Citrus mangshanensis pollen confers a xenia effect on linalool oxide

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Agricultural and Environmental Chemistry

Citrus mangshanensis pollen confers a xenia effect on linalool oxide accumulation in pummelo fruit by enhancing the expression of a cytochrome P450 78A7 gene CitLO1 haipeng zhang, Cuihua Liu, Jia-Long Yao, Cecilia Hong Deng, Shilin Chen, Jiajing Chen, Zhenhua Wang, Qiaoming Yu, Yunjiang Cheng, and Juan Xu J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b03158 • Publication Date (Web): 05 Aug 2019 Downloaded from pubs.acs.org on August 10, 2019

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Journal of Agricultural and Food Chemistry

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Citrus mangshanensis pollen confers a xenia effect on linalool oxide accumulation in

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pummelo fruit by enhancing the expression of a cytochrome P450 78A7 gene CitLO1

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Authors: Haipeng Zhang1#, Cuihua Liu2#, Jia-Long Yao3, Cecilia Hong Deng3, Shilin Chen4,

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Jiajing Chen1, Zhenhua Wang1, Qiaoming Yu1, Yunjiang Cheng1, Juan Xu1*

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1Key

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and Forestry, Huazhong Agricultural University, Wuhan 430070, PR China

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2College

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3The

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1142, New Zealand

Laboratory of Horticultural Plant Biology (Ministry of Education), College of Horticulture

of Horticulture, Northwest A&F University, Yangling, Shaanxi 712100, PR China

New Zealand Institute for Plant & Food Research Limited, Private Bag 92169, Auckland

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4Agricultural

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#Contribute

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Authors and Emails addresses:

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Haipeng Zhang: [email protected];

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Cuihua Liu: [email protected];

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Jia-Long Yao: [email protected];

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Cecilia Hong Deng: [email protected];

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Shilin Chen: [email protected];

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Jiajing Chen: [email protected];

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Zhenhua Wang: [email protected];

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Qiaoming Yu: [email protected];

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Yunjiang Cheng: [email protected];

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Juan Xu: [email protected];

Bureau of Yichang District, Yiling 443310, PR China

equally to this article.

ACS Paragon Plus Environment


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*Corresponding author:

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Juan Xu Key Laboratory of Horticultural Plant Biology (Ministry of Education), College of

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Horticulture and Forestry, Huazhong Agricultural University, Wuhan 430070, PR China

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Tel: +86-027-87286965

E-mail: [email protected];

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Abstract The aroma quality of citrus fruit is determined by volatiles that are present at extremely

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low levels in the citrus fruit juice sacs, it can be greatly improved by increasing volatiles. In this

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study, we showed that the contents of cis- and trans-linalool oxides were significantly increased in

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the juice sacs of three pummelos artificially pollinated with the Citrus mangshanensis (MS) pollen.

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A novel cytochrome P450 78A7 gene (CitLO1), was significantly upregulated in the juice sacs of

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‘Huanong Red’ pummelo pollinated with MS pollen in comparison with open pollination.

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Comparing to wild type tobacco Bright-Yellow2 cells, transgenic cells over-expressing CitLO1

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promoted 3–4 folds more conversion of (–)-linalool to cis- and trans-linalool oxides. Overall, our

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results suggest that MS pollen has a xenia effect on pummelo fruit aroma quality, and CitLO1 is a

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linalool oxide synthase gene that played an important role in the xenia effect.

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KEYWORDS: Volatile; Citrus mangshanensis; Pummelo (C. grandis); Xenia effect; CitLO1;

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Linalool oxides

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INTRODUCTION

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Citrus is an important fruit crop in the world, and is mainly consumed as fresh fruit although

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it may be processed into canned and juice-based products. Citrus fruit contain rich bioactive

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compounds such as carotenoids, flavonoids, limonoids, nomilins, organic acids, soluble sugars and

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volatile compounds.1-4 These bioactive compounds can affect the quality of fruit and the processed

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products. Organic acids and soluble sugars contribute to the taste, while flavonoids, volatiles

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and/or carotenoids contribute to the taste, flavor and also color of fruit.

