Comparative analysis of volatile compounds in thirty nine melon cultivars by headspace solid-phase microextraction and gas chromatography-mass spectrometry

Journal Pre-proofs Comparative analysis of volatile compounds in thirty nine melon cultivars by headspace solid-phase microextraction and gas chromatography-mass spectrometry Jianda Shi, Haibo Wu, Mu Xiong, Yanjun Chen, Jihao Chen, Bo Zhou, Hui Wang, Liangliang Li, Xiaofa Fu, Zhilong Bie, Yuan Huang PII: DOI: Reference:

S0308-8146(20)30196-5 https://doi.org/10.1016/j.foodchem.2020.126342 FOCH 126342

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Food Chemistry

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Please cite this article as: Shi, J., Wu, H., Xiong, M., Chen, Y., Chen, J., Zhou, B., Wang, H., Li, L., Fu, X., Bie, Z., Huang, Y., Comparative analysis of volatile compounds in thirty nine melon cultivars by headspace solid-phase microextraction and gas chromatography-mass spectrometry, Food Chemistry (2020), doi: https://doi.org/10.1016/ j.foodchem.2020.126342

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Comparative analysis of volatile compounds in thirty nine melon cultivars by headspace solid-phase microextraction and gas chromatography-mass spectrometry Jianda Shi1†, Haibo Wu2†, Mu Xiong1, Yanjun Chen3, Jihao Chen2, Bo Zhou2, Hui Wang4, Liangliang Li4, Xiaofa Fu2, Zhilong Bie1, Yuan Huang1* 1College

of Horticulture and Forestry Sciences, Huazhong Agricultural

University and Key Laboratory of Horticultural Plant Biology, Ministry of Education, Wuhan, 430070, P. R. China 2Hainan

Sanya Crop Breeding Experimental Center, Xinjiang Academy of

Agricultural Sciences, Sanya, 572000, P. R. China 3College

of Plant Science and Technology, Huazhong Agricultural University,

Wuhan, 430070, P. R. China 4Hainan †These

Nanfan Administration Office, Sanya, 572000, P. R. China

authors contributed equally to this work

*To whom correspondence should be addressed Yuan Huang E-mail: [email protected], Tel: +86 27 87282010, Fax: +86 27 87282010

1

Abstract The types and amounts of volatiles in the fruits of 39 melon cultivars were determined. We identified 146 volatiles, including 55 esters, 23 aldehydes, 30 alcohols, 15 ketones, 6 acids and 17 others. Ethyl acetate, (Z)-6-nonenal and 3,6-(E,Z)-nonadien-1-ol were the most three abundant volatiles (average content > 50 µg/kg FW). Aroma profiles showed significant differences among cultivars. Zhongtian49 and Zhongtian20 had the most abundant aroma components (76) and Jinguniang exhibited the least (23). One non-climacteric inodorus cultivar (Xizhoumi25) had the highest content of total volatiles (1840 µg/kg FW). Principal component analysis clustered the 39 melon cultivars into five groups. This work describes the comparative diversity of melon fruit volatiles for a large number of cultivars. Furthermore, this study could support the selection of cultivars with a flavor that suits the public and also future breeding work towards the genetic improvement of melon flavor. Keywords: aroma volatiles; cucumis melo; fruit quality; HS-SPME-GC-MS

1. Introduction Melon (Cucumis melo L.) is one of the most economically important and widely cultivated crops in the world, with 40% of the total amount being produced in China (Luo, Pang, Xu, Bi, Zhang, & Wu, 2018). Volatile compounds are major determinants of fruit quality perceived by consumers (Kourkoutas, Elmore, & Mottram, 2006; Amaro, Beaulieu, Grimm, Stein, & Almeida, 2012; Vallone et al., 2013; Ye et al., 2017; Poverenov et al., 2018). Melons accumulate a large 2

number of volatile aroma compounds, according to the review presented in melon samples reported, 291 volatile compounds have been identified (Fredes, Sales, Barreda, Valcarcel, Rosello, & Beltran, 2016). Melon aroma is strongly dependent on the genotypes (Verzera, Dima, Tripodi, Ziino, Lanza, & Mazzaglia, 2011; Bernillon et al., 2013; Lignou, Parker, Oruna-Concha, & Mottram, 2013; Chen, Cao, Jin, Tang, & Qi, 2016; Esteras et al., 2018; Kende et al., 2019). Around 240 compounds (mainly esters) have been identified in climacteric Galia or Cantaloupe melons, but a considerable reduction in the aroma profile have been identified in non-climacteric melons such as Rochet, Piel de Sapo, honeydew or Casaba type (Beaulieu & Grimm, 2001; Kourkoutas et al., 2006; Obando-Ulloa et al., 2008; Perry, Wang, & Lin, 2009; Dos-Santos, Bueso, & Fernandez-Trujillo, 2013; Tang, Zhang, Cao, Wang, & Qi, 2015; Chaparro-Torres, Bueso, & Fernandez-Trujillo, 2016). However, the diversity of fruit volatiles has been confirmed mainly in a small number of melon cultivars and breeding lines (Aubert & Bourger, 2004; Obando-Ulloa, Ruiz, Monforte, & Fernandez-Trujillo, 2010; Esteras et al., 2018; Kende et al., 2019), little information is available on the comparative analysis of volatile compounds in a large number of commercial hybrid cultivars. China is the largest producer and consumer country for melon, and a lot of hybrid cultivars are cultivated; however, the information of the fruit volatile compounds is largely unknown. In this study, headspace solid-phase microextraction

(HS-SPME)

combined 3

with

gas

chromatography-mass

spectrometry (GC-MS) was applied to determine the types and contents of fruit volatile compounds in 39 melon cultivars. The aim of this study is to evaluate the aroma quality of the melon at the cultivar level and to provide a reference for future cultivation and breeding. 2. Materials and methods 2.1. Plant materials In this study, 39 melon cultivars were grown in the plastic greenhouse at Hainan Sanya Crop Breeding Experimental Center, Xinjiang Academy of Agricultural Sciences, China. Details and pictures of mature fruit of the 39 cultivars were listed in Supplementary Table S1 and Fig. 1. The 39 cultivars were composed of typical hybrid cultivars widely cultivated in China. The plants were managed with the same level of irrigation, pruning, disease control, and fertilization. One fruit was kept in each plant. Fruits were sampled at full ripening, as determined on the maturity standard of each cultivar, generally the total soluble solid content was above 12oBrix (Supplementary Table S1). Soluble solid is an important indicator to measure melon fruit quality, is often shown by the Brix value (Flores, Sanchez, Perez-Marin, Lopez, Guerrero, & Garrido-Varo, 2008). Nine fruits for each cultivar were harvested, each replicate containing 3 fruits. The melon fruit was cut longitudinally into halves, and then flesh (after peel and seeds removal) was chopped into small pieces and was frozen in liquid nitrogen. All samples were stored at -80 °C until analysis. 2.2. HS-SPME analysis 4

