Bola De Oro Research Articles

Insights into the early transcriptomic response against watermelon mosaic virus in melon

Background Watermelon mosaic virus (WMV) is one of the most prevalent viruses affecting melon worldwide. Recessive resistance to WMV in melon has previously been reported in the African accession TGR-1551. Moreover, the genomic regions associated to the resistance have also been described. Nevertheless, the transcriptomic response that might infer the resistance to this potyvirus has not been explored.ResultsWe have performed a comparative transcriptomic analysis using mock and WMV-inoculated plants of the susceptible cultivar “Bola de oro” (BO) and a resistant RIL (Recombinant inbred line) derived from the initial cross between “TGR-1551” and BO. In total, 616 genes were identified as differentially expressed and the weighted gene co-expression network analysis (WGCNA) detected 19 gene clusters (GCs), of which 7 were differentially expressed for the genotype x treatment interaction term. SNPs with a predicted high impact on the protein function were detected within the coding regions of most of the detected DEGs. Moreover, 3 and 16 DEGs were detected within the QTL regions previously described in chromosomes 11 and 5, respectively. In addition to these two specific genomic regions, we also observde large transcriptomic changes from genes spread across the genome in the resistant plants in response to the virus infection. This early response against WMV implied genes involved in plant-pathogen interaction, plant hormone signal transduction, the MAPK signaling pathway or ubiquitin mediated proteolysis, in detriment to the photosynthetic and basal metabolites pathways. Moreover, the gene MELO3C021395, which coded a mediator of RNA polymerase II transcription subunit 33A (MED33A), has been proposed as the candidate gene located on chromosome 11 conferring resistance to WMV.ConclusionsThe comparative transcriptomic analysis presented here showed that, even though the resistance to WMV in TGR-1551 has a recessive nature, it triggers an active defense response at a transcriptomic level, which involves broad-spectrum resistance mechanisms. Thus, this study represents a step forward on our understanding of the mechanisms underlaying WMV resistance in melon. In addition, it sheds light into a broader topic on the mechanisms of recessive resistances.

Advanced Genetic Studies on Powdery Mildew Resistance in TGR-1551.

Cucurbits powdery mildew (CPM) is one of the main limiting factors of melon cultivation worldwide. Resistance to races 1, 2, and 5 has been reported in the African accession TGR-1551, whose resistance is controlled by a dominant–recessive epistasis. The dominant and recessive quantitative trail loci (QTL) have previously been located in chromosomes 5 and 12, respectively. We used several densely genotyped BC3 families derived from the cross between TGR-1551 and the susceptible cultivar ‘Bola de Oro’ to finely map these resistance regions. The further phenotyping and genotyping of the selected BC5, BC5S1, BC5S2, BC4S1, BC4xPS, and (BC4xPS) S1 offspring allowed for the narrowing of the candidate intervals to a 250 and 381 kb region in chromosomes 5 and 12, respectively. Moreover, the temperature effect over the resistance provided by the dominant gene has been confirmed. High resolution melting markers (HRM) were tightly linked to both resistance regions and will be useful in marker-assisted selection programs. Candidate R genes with variants between parents that caused a potential modifier impact on the protein function were identified within both intervals. These candidate genes provide targets for future functional analyses to better understand the resistance to powdery mildew in melons.

Melon Genome Regions Associated with TGR-1551-Derived Resistance to Cucurbit yellow stunting disorder virus.

Cucurbit yellow stunting disorder virus (CYSDV) is one of the main limiting factors of melon cultivation worldwide. To date, no commercial melon cultivars resistant to CYSDV are available. The African accession TGR-1551 is resistant to CYSDV. Two major quantitative trait loci (QTLs) have been previously reported, both located near each other in chromosome 5. With the objective of further mapping the gene or genes responsible of the resistance, a recombinant inbred line (RIL) population derived from the cross between TGR-1551 and the susceptible cultivar ‘Bola de Oro’ was evaluated for resistance to CYSDV in five different assays and genotyped in a genotyping by sequencing (GBS) analysis. The major effect of one of the two QTLs located on chromosome 5 was confirmed in the multienvironment RIL assay and additionally verified through the analysis of three segregating BC1S1 populations derived from three resistant RILs. Furthermore, progeny test using the offspring of selected BC3 plants allowed the narrowing of the candidate interval to a 700 kb region. The SNP markers identified in this work will be useful in marker-assisted selection in the context of introgression of CYSDV resistance in elite cultivars.

