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BACKGROUND. A near-isogenic line (NIL) of melon (SC10-2) with introgression in linkage group X was studied from harvest (at firm-ripe stage of maturity) until day 18 of postharvest storage at 20.5 °C together with its parental control (‘Piel de Sapo’, PS). RESULTS. SC10-2 showed higher flesh firmness and whole fruit hardness but lower juiciness than its parental. SC10-2 showed a decrease in respiration rate accompanied by a decrease in ethylene production during ripening, both of which fell to a greater extent than in PS. The introgression affected 11 volatile organic compounds (VOCs), the levels of which during ripening were generally higher in SC10-2 than in PS. Transcriptomic analysis from RNA-Seq revealed differentially expressed genes (DEGs) associated with the effects studied. For example, 909 DEGs were exclusive to the introgression, and only 23 DEGs were exclusive to postharvest ripening time. Major functions of the DEGs associated with introgression or ripening time were identified by cluster analysis. About 37 genes directly and/or indirectly affected the delay in ripening of SC10-2 compared with PS in general and, more particularly, the physiological and quality traits measured and, probably, the differential non-climacteric response. Of the former genes, we studied in more detail at least five that mapped in the introgression in linkage group (LG) X, and 32 outside it. CONCLUSION. There is an apparent control of textural changes, VOCs and fruit ripening by an expression quantitative trait locus located in LG X together with a direct control on them due to genes presented in the introgression (CmTrpD, CmNADH1, CmTCP15, CmGDSL esterase/lipase, and CmHK4-like) and CmNAC18.

This is the peer reviewed version of the following article: Zarid, M., García-Carpintero, V., Esteras, C., Esteva, J., Bueso, M.C., Cañizares, J., Picó, M.B., Monforte, A.J. and Fernández-Trujillo, J.P. (2021), Transcriptomic analysis of a near-isogenic line of melon with high fruit flesh firmness during ripening. J Sci Food Agric, 101: 754-777, which has been published in final form at https://doi.org/10.1002/jsfa.10688. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving., [EN] BACKGROUND A near-isogenic line (NIL) of melon (SC10-2) with introgression in linkage group X was studied from harvest (at firm-ripe stage of maturity) until day 18 of postharvest storage at 20.5 degrees C together with its parental control ('Piel de Sapo', PS). RESULTS SC10-2 showed higher flesh firmness and whole fruit hardness but lower juiciness than its parental. SC10-2 showed a decrease in respiration rate accompanied by a decrease in ethylene production during ripening, both of which fell to a greater extent than in PS. The introgression affected 11 volatile organic compounds (VOCs), the levels of which during ripening were generally higher in SC10-2 than in PS. Transcriptomic analysis from RNA-Seq revealed differentially expressed genes (DEGs) associated with the effects studied. For example, 909 DEGs were exclusive to the introgression, and only 23 DEGs were exclusive to postharvest ripening time. Major functions of the DEGs associated with introgression or ripening time were identified by cluster analysis. About 37 genes directly and/or indirectly affected the delay in ripening of SC10-2 compared with PS in general and, more particularly, the physiological and quality traits measured and, probably, the differential non-climacteric response. Of the former genes, we studied in more detail at least five that mapped in the introgression in linkage group (LG) X, and 32 outside it. CONCLUSION There is an apparent control of textural changes, VOCs and fruit ripening by an expression quantitative trait locus located in LG X together with a direct control on them due to genes presented in the introgression (CmTrpD,CmNADH1,CmTCP15,CmGDSL esterase/lipase, andCmHK4-like) and CmNAC18.

A melon non-climacteric near-isogenic line (NIL) SC10-2 and its parental ‘Piel de Sapo’ (PS) line were studied in two different seasons to analyse the seasonal effects of an introgression (located in LG X) on texture and aroma volatiles at harvest. Several univariate and multivariate statistical techniques were used to determine the association between the aroma volatile levels and differences in melon texture. Three methodologies involving different treatments of null values were applied to analyse the effects of introgression on volatile organic compounds (VOCs). Substitution of the zeros with the minimum value of the same VOC variable per season and line is proposed for the analysis of VOC data. SC10-2 showed higher whole fruit hardness, flesh firmness, and fibrousness, but lower juiciness than PS. One hundred sixteen VOCs were identified to evaluate the introgression effect, in which 60 VOCs were detected in two seasons with very different climatic conditions during fruit set and ripening; twenty-eight VOCs were involved in the differences on aroma production in the comparison of the NIL SC10-2 with PS. The environmental effects were more significant than the introgression effect. The environment particularly affected VOCs rather than textural traits, and at least one potential VOC quantitative trait locus (QTL) located in LG X alone and another interacting with the environment are proposed to affect VOC fruit metabolism.

