Caracterització molecular d'una colecció d'especies silvestres relacionades amb l'albergnia (S. melongena) emprant les noves tecnologíes de següenciació

[CA] L’albergínia (Solanum melongena) ha sigut cultivada durant segles a tot arreu del món i actualment és un dels cultius amb més importància econòmica. La relació entre la versió salvatge, semi-domèstica i cultivada de Solanum melongena ha sigut sempre motiu de controvèrsia. És versemblant que l’albergínia que es consumeix en l’actualitat siga conseqüència de la domesticació de l’espècie silvestre Solanum incanum. Per una altra part, la existència d’una successió continua de formes fa que els límits entre taxons moltes vegades no siga massa clar entre albergínia i les seues espècies relacionades. La domesticació de les espècies cultivades ha provocat un empobriment genètic fins al punt en que l’albergínia d’avui dia és susceptible a nombroses malalties i plagues. És en aquest context on les espècies silvestres relacionades tenen un paper interessant, i és que aquestes han mostrat ser una reserva important de variabilitat genètica i variació al·lèlica per a caràcters d’interès agronòmic i característiques qualitatives tant de la planta com del fruit. La caracterització molecular detallada de col·leccions de germoplasma ens permetrà estudiar la diversitat a més de permetre’ns identificar accessions potencialment interessants per a la selecció i millora a banda de l’elaboració d’estratègies per a la conservació i maneig del germoplasma. En aquest treball s’han caracteritzat 48 accessions pertanyents a les espècies S. aethiopicum, S. anguivi, S. macrocarpon, S. dasyphyllum i S. incanum mitjançant l’ús de 39 marcadors SNPs. Els anàlisis de PCoA i clúster diferencien les espècies entre elles a més de mostrar la variabilitat genética existent dins d’elles. D’altra banda, els resultats obtinguts en aquest treball també confirmen que els SNPs seleccionats a partir dels transcriptomes de S. incanum i S. aethiopicum efectivament són reals, el que ens permetrà en un futur utilitzar-los en plataformes de genotipat massiu com pot ser SNPplex.[ES] La berenjena (Solanum melongena) ha sido cultivada durante siglos en todo el mundo y actualmente es uno de los cultivos más importantes economicamente hablando. La relación entre la versión salvaje, semi domestica y cultivada de Solanum melongena ha sido siempre motivo de controvèrsia. Es plausible que la berenjena que se consume hoy en día sea la consecuencia de la domesticación de la especie silvestre Solanum incanum. Por otra parte, la exististencia de una sucesión continua de formes hace que los límites entre taxones muchas veces no sea demasiado claro entre la berenjena y sus especies relacionades. La domesticación de las especies cultivades ha provocado un empobrecimiento genético hasta tal punto en que la berenjena de hoy en día sea susceptible a diferentes enfermedades y plagas. Es en este contexto donde las especies silvestres relacionades tienen un papel interesante, y es que estas han mostrado er una reserva importante de variavilidad genètica y variación alélica para catacteres de interès agronómicos y características cualitativas tanto de la planta como del fruto. La caracterización molecular detallada de col·lecciones de germoplasma nos permitirá estudiar la diversidad además de permitirnos identificar accesiones potencialmente interessantes para la selección y mejora además de la elaboración de estrategias para la conservación y manejo de germoplasma. En este Trabajo se han caracterizado 48 accesiones pertenecientes a S. aethiopicum, S. anguivi, S. macrocarpon, S. dasyphyllum y S. incanum mediante el uso de 39 marcadores SNPs. El anàlisis de PCoA y de clúster diferencia las especies entre ellas y muestra también la variabilidad genética existente entre elles. Por otra parte, los resultados obtenidos en este Trabajo también confirman que los SNPs seleccionados a partir de los transcriptomes de S. incanum y S. aethiopicum efectivamente son reales, lo cual nos permitirá en un futuro utilizarlos en plataformas de genotipado masivo como puede ser SNPplex.[EN] The eggplant (Solanum melongena) has been cultivated for centuries all over the world and nowadays is one of the most economical important cultivars. The relationship between the wild and the cultivated version of Solanum melongena has been controversial. It is likely that the consumed eggplant today is the result of the domestication of wild species Solanum incanum. On the other hand the existence of a flux of forms makes unclear the limits between Solanum melongena and its wild relative taxons. The domestication of the cultivated species has ended with a genetic impoverishment and therefore making the eggplant susceptible to many diseases. In this context the wild related species have an interesting role as they are a source of genetic and allelic variation for different important traits. The detailed molecular characterization of germplasm collections will allow us to study the diversity and even identifying potentially interesting accessions for breeding and also to improve the conservation and handle germplasm strategies design. Here we have characterized 48 accessions belonging to the species S. aethiopicum, S. anguivi, S. macrocarpon, S.dasyphyllum and S. incanum by the use of 39 SNPs markers. The PCoA and cluster analysis are able to distinguish the species between and within them. Moreover, the obtained results also confirm that the SNPs selected from the transcriptomes of S. incanum and S. aethiopicum are indeed real, which in the future will allow us to use them in massive genotyping platforms such SNPplex.Borràs Palomares, D. (2014). Caracterització molecular d'una colecció d'especies silvestres relacionades amb l'albergnia (S. melongena) emprant les noves tecnologíes de següenciació http://hdl.handle.net/10251/46169.Archivo delegad