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The content of volatiles varies largely among different tissues in various citrus germplasms.

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They are the highest amount in fruit peels, followed by the leaves, flowers and fruit juice sacs.4 In

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our previous study, as many as 198 volatile compounds were identified in citrus.4 However, most

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of volatile compounds are as background aroma, only one or a few volatile compounds are mainly

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responsible for the characteristic aroma trait of citrus. For example, d-limonene, the most

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abundant compound in most citrus germplasms, may serve as the background aroma.5 Cis- and

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trans-linalool oxides are the main characteristic aroma compounds in C.mangshanensis (MS) fruit.

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Valencene together with ethyl butyrate and hexyl hexanoate may be the characteristic aroma

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compounds and are specifically accumulated in sweet oranges.6,7 β-pinene, γ-terpinene, linalyl

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acetate and linalool can largely reconstruct the aroma of lemon.8 β-citronellal, nerol acetate and

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geranyl acetate contribute to the characteristic aroma of lime fruit.9 Linalool and its derivatives are

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mainly responsible for the sweet and fragrant aroma of Miyamoto Satsuma mandarin (C.

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reticulata).10 Notably, some terpenoid derivatives (terpenoid alcohols, terpenoid aldehydes and

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terpenoid oxides) at relatively low contents may also play important roles in the characteristic

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aroma of citrus fruit.5,6,10


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Monoterpenes and sesquiterpenes are important volatile compounds accumulated in citrus

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fruit, and are mainly biosynthesized via 2-C-methyl-d-erythritol 4-phosphate (MEP) and

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mevalonate (MVA) pathway, respectively.11 Some of them are further metabolized to various

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oxidized or cyclised derivatives by cytochrome P450 (CYP450) enzymes.12 For example,

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CYP71B31 functions in the production of various oxygenated linalool derivatives; CYP76C3

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metabolizes linalool into 8-hydroxylinalool and an unknown linalool derivative in Arabidopsis13.

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CYP76C1 is a multifunctional enzyme and catalyzes a cascade of oxidation reactions to produce

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8-hydroxy, 8-oxo, and 8-COOH-linalool in Arabidopsis.14 Lots of volatile compounds, such as

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d-limonene, nerol, geraniol, germacrene A and valencene, can be partially metabolized to yield

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corresponding derivatives by CYP450 enzymes.12,15

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Citrus volatile compounds are mainly accumulated in the oil sacs of the outer peel layer of

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citrus fruit.4,5,16 Citrus juice sacs are the most important tissues for fresh fruit consumption or

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canned products, but they contain only a small amount volatile compounds.4 Thus, the increases in

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the contents or changes in the profile of volatiles in the juice sacs may greatly improve citrus fruit

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quality.

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The term xenia was coined by Wilhelm Focke in 188117 and refers to the effect of pollen on

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maternal tissues, including the seed coat and pericarp. This effect is separate from the genetic

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contribution of the pollen towards the next generation. Xenia are shown to not only affect fruit

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size, color and maturation date,18 but also to influence the metabolites related to fruit qualities,

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such as soluble sugars, amino acids, proteins, and tannins in pear, plum, blueberry and

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kiwifruit.19-23 In citrus, xenia effects change the number and weight of seeds, size of fruit and

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fruiting rate.24,25 To date, many reports have descripted different xenia phenomenon, but no



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reports describe any xenia effect on the volatile compounds in fruit. Some studies have indicated

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that pollination of a certain apple species with pollen from different species could result in

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different fruit shape indexes, fruit mass, vitamin C and anthocyanin mass fraction.26,27 Most of

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pummelo germplasms are self-incompatible, and thus need pollination from other sources.28

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Hence, the pollen may affect the internal quality of pummelo fruit.

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MS is a wild citrus species native to China and produces inedible fruit containing large

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amounts ofvolatiles.5 The characteristic volatile compounds in MS are cis- and trans-linalool

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oxides and β-myrcene, conferring a special balsamic and floral aroma.5 Interestingly, the

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characteristic aroma of MS has been identified in fruit of ‘Citrange’ (Poncirus trifoliata × C.