After freeze-thawing at 5°C, the flesh was used to prepare juice for volatile analysis. For each extraction, a 6 mL of the juice was pipetted into a vial and 2 g of NaCl and 10 µL 3-octanol solution (32.75 mg L-1, added as internal standard) were added. The vials were sealed tightly and placed in the Triplus auto-sampler (Thermo Fisher Scientific, Waltham, MA, USA). Headspace (HS) volatiles were extracted by exposing the melon juice sample to a 2 cm long Supelco SPME fiber composed of 50/30 μm carboxen divinylbenzene polydimethylsiloxane (CAR/DVB/PDMS) (Supelco Inc., Bellefonte, PA, USA). Samples were stirred at 50°C for 30 min to equilibrate the solution and headspace. The fiber was exposed to the headspace of the capped vial to adsorb volatile substances for 30 min, then withdrawn, and introduced into a heated injection port of the gas chromatography (GC) for desorption at 250oC for 5 min. 2.3. GC-MS analysis A GC-MS system (Trace DSQII, Thermo Fisher Scientific, Waltham, MA, USA) equipped with a HP-5 MS capillary column (30 m × 0.25 mm id with 0.25 μm film thickness; J & W Scientific, Folsom, CA, USA) were used for analysis. The volatiles were desorbed from the SPME fiber at 250°C in the injection port. The GC temperature was programmed as described by Liu et al. (2012): held at 35°C for 5 min, increased to 150°C at 5°C/min, and held for 3 min, increased to 190°C at 8°C/min and held for 1 min, and increased to 250°C at 30°C/min and held for 5 min. Both the ion source and MS transfer line temperatures were maintained at 250°C. Helium was used as carrier gas with a flow rate of 1 mL/min, and the 5

injection was in a splitless mode. The mass spectrometer was operated in electron ionization (EI) mode at 70 eV with a scan range from m/z 35 to 400. 2.4. Qualitative and quantitative analysis The data collected from the GC-MS were processed using Xcalibur software, and volatile compounds were identified according to the database of NIST/EPA/NIH Mass Spectral Library (NIST 2014). Retention index (RI) of compounds were calculated from a series of n-alkane (C6-C40) (Sigma, St. Louis, MO, USA), which had the same GC-MS analysis program as that applied to the sample. The relative content of each volatile was quantified as 3-octanol equivalent (internal standard) by the GC peak area (Supplementary Fig. S1). 2.5. Statistical analysis All the data were the mean of three replicates. Hierarchical cluster analysis and heatmap were constructed using major volatiles (average content > 15 µg/kg FW) in the fruits of 39 melon cultivars, using the euclidean distance as the dissimilarity measure. The mean values of volatile contents were used as input data in the principal component analysis (PCA). R programming language (Version 3.6.0, https://cran.r-project.org/) was used to perform statistical analyses. 3. Results and discussion 3.1. Volatile compound identification Identification of volatile compounds was based on the retention time and retention index (Tables 1-3), as obtained from GC-MS chromatograms 6

(Supplementary Fig. S1). One hundred and forty six volatile compounds were detected, including 55 esters, 23 aldehydes, 30 alcohols, 15 ketones, 6 acids and 17 other compounds (Tables 1-3). An average of 53 kinds of volatile compounds existed in each cultivar (Supplementary Table S3). Only one compound (A15, benzyl alcohol) existed in all cultivars (Supplementary Table S4). ZT49 and ZT20 contained the highest number of volatile compound types at 76, while the lowest value was 23 in JGN (Supplementary Table S3). Therefore, this study suggested that the volatile composition is strongly dependent on the cultivars, which is in agreement with previous studies (Kourkoutas et al., 2006; Obando-Ulloa et al., 2008; Dos-Santos et al., 2013; Tang et al., 2015; Chaparro-Torres et al., 2016). For the most (35 out of 39) cultivars, the main types of volatile compounds were ester, aldehyde and alcohol (Supplementary Table S5), which contributed to 90% of the total volatiles content (Supplementary Table S6). By contrast, other 4 cultivars showed a different accumulation pattern of volatiles, XZM25 and QCCM

had

higher

content

of

ketones

(mainly

6,10-dimethyl-5,9-undecadien-2-one), occupied 19.45% and 15.90% of total volatiles, respectively (Supplementary Table S6). In addition, cultivars ZT20 and ZT27 had higher content of other compounds (eucalyptol, 10% of total volatiles content,

Supplementary

Tables

S2,

6).

The

compound

6,10-dimethyl-5,9-undecadien-2-one, also known as geranylacetone, is classified as a norisoprenoid derived from the degradation of long chain terpenes 7

(β-carotene and lycopene), conferring a floral aroma to ripe fruit (Lewinsohn et al., 2005). Eucalyptol (1, 8-cineole) is the active ingredient of the eucalyptus oil, responsible for its various pharmacological actions (Trivedi & Hotchandani, 2004). Therefore, our results uncover interesting cultivars that had diverse volatile profiles, which could be useful for the selection of cultivars. 3.2. Total volatiles content In this study, differences in total volatiles content between cultivars were observed (Supplementary Table S3). XZM25 had the highest volatile content (1840 µg/kg FW), followed by WM and XZM21 at 1498 and 1368 µg kg-1 FW, respectively; by contrast, ZT10 had the lowest volatile content (113 µg kg-1 FW), followed by ZT384 and SXY at 124 and 139 µg kg-1 FW, respectively (Supplementary Table S3). 3.3. Volatile composition The average volatile contents and their distribution ranges in the 39 melon cultivars were shown in Table 1. Major volatiles (12 types, average content >15 µg/kg FW) were presented in Fig. 2. 3.3.1. Esters Fifty five ester compound types were identified, which contributed to 0.74-88.69% of the total volatiles content, the average value is 30.34% (Table 1). The highest ester content (Supplementary Table S2) existed in cultivar ZT7 (1001.32 µg kg-1 FW, 89% of total volatiles content), followed by WM (955.16 µg kg-1 FW, 64% of total volatiles content) and CM (936.26 µg kg-1 FW, 80% of total volatiles 8

