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[EN] Tomato leaf curl New Delhi virus (ToLCNDV) is a whitefly-transmited virus that causes serious damage to different cultivated plant species mainly belonging to the Solanaceae and Cucurbitaceae families. The complex epidemiological factors associated with this disease make the seeking and use of genetically resistant varieties as one of the most efficient, sustainable and frequently employed strategies to control viral infections in the fields. In the case of melon, resistance to mechanical transmission of ToLCNDV has been identified in Cucumis melo subsp. agrestis, in three accessions of the Momordica group and in two wild accessions. The genetic control of the resistance derived from one of the wild sources has been studied recently and a major locus in chromosome 11 has been found, along with two modifier regions located in chromosomes 2 and 12. Previous studies have reported the active role of different genes regarding ToLCNDV resistance in different species. In this work we have validated by qPCR twelve of these genes by determining the transcriptomic changes in leaves of a resistant Momodica and a susceptible Piel de Sapo genotypes along a time course experiment after infection with ToLCNDV. The transcript amount of the genes Cm ARP4 and Cm NAC domain protein, was differentially higher in the inoculated susceptible genotype when compared to the inoculated resistant one. For other validated genes, the resistant accession showed higher levels of expression. The location of Cm ARP4 in a modifier QTL of chomosome 2, suggest that the accumulation of transcripts of this gene is associated to the level of ToLCNDV accumulation and degree of symptoms development. The molecular bases of ToLCNDV resistance regarding the expression results for these candidate genes in melon are discussed as well and their potential use in TILLING platforms and breeding strategies of the species.

[EN] Tomato leaf curl New Delhi virus (ToLCNDV) is a whitefly-transmited virus that causes serious damage to different cultivated plant species mainly belonging to the Solanaceae and Cucurbitaceae families. The complex epidemiological factors associated with this disease make the seeking and use of genetically resistant varieties as one of the most efficient, sustainable and frequently employed strategies to control viral infections in the fields. In the case of melon, resistance to mechanical transmission of ToLCNDV has been identified in Cucumis melo subsp. agrestis, in three accessions of the Momordica group and in two wild accessions. The genetic control of the resistance derived from one of the wild sources has been studied recently and a major locus in chromosome 11 has been found, along with two modifier regions located in chromosomes 2 and 12. Previous studies have reported the active role of different genes regarding ToLCNDV resistance in different species. In this work we have validated by qPCR twelve of these genes by determining the transcriptomic changes in leaves of a resistant Momodica and a susceptible Piel de Sapo genotypes along a time course experiment after infection with ToLCNDV. The transcript amount of the genes Cm ARP4 and Cm NAC domain protein, was differentially higher in the inoculated susceptible genotype when compared to the inoculated resistant one. For other validated genes, the resistant accession showed higher levels of expression. The location of Cm ARP4 in a modifier QTL of chomosome 2, suggest that the accumulation of transcripts of this gene is associated to the level of ToLCNDV accumulation and degree of symptoms development. The molecular bases of ToLCNDV resistance regarding the expression results for these candidate genes in melon are discussed as well and their potential use in TILLING platforms and breeding strategies of the species.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan (R) MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidenialis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.

A quantitative real-time reverse transcription-polymerase chain reaction (RT-qPCR) procedure using a general primer set and three TaqMan (R) MGB probes was developed for general and genotype-specific detection and quantitation of the genomic M segment of Tomato spotted wilt virus (TSWV). Standard curves using RNA transcripts homologous to the three probes allowed reproducible quantitative assays with a wide dynamic range (10(3)-10(10) TSWV M segment RNA copies/ng of total RNA) and high sensitivity. This protocol was assayed with a battery of TSWV isolates, covering the range of the present known genetic variation, in single and/or mix infections in three plant hosts, as well as in the thrips vector Frankliniella occidenialis. This quantitative detection assay will be a valuable tool for molecular biology and epidemiology studies, diagnosis and disease control.