Fine mapping of wmv1551, a resistance gene to Watermelon mosaic virus in melon

Recessive resistance to Watermelon mosaic virus (WMV) in melon has previously been reported in the African accession TGR-1551. Using a population of recombinant inbred lines (RIL), derived from a cross between TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’ (BO), a major quantitative trait locus (QTL) controlling the resistance was previously mapped to a region of approximately 760 kb in chromosome 11. Minor QTLs were also reported with lower effects, dependent on the environmental conditions. A genotyping by sequencing (GBS) analysis of the RIL population has provided new information that allowed the better location of the major QTL in chromosome 11. Moreover, three minor QTLs in chromosomes 4, 5, and 6 were identified. Generations derived from the RIL population were subsequently phenotyped for resistance and genotyped with SNP markers to fine map the resistance derived from TGR-1551. The results obtained have allowed to narrow the position of the resistance gene on chromosome 11, designated as wmv1551, to a 141-kb region, and the confirmation of a minor QTL in chromosome 5. The effect of the minor QTL in chromosome 5 was significant in heterozygote plants for the introgression in chromosome 11. The SNP markers linked to both QTLs will be useful in breeding programs aimed at the introgression of WMV resistance derived from TGR-1551. Future work will be directed to identifying the resistance gene, wmv1551, in the candidate region on chromosome 11.

Influence of temperature on powdery mildew development in TGR-1551

Several plants of 'TGR-1551', a Zimbabwean melon accession resistant to races 1, 2 and 5 of Podosphaera xanthii, and the susceptible Spanish cultivar 'Bola de Oro' were inoculated with powdery mildew race 2 at two different temperatures (18-22 and 25-30°C) in order to study the plant-pathogen interaction. Fungal development was evaluated at microscopic level through several parameters such as the number of penetrated points, detected by callose accumulation with the double aniline-blue-calcofluor staining technique, at 48, 72, 96 and 120 h post inoculation (hpi), as well as the number of conidiophores at 120 hpi stained with an ink/acetic acid solution. The melon accessions 'PMR 45' and 'WMR 29', susceptible and resistant to race 2 respectively, were used as control lines at 48 and 120 hpi. At low temperature, 'TGR-1551' showed a non-resistant phenotype since the number of penetration points increased with time, as it is also observed for susceptible lines. Likewise, a high number of conidiophores were detected, although in lower quantities than in the susceptible lines. At high temperature, however, most of fungal conidia deposited on 'TGR-1551' plants stopped its development at germ tube or primary hyphae stage, and no conidiophores were observed at 120 hpi. A similar behavior was recorded for 'WMR 29'. The susceptible melon lines, 'Bola de Oro' and 'PMR 45', showed a great fungal development at both temperatures, being the greatest values observed at high temperature. This environmental factor might be affecting significantly the resistance of several melon genotypes to powdery mildew.

Inheritance and QTL mapping of glandular trichomes type I density in Cucumis melo L.

To determine the genetic basis of the density of glandular trichomes type I in melon, a six-generation family and a recombinant inbred lines (F7) population have been tested for the character; both genetic groups have been obtained from a cross between the Zimbabwean melon line TGR-1551 (high density of glandular trichomes) and the Spanish cultivar ‘Bola de Oro’ (low density of glandular trichomes). Classic quantitative genetic analysis indicated an oligogenic model for the inheritance of the character with an important additive component and strong epistatic relationships between loci. The quantitative trait locus (QTL) analyses revealed that the character is controlled by one major QTL, tric11, which explained between 23.8 and 58.7 % of its phenotypic variance; other minor QTLs of weaker effect and dependent of the assayed growing conditions were also found to be involved in the character expression. A codominant marker, the microsatellite ECM63, was found to be linked to the major QTL tric11. The validation of this marker in advanced backcrosses confirmed that the TGR-1551 alleles for this QTL increased glandular trichome density. The validation of the detected QTL was also done in other genetic backgrounds, which indicated that ECM63 could be effective in the selection of plants with high density of glandular trichomes type I.

