYB, lycopene beta-cyclase; LCYE, lycopene epsilon-cyclase; NCED, 9-cisepoxycarotenoid dioxygenase; PDS, phytoene desaturase; PSY, phytoene synthase; PTOX, alternative oxidase; VDE, violaxanthin deepoxidase; ZDS, zeta-carotene desaturase; ZEP, zeaxanthin epoxidase; ZISO, 9, 15, 9 -tri-cis-zeta-carotene isomerase. (b) Kiwifruit gene families in which expansions occurred are shown with the comparison of gene members in Arabidopsis (cyan), tomato (red), grape (purple), sweet orange (dark orange) and kiwifruit (green). Heatmaps representing expression levels of kiwifruit genes in leaf, immature fruit, mature green fruit and ripe fruit (from left to right) are shown on the right. Log2-transformed gene expression values were used to generate heatmaps. 9

Supplementary Figure S10. Biosynthesis pathway of anthocyanins. Compared to Arabidopsis, tomato, grape, and sweet orange, gene families with expanded members in kiwifruit are shown in red.

10 Supplementary Figure S10. Biosynthesis pathway of anthocyanins. Compared to Arabidopsis, tomato, grape, and sweet orange, gene families with expanded members in kiwifruit are shown in red. CHS, Chalcone synthase; CHI, Chalcone isomerase; F3H, Flavanone 3-hydroxylase; F3 H, Flavonoid 3 - hydroxylase; F3 5 H, Flavonoid 3 5 -hydroxylase; DFR, Dihydroflavonol 4-reductase; LDOX, Leucoanthocyanidin dioxygenase; ANS, Anthocyanidin synthase; UFGT, UDP-glucose flavonoid 3-Oglucosyltransferase; FLS, Flavonol synthase. 10

11 Supplementary Table S1. Physical or physiological parameters of tissues/organs of kiwifruit cv. Hongyang fruit (20 DAP) fruit (120 DAP) fruit (127 DAP) leaf Weight per fruit or leaf (g) 2.83± ± ± ±0.41 Tranverse diameter (cm) 1.39± ± ± ±0.56 Longitudinal diameter (cm) 1.85± ± ± ±1.10 Brix ± ± Starch (mg/g FW) 0.63± ± ± ±0.003 Ascorbic acid (Vitamin C) (mg/100g FW) ± ± ± ±2.24 Total carotenoids (mg/100g FW) 1.40± ± ± ±0.71 Total anthocyanins (mg/100g FW) 1.91± ± ± ±0.06 Total flavonoids (mg/g FW) 0.90± ± ± ±0.01 Chlorophyll a (mg/100g FW) 4.82± ± ± ±10.99 Chlorophyll b (mg/100g FW) 2.27± ± ± ±4.81 Total Chlorophyll (mg/100g FW) 7.08± ± ± ±15.78 Reducing Sugar (mg/g FW) 6.44± ± ± ±0.81 Total sugar (mg/g FW) 22.47± ± ± ±0.78 a Data is shown as the mean ± standard error of triplicated samples 11

12 Supplementary Table S2. Summary of kiwifruit genome DNA sequencing data Sequence data Library insert size Library number Base numbers Read length(bp) Base number after filtering Coverage 180 bp 1 29,993,534, ,604,783, bp 1 22,101,901, ,131,288, bp 1 36,446,507, ,895,628, Illumina reads 500 bp* 1 37,992,919, ,452,582, kb 1 13,687,594, ,552,927, kb 1 5,348,257, ,785,837, kb 1 2,440,967, ,230,080, kb 1 4,078,442, ,646,350, Total 7 114,097,206, ,846,896, * This library was only used for the quality evaluation of the genome assembly and not used for assembly and counting total bases and coverage. 12