[SPA] Con el fin de comparar la expresión génica de un melón cerca de la línea isogénica (NIL) SC10-2 y su parental Piel de Sapo (PS) durante la maduración y para comprender los mecanismos de diferenciación, se realizó una secuenciación transcriptoma. Dos genes de CmGGP (GDP-L-galactosa fosforilasa 1) y CmRAP2-11 (factor de transcripción sensible al etileno RAP2-11) mostraron menor expresión relativa en la NIL SC10 -2 versus PS debido a la introgresión en LG X. Sin embargo, no existieron diferencias en expresión de CmAP2-like X1 (factor de transcripción sensible al etileno, similar a AP2 TOE3 isoforma X1). En consecuencia, la expresión de genes que mapearon en el grupo de ligamiento X como un factor de transcripción de respuesta a etileno o del metabolismo del ácido ascórbico estuvieron probablemente asociados con el retraso de maduración. [ENG] The expression of selected genes during ripening was studied considering a melon Near-isogenic Line (NIL) SC10-2 and its parental “Piel de Sapo” (PS). The expression of CmGGP (GDP-L-galactose phosphorylase 1), CmAP2-like X1 (AP2-like ethylene-responsive transcription factor TOE3 isoform X1) and CmRAP2-11 (ethylene-responsive transcription factor RAP2-11) were differentially expressed in the NIL SC10-2 compared with PS. Consequently, expression of genes that mapped in LG X such as one ethylene response transcription factors or ascorbic acid metabolism gene were probably associated with delayed ripening.

Quality in melons (Cucumis melo L.) is an important criterion for consumer’s acceptance. Like any other fruit, melons development and ripening process are linked to a series of biochemical and physiological changes. The goal of these researches has been focused on determination of the global quality and aroma profile of a non-climacteric near-isogenic line (NIL) SC10-2 of melon developed from the Spanish cultivar T111 (ssp. melo) of the ‘Piel de Sapo’ type (PS) and the Korean accession PI 161375 Songwhan Charmi (ssp. agrestis) (SC), with different texture than its non-climacteric parental PS, in order to map QTLs associated with fruit quality and, particularly, aroma volatiles. A methodology for a consistent study of outliers of non-climacteric melon volatile organic compounds at harvest was reported. For extreme outliers, we proposed the study of compound classes in percentage of total area counts by principal component analysis followed by a further verification with box-whisker plot. The presence of esters usually at higher proportions than expected, or an excess of minority compounds such as ketones, alcohols, terpenes or others are usually a sign of volatile results that can be considered outliers. A comparison was performed in two seasons between the melon near-isogenic line SC10-2 and its parental PS in order to identify the most discriminant flesh aroma volatiles at harvest as potential biomarkers of textural differences (especially flesh firmness) due to introgressions in melon chromosome X, examining seasonal effects on these the former traits. Several univariate and multivariate statistical techniques were used to determine the association between the aroma volatile levels and differences in melon texture. Three methodologies involving different treatments of null values were applied to analyse the effects of introgression on volatile organic compounds (VOCs). Substitution of the zeros with the minimum value of the same VOC variable per season and line is

[SPA] Con el fin de comparar la expresión génica de una línea casi isogénica (NIL) SC10-2 de melón y su Piel de Sapo (PS) parental durante la maduración y para comprender los mecanismos de diferenciación, se realizó una secuenciación de transcriptoma. Los genes CmTCP15 (Factor de actividad de transcripción) y CmGDSL (actividad de la esterasa y la lipasa) tenían una alta expresión diferencial en el NIL SC10-2 en comparación con el PS debido a la introgresión en LG X. En consecuencia, algunos atributos de calidad de fruto como el aroma, dulzura y, probablemente otros pueden estar afectados por tales genes. [ENG] In order to compare the gene expression of a melon Near-isogenic Line (NIL) SC10-2 and its parental Piel de Sapo (PS) during ripening and to understand the differentiate mechanisms, a transcriptome sequencing was performed. CmTCP15 (Transcription factor activity) and CmGDSL (Esterase and lipase activity) genes were high differentially expressed in the NIL SC10-2 compared with PS due to the introgression in LG X. Consequently, some fruit quality traits such as aroma, sweetness and probably others can be affected by such genes.

A methodology for making consistent studies of outliers of non-climacteric melon volatile organic compounds at harvest is reported. The juice was squeezed from the fruit of the ‘Piel de sapo’ cultivar harvested during two consecutive seasons and the aroma volatiles were extracted by headspace solid phase microextraction and measured by gas chromatography coupled to mass-spectrometry. A deconvolution analysis was performed to obtain volatile organic compounds. For multivariate the reliable identification of outliers, compound classes were studied as a percentage of total area counts of the melon compounds identified in the chromatogram by principal component analysis and partial least-squares discriminant analysis, and then verified by correlation analysis, box-whisker plot, and formal tests for univariate outliers. Principal component analysis was the key methodology for selecting outliers in variables that mostly did not follow a normal distribution. The presence of an excess in terms of relative percentage of area and the diversity of minor compounds such as alcohols, terpenes, acids, among others, are usually a sign of anomalous data that can be considered outliers in the aroma of this non-climacteric cultivar. This multivariate approach removed outliers, but kept the variability of aroma among the samples of every cultivar.