content). However, cultivars XZM25 and K1710 had lower ester content, less than 1% of total volatiles (Supplementary Table S6). Ethyl acetate (E2) existed as the highest ester content at 50.45 µg kg-1 FW (Table 1), and is a major ester in climacteric and highly aromatic varieties of melon (Obando-Ulloa et al., 2008). As the second highest ester content, 2,3-butanediol diacetate (E31) with a content of 16.03 µg kg-1 FW (Table 1). 2,3-butanediol diacetate had an earthy, soily odour, and were also identified in Japanese melon (cv. Golden Crispy) (Lignou, Parker, Baxter, & Mottram, 2014). ZTCX, WM, CM and ZT7 had high level of ethyl acetate (E2), more than 200 µg kg-1 FW (Fig. 2). WM and ZT7 had high content of 2,3-butanediyl diacetate (E31), more than 150 µg kg-1 FW (Fig. 2). On the contrary, XZM25, K1710, HM8, QCCM and HMC had not detected ethyl acetate and 2,3-butanediyl diacetate (Fig. 2). 3.3.2. Aldehydes In this study, 23 aldehyde compounds were identified, which contributed to 1.02-75.99% of the total volatiles content, the average value is 32.91% (Table 2). The highest aldehyde content existed in XZM25 (928.84 µg kg-1 FW, 50% total volatiles), whereas the lowest value existed in ZT7 (11.5 µg kg-1 FW, 1% total volatiles) (Supplementary Tables S5, 6). Octanal (L7), (Z)-6-nonenal (L12), nonanal (L13), (E, Z)-2,6-nonadienal (L14) and (E)-2-nonenal (L15) were the major aldehydes (Table 2, > 15 µg/kg FW). These compounds accounted for 86% of the total aldehyde content (Table 2). As 9

shown in Fig. 2, NSM had the highest octanal (L7) content, XZM21, XZM17, XZM25 and HM8 had higher level of (Z)-6-nonenal (L12), more than 180 µg kg-1 FW, the highest content of nonanal (L13), (E,Z)-2,6-nonadienal (L14) and (E)-2-nonenal (L15) was observed in XZM25. Octanal is an aldehyde previously reported as a major component in the flesh of Queen Ann’s pocket melons (Aubert & Pitrat, 2006) and fresh-cut Cantaloupe melons (Beaulieu & Lancaster, 2007). Previous studies suggest that among aldehydes, (Z)-6-nonenal, nonanal, (E,Z)-2,6-nonadienal and (E)-2-nonenal prevailed in the inodorus samples (Verzera et al., 2011). In addition, (E)-2-nonenal is associated with a strong cucumber-like odor, and is consistently detected in inodorus melons (Buescher & Buescher, 2001; Esteras et al., 2018). In this study, we also detected (E)-2-nonenal in high quantities in inodorus melon cultivars (Supplementary Table S1). 3.3.3. Alcohols In this study, 30 alcohol compounds were identified (Table 2), which contributed to 7.07-67.5% of the total volatiles content, the average value was 31.54% (Table 2). The highest alcohol content existed in HMC (835.27 µg kg-1 FW, 67% total volatiles), whereas the lowest value existed in ZT10 (28.47 µg kg-1 FW, 25% total volatiles) (Supplementary Tables S5, 6). Benzyl alcohol (A15), (Z)-3-nonen-1-ol (A18), 3,6-(E,Z)-nonadien-1-ol (A20), 6-nonen-1-ol (A24) and 1-nonanol (A25) were the major alcohols (Table 2, > 15 µg/kg FW). Benzyl alcohol (A15) was the only compound identified in all 10

cultivars (Supplementary Table S4), indicating that it is an important volatile for melon aroma. CM had the highest content of benzyl alcohol (A15, 98.35 µg kg-1 FW). The highest content of (Z)-3-nonen-1-ol (A18, 295.96 µg kg-1 FW) was observed in XZM17, followed by HMC (243.15 µg kg-1 FW) and M3 (185.40 µg kg-1 FW). XZM21 had the highest content of 3,6-(E,Z)-nonadien-1-ol (A20, 324.33 µg kg-1 FW) and 6-nonen-1-ol (A24, 315.02 µg kg-1 FW) (Fig. 2). The highest content of 1-nonanol (A25) was observed in HMC (Fig. 2). 3.3.4. Ketones Fifteen ketone compounds were identified, which contributed to 0.02-19.45% of the total volatiles content, the average value was 2.85% (Table 3). The highest ketone content existed in XZM25 (357.98 µg kg-1 FW, 19% total volatiles); 6, 10-dimethyl-5,9-undecadien-2-one

(K9)

4-(2,6,6-trimethyl-1-cyclohexen-l-yl)-3-buten-2-one

(K10)

and were

the

main

ketones, they comprised 90% of the total ketone compounds in XZM25 (Supplementary Table S2). 3.3.5. Acids Six acid compounds were detected, including 2-hydroxy-2-methylbutyric acid (C1), 2-ethylhexanoic acid (C2), 4-tert-butylcyclohexanecarboxylic acid (C3), 6-methyl-5-octenoic acid (C4), benzothiazole-2-carboxylic acid (C5) and nonanoic acid (C6), which accounted for 0-0.69% of the total volatiles content, the average value was 0.12% (Table 3). XZM21 had the highest content of