13 Supplementary Table S3. Potential structure variations (misassemblies) identified in the kiwifruit genome assembly Scaffold Scaffold size Left anchor start Left anchor end Right anchor start Right anchor end SV type 13 No. supporting read pairs scaffold_ DELETION 13 scaffold_ DELETION 13 scaffold_ DELETION 14 scaffold_ DELETION 15 scaffold_ DELETION 18 scaffold_ DELETION 20 scaffold_ INVERSIO 20 scaffold_ DELETION N 22 scaffold_ DELETION 23 scaffold_ DELETION 23 scaffold_ DELETION 24 scaffold_ DELETION 27 scaffold_ DELETION 30 scaffold_ DELETION 31 scaffold_ DELETION 32 scaffold_ DELETION 37 scaffold_ DELETION 37 scaffold_ DELETION 41 scaffold_ DELETION 44 scaffold_ DELETION 46 scaffold_ DELETION 47 scaffold_ DELETION 51 scaffold_ DELETION 54 scaffold_ DELETION 63

14 Supplementary Table S4. Summary of statistics of EST mapping to the kiwifruit genome assembly Dataset Number Total length (bp) Base pair covered >90% of sequence covered by one scaffold by one scaffold >50% of sequence coverred by one scaffold by one scaffold Length Percent Number Percent Number Percent EST from A. chinese >=100 47,357 26,302,430 25,842, , , >=500 34,596 21,323,296 20,928, , , >= ,563 3, EST from A. deliciosa >=100 56,509 26,004,153 24,539, , , >=500 27,412 16,259,283 15,288, , , >= ,848 4, EST from A. eriantha >=100 12,336 6,117,919 5,868, , , >=500 7,236 4,403,289 4,230, , , >= ,218 3,

15 Supplementary Table S5. Repetitive sequences in the kiwifruit genome Element Type Number (copies) Lengths (bp) LTR/Copia 75,720 33,834,772 LTR/Gypsy 76,771 40,764,135 LTR/BEL 1, ,885 LTR/ERV1 16,034 3,674,207 LTR/ERV2 4, ,049 LTR/ERV3 4, ,031 Caulimoviridae 2,002 1,203,627 Endogenous 3, ,198 Class I LTR/other ,071 SINE/5S_rRNA 32 87,467 SINE/45S_rRNA ,219 SINE/other 5,102 1,945,511 LINE/CR1 7,976 2,459,485 LINE/L1 6,735 1,445,426 LINE/L ,532 LINE/LINE 3, ,017 LINE/R1 7 1,750 LINE/Penelope 1, ,573 DNA/MuDR 18,664 4,239,939 DNA/PIF_Harbinger 2, ,171 DNA/hAT 20,786 6,348,660 DNA/Helitron 16,518 3,256,637 DNA/P ,446 Class II DNA/EnSpm 31,928 7,716,352 DNA/Kolobok 3, ,798 DNA/Mariner/Tc1 4,413 1,086,953 DNA/Chapaev 3, ,307 DNA/other 18,641 4,549,450 Satellite/Satellite ,823 Other repeat 128,956 35,487,734 Unclassified 242,989 69,439,758 Total 698,275 ~222M 15

16 Supplementary Table S6. Enriched GO terms in gene clusters specific to flesh fruits (kiwifruit, tomato and grape) Gene Ontology term Enrichment FDR RNA-dependent DNA replication 7.94E-07 response to gibberellin stimulus 1.56E-05 response to UV-B 4.10E-05 flavonoid biosynthetic process response to cyclopentenone regulation of gibberellic acid mediated signaling pathway flavonoid metabolic process regulation of chlorophyll biosynthetic process regulation of cellular biosynthetic process phenylpropanoid metabolic process regulation of biosynthetic process regulation of nitrogen compound metabolic process response to toxin regulation of chlorophyll metabolic process phenylpropanoid biosynthetic process nucleic acid metabolic process raffinose family oligosaccharide biosynthetic process response to stress response to osmotic stress regulation of cellular metabolic process anthocyanin-containing compound biosynthetic process response to auxin stimulus response to light stimulus negative regulation of heterochromatin assembly negative regulation of chromatin assembly or disassembly toxin catabolic process anthocyanin-containing compound metabolic process response to salt stress primary metabolic process toxin metabolic process regulation of cofactor metabolic process regulation of secondary metabolic process defense response response to radiation oligosaccharide biosynthetic process response to organic cyclic compound