Genetic structure of Cannabis sativa var. indica cultivars based on genomic SSR (gSSR) markers: implications for breeding and germplasm management

[EN] Cannabis saliva L. is cultivated for its fiber or seeds (var. saliva; hemp), or for its high content in cannabinoids (var. indica; marijuana). Knowledge of the genetic structure of C. sativa var. indica is important for selection and breeding of cultivars with medicinal interest. We used six genomic SSRs (gSSRs) for genotyping 154 individual plants of 20 cultivars of C. sativa var. indica, plus two cultivars of C. sativa var. sativa. A very high polymorphism was observed, with an average of 17 alleles and 23.8 genotypes per locus. Expected (H-e) and observed (H-o) heterozygosities were high, with average values of 0.753 and 0.429, respectively. In some cultivars H-e and H-o presented similar values, while in others H-e was considerably higher than H-o suggesting that consanguinity and fixation had taken place during its development. In addition, some cultivars had a reduced number of alleles per locus (in some cases only two) indicating that a genetic bottleneck had taken place during its development. Gene flow (Nm) between both botanical varieties was high, with Nm = 1.736. The molecular analysis of variance (AMOVA) revealed that only 31.94% of the molecular variation observed was caused by differences among cultivars, while the variation among plants of the same cultivar was of 37.11%, and within individual variation, due to heterozygosity, was of 30.96%. This indicates that a large variation exists within cultivars, which can be exploited for selection, but also complicates germplasm management and regeneration. The population structure analysis identified 14 genetic clusters, with most individuals of a single cultivar clustering together. This analysis, together with UPGMA cluster analysis shows that the two C. saliva var. saliva cultivars studied are differentiated from C. sativa var. indica, and that some cultivars of C. saliva var. indica seem to represent different selections from a common original cultivar. Our results represent the first comprehensive study of intra-varietal diversity in C. sativa var. indica and provide information of relevance for selection, breeding, and germplasm conservation, as well as for forensic studies in this crop.Soler Aleixandre, S.; Gramazio, P.; Figás-Moreno, MDR.; Vilanova Navarro, S.; Rosa-Martínez, E.; Llosa, ER.; Borràs-Palomares, D.... (2017). Genetic structure of Cannabis sativa var. indica cultivars based on genomic SSR (gSSR) markers: implications for breeding and germplasm management. Industrial Crops and Products. 104:171-178. doi:10.1016/j.indcrop.2017.04.043S17117810

Molecular characterization of scarlet and gboma eggplants based on single nucleotide polymorphisms