11

benzothiazole-2-carboxylic acid (C5); the value was 5.31 µg kg-1 FW (100% of total acids content) (Supplementary Table S2). 3.3.6. Others Other 17 kinds of compounds formed 0.03-10.44% of the total volatile substances, the average value was 2.23% (Table 3). The two main components were eucalyptol (O5, 8.45 µg kg-1 FW) and 2,4-di-tert-butylphenol (O17, 1.85 µg kg-1 FW) (Table 3). The highest content of eucalyptol was observed in ZT20 (67.36 µg kg-1 FW), followed by XZM25 (63.30 µg kg-1 FW) and QCCM (57.64 µg kg-1 FW). XZM25 had the highest content of 2,4-di-tert-butylphenol (19.12 µg kg-1 FW). 3.4. Principal component analysis (PCA) Principal component analysis (PCA) was used to extract important information from 146 volatiles detected in 39 melon cultivars. The first two principal components (PCs) were extracted by PCA, PC1 represented 35% of the total variance and PC2 represented 20% of the total variance (Fig. 3A, B). As shown in Fig. 2 and Fig. 3A, B, 39 melon cultivars can be divided into five groups according to the location of plotted data: (1) 4 cultivars (ZTCX, CM, WM and ZT7) had high ethyl acetate (E2) and 2,3-butanediol diacetate (E31) contents, (2) 6 cultivars (XZM25, HM8, YT, K1710, JGN and ZT9) had high (Z)-6-nonenal (L12), nonanal (L13), (E,Z)-2,6-nonadienal (L14), (E)-2-nonenal (L15) and 3,6-(E,Z)-nonadien-1-ol (A20) contents, (3) 1 cultivar (Xizhoumi21) had high (Z)-3-nonen-1-ol

(A18),

diisobutyl

ester 12

(E54),

6-nonen-1-ol

(A24),

(Z)-6-nonenal (L12) and 3,6-(E,Z)-nonadien-1-ol (A20) contents, (4) 2 cultivars (HMC and XZM17) had high 1-nonanol (A25), (Z)-3-nonen-1-ol (A18), (Z)-6-nonenal (L12), 3,6-(E,Z)-nonadien-1-ol (A20) contents, and (5) the other 26 cultivars. We should mention that the cultivars in certain group are not always belonging to the same botanical group, for example, in group 1, ZTCX belongs to chandalak, CM and ZT7 belong to reticulatus, and WM belong to chinensis, demonstrating the complex relationship between aroma composition and melon varieties. Similar result was also observed by Esteras et al. (2018). As previously reported, climacteric and non-climacteric accessions presented important differences in the content of volatiles, with higher production of esters in climacteric aromatic types and more lipid-derived aldehydes in the non-climacteric and non-aromatic types (Obando-Ulloa et al., 2008; Chen et al., 2016; Esteras et al., 2018). Therefore, in this study, 17 cultivars (JGN, YT, HM8, XZM25, K1710, SG, SHG, ZT31, QCCM, JT, HMC, ZT23, M2, M3, NSM, SXY and XZM17) could be regarded as typical non-climacteric types, as the aldehydes content/esters content ratio was relatively higher (> 2, Supplementary Table S6), on the contrary, 11 cultivars (ZT5, SG5, ZT7, ZT10, HZX, CM, ZT6, WM, YJM, ZT20 and ZT384) could be regarded as typical climacteric types, as the aldehydes content/esters content ratio was relatively lower (< 0.5, Supplementary Table S6). The remaining 11 cultivars could be regarded as the intermediates between climacteric and non- climacteric types.

13

Among the 35 cultivars, XZM25 is an interesting cultivar, which belongs to the inodorus group. The aroma profile of non-climacteric inodorous melons has received little attention in the literature, but these melons have great interest as intact, fresh-cut or processed fruit due to their longer shelf-life than the climacteric ones (Kourkoutas et al., 2006; Esteras et al., 2018). XZM25 had the highest total content of volatiles, typically aldehydes, but with less esters accumulation (Supplementary Tables S5, 6), suggesting that non-climacteric and non-aromatic cultivar can still accumulate higher contents of volatiles. XZM25 is one of the most appreciated melon cultivars planted in the Xinjiang Uyghur Autonomous Region of China for both the foreign and local markets. Identification of the aroma profile provides the theoretical basis for the cultivation of this cultivar. Previous study suggested that there may be a relationship between carotenoid metabolism and volatile compound production in melon (Kyriacou, Leskovar, Colla, & Rouphael, 2018). Therefore, in the future, the link between carotenoid profiles and volatile fractions could be investigated using the contrasting cultivars in this study. 4. Conclusions This study showed that the compositions and content in the melon varied largely with cultivars. The 39 melon cultivars can be clustered into five groups: (1) Zhongtiancuixue, Cuimi, Weimi and Zhongtian7 had high ethyl acetate and 2,3-butanediyl diacetate contents, (2) Xizhoumi25, Haimi8, Yutong, K1710, 14

Jinguniang

and

Zhongtian9

had

high

(Z)-6-nonenal,

nonanal,

(E,Z)-2,6-nonadienal, (E)-2-nonenal and 3,6-(E,Z)-nonadien-1-ol contents, (3) Xizhoumi21 had high (Z)-3-nonen-1-ol, diisobutyl ester, 6-nonen-1-ol, (Z)-6-nonenal and 3,6-(E,Z)-nonadien-1-ol contents, (4) Huangmengcui and Xizhoumi17

had

high

1-nonanol,

(Z)-3-nonen-1-ol,

(Z)-6-nonenal,

3,6-(E,Z)-nonadien-1-ol contents, and (5) the other 26 cultivars. Ethyl acetate, (Z)-6-nonenal and 3,6-(E,Z)-nonadien-1-ol were the most three abundant volatiles among the identified 146 compounds. One non-climacteric inodorus cultivar (Xizhoumi25) had the highest content of total volatiles. This work could support selection of cultivars with a flavor that suits the public and future breeding work towards the genetic improvement of melon flavor. Acknowledgements This work was supported by the National Key Research and Development Program of China (2019YFD1001900), China Agriculture Research System (CARS-25), Fundamental Research Funds for the Central Universities (2662018JC037, 2662018PY039), Natural Science Foundation of Hainan Province (318MS116), and the Innovative Research Group Project of Natural Science Foundation of Hubei Province, China (2019CFA017). Author contributions Y.H designed the experiments. JD.S, HB.W, M.X, YJ.C, JH.C, B.Z, H.W, LL.L, and XF.F performed the experiments. JD.S, YJ.C and ZL.B analyzed the data. Y.H, JD.S and HB.W wrote the article. All authors read and approved the final 15

manuscript.