[SPA] Se realizó una secuenciación de transcriptoma para analizar los genes implicados en la formación de aromas expresados durante la maduración y para comprender los mecanismos moleculares que diferencian una línea casi isogénica (NIL) SC10-2 de melón y su parental Piel de Sapo (PS). El gen CmLOX18 (similar a la lipoxigenasa 4) se expresó diferencialmente comparando la NIL SC10-2 y PS y se asoció a la producción de hexanal, un compuesto diana e indicador del proceso de maduración no climatérica. La expresión del gen de la CmACO1 (1-aminocyclopropane-1-carboxylate oxidase 1) implicado en la biosíntesis de etileno no manifestó diferencias durante la maduración. La introgresión en LG X estuvo asociada a la diferente producción de hexanal entre la NIL y PS. Se propone un eQTL en el LG X que controla la producción de aromas del gen CmLOX18 localizado en LG I. [ENG] Transcriptome sequencing was performed in order to analyze the genes associated to volatile synthesis expressed during ripening and to understand the molecular mechanisms that differentiate a melon Near-isogenic Line (NIL) SC10-2 and its parental Piel de Sapo (PS). CmLOX18 gene (Similar to Lipoxygenase 18) was differentially expressed in the NIL SC10-2 compared with PS associated with the aroma volatile compound hexanal as a target compound of the non-climacteric ripening. The expression of CmACO1 (1-aminocyclopropane-1-carboxylate oxidase 1) gene associated with ethylene biosynthesis did not change during ripening. The introgression in LG X was associated with the differential hexanal production of the NIL and PS. An eQTL located in LG X is probably controlling the production of aroma volatiles due to CmLOX18 in LG I.

[SPA] La calidad del fruto de melón (Cucumis melo L.) es un criterio importante para la aceptación por parte del consumidor. El desarrollo del fruto y el proceso de maduración están vinculados a una serie de cambios bioquímicos y fisiológicos. Este trabajo compara clases de compuestos químicos del aroma del fruto de melón de una línea casi isogénica (NIL) SC7-2 durante dos campañas (S1, S2). El objetivo de este trabajo fue determinar la influencia de la campaña en el uso de los volátiles como potenciales biomarcadores de diferencias de textura (especialmente firmeza de la pulpa), debido a la introgresión en el melón LG VII. En la campaña S2, con mejores condiciones ambientales para el desarrollo del fruto en comparación con S1, se obtuvo un fruto con mejores atributos de calidad en general y con una firmeza de pulpa ligeramente inferior. Las diferencias entre temporadas fueron que el porcentaje de ésteres de acetato totales y otros compuestos aumentaron en S2, mientras que el porcentaje de aldehídos, alcoholes o alcanos típicos de los melones no climatéricos disminuyó. Nuestra hipótesis es que la biosíntesis de ésteres de acetato aumentó en los melones no climatéricos en mayor medida cuando las condiciones de crecimiento fueron favorables. [ENG] Fruit quality in melon (Cucumis melo L.) is an important criterion for consumer’s acceptance. Melons development and ripening process are linked to a series of biochemical and physiological changes. This work compared aroma compound classes of a melon near-isogenic line (NIL) SC7-2 during two seasons (S1, S2). The goal of this work was tracking the seasonal influence on the volatiles as potential biomarkers of textural differences (especially flesh firmness) due to introgression in melon LG VII. S2 with a better growing conditions compared with S1 resulted in enhanced quality traits with a slightly lower flesh firmness. Differences between seasons were that the percentage of total acetate esters and other compounds increased

[SPA] La calidad en los melones (Cucumis melo L.) es un criterio importante para la aceptación del consumidor. Al igual que en cualquier otra fruta, el proceso de desarrollo y maduración involucra una serie de cambios bioquímicos y fisiológicos como la descomposición de la clorofila, la degradación de la pared celular (que resulta desde ablandamiento a cambios en la ultraestructura de la pulpa y del fruto entero), el aumento en el contenido de azúcares y el contenido de sólidos solubles (SSC), la biosíntesis de pigmentos, aromas y la generación de sabor, etc. El objetivo de la tesis doctoral será determinar la calidad global y perfil aromático de al menos una línea casi isogénica (NIL) de melón con diferencias en textura respecto a su parental no climatéricos (PS) para mapear QTLs asociados a la calidad especialmente aromática y textural de la fruta. Utilizando resultados de expresión génica diferencial PS-NIL y el mapa genético de melón y las secuencias previamente almacenada en el GenBank estaremos posicionando genes candidatos y relacionando esta información con el fenotipo. [ENG] Quality in melons (Cucumis melo L.) is an important criterion for consumer’s acceptance. Like any other fruit, melons development and ripening process are linked to a series of biochemical and physiological changes including chlorophyll breakdown, cell wall degradation (leading from flesh to whole fruit softening to changes in ultrastructure), increase in the sugar content and soluble solids content (SSC), pigment biosynthesis, aroma and flavour generation, etc. The goal of the PhD will be to determine global quality and aroma profile at least one near-isogenic lines (NILs) of melon with different texture than its non-climacteric parental PS in order to map QTLs associated with fruit quality and particularly aroma volatiles. Using some results of differential gene expression and the genetic map of melon and the sequences stored in GenBank some candidate genes will be suggested and linki