[EN] The common eggplant (Solanum melongena) is one of the most economically important specie for cultivated aubergine . The relationship between eggplant and it the wild relatives has been controversial. The domestication of the cultivated species has resulted in a narrow genetic base, and this difficults further improvements. Here we have characterized 48 accessions belonging to the cultivated scarlet eggplant (S. aethiopicum) and it wild ancestor (S. anguivi), the cultivated gboma eggplant (S. macrocarpon) and its wild ancestor (S. dasyphyllum), including also one accession of cultivated eggplant and one of its wild ancestor (S. incanum) using 39 single nucleotide polymorphism (SNP) markers. A total of 82 alleles were detected with an average of 2.34 alleles/locus. A good differentiation between the three groups corresponding to common, scarlet and gboma eggplants was obtained with a PCoA analysis. These results are of interest for the enhancement of genetic resources of eggplants.Borràs, D.; Plazas Ávila, MDLO.; Andújar Pérez, I.; Gramazio, P.; Herraiz García, FJ.; Prohens Tomás, J.; Vilanova Navarro, S. (2015). Molecular characterization of scarlet and gboma eggplants based on single nucleotide polymorphisms. Bulletin of University of Agricultural Sciences and Veterinary Medicine Cluj-Napoca : Horticulture. 448-449. doi:10.15835/buasvmcn-hort:11408S44844

Inoculation of cucumber, melón and zucchini varieties with Tomato leaf curl New Delhi virus (ToLCNDV) and evaluation of infection using different methods

This is the peer reviewed version of the following article: Figás-Moreno, MDR.; Alfaro Fernández, AO.; Font San Ambrosio, MI.; Borràs Palomares, D.; Casanova-Calancha, C.; Hurtado Ricart, M.; Plazas Ávila, MDLO.... (2017). Inoculation of cucumber, melón and zucchini varieties with Tomato leaf curl New Delhi virus (ToLCNDV) and evaluation of infection using different methods. Annals of Applied Biology. 170(3):405-414. doi:10.1111/aab.12344, which has been published in final form at http://doi.org/10.1111/aab.12344. This article may be used for non-commercial purposes in accordance with Wiley Terms and Conditions for Self-Archiving.[EN] The disease caused by Tomato leaf curl New Delhi virus (ToLCNDV), which is naturally transmitted by the whitefly Bemisia tabaci, causes important economic losses in cucurbit crops. The availability of simple and efficient inoculation protocols and detection methods is necessary for screening varieties and germplasm collections as well as for breeding populations. We evaluated the infectivity of ToLCNDV inocula prepared using three different buffers for mechanical sap inoculation in a susceptible variety of zucchini. We found that inoculum prepared with buffer III, which contains polyvinylpyrrolidone, is highly efficient for mechanical inoculation, with 100% of plants displaying severe symptoms 21 days post-inoculation. Using this buffer, we mechanically inoculated 19 commercial varieties of cucurbit crops (six of cucumber, six of melon and seven of zucchini), evaluated the evolution of symptoms and diagnosed infection using nine different ToLCNDV detection methods (four based on serology, four based on molecular hybridization and one based on PCR detection). The results revealed that all varieties are susceptible, although cucumber varieties display less severe symptoms than those of melon or zucchini. All detection methods were highly efficient (more than 85% of plants testing positive) in melon and zucchini, but in cucumber, the percentage of positive plants detected with serology and molecular hybridization methods ranged from 20.4% with Squash leaf curl virus (SLCV) antiserum, to 78.5% with DNA extract hybridization. Overall, the best detection results were obtained with PCR, with 92.6%, 92.4% and 98.4% cucumber, melon and zucchini plants, respectively, testing positive. When considering the overall results in the three crops, the best serology and molecular hybridization methods were those using Watermelon chlorotic stunt virus (WmCSV) antiserum and DNA extract, respectively. The inoculation methodology developed and the information on detection methods are of great relevance for the selection and breeding of varieties of cucurbit crops that are tolerant or resistant to ToLCNDV.Figás-Moreno, MDR.; Alfaro Fernández, AO.; Font San Ambrosio, MI.; Borràs Palomares, D.; Casanova-Calancha, C.; Hurtado Ricart, M.; Plazas Ávila, MDLO.... (2017). Inoculation of cucumber, melón and zucchini varieties with Tomato leaf curl New Delhi virus (ToLCNDV) and evaluation of infection using different methods. Annals of Applied Biology. 170(3):405-414. doi:10.1111/aab.12344S405414170

Comparison of transcriptome-derived simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers for genetic fingerprinting, diversity evaluation, and establishment of relationships in eggplants

