Genetic and biochemical characterization of fruit from different apricot accessions highlights apricot as a rich source of and , and indicates CCD4 gene as a potential regulator of fruit color

Tal Isaacson, Fruit tree sciences, Newe Ya’ar, ARO, RGC8 ISRAEL June 21 -24 th , 2016 ∑Essential components in , serve as precursors to phytohormones, furnish many fruit and flowers with bright colors and degredation products are important aroma compounds. ∑Essential in our diet as a source for vitamin A and considered as protective agents decreasing the risk of different diseases such as cancer and cardiovascular disorders. Important in determining fruit color, taste and nutritional value OPP + PPO GGPP GGPP Psy Carotenoids in green tissues Pds 15-cis Phytoene

9,15-cis Phytofluene Pds

9,15,9’-cis ζ- Z-Iso

9, 9’- ζ-carotene cis Zds

7, 9, 9’-cis Zds

7, 9, 7’, 9’-cis (Prolycopene) CrtISO

All- Lycopene Lcy-b Lcy-e trans

δ-Carotene Lcy-b γ-Carotene Lcy-b

α-Carotene CrtR-b β-Carotene CrtR-b CrtR-e OH

HO HO β-Cryptoxanthin CrtR-b OH

HO Zep OH

O HO Zep OH O

O HO Nxs? OH O Nced Xanthoxin .OH HO OPP + PPO GGPP GGPP Psy Carotenoids in colored Pds 15- Phytoene cis tissues

9,15-cis Phytofluene Pds

9,15,9’-cis ζ-carotene Z-Iso

9, 9’- ζ-carotene cis Zds

7, 9, 9’-cis Neurosporene Zds

7, 9, 7’, 9’-cis Lycopene (Prolycopene) CrtISO

All- Lycopene Lcy-b Lcy-e trans

δ-Carotene Lcy-b γ-Carotene Lcy-b

α-Carotene CrtR-b β-Carotene CrtR-b CrtR-e OH

HO HO Lutein β-Cryptoxanthin CrtR-b OH

HO Zeaxanthin Zep OH

O HO Antheraxanthin Zep OH O

O HO Violaxanthin Nxs? OH O Nced Neoxanthin Abscisic acid Xanthoxin .OH HO Apricot germplasm collection in Newe-Ya’ar

Apricot germplasm collection includes about 85 accessions (imported and ‘local’) and dozens of hybrid lines. The collection exhibit a wide range of phenotypes. Carotenoids content and composition of fruit from the Newe-Ya’ar apricot germplasm collection Fruit samples were collected from all accessions and analyzed by HPLC

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0 Carotenoid μg/g Weight Fresh content phytoene phytofloune isomer ζ-carotene neurosporene lycopene isomer γ- carotene β-carotene β-cryptoxanthin esters unknown Carotenoids content and composition of fruit from the Newe-Ya’ar apricot germplasm collection Fruit samples were collected from all accessions and analyzed by HPLC

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0 Carotenoid μg/g Weight Fresh content phytoene phytofloune isomer ζ-carotene neurosporene lycopene isomer γ- carotene β-carotene β-cryptoxanthin esters unknown Carotnoids composition in apricot fruit is unusual

unknown Phytoene 3% β-cryptoxanthin 5% Phytofluene 1% ζ-Carotene phytoene Neurosporene 26% Lycopene

δ-Carotene γ-Carotene

α-Carotene β-Carotene β-carotene 31% Lutein β-Cryptoxanthin

Zeaxanthin phytofloune Antheraxanthin 25%

Violaxanthin γ- carotene Neoxanthin 2% lycopene isomer ζ-carotene 5% 2% Carotnoids composition in Prunus fruit

Phytoene

Phytofluene

ζ-Carotene Apricot Neurosporene

Lycopene

δ-Carotene γ-Carotene

α-Carotene β-Carotene Plum Lutein β-Cryptoxanthin

Zeaxanthin

Antheraxanthin Peach Violaxanthin

Neoxanthin Pictures: chefscottmcmillen.wordpress.com americanathleticinstitute.org Choosing representing accessions for RNA-Seq