17 Supplementary Table S7. Enriched GO terms in gene clusters specific to kiwifruit Gene Ontology term Enrichment FDR RNA-dependent DNA replication 9.88E-15 DNA integration 7.40E-12 pollen tube reception 2.02E-06 intraspecies interaction between organisms 1.72E-05 steroid hormone mediated signaling pathway response to brassinosteroid stimulus positive regulation of protein export from nucleus brassinosteroid mediated signaling pathway cellular response to brassinosteroid stimulus positive regulation of cell fate commitment specification of floral organ identity formation of organ boundary formation of anatomical boundary regulation of cell fate commitment cellular response to steroid hormone stimulus

18 Supplementary Table S8. Comparative analysis of gene families in the vitamin C biosynthesis and recycling pathway Number of genes Gene Description Sweet Arabidopsis Kiwifruit Tomato Grape orange Alase aldonolactonase AO L-ascorbate oxidase APX L-ascorbate peroxidase DHAR dehydroascorbate reductase GalLDH L-galactono-1,4-lactone dehydrogenase GalUR D-galacturonic acid reductase GDH L-galactose dehydrogenase GGP GDP-L-galactose phosphorylase GLOase L-galactono-1,4-lactone dehydrogenase GME GDP-D-mannose-3,5-epimerase GMP GDP-D-mannose pyrophosphorylase GPP L-galactose-1-phosphate phosphatase IPS inositol-3-phosphate synthase MDHAR monodehydroascorbate reductase MIOX myo-inositol oxygenase PGI glucose-6-phosphate isomerase PMI mannose-6-phosphate isomerase PMM phosphomannomutase

19 Supplementary table S9. Expression of kiwifruit genes in the vitamin C biosynthesis and recycling pathway Normalized expression (FPKM) Gene Description Gene ID Immature Mature green Ripe Leaf fruit fruit fruit Alase aldonolactonase Achn Achn Achn Achn Achn Achn Achn AO L-ascorbate oxidase Achn Achn Achn Achn Achn Achn Achn APX L-ascorbate peroxidase Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn DHAR dehydroascorbate reductase Achn Achn GalLDH L-galactono-1,4-lactone dehydrogenase Achn GalUR D-galacturonic acid reductase Achn Achn Achn

20 Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn GDH L-galactose dehydrogenase Achn GGP GDP-L-galactose phosphorylase Achn Achn GLOase L-galactono-1,4-lactone dehydrogenase Achn GME GDP-D-mannose-3,5-epimerase Achn Achn GMP GDP-D-mannose pyrophosphorylase Achn Achn Achn GPP L-galactose-1-phosphate phosphatase Achn Achn IPS inositol-3-phosphate synthase Achn Achn Achn Achn MDHAR monodehydroascorbate reductase Achn Achn Achn Achn Achn Achn

21 Achn MIOX myo-inositol oxygenase Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn PGI glucose-6-phosphate isomerase Achn Achn Achn Achn PMI mannose-6-phosphate isomerase Achn Achn Achn PMM phosphomannomutase Achn

22 Supplementary Table S10. Comparative analysis of gene families in the carotenoid biosynthesis pathway Number of genes Gene Description Sweet Arabidopsis Kiwifruit Tomato Grape orange CCD carotenoid cleavage enzymes CHY non-heme hydroxylases CrtISO 7,9,7',9'-tetra-cis-lycopene isomerase CYCB lycopene beta-cyclases CYP P450 hydroxylases LCYB lycopene beta-cyclases LCYE lycopene epsilon-cyclase NCED carotenoid cleavage enzymes PDS phytoene desaturase PSY phytoene synthases PTOX alternative oxidase VDE violaxanthin deepoxidase ZDS zeta-carotene desaturase ZEP zeaxanthin epoxidase ZISO 9,15,9'-tri-cis-zeta-carotene isomerase

23 Supplementary Table S11. Comparative analysis of gene families in the flavonoid and anthocyanin biosynthesis pathway Number of genes Gene Description Sweet Arabidopsis Kiwifruit Tomato Grape orange CHS Chalcone synthase CHI Chalcone isomerase F3H Flavanone 3-hydroxylase F3 H Flavonoid 3 -hydroxylase DFR Dihydroflavonol 4-reductase LDOX / ANS Leucoanthocyanidin dioxygenase / Anthocyanidin synthase UFGT UDP-glucose flavonoid 3-O-glucosyltransferase FLS Flavonol synthase