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sinensis) plants that are growing near MS in our experimental field. Hence, we speculate that MS

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pollen possibly has a xenia effect on the ‘Citrange’ fruit aroma.

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To test the above speculation in this study, flowers of nine citrus cultivars were artificially

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pollinated with MS pollen. Xenia effect was found on the fruit of three pummelo cultivars,

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showing increased levels of cis- and trans-linalool oxides in fruit juice sacs, changed aroma and

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up-regulated expression level of a cytochrome P450 78A7 gene CitLO1 (cs3g22160). CitLO1 was

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demonstrated to be functional in the production of cis- and trans-linalool oxides in transgenic

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tobacco Bright-Yellow2 (BY2) cells.

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MATERIALS AND METHODS

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Materials

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Fruit on ‘Citrange’ plants grown near MS plants (CM) were collected in 2010 from the

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National Citrus Breeding Center (NCBC) in Huazhong Agricultural University (Wuhan, Hubei)


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for Gas Chromatography-Mass Spectrometry-Olfactometry (GC-MS-O) analysis.

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Flowers of ‘Citrange’, four orange (C. sinensis) cultivars, ‘Early-gold’, ‘Newhall’, ‘Jincheng’,

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‘Lane late’, three pummelo (C.grandis) cultivars, ‘Huanong Red’, ‘Hirado Buntan’ (HB), ‘Feicui’,

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and ‘Clementine’ mandarin (C. clementina) were pollinated with MS pollen separately, in five

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years 2011−2015 (Table 1). Mature fruit resulted from these pollinations were collected and

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washed with tap water. The whole fruit of ‘Huanong Red’ pummelo was used for sensory

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evaluation. The flavedo, albedo, segment membrane and juice sacs of all nine citrus cultivars were

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separated with a scalpel, immediately placed in liquid nitrogen, and stored in ultra-low

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temperature freezers (−80°C) for further analyses.

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Artificial Pollination

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Pollen collection and artificial pollination were conducted as previously described.29 Flowers

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of MS were collected before flower opening. The pollens were collected from dry and open

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anthers under a dim light environment, and then placed in a 28°C oven for drying in dark

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environment. Then, the dry pollens were placed in a centrifuge tube wrapped with foil and stored

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in the desiccator with silica gel as desiccant. Healthy balloon stage flowers of the nine cultivars

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were selected and emasculated, pollinated with MS pollen separately, and subsequently bagged.

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After two days, the bag was removed. The fruit were collected at mature stage for Gas

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Chromatography-Mass Spectrometry (GC-MS) analysis. The numbers of pollinated flowers and

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harvested fruit were shown in Table 1.

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GC-MS-O Analysis

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The GC-MS-O analysis was carried out with an Agilent 6890 GC coupled with an Agilent

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5973 Network mass spectrometer (Agilent Technologies, Palo Alto, CA, USA) equipped with an



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olfactory detected port Gerstel ODP-2 (Gerstel AG Enterprise, Mülheim an der Ruhr, Germany).

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The GC was fitted with an HP-Innowax column (60m × 0.25mm × 0.25μm, Agilent, Palo Alto,

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CA,USA). Other parameters used were the same as described by Liu et al.5

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Extraction and Analysis of Volatiles, Soluble Sugars, Organic Acids and Carotenoids

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Volatiles were extracted as described by Liu et al.5 and quantified using a TRACE GC Ultra

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GC coupled with a DSQ II mass spectrometer (Thermo Fisher Scientific, Waltham, MA) with a

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TRACE TR-5 MS column (30 m × 0.25 mm × 0.25 μm; Thermo Scientific, Bellefonte, PA)

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with a split ratio of 50:1 for the flavedo and a splitless mode for the albedo, segment membrane

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and juice sacs. For identification of volatile compounds, 22 authentic standards were used

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(Supplementary Table 1), and the rest volatile compounds were identified using Xcalibur software

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based on NIST, EPA/NIH Mass Spectral Library (NIST 2008) and the Wiley Registry of Mass

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Spectral Data (8th ed.). The concentrations of volatile compounds were quantified based on the

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contents of internal standard (Methyl nonoate, Sigma, St. Louis, Mo, USA).