Resistance to Cucurbit aphid-borne yellows virus in Melon Accession TGR-1551.

The genetic control of resistance to Cucurbit aphid-borne yellows virus (CABYV; genus Polerovirus, family Luteoviridae) in the TGR-1551 melon accession was studied through agroinoculation of a genetic family obtained from the cross between this accession and the susceptible Spanish cultivar 'Bola de Oro'. Segregation analyses were consistent with the hypothesis that one dominant gene and at least two more modifier genes confer resistance; one of these additional genes is likely present in the susceptible parent 'Bola de Oro'. Local and systemic accumulation of the virus was analyzed in a time course experiment, showing that TGR-1551 resistance was expressed systemically as a significant reduction of virus accumulation compared with susceptible controls, but not locally in agroinoculated cotyledons. In aphid transmission experiments, CABYV inoculation by aphids was significantly reduced in TGR-1551 plants, although the virus was acquired at a similar rate from TGR-1551 as from susceptible plants. Results of feeding behavior studies using the DC electrical penetration graph technique suggested that viruliferous aphids can salivate and feed from the phloem of TGR-1551 plants and that the observed reduction in virus transmission efficiency is not related to reduced salivation by Aphis gossypii in phloem sieve elements. Since the virus is able to accumulate to normal levels in agroinoculated tissues, our results suggest that resistance of TGR-1551 plants to CABYV is related to impairment of virus movement or translocation after it reaches the phloem sieve elements.

La madera deTrema micrantha (L.) Blume de Veracruz, México

Se estudió la anatomía microscópica, los índices hidráulicos y mecánicos de la madera de 28árboles de Trema micrantha de cuatro localidades ubicadas en los municipios de Xico y Coatepec enel estado de Veracruz. Adicionalmente, se determinaron las características macroscópicas, ladensidad relativa, el contenido de humedad y algunas pruebas de carpintería en probetas de diferentestamaños de la madera de tres árboles, con el fin de proponer usos alternativos en zonasrurales. Los resultados mostraron que T. micrantha presenta un xilema mesomórfico, poco resistentea la cavitación, de baja densidad relativa, con valores bajos de índices de colapso de vaso y fibras yaltos para el índice de vulnerabilidad. Los análisis de varianza confirman que de todas las característicasestudiadas solamente el índice de colapso de los vasos es similar entre indivíduos y poblaciones.Las comparaciones múltiples arrojan que los caracteres que separan a las localidades Bola de Oro yLos Barrales de las otras son el grosor de la pared y el número de radios. Las pruebas tecnológicasdescriben una madera que no se raja al clavado, responde bien al ensamblado y al torneado por loque se recomienda para su uso en la carpintería rural.

Simple sequence repeat markers linked to QTL for resistance to Watermelon mosaic virus in melon

A population of recombinant inbred lines (RIL) derived from a cross between the Watermelon mosaic virus (WMV) resistant genotype TGR-1551 and the susceptible Spanish cultivar 'Bola de Oro' has been evaluated for WMV resistance in spring, fall and growth chamber conditions. The quantitative trait loci (QTL) analyses detected one major QTL (wmv) on linkage group (LG) XI close to the microsatellite marker CMN04_35. This QTL controls the resistance to WMV in the three environmental conditions evaluated. Other minor QTLs affecting the severity of viral symptoms were identified, but they were not detected in all the assayed environments. The screening of the marker CMN04_35 in an F(2) progeny, derived from the same cross, confirmed the effect of this QTL on the expression of WMV resistance also in early generations, which evidences the usefulness of this marker for a marker assisted selection program.