24 Supplementary Table S12. Comparative analysis of gene families in Chlorophyll biosynthesis and degradation pathway Gene Description Number of genes Arabidopsis Kiwifruit Tomato Grape CAO chlorophyll a oxygenase GluTR glutamyl-trna reductase LHCB light harvesting complex photosystem II RBCS Rubisco small subunit CBR chlorophyll b reductase CLH chlorophyllase CLS chlorophyll synthase PAO pheophorbide a oxygenase PPH pheophytin pheophorbide hydrolase SGR chloroplast stay-green protein

25 Supplementary Table S13. Expression of kiwifruit genes in the carotenoid biosynthesis pathway Normalized expression (FPKM) Gene Description Gene ID Immature Ripe Leaf Mature green fruit fruit fruit CCD carotenoid cleavage enzymes Achn Achn Achn Achn Achn Achn Achn Achn CHY non-heme hydroxylases Achn Achn CrtISO 7,9,7',9'-tetra-cis-lycopene isomerase Achn CYCB lycopene beta-cyclases Achn Achn Achn CYP P450 hydroxylases Achn Achn LCYB lycopene beta-cyclases Achn Achn Achn LCYE lycopene epsilon-cyclase Achn Achn NCED carotenoid cleavage enzymes Achn Achn Achn Achn Achn PDS phytoene desaturase Achn Achn PSY phytoene synthases Achn Achn

26 Achn PTOX alternative oxidase Achn VDE violaxanthin deepoxidase Achn Achn ZDS zeta-carotene desaturase Achn ZEP zeaxanthin epoxidase Achn ZISO 9,15,9'-tri-cis-zeta-carotene isomerase Achn

27 Supplementary Table S14. Expression of genes involved in flavonoid and anthocyanin biosynthetic pathway in kiwifruit Gene expression value (FPKM) Gene Description Gene ID immature mature green ripe leaf fruit fruit fruit CHS Chalcone synthase Achn Achn Achn Achn Achn Achn CHI Chalcone isomerase Achn Achn Achn Achn Achn Achn F3H Flavanone 3-hydroxylase Achn Achn Achn Achn F3 H Flavonoid 3 -hydroxylase Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn Achn

28 Achn Achn Achn DFR Dihydroflavonol 4-reductase Achn Achn Achn Achn Achn Achn Achn LDOX / ANS Leucoanthocyanidin dioxygenase / Anthocyanidin synthase Achn Achn Achn Achn Achn UFGT UDP-glucose flavonoid 3-O-glucosyltransferase Achn Achn Achn Achn Achn Achn Achn Achn

29 Supplementary Table S15. Expression of kiwifruit genes in Chlorophyll biosynthesis and degradation pathway Gene Description Gene ID Normalized expression (FPKM) Leaf Immature fruit Mature green fruit Ripe fruit CAO chlorophyll a oxygenase Achn GluTR glutamyl-trna reductase Achn Achn Achn Achn LHCB light harvesting complex photosystem II Achn Achn Achn Achn RBCS Rubisco small subunit Achn Achn CBR chlorophyll b reductase Achn Achn CLH chlorophyllase Achn Achn CLS chlorophyll synthase Achn Achn PAO pheophorbide a oxygenase Achn Achn Achn Achn Achn Achn PPH pheophytin pheophorbide hydrolase Achn Achn Achn SGR chloroplast stay-green protein Achn Achn Achn Achn

Flavonoids in grapes. Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey. ASVO SEMINAR : MILDURA, 24 July 2014 AGRICULTURE FLAGSHIP

Flavonoids in grapes. Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey. ASVO SEMINAR : MILDURA, 24 July 2014 AGRICULTURE FLAGSHIP Flavonoids in grapes Simon Robinson, Mandy Walker, Rachel Kilmister and Mark Downey ASVO SEMINAR : MILDURA, 24 July 2014 AGRICULTURE FLAGSHIP Flavonoids in grapes Grape Flavonoids Flavonoids are important