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The soluble sugars and organic acids were determined according to Liu et al.30 using a GC

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(Agilent 7890A, Agilent Technologies, Santa Clara, CA,USA) coupled with a HP-5 capillary

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column. Standards of three soluble sugars and three organic acids were used for the qualitative

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analysis, and β-D-phenyl glucopyranoside was used as an internal standard for quantitative

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analysis. All of these authentic standards were purchased from Sigma-Aldrich (St. Louis, Missouri,

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United States). The carotenoids in different citrus tissues were measured by high-performance

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liquid chromatography (HPLC). The method for extraction and parameters for analysis were

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according to Liu et al.30 The standards of violaxanthin, phytoene, α-carotene and lutein were

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purchased from CartoNature (Lupsingen, Switzerland), and β-carotene and lycopene were


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purchased from Sigma-Aldrich.

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Sensory Evaluation of ‘Huanong Red’ Pummelo Fruit

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The whole fruit of ‘Huanong Red’ pummelo resulted from pollination with MS pollen (HM)

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and open pollination (HW) was evaluated by 11 sensory evaluators. A triangle sensory evaluation

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method was adopted to distinguish HM from HW. Four groups of fruit, each including two HM

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and one HW fruit or two HW and one HM fruit, were presented to the sensory evaluators who

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were asked to identify the one fruit showing a different smell from other two fruit. The results

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were analyzed using the one-way analysis of variance (ANOVA).

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RNA-Sequencing and Analysis

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Total RNA was extracted using method as described by Liu et al.31 from HM and HW fruit

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collected in 2012 and 2013. For each sample, mRNA was purified from 2 μg total RNA by

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oligo-dT beads, used to construct RNA-Seq libraries with TruSeq® RNA Sample Prep Kit v2

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(Illumina, SanDiego, CA,USA). The quality of the libraries was checked using a Qubit 3.0

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fluorometer and Quant-iT™ PicoGreen® dsDNA Assay Kit (Invitrogen). The libraries were

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sequenced on the Illumina HiSeq 2000 platform in a paired-end mode.

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The raw RNA-Seq data were cleaned by removing adaptor sequences and low-quality reads

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using Cutadapt v1.18 and Trimmomatic-0.36 software respectively.32,33 The clean data were

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mapped to the reference sweet orange genome assembly (http://citrus.hzau.edu.cn/orange/

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index.php) using the TopHat program.34 Based on the mapping results, transcript abundance for

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each gene was estimated using HTSeq,35 and differentially expressed genes were identified using

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edgeR package.36 Gene annotation was performed using PlabiPD (Mercator sequence annotation,

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http://www.plabipd.de/portal/web/guest/mercator-sequence-annotation). The raw data were upload



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to NCBI SRA database (BioProject ID: PRJNA549576). The Pearson correlation coefficients

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between biological replicateswere calculated by R package‘‘corrplot’’.37 The heatmap was

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constructed using TBtools.38

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Quantitative Reverse Transcription Polymerase Chain Reaction (qRT-PCR)

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The total RNA extracted from the HW and HM fruit was used for single-strand cDNA

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synthesizing using the RevertAid First Strand cDNA Synthesis Kit (K1621, Thermo Scientific).

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The cDNA was used as template in PCR together with gene-specific primers (Supplementary

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Table 2) designed using Primer Express 3.0 (Applied Biosystems, Foster City, CA). The PCR was

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performed using the ABI 7900 Fast Real Time System (PE, Applied Biosystems, Foster City, CA,

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USA). Actin gene was as an internal control to normalize expression between different samples.