Supplementary data Supplementary Tables Supplementary Table S1. Melon cultivars used in this study Supplementary Table S2. Relative contents (µg/kg FW) of identified volatiles in the fruits of 39 melon cultivars Supplementary Table S3. Number of volatiles and total content of volatiles identified in the fruits of 39 melon cultivars Supplementary Table S4. Number of melon cultivars for each identified volatile compound Supplementary Table S5. Total content (µg/kg) of each type of volatiles in 39 melon cultivars Supplementary Table S6. Percentage (%) of each type of volatiles in 39 melon cultivars Supplementary Figures Supplementary Fig. S1. Representative GC-MS chromatograms of volatiles in melon fruits Compliance with ethical standards Conflict of interest: The authors have declared that no competing interests exist

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species reveals unexploited variability for future genetic breeding. Journal of the Science of Food and Agriculture, 98(10), 3915-3925. 13. Flores, K., Sanchez, M. T., Perez-Marin, D. C., Lopez, M. D., Guerrero, J. E., & Garrido-Varo, A. (2008). Prediction of total soluble solid content in intact and cut melons and watermelons using near infrared spectroscopy. Journal of Near Infrared Spectroscopy, 16(2), 91-98. 14. Fredes, A., Sales, C., Barreda, M., Valcarcel, M., Rosello, S., & Beltran, J. (2016). Quantification of prominent volatile compounds responsible for muskmelon and watermelon aroma by purge and trap extraction followed by gas chromatography-mass spectrometry determination. Food Chemistry, 190, 689-700. 15. Kende, A., Lim, P. P., Lai, F., Jessop, M., Swindale, L., Oliver, M., Hurr, B., Rickett, D., & Baxter, C. (2019). High throughput quantitative volatile profiling of melons with silicone rod extraction-thermal desorption-GC-MS for plant breeding line selection. Food Chemistry, 270, 368-374. 16. Kourkoutas, D., Elmore, J. S., & Mottram, D. S. (2006). Comparison of the volatile compositions and flavour properties of cantaloupe, Galia and honeydew muskmelons. Food Chemistry, 97, 95-102. 17. Kyriacou, M. C., Leskovar, D. I., Colla, G., & Rouphael, Y. (2018). Watermelon and melon fruit quality: The genotypic and agro-environmental factors implicated. Scientia Horticulturae, 234, 393-408. 18. Lewinsohn, E., Sitrit, Y., Bar, E., Azulay, Y., Ibdah, M., Meir, A., Yosef, E., 19

Zamir, D., & Tadmor, Y. (2005). Not just colors-carotenoid degradation as a link between pigmentation and aroma in tomato and watermelon fruit. Trends in Food Science & Technology, 16(9), 407-415. 19. Lignou, S., Parker, J. K., Oruna-Concha, M. J., & Mottram, D. S. (2013). Flavour profiles of three novel acidic varieties of muskmelon (Cucumis melo L.). Food Chemistry, 139, 1152-1160. 20. Lignou, S., Parker, J. K., Baxter, C., & Mottram, D. S. (2014). Sensory and instrumental analysis of medium and long shelf-life Charentais cantaloupe melons (Cucumis melo L.) harvested at different maturities. Food chemistry, 148, 218-229. 21. Liu, C., Zhang, H., Dai, Z., Liu, X., Liu, Y., Deng, X., Chen, F., & Xu, J. (2012). Volatile chemical and carotenoid profiles in watermelons [Citrullus vulgaris (Thunb.) Schrad (Cucurbitaceae)] with different flesh colors. Food Science and Biotechnology, 21(2), 531-541. 22. Luo, D., Pang, X., Xu, X., Bi, S., Zhang, W., & Wu, J. (2018). Identification of cooked off-flavor components and analysis of their formation mechanisms in melon juice during thermal processing. Journal of Agricultural and Food Chemistry, 66, 5612-5620. 23. Obando-Ulloa, J. M., Moreno, E., Garcia-Mas, J., Nicolai, B., Lammertyn, J., Monforte, A. J., & Fernandez-Trujillo, J. P. (2008). Climacteric or non-climacteric behavior in melon fruit: 1. Aroma volatiles. Postharvest Biology and Technology, 49(1), 27-37. 20

24. Obando-Ulloa, J. M., Ruiz, J., Monforte, A. J., & Fernandez-Trujillo, J. P. (2010). Aroma profile of a collection of near-isogenic lines of melon (Cucumis melo L.). Food Chemistry, 118, 815-822. 25. Perry, P. L., Wang, Y., & Lin, J. (2009). Analysis of honeydew melon (Cucumis melo var. inodorus) flavour and GC-MS/MS identification of (E,Z)-2, 6-nonadienyl acetate. Flavour and Fragrance Journal, 24(6), 341-347. 26. Poverenov, E., Arnon-Rips, H., Zaitsev, Y., Bar, V., Danay, O., Horev, B., Bilbao-Sainz, C., McHugh, T., & Rodov, V. (2018). Potential of chitosan from mushroom waste to enhance quality and storability of fresh-cut melons. Food Chemistry, 268, 233-241. 27. Tang, Y., Zhang, C., Cao, S., Wang, X., & Qi, H. (2015). The effect of CmLOXs on the production of volatile organic compounds in four aroma types of melon (Cucumis melo). PLoS One, 10(11), e0143567. 28. Trivedi, N. A., & Hotchandani, S. C. (2004). A study of the antimicrobial activity of oil of Eucalyptus. Indian Journal of pharmacology, 36(2), 93. 29. Vallone, S., Sivertsen, H., Anthon, G. E., Barrett, D. M., Mitcham, E. J., Ebeler, S. E., & Zakharov, F. (2013). An integrated approach for flavour quality evaluation in muskmelon (Cucumis melo L. reticulatus group) during ripening. Food Chemistry, 139, 171-183. 30. Verzera, A., Dima, G., Tripodi, G., Ziino, M., Lanza, C. M., & Mazzaglia, A. (2011). Fast quantitative determination of aroma volatile constituents in melon fruits by headspace-solid-phase microextraction and gas chromatography-mass 21

spectrometry. Food Analytical Methods, 4(2), 141-149. 31. Ye, L., Yang, C., Li, W., Hao, J., Sun, M., Zhang, J., & Zhang, Z. (2017). Evaluation of volatile compounds from Chinese dwarf cherry (Cerasus humilis (Bge.) Sok.) germplasms by headspace solid-phase microextraction and gas chromatography-mass spectrometry. Food chemistry, 217, 389-397. Y.H designed the experiments. JD.S, HB.W, M.X, YJ.C, JH.C, B.Z, H.W, LL.L, and XF.F performed the experiments. JD.S, YJ.C and ZL.B analyzed the data. Y.H, JD.S and HB.W wrote the article. All authors read and approved the final manuscript. 32. Declaration of interests ☒ The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper.