PRELIMINARY STUDY OF RESISTANCE AGAINST BEMISIA TABACI GENN. IN TGR-1551 MELON GENOTYPE

The Zimbabwean genotype TGR-1551 had been found to be resistant to Bemisia tabaci Genn. in previous studies. In order to have a better knowledge of this resistance and determine the most suitable character to evaluate the genetic resistance to B. tabaci, three whitefly life-parameters have been tested in a RIL population (F 7 ) derived from a cross between 'TGR-1551' and the susceptible Spanish cultivar 'Bola de Oro'. Plants of each parent, their F 1 and 62 RIL were grown in a greenhouse under natural conditions of whitefly infestation. In each plant, the number of adults of B. tabaci, ovoposition and number of empty pupal cases were recorded. Significant differences were found between parents for each whitefly life-parameter: TGR-1551 showed a high level of resistance whatever the parameter was considered, and 'Bola de Oro' behaved as highly susceptible to B. tabaci. The low number of adults and low ovoposition found on leaves of TGR-1551 suggested the presence of non-preference mechanisms in this genotype. In the F 1 population, the number of adult whiteflies and the ovoposition was similar to 'Bola de Oro', while the number of empty pupal cases was similar to the number found in TGR-1551. These results pointed to the existence of antibiotic mechanisms in the resistant genotype which impede B. tabaci to reach its adult stage. Highly significant differences among RIL were observed for the three parameters, mainly for the number of empty pupal cases, explaining the 43 % of the variance observed. Some RIL behaved like TGR-1551 and some like the F 1 , which suggested a different genetic control of antixenosis and antibiosis mechanisms in TGR-1551. Dominance effects seem to be involved in antibiosis, while antixenosis, measured as ovoposition, should be regulated by recessive factors. These results confirm the usefulness of the number of empty pupal cases in the selection of B. tabaci resistance and for genetic studies.

CONSTRUCTION OF A GENETIC LINKAGE MAP AND IDENTIFICATION OF A MAJOR QTL CONFERRING RESISTANCE TO POWDERY MILDEW IN THE MELON GENOTYPE TGR-1551

Melon (Cucumis melo L.) is a valuable crop grown in temperate and tropical regions worldwide. Powdery mildew caused by Podosphaera xanthii is one of the most frequent diseases of melon. The genotype TGR-1551 is resistant to races 1, 2, and 5 of powdery mildew. We constructed a genetic linkage map using an F 2 population derived from a cross between TGR-1551 and the susceptible Spanish cultivar 'Bola de Oro'. The software JoinMap ® 4.0 was used to locate on the linkage map a total of 429 loci: 241 AFLP, 55 RAPD, 117 SSR, 14 SCAR/CAPS/dCAPS, and 2 phenotypic traits (Vat and a loci), which were distributed among 12 linkage groups with a LOD score ≥6.0. The map spans 1318.2 cM, with an average of 3.1 cM/ marker. Powdery mildew resistance was evaluated as a quantitative trait and then, QTL analyses were performed using interval mapping and multiple QTL model mapping with MapQTL ® 5.0 software. Both methods detected a dominant QTL for resistance to either 1, 2 or 5 races of powdery mildew, all located at the same genomic region of G4. Therefore, these QTL were considered as a unique QTL designated as Pm-R. This QTL was supported by LOD scores of 26.5, 33.3, and 36.2, explaining 53.6, 61.9, and 64.9% of the phenotypic variance in the mapping population for resistance to powdery mildew races 1, 2, and 5, respectively. In addition, four codominant markers, previously described as linked to the dominant powdery mildew resistance gene, were linked to the Pm-R QTL, which suggests the correspondence between this QTL and the dominant resistance gene. One of those codominant markers, PM3-CAPS, co-segregated with the Pm-R QTL, and the markers PM2-CAPS and PM4-dCAPS flanked this QTL. These markers could be used to select resistance to powdery mildew in melon breeding.

Role of leaf glandular trichomes of melon plants in deterrence of Aphis gossypii Glover

External characteristics of the leaf epidermis and their effects on behaviour of Aphis gossypii Glover were evaluated in two Cucumis melo L. genotypes, 'Bola de Oro' (aphid susceptible) and TGR-1551 (aphid resistant) in order to explore their role in the early rejection of TGR-1551 by this aphid. No differential effects of epicuticular waxes on aphid behaviour were observed. The type, distribution and number of trichomes on melon leaves were also studied. Pubescence in melon, measured as the number of non-glandular trichomes per cm(2), was not sufficient to prevent aphid settling. However, there was a high density of type I glandular trichomes on leaves of the aphid-resistant genotype. According to microscopic observations and stain testing, these trichomes store and secrete phenols and flavonoids. Free-choice tests were conducted to determine the effect of these glandular trichomes on A. gossypii preference, revealing that aphids reject leaf disks of TGR-1551 from the onset of the experiment. Additional experiments after removal of leaf type I glandular trichome exudates showed that A. gossypii preferred washed TGR-1551 leaf disks over unwashed disks, while this effect was not observed in experiments using washed and unwashed 'Bola de Oro' leaf disks. These results suggest that a high density of glandular trichomes and chemicals secreted by them deter A. gossypii and disturb aphid settling on TGR-1551.