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The Actin primers and data analysis protocol were the same as described by to Liu et al.39

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Gene Isolation and Sequence Analysis

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The CDS ofCitLO1genewas amplified from the cDNA of HM fruit using a pair of primers

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(F:ATGGGTCGAATCCCCATCCCCGG, R: CCGGGGATGGGGATTCGACCCAT) and the

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Phanta® Max Super-Fidelity DNA Polymerase (P505, Vazyme, Nanjing, China), then cloned into

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a pTOPO-Blunt vector (CV1702, Aidlab, Co., Beijing, China). The protein sequences for 14

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CYP450and five putative citrus CYP450 genes identified in this study were download from NCBI

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database and sweet orange reference genome respectively. Those sequences were used to construct

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a phylogenetic tree by MEGA7 with the neighbor-joining method based on ClustalW analysis.40

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The alignment of 11 CYP78A7 and CitLO1 protein sequences was conducted using the GeneDoc

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software version 2.7.

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Expression of CitLO1 in Tobacco BY2 Cells


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The CitLO1 CDS was transferred into pDONR201 vector by the Gateway BP reaction to

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produce an entry clone. The CitLO1 CDS was further transferred from the entry clone into the

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binary vector PH7WG2D by Gateway LR reaction to form a translational fusion with green

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fluorescent protein (GFP) reporter gene under the control of CaMV-35S promoter.41 Then the

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destination vector was electroporated into Agrobacterium tumefaciens (GV3101). A single

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positive GV3101 colony containing the CitLO1 CDS was used for inoculation in 50 mL liquid LB

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medium and the liquid culture was grown at 28°C until its absorbance reached 0.6–0.8 at 600 nm.

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The GV3101 cultures were used to transform tobacco BY2 cells according to a previously

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reported

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stereomicroscope with the GFP filter of a 488-nm excitation and confirmed by PCR using CitLO1

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gene specific primers.

method.42

Transgenic

BY2

calli

were

identified

using

an

epifluorescence

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Fresh BY2 cultures (2 g each) of wild-type (WT) and transgenic (BY2-CitLO1) cells were

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grown on agar medium for 7 d. Then, 1 g fresh cells were cultured in 20 ml liquid medium for

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another 4 d, followed by the addition of 50 μM (–)-linalool (95.0%, CAS126-91-0, Aldrich,

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Spain)as the substrate. At 48 h after substrate addition, the cells were collected by centrifugation

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and extracted for volatiles that were analyzed by GC-MS. Linalool oxides in the volatile mixture

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were quantified using three biological replicates and the standard curves of pure cis- and

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trans-linalool oxides according to Liu et al,5 The profiles of cis- and trans-linalool oxides shared a

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common specific ion (m/z=59). The volatiles of BY2-CitLO1 cells were analyzed using the total

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ion current (TIC) and selective ion monitoring (SIM) (m/z=59) mode. The equations of standard

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curves obtained from the TIC mode (Table 1) based on 4–6 concentrations were used for

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quantifying the contents of cis- and trans-linalool oxides.



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Data Analysis

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The contents of volatiles, soluble sugars, organic acids and carotenoids were quantified based

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on the contents of internal standards. In ‘Huanong Red’ pummelo fruit, the contents of each

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compound in HM fruit were set as 1 for normalization. SAS software (SAS Institute Inc., Cary,

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NC, USA) was used for analysis of variance with ANOVA (P< 0.05).

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RESULTS

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Identification of Characteristic Aroma of C.mangshanensis in ‘Citrange’ Fruit

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C.mangshanensis (MS) has a special aroma contributed by some aromatic volatiles that are

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not normally found in other citrus species5. Interestingly, our preliminary experiments identified

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these especial aromatic compounds in the fruit of ‘Citrange’ plants that were grown near MS

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(termed as CM fruit). Thirty aromatic compounds were identified in the peel of the CM fruit by

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GC-MS-O analysis (Table 2). Of them, 20 were also identified in the peel of MS fruit (Table 2),

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including the three compounds, β-myrcene, cis- and trans-linalool oxides, contributing to MS

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special aroma. The β-myrcene content in the CM fruit (520.83 ± 58.84 ng/g) was significantly

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higher than that in normal ‘Citrange’ fruit (283.02 ± 102.92 ng/g) that was not near MS plants.