☐The authors declare the following financial interests/personal relationships which may be considered as potential competing interests:

Authors Jianda Shi, Haibo Wu, Mu Xiong, Yanjun Chen, Jihao Chen, Bo Zhou, Hui Wang, Liangliang Li, Xiaofa Fu, Zhilong Bie, Yuan Huang

22

Oct 4, 2019

33.

Fig. 1. Fruits of 39 cultivars used in this study. The codes in Fig. 1 correspond to the codes of cultivars in supplementary Table S1.

23

Cultivars

Volatiles Fig. 2. Contents (as shown by heatmap) and hierarchical cluster analysis of the major volatiles (average content > 15 µg/kg FW) in the fruits of 39 melon cultivars. The codes in Fig. 2 correspond to the codes of cultivars in supplementary Table S1 and volatiles in Tables 1-2.

24

Fig. 3. Principal component analysis (PCA) of 39 melon cultivars. Fig. 3A shows the PCA scores scatter plot. Fig. 3B shows a PCA loading plot. Fig. 3A codes correspond to cultivar codes in supplementary Table S1. The codes in Fig. 3B correspond to the codes of volatiles in Tables 1-3. 34. Table 1. Average contents of esters (n = 3, equivalent of 3-octanol) and their distribution ranges (in parenthesis) in the fruits of 39 melon cultivars Codea

CAS No

Compounds

RTb

RIc

Content (µg/kg FW)

Esters E1 E2 E3 E4 E5 E6 E7 E8 E9 E10 E11 E12 E13 E14 E15 E16 E17 E18 E19 E20 E21 E22 E23 E24 E25 E26 E27 E28 E29 E30 E31 E32 E33 E34 E35 E36

79-20-9 141-78-6 547-63-7 105-37-3 109-60-4 110-45-2 97-62-1 110-19-0 868-57-5 105-54-4 123-86-4 638-11-9 2432-51-1 7452-79-1 624-41-9 590-01-2 628-63-7 1191-16-8 106-70-7 42075-43-4 37064-20-3 2438-20-2 109-21-7 123-66-0 72237-36-6 142-92-7 2445-69-4 13532-18-8 623-84-7 NA 1114-92-7 110318-09-7 93-58-3 13327-56-5 2442-10-06 16630-55-0

methyl acetate ethyl acetate methyl isobutyrate ethyl propanoate n-propyl acetate 3-methylbutyl formate ethyl 2-methylpropanoate isobutyl acetate methyl 2-methylbutanoate ethyl butanoate butyl acetate isopropyl butyrate s-methyl butanethioate ethyl 2-methylbutanoate 2-methyl-1-butanol acetate butyl propanoate pentyl acetate 3-methyl-2-buten-1-yl acetate methyl hexanoate s-methyl pentanethioate propyl 2-methylbutanoate 2-methylbutyl propanoate butyl butanoate ethyl hexanoate 4-hexenyl acetate hexyl acetate 2-methylbutyl 2-methylpropanoate methyl 3-(methylthio)propionate propane-1,2-diol diacetate phenacyl 11-octadecenoate 2,3-butanediol diacetate ethyl sorbate methyl benzoate ethyl 3-(methylthio)propionate 1-octen-3-yl-acetate 3-(methylthio)propyl acetate

2.11 2.74 3.80 4.38 4.42 5.00 6.10 6.38 6.46 7.63 7.89 8.98 9.18 9.29 10.53 11.52 11.72 11.98 12.08 12.60 12.85 13.80 14.64 14.76 14.96 15.20 15.33 15.55 15.59 16.88 17.03 17.85 17.87 18.07 18.34 18.76

506 606 651 676 677 702 749 761 764 808 815 843 848 851 883 910 915 922 925 940 946 973 996 999 1005 1012 1016 1023 1024 1063 1068 1093 1093 1099 1108 1122

3.64 (0-19.72) 50.45 (0-407.67) 0.54 (0-7.39) 2.51 (0-33.47) 1.14 (0-19.63) 0 (0-0.02) 1.42 (0-16.17) 6.4 (0-70.64) 10.64 (0-85.7) 8.4 (0-139.58) 7.25 (0-68.78) 0.06 (0-0.56) 0.28 (0-9.68) 11.93 (0-143.36) 13.34 (0-111.05) 0.11 (0-2.74) 0.59 (0-10.75) 0.21 (0-3.53) 0.74 (0-10.64) 7.23 (0-82.71) 0.15 (0-2.15) 0.28 (0-7.34) 0.07 (0-1.55) 4.49 (0-68.9) 1.89 (0-29) 11.79 (0-73.86) 0.17 (0-4.34) 0.15 (0-3.7) 0.72 (0-6.71) 0.03 (0-0.39) 16.03 (0-226.4) 0.28 (0-10.39) 0.99 (0-7.4) 2.23 (0-52.27) 0.43 (0-8.27) 2.25 (0-13.47)

25

E37 E38 E39 E40 E41 E42 E43 E44 E45 E46 E47 E48 E49 E50 E51 E52 E53 E54 E55

108-84-9 140-11-4 138234-61-4 93-92-5 2639-63-6 4166-44-3 112-14-1 103-45-7 6290-37-5 17369-57-2 92618-89-8 76649-26-8 34912-29-3 80-26-2 74367-31-0 25779-85-5 112-39-0 84-69-5 56875-67-3

4-methyl-2-pentyl acetate benzyl acetate ethyl 4-octenoate α-methyl-benzenemethanol acetate hexyl butanoate 2,2-dimethyl-3-hexanol acetate octyl acetate 2-phenylethyl acetate 2-phenylethyl hexanoate 3-methylbenzyl acetate bicyclo[2.2.1]heptan-2-ol,1,7,7-trimethyl-, 2-acetate 3-6-nonadien-1-yl-acetate 2-ethoxyethyl hexanoate α-terpinyl acetate 2-ethyl-3-hydroxyhexyl 2-methylpropanoate methyl dihydrohydnocarpate methyl hexadecanoate diisobutyl ester (Z)-methyl hexadec-7-enoate Subtotal Subtotal (%) aCompound codes, bRetention time (min), cRetention index. FW, fresh weight.