Genetic linkage map of melon (Cucumis melo L.) and localization of a major QTL for powdery mildew resistance

Powdery mildew caused by Podosphaera xanthii has become a major problem in melon since it occurs all year round irrespective of the growing system. The TGR-1551 melon genotype was found to be resistant to several melon diseases, among them powdery mildew. However, the corresponding resistance genes have been never mapped. We constructed an integrated genetic linkage map using an F2 population derived from a cross between the multi-resistant genotype TGR-1551 and the susceptible Spanish cultivar ‘Bola de Oro’. The map spans 1,284.9 cM, with an average distance of 3.6 cM among markers, and consists of 354 loci (188 AFLP, 39 RAPD, 111 SSR, 14 SCAR/CAPS/dCAPS, and two phenotypic traits) distributed in 14 linkage groups. QTL analysis identified one major QTL (Pm-R) on LG V for resistance to races 1, 2, and 5 of powdery mildew. The PM4-CAPS marker is closely linked to the Pm-R QTL at a genetic distance of 1.9 cM, and the PM3-CAPS marker is located within the support interval of this QTL. These codominant markers, together with the map information reported here, could be used for melon breeding, and particularly for genotyping selection of resistance to powdery mildew in this vegetable crop species.

Inheritance of resistance to races 1, 2 and 5 of powdery mildew in the melon TGR-1551

With 1 figure and 2 tables The inheritance of resistance to races 1, 2 and 5 of Podosphaera xanthii in the melon TGR-1551 was studied in a cross with the susceptible cultivar ‘Bola de oro’. Under artificial inoculation conditions, four phenotypic classes were defined according to the level of fungal sporulation. The segregation ratios for resistance/susceptibility observed in the different populations and the results of the progeny test indicated that resistance to races 1, 2 and 5 of P. xanthii in TGR-1551 was governed by two independent genes, one dominant and one recessive, which means that the genetic control is a dominant–recessive epistasis. This is the first time that a recessive gene that confers resistance to more than one race of powdery mildew has been identified in melon. TGR-1551 could be considered a good genetic source for the development of powdery mildew resistant breeding lines.

New source of resistance to mosaic virus transmission by Aphis gossypii in melon

The melon accession ‘TGR-1551’ shows a clear and total resistance to the transmission of several isolates of CMV and ZYMV when A. gossypii was used as the vector. The genetic analysis of the progenies obtained crossing this accession with the susceptible Spanish cultivar ‘Bolade Oro’ showed that the resistance to mosaic virus transmission is conferred by the presence of a dominant gene. There are also evidences that this dominant gene is different from ‘Vat’ gene.

Spanish Melon necrotic spot virus Isolate Overcomes the Resistance Conferred by the Recessive nsv Gene of Melon.