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The contents for cis- and trans-linalool oxides were too low in both types of fruits to show any

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significant difference between them. Nevertheless, this result indicated a possible xenia effect of

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MS pollen on ‘Citrange’ fruit chemical composition.

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Effect of C.mangshanensis Pollen on Fruit Volatile Profiles

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To confirm the above xenia effect, we compared two groups of ‘Huanong Red’ pummelo

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fruit, obtained from pollination with MS pollen (HM) or obtained from open pollination (HW). A


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sensory evaluation result showed that the aroma was significantly different between HM and HW

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fruit (P=0.0007) (Table 3), indicating that the xenia effect of MS pollen changed the olfactory

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quality of ‘Huanong Red’ pummelo fruit.

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The above fruit were further shown to contain at least 16 volatile compounds in their juice

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sacs, including four monoterpenes, two monoterpene oxides, one monoterpene aldehyde, five

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sesquiterpenes, one sesquiterpene alcohol, one sesquiterpene ketone and two aldehydes. The

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contents of the two monoterpene oxides (cis- and trans-linalool oxides) in HM were significantly

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higher than those in HW in two harvest seasons (Supplementary Table 3).

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In the flavedo of HM and HW fruit, 50 volatile compounds were detected in two harvest

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seasons, including 29 monoterpenes and their derivatives, 16 sesquiterpenes and their derivatives,

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four aldehydes and one ester (Supplementary Table 4). Five volatile compounds (sabinene hydrate,

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citronellal, (E,E)-α-farnesene, methyl palmitate and octanal) were detected only in 2012. The

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concentrations of thirteen monoterpene compounds (α-thujene, α-pinene, camphene, sabinene,

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β-pinene,

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trans-β-ocimene, γ-terpinene and terpinolene) were significantly higher in HW than in HM in

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2012; however, only one monoterpene (α-thujene) was significantly different between HM and

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HW in 2013. The total content of monoterpenes in HW was significantly higher than that in HM,

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especially in 2012. Four monoterpene oxides were detected, and cis- and trans-linalool oxides

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were significantly lower in HM than in HW in two seasons. Twelve and thirteen sesquiterpenes

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were detected in 2012 and 2013, respectively. The concentrations of eight (2012) and five (2013)

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sesquiterpenes were significantly higher in HW than in HM (Supplementary Table 4).

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β-myrcene,

pseudolimonene,

α-phellandrene,

d-limonene,

cis-β-ocimene,

In the albedo, 24 volatile compounds were detected, with 15 and 18 compounds being found



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in 2012 and 2013, respectively. Among the six monoterpenes, two monoterpenes showed

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significantly lower concentrations in HM than in HW in 2012, but no difference was observed for

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all the monoterpenes in 2013. As for monoterpene oxides, the concentrations of cis- and

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trans-linalool oxides in HM were significantly higher than those in HW in 2013, but no difference

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was observed in 2012. Only one monoterpene alcohol (cis-carveol) was significantly higher in

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HM than in HW based on concentration. For sesquiterpenes, only one showed significant

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differences between HW and HM (Supplementary Table 5).

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In the fruit segment membrane, 18 volatile compounds were detected, with 10 and 14

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compounds being detected in 2012 and 2013, respectively. Among the 18 compounds, four

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monoterpenes (α-pinene, β-pinene, β-myrcene and d-limonene) were detected, but they showed no

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significant differences between HM and HW. There were three monoterpene oxides, and cis- and

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trans-linalool oxides were significantly higher in HM than in HW. A total of four monoterpene

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alcohols and three sesquiterpenes were found, but only limonene-diol was significantly higher in

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HM than in HW (Supplementary Table 6).