19.64 19.99 20.80 20.85 20.93 21.21 21.50 22.73 22.74 22.98 23.69 23.88 24.04 25.40 25.93 35.47 35.54 37.85 38.34

1151 1162 1189 1190 1193 1202 1213 1256 1256 1265 1289 1296 1302 1353 1372 1725 1728 1966 2005

0.55 (0-17.77) 6.88 (0-57.08) 0.16 (0-6.21) 0.91 (0-6.49) 0.36 (0-9.21) 0.8 (0-7) 0.6 (0-5.99) 0.23 (0-7.12) 0.16 (0-2.87) 0.06 (0-1.96) 0.05 (0-0.8) 0.1 (0-1.41) 0.18 (0-7.05) 1.72 (0-40.77) 0.83 (0-7.41) 0.14 (0-1.81) 5.12 (0-74.9) 5.37 (0-101.28) 0.28 (0-8.12) 193.33 30.34 (0.74-88.69) (4.52-1001.32)

Table 2. Average contents of aldehydes and alcohols (n = 3, equivalent of 3-octanol) and their distribution ranges (in parenthesis) in the fruits of 39 melon cultivars Codea

CAS No

Aldehydes L1 66-25-1 L2 6728-26-3 L3 6728-31-0 L4 111-71-7 L5 NA L6 100-52-7 L7 124-13-0 L8 4313-03-5 L9 122-78-1 L10 21944-83-2 L11 2277-16-9 L12 2277-19-2 L13 124-19-6 L14 557-48-2 L15 18829-56-6 L16 13019-16-4 L17 112-31-2 L18 21662-16-8 L19 432-25-7 L20 472-66-2 L21 106-26-3 L22 5392-40-5 L23 21632-06-4

Alcohols A1 A2 A3 A4 A5 A6 A7 A8 A9

137-32-6 5271-38-5 544-12-7 111-27-3 6191-71-5 111-70-6 505-10-2 3391-86-4 104-76-7

RTb

RIc

Content (µg/kg FW)

hexanal (E)-2-hexenal (Z)-4-heptenal heptanal isovanillin benzaldehyde octanal (E,E)-2,4-heptadienal benzeneacetaldehyde (Z,Z)-3,6-nonadienal (Z)-4-nonenal (Z)-6-nonenal nonanal (E,Z)-2,6-nonadienal (E)-2-nonenal 2-butyl-2-octenal decanal (E,E)-2,4-dodecadienal β-cyclocitral 2,6,6-trimethyl-1-cyclohexene-1-acetaldehyde (Z)-3,7-dimethyl-2,6-octadienal citral α,2,6,6-tetramethyl-1-cyclohexene-1-butanal Subtotal Subtotal (%)

7.32 9.31 11.10 11.22 13.12 13.31 14.91 15.13 16.20 17.95 18.01 18.18 18.24 19.73 19.96 21.32 21.32 21.64 21.80 22.81 23.12 23.15 29.26

800 852 898 901 954 959 1004 1010 1043 1096 1098 1103 1105 1154 1161 1206 1206 1218 1223 1259 1269 1271 1497

5.12 (0-39.41) 1.51 (0-15.6) 0.02 (0-0.26) 0.4 (0-5.96) 0.42 (0-2.96) 8.44 (0-37.18) 17.77 (0-191.64) 0.13 (0-1.11) 0.3 (0-1.64) 0.06 (0-1.38) 0.18 (0-1.52) 53.51 (0-192.08) 34.9 (0-132.56) 44.07 (0-319.01) 31.02 (0-198.4) 0.41 (0-15.83) 4.78 (0-40.76) 0.04 (0-0.29) 2.25 (0-30.24) 0.33 (0-1.49) 0.36 (0-4.81) 0.37 (0-5.99) 3.26 (0-41.42) 209.65 (11.5-928.84) 32.91 (1.02-75.99)

2-methyl-1-butanol 2-(methylthio)ethanol 3-hexen-1-ol 1-hexanol cis-hept-4-enol 1-heptanol 3-(methylthio)-1-propanol 1-octen-3-ol 2-ethyl-1-hexanol

5.57 8.70 9.45 10.05 13.55 13.75 13.97 14.11 15.76

726 836 855 871 966 971 977 981 1019

3.56 (0-21.54) 0.13 (0-2.02) 0.83 (0-11.17) 7.18 (0-60.59) 0.14 (0-0.97) 0.46 (0-3.61) 0.17 (0-3.67) 10.41 (0-147.59) 2.96 (0-24.26)

Compounds

26

A10 A11 A12 A13 A14 A15 A16 A17 A18 A19 A20 A21 A22 A23 A24 A25 A26 A27 A28 A29 A30

69668-82-2 5340-36-3 NA 111-87-5 5989-33-3 100-51-6 60-12-8 NA 10340-23-5 10339-61-4 56805-23-3 122-97-4 7786-44-9 22104-79-6 31502-19-9 143-08-8 2216-51-5 562-74-3 38049-26-2 10482-56-1 121841-67-6

aCompound

3,5-octadien-2-ol 3-methyl-3-octanol 5-octen-2-yn-4-ol 1-octanol cis-5-ethenyltetrahydro-α,α,5-trimethyl-2-furanmethanol benzyl alcohol phenylethyl alcohol (2Z)-3-pentyl-2,4-pentadien-1-ol (Z)-3-nonen-1-ol E-non-3-en-1-ol 3,6-(E,Z)-nonadien-1-ol 3-phenylpropanol 2,6-nonadien-1-ol 2-nonen-1-ol 6-nonen-1-ol 1-nonanol levomenthol terpinen-4-ol dihydrocarveol α-terpineol 2,2,6,7-tetramethyl-10-oxatricyclo[4.3.1.0(1,6)]decan-5-ol Subtotal Subtotal (%)

16.06 16.38 16.48 17.13 17.67 18.11 18.44 19.59 19.72 19.72 19.84 20.01 20.06 20.18 20.23 20.31 20.56 20.62 20.96 21.04 25.07

1038 1048 1051 1070 1087 1101 1111 1149 1153 1153 1157 1163 1165 1169 1170 1173 1181 1183 1194 1197 1340

0.04 (0-0.6) 0.32 (0-1.74) 0 (0-0.06) 0.17 (0-2.97) 0.02 (0-0.3) 20.97 (2.19-98.35) 2.16 (0-11.36) 0.13 (0-2.41) 30.97 (0-295.96) 2.18 (0-67.41) 50.49 (0-324.33) 0.19 (0-4.96) 4 (0-31.08) 2.77 (0-48.95) 34.97 (0-315.02) 21.24 (0-187.15) 0.02 (0-0.66) 0.15 (0-2.59) 0.47 (0-5.03) 1.35 (0-13.87) 0.09 (0-1.5) 198.56 (28.47-835.27) 31.54 (7.07-67.5)

codes, bRetention time (min), cRetention index. FW, fresh weight.