Melon necrotic spot virus (MNSV, genus Carmovirus, family Tombusviridae) is a worldwide natural pathogen that can cause significant economic losses in cucurbit crops (2). Use of commercial cultivars that carry the recessive nsv gene, the only resistance to MNSV described until now (1), is an effective means of controlling MNSV on melon crops. We report here the appearance of MNSV isolates able to overcome nsv resistance. Typical MNSV symptoms were observed in plants with an nsv/nsv genotype during a survey of melon in Almería (southeastern Spain) in 1999. The presence of MNSV in symptomatic plants was confirmed by standard double-antibody sandwich enzyme-linked immunosorbent assay (DAS-ELISA) using commercial antisera (Loewe Biochemica GmbH, Sauerlach, Germany). MNSV was isolated from these samples by three serial single-lesion inoculation passages in melon (cv. Bola de Oro). The isolate obtained (MNSV-264) was characterized by sequencing a fragment of its RNA genome corresponding to most of the capsid protein (CP) gene. Briefly, total RNA extracts (TRI reagent, Sigma Chemical, St. Louis, MO) of MNSV-264-infected plants were used in a reverse transcription polymerase chain reaction (RT-PCR) in which the oligonucleotide for the reverse transcription was 5'-TTAGGCGAGGTAAGCAGTTTC-3' (MA34), and the oligonucleotides for the PCR were MA34 and 5'-ATGGCGATGGTTAAACGC-3' (MA32). MA32 and MA34 were designed based on an alignment of nucleotide sequences of CP genes of diverse MNSV isolates. A DNA product of approximately 1.1 kbp was obtained, purified, and sequenced (GenBank Accession No. AF488692). Sequence comparisons (GCG Software Package, Madison, WI) of MNSV-264 and MNSV-Dutch (2) showed a 93% nucleotide sequence identity and a 96% similarity of the deduced amino acid sequence. Therefore, both isolates appear to be closely related. MNSV-264 was inoculated on melon accessions known to be susceptible (cvs. Panal and Bola de Oro) or resistant (cvs. Primal F1, Planters Jumbo, and PI161375) to common MNSV strains. Inoculations with MNSV-Dutch were used as a control for this experiment. The results showed that MNSV-264 was able to infect plants of all genotypes, whereas MNSV-Dutch infected only plants of the susceptible genotypes. Therefore, MNSV-264 is an isolate able to overcome the resistance conferred by nsv. The genetic determinant of the MNSV-264 virulence on resistant genotypes is currently under investigation. From 55 MNSV isolates obtained from fields in southeastern Spain during the last 3 years, only one could overcome nsv, like MNSV-264. Therefore, resistance-breaking isolates are not frequent in the MNSV populations in Spain. It is plausible that the situation may change as a consequence of the widespread use of commercial melon cultivars with the nsv genotype. A search for new sources of genetic resistance to MNSV isolates like MNSV-264 is in progress in our laboratory.

Resistance ofCucumis meloAgainstBemisia tabaci(Homoptera: Aleyrodidae)

The life history parameters of Bemisia tabaci (Gennadius), net reproduction, adult longevity, adult survival, and length of the developmental time have been evaluated in Cucumis melo L. and wild relatives looking for sweetpotato whitefly resistance. Under free-choice conditions, whitefly net reproduction on C. melo variety agrestis cultivar ‘87’ and C. melo ‘TGR-1551’ was lower than on the susceptible C. melo ‘Bola de Oro’. Under no-choice conditions the net reproduction of B. tabaci on C. melo variety agrestis 87 and C. melo TGR-1551 was also lower than on the susceptible control cultivar C. melo Bola de Oro. Length of developmental time, adult survival, and adult longevity were not good life history parameters to differentiate resistant and susceptible genotypes against whitefly. The results seem to indicate the existence of genetic resistance to B. tabaci in both genotypes, C. melo variety agrestis 87 and C. melo TGR-1551.

Resistance mechanisms of Cucumis melo var. agrestis against Trialeurodes vaporariorum and their use to control a closterovirus that causes a yellowing disease of melon

One of the yellowing diseases that affects melon (Cucumis melo) crops cultivated under plastic greenhouses in southern Europe is caused by a closterovirus transmitted by the greenhouse whitefly Trialeurodes vaporariorum. A Cucumis melo var. agrestis accession shows a certain level of resistance to that virus. Under free‐choice conditions, the reproduction of T. vaporariorum on this accession was significantly lower than on the susceptible control accession C. melo cv. ‘Bola de Oro’, and this suggests that T. vaporariorum prefers to colonize other accessions before C. melo var. agrestis. Under no‐choice conditions, we observed that T. vaporariorum reproduction on C. melo var. agrestis was less than on the susceptible controls. The results showed the participation of both antixenosis and antibiosis resistance mechanisms against T. vaporariorum in C. melo var. agrestis. It is difficult to cultivate melon completely free from T. vaporariorum and yellowing disease. However, success in breeding commercial melon cultivars which incorporate the antixenosis and antibiosis found in C. melo var. agrestis together with biological or chemical insect control, or a combination of these, would reduce the insect population and, therefore, the melon‐yellowing disease would occur later and in less severe form.