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The above data obtained with ‘Huanong Red’ pummelo clearly showed a xenia effect. To

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further test whether the xenia effect work in different pummelo cultivars, two other pummelo

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cultivars, ‘HB’and ‘Feicui’, were pollinated with MS pollen in Yichang (Hubei province) and

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Lishui (Zhejiang province) in 2015 (Table 1). Fourteen ‘HB’ fruit (HBM) and three ‘Feicui’ fruit

280

(FM) obtained with MS pollen were collected at maturity stage, and six ‘HB’ fruit (HB) and four

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‘Feicui’ (FC) fruit obtained with open pollination were collected at maturity stage. Analysis of

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volatiles showed that the contents of cis- and trans-linalool oxides in the juice sacs of pummelo

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fruit were significantly increased by the pollination with MS pollen (Fig.1), conforming that the


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xenia effect worked in different pummelo cultivars.

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We further tested if the xenia effect could be present on non-pummelo cultivars. Fruit of one

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‘Citrange’, four orange (‘Early-gold’, ‘Newhall’, ‘Jincheng’, ‘Lane late’) and one ‘Clementine’

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mandarin were produced by pollination with the MS pollen. The volatile profiles in juice sacs of

288

the fruit were analyzed. The result showed a low level of total volatile contents, non-detectable

289

amount of cis- and trans-linalool oxides and no significantly difference to fruit obtained from

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open pollination

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detected in these three types of non-pummelo citrus cultivars.

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Content Changes of Other Metabolites in HM and HW Fruit

(Supplementary Table 7). This result indicated that the xenia effect could not be

293

Totally, three main organic acids and three main soluble sugars were analyzed in different

294

tissues of ‘Huanong Red’ pummelo (Supplementary Table 8). In 2013, the total content of organic

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acids in flavedo was lower than that in 2012, while that of soluble sugars was higher. The content

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of malic acid was significantly higher in HM than in HW, and it was the opposite case for fructose

297

and glucose in 2012. However, there were no significant differences between HM and HW in

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2013, due to the large differences in biological replicates (Supplementary Table 8). The content of

299

citric acid in albedo in HM was significantly higher than that in HW in 2013, but not detected in

300

neither HW nor HM in 2012. The content of quinic acid in the segment membranes was

301

significantly higher in HM than that in HW in 2013, but was not significantly different between

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the two types of fruits in 2012. In juice sacs, the contents of the six compounds were not

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significantly different between HM and HW in 2012, even three soluble sugars in HM were

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significantly lower compared with those in HW in 2013 (Supplementary Table 8). Compared with

305

HW fruit, HM fruit appeared to show an increased level of organic acids but a decreased level of



306

soluble sugars.

307

Among the 14 carotenoids detected in the flavedo, phytoene was the most abundant

308

compound and did not show significant differences between HM and HW in two seasons. Seven

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compounds (violaxanthin, luteoxanthin, 9-cis-violaxanthin, lutein, zeaxanthin, β-crytoxanthin and

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β-carotene) were significantly lower in HM than in HW in 2012, but only two compounds

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(α-carotene and cis-β-carotene) were significantly lower in HM than in HW in 2013. Three

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(β-carotene, γ-carotene and lycopene) and five compounds (phytofluene a, phytofluene b,

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β-carotene, γ-carotene and lycopene) were detected in the albedo and juice sacs respectively in

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two seasons, but they showed no significant differences between HW and HM. Three compounds

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(β-carotene, γ-carotene and lycopene) were detected in the segment membrane, among which

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β-carotene and γ-carotene were significantly higher in HM than in HW. Therefore, MS xenia

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mainly affects the carotenoids in flavedo and segment membrane, but has no significant effect on

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thecarotenoids in other fruit tissues (Supplementary Table 9).

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Candidate Genes for Production of Cis- and Trans-linalool Oxides

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To identify the genes contributing to the increased biosynthesis of cis- and trans-linalool

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oxides in ‘Huanong Red’ pummelo pollinated with MS, the juice sacs of HW and HM collected in

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2012 (HW-1 and HM-1) and in 2013 (HW-2 and HM-2) were used for RNA-Seq analyses.

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Pearson correlation coefficient was 0.92 between HW-1 and HW-2, while was 0.94 between

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HM-1 and HM-2 (Supplementary Fig.1), which indicated a good repeatability between biological

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replicates. A total of 375 differentially expressed genes (|log2FC| > 2, P

Citrus mangshanensis pollen confers a xenia effect on linalool oxide

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