Table 3. Average contents of ketones, acids and other compounds (n = 3, equivalent of 3-octanol) and their distribution ranges (in parenthesis) in the fruits of 39 melon cultivars Codea

RTb

RIc

CAS No

Compounds

Content (µg/kg FW)

K1

4906-24-5

3-acetoxy-2-butanone

10.75

889

1.02 (0-37.52)

K2

313253-65-5

4-cyclohexylidene-3,3-diethyl-2-pentanone

13.22

957

1.4 (0-20.8)

K3

110-93-0

6-methyl-5-heptene-2-one

14.24

985

0.75 (0-5.4)

K4

78-59-1

isophorone

16.72

1058

1.09 (0-9.47)

K5

107-70-0

4-methyl-4-methoxy-2-pentanone

19.08

1132

0.16 (0-1.7)

K6

121747-63-5

2,2,6,7-tetramethyl-10-oxatricyclo[4.3.0.1(1,7)]decan-5-one

25.30

1349

1.14 (0-16.63)

K7

20201-45-0

1-(2-desoxy-β-d-ribofuranosyl)-4-methylthio-5-fluoropyrimidin-2-one

26.13

1380

0.12 (0-2.58)

K8

17283-81-7

4-(2,6,6-trimethyl-1-cyclohexen-1-yl)-2-butanone

27.69

1438

0 (0-0.04)

K9

689-67-8

6,10-dimethyl-5,9-undecadien-2-one

27.84

1445

11.43 (0-256.97)

K10

14901-07-6

4-(2,6,6-trimethyl-1-cyclohexen-l-yl)-3-buten-2-one

28.93

1485

3.73 (0-65.33)

K11

23267-57-4

4-(2,2,6-trimethyl-7-oxabicyclo[4.1.0]hept-1-yl)-3-buten-2-one

29.08

1490

1.23 (0-17.93)

K12

83406-41-1

cis-hexahydro-8a-methyl-1,8(2H,5H)-naphthalenedione

29.83

1515

0.36 (0-10.24)

K13

17092-92-1

(R)-5,6,7,7a-tetrahydro-4,4-7a-trimethyl-2-(4H)-benzofuranone

30.44

1534

0.53 (0-7.27)

K14

NA

2-imino-1,2-diphenyl-semicarbazone ethanone

36.55

1778

0.04 (0-0.53)

K15

7297-85-0

4-[3,3-bis(4-hydroxyphenyl)butyl]-5,5-dimethyloxolan-2-one

40.51

2320

0.23 (0-3.06)

Ketones

Subtotal

23.23 (0.14-357.98)

Subtotal (%)

2.85 (0.02-19.45)

Acids C1

3739-30-8

2-hydroxy-2-methylbutyric acid

13.00

951

0.03 (0-0.41)

C2

149-57-5

2-ethylhexanoic acid

18.66

1119

0.01 (0-0.21)

C3

5451-55-8

4-tert-butylcyclohexanecarboxylic acid

18.94

1128

0.19 (0-3.32)

C4

103263-58-7

6-methyl-5-octenoic acid

19.01

1130

0.07 (0-1.6)

C5

3622-04-06

benzothiazole-2-carboxylic acid

21.85

1225

0.28 (0-5.31)

C6

112-05-0

nonanoic acid

23.26

1274

0.1 (0-1.28)

Subtotal

0.68 (0-5.31)

Subtotal (%)

0.12 (0-0.69)

Others

27

O1

16745-94-1

3,4-dimethyl-1-hexene

5.61

728

0.16 (0-6.37)

O2

17669-40-8

2-methyl-1-hepten-3-yne

10.41

880

0.02 (0-0.87)

O3

4430-91-5

2,3-dimethyl-cyclohexa-1,3-diene

12.40

934

0.05 (0-1.26)

O4

100-84-5

1-methoxy-3-methylbenzene

15.40

1018

0.87 (0-7.43)

O5

470-82-6

eucalyptol

15.84

1032

8.45 (0-67.36)

O6

646-14-0

1-nitrohexane

16.25

1044

0.13 (0-4.46)

O7

126-39-6

2-methyl-2-ethyl-1,3-dioxolane

18.88

1126

0.61 (0-3.36)

O8

91-20-3

naphthalene

20.68

1185

0.6 (0-4.24)

O9

494-99-5

3,4-dimethoxytoluene

22.22

1238

0.1 (0-4.03)

O10

2801-68-5

2,5-dimethoxy-α-methyl-benzeneethanamine

22.45

1246

0 (0-0.08)

O11

24599-58-4

1,4-dimethoxy-2-methylbenzene

22.56

1250

0.11 (0-1.3)

O12

501-92-8

4-(2-propenyl)phenol

22.66

1253

0.05 (0-2.05)

O13

2051-49-2

hexanoic anhydride

22.83

1259

0.07 (0-2.71)

O14

93-15-2

methyleugenol

26.80

1405

0.04 (0-1.52)

O15

87-44-5

caryophyllene

27.44

1429

0.02 (0-0.75)

O16

502-61-4

trans-α-farnesene

28.29

1461

0.54 (0-19.1)

O17

96-76-4

2,4-di-tert-butylphenol

29.91

1518

1.85 (0-19.12)

aCompound

Subtotal

13.68 (0.22-86.66)

Subtotal (%)

2.23 (0.03-10.44)

codes, bRetention time (min), cRetention index. FW, fresh weight.

35.

Highlights 1. A total of 146 volatiles were identified in the fruits of 39 melon cultivars. 2. (Z)-6-nonenal was the most abundant volatile. 3. Principal component analysis clustered the 39 melon cultivars into five groups. 4. One inodorus cultivar (Xizhoumi25) had the highest content of total volatiles. 36.

28