Mustakawi Canino Paz

80.0

60.0

40.0

20.0 µg/gFW

0.0 β-cryptoxanthin lycopene isomer γ- carotene neurosporene ζ-carotene β-carotene phytofloune phytoene xanthophylls others RNA-Seq

4 3 2 1 RNA Extraction RNAseq

Sample ID # Reads % of raw clusters per lane Mean Quality Score (PF) Mustakawi 1 33,363,354 8.19 35.36 Mustakawi 2 27,301,292 6.7 35.46 Mustakawi 3 34,043,542 8.36 35.41 Mustakawi 4 44,934,796 11.03 35.43 canino1 31,405,632 7.71 35.39 canino2 32,970,584 8.1 35.53 canino3 30,453,924 7.48 35.58 canino4 35,265,574 8.66 35.6 PAZ1 26,353,976 6.47 35.46 PAZ2 33,280,120 8.17 35.52 PAZ3 38,336,006 9.41 35.5 PAZ4 34,259,110 8.41 35.33 Undetermined 5,300,702 1.3 29.59 Higher expression of Psy-1 in ‘Paz’ fruit Ggpp*2 Psy Phytoene 1500 Pds Psy Phytofluene 1000 Pds ζ-Carotene 500 Zds Neurosporene 0 40 Zds Pds Lycopene 30

δ-Carotene γ-Carotene 20

α-Carotene β-Carotene 10

β-Cryptoxanthin 0 Lutein 1000 Zds Zeaxanthin Antheraxanthin 500 Violaxanthin ABA 1 23 4 1 23 4 1 2 3 4 Neoxanthin 0 Mustakawi Canino Paz Higher expression of genes encoding carotenoid cleavage enzymes in ‘Mustakawi’ Ggpp*2

Phytoene 500 450 Phytofluene 400 350 NCED 300 ζ-Carotene 250 200 Neurosporene 150 100 50 Lycopene 0 123412341234 δ-Carotene γ-Carotene Mustakawi canino paz α-Carotene β-Carotene CCD4

Apocarotenoids 40 β-Cryptoxanthin Lutein 35 CCD4 30 Zeaxanthin 25 20 15 Antheraxanthin 10 5 Violaxanthin 0 ABA NCED 123412341234 Neoxanthin Mustakawi canino paz Genetic Association Studies for carotenoid traits in the Apricot Germplasm Collection

% µg\gFW

β Ph Phf Ly Tr β Ph Phf Ly Vis_c To_c Ccd4-1 0.0008 0.0003 0.0026 0.0026 0.0001 0.0018 0.0001 0.0028 Ccd4-2 0.0016 0.0046 0.0009 0.0096 0.0009 0.0003 0.0012 0.0003 0.0083 CrtISO 1 CrtR-b1-2 0.0001 0.0034 0.002 0.0001 0.0037 0.002 Cyc1-1 0.0075 0.0065 0.0095 Cyc1-2 0.0036 0.0023 Cyc2-1 0.0045 0.0039 Cyc2-2 0.0029 0.0021 Ipi 1 Pds1 0.0038 0.0078 0.0002 0.0078 Pds2 Psy1-1 0.0088 Ptox SDG8-1 0.0007 0.0038 0.0056 0.0056 0.0006 0.0023 0.0003 Zep Ziso1 0.0045 Summary HPLC analysis of fruit from more than 120 accessions: ∑ Great variation in carotenoid profile among accessions ∑ Apricot fruit exhibit unique carotenoid composition with high amounts of colorless carotenoids and unusual isomers of lycopene. RNA-Seq of ripening fruit series: ∑ Elevated expression of genes encoding the first enzymatic steps in the biosynthesis pathway might contribute to high carotenoid content in the fruit. ∑ Elevated expression of genes encoding carotenoid cleavage enzymes might contribute to low carotenoid accumulation in pale yellow fruit. Genetic association studies: ∑ Suggest CCD4 as a potential regulator of Fruit carotenoid content. Acknowledgment

Matat Zohar Efraim Lewinsohn Ksenija Gasic (Clemson University) Taly Trainin Einat Bar Keren Shemesh Einav Shor-Shimoni

Adi Faigenboim

Doron Holland Irit Bar-Yaakov Kamel Hatib Rotem Beja Zohar Ben-Shimhon

Amir Sherman Ada Rozen