[EN] Simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers are amongst the most common markers of choice for studies of diversity and relationships in horticultural species. We have used 11 SSR and 35 SNP markers derived from transcriptome sequencing projects to fingerprint 48 accessions of a collection of brinjal (Solanum melongena), gboma (S. macrocarpon) and scarlet (S. aethiopicum) eggplant complexes, which also include their respective wild relatives S. incanum, S. dasyphyllum and S. anguivi. All SSR and SNP markers were polymorphic and 34 and 36 different genetic fingerprints were obtained with SSRs and SNPs, respectively. When combining both markers all accessions but two had different genetic profiles. Although on average SSRs were more informative than SNPs, with a higher number of alleles, genotypes and polymorphic information content (PIC), and expected heterozygosity (He) values, SNPs have proved highly informative in our materials. Low observed heterozygosity (Ho) and high fixation index (f) values confirm the high degree of homozygosity of eggplants. Genetic identities within groups of each complex were higher than with groups of other complexes, although differences in the ranks of genetic identity values among groups were observed between SSR and SNP markers. For low and intermediate values of pair-wise SNP genetic distances, a moderate correlation between SSR and SNP genetic distances was observed (r(2) = 0.592), but for high SNP genetic distances the correlation was low (r(2) = 0.080). The differences among markers resulted in different phenogram topologies, with a different eggplant complex being basal (gboma eggplant for SSRs and brinjal eggplant for SNPs) to the two others. Overall the results reveal that both types of markers are complementary for eggplant fingerprinting and that interpretation of relationships among groups may be greatly affected by the type of marker used.This work has been funded by European Union's Horizon 2020 Research and Innovation Programme under Grant Agreement No 677379 (G2P-SOL project: Linking genetic resources, genomes and phenotypes of Solanaceous crops) and by Spanish Ministerio de Economia y Competitividad and Fondo Europeo de Desarrollo Regional (Grant AGL2015-64755-R from MINECO/FEDER). Pietro Gramazio is grateful to Universitat Politecnica de Valencia for a pre-doctoral contract (Programa FPI de la UPV-Subprograma 1/2013 call). Mariola Plazas is grateful to Spanish Ministerio de Economia, Industria y Competitividad for a post-doctoral grant within the Juan de la Cierva-Formacion programme (FJCI-2015-24835).Gramazio, P.; Prohens Tomás, J.; Borras, D.; Plazas Ávila, MDLO.; Herraiz García, FJ.; Vilanova Navarro, S. 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Mejora genética de la "Tomaca Valenciana d'El Perelló" para la resistencia al virus del mosaico del tomate (Tomato mosaic virus, ToMV)

[EN] The traditional valencian tomato is appreciated due to its organoleptic properties which confer to this tomato a high commercial interest. Specifically the ¿Tomaca Valenciana d¿El Perelló¿ is increasingly appreciated by superstores. However it presents a high susceptibility to the Tomato Mosaic Virus (ToMV). So, it is very interesting to obtain varieties of valencian tomato with resistance to ToMV. In this study a plant breeding program is made through Back Crosses Method in which the introgression of the Tm22 gene is pretended. The selection of resistance carrier is made by Molecular Assisted Selection and checked by inoculation and ELISA analysis. In this moments the second backcross generation is available.[CA] La tomaca tradicional valenciana és apreciada per les seues propietats organolèptiques que li atorguen un alt interès comercial. En concret la ¿Tomaca Valenciana d¿El Perelló¿ és cada vegada més apreciada en les superfícies comercials. No obstant, presenta una elevada susceptibilitat al virus del mosaic de la tomaca (ToMV). Així, és molt interessant obtenir varietats de tomaca valenciana resistents a ToMV. En aquest treball és du a terme un programa de millora genètica mitjançant el Mètode de Retrocreuaments amb el qual es pretén introgressar el gen de resistència a ToMV (Tm22). La selecció dels individus portadors de la resistència es realitza mitjançant Selecció Assistida per Marcadors i es comprova mitjançant inoculació i anàlisi ELISA. En aquests moments es disposa de la segona generació de retrocreuament (RC2).[ES] El tomate tradicional valenciano es apreciado por sus propiedades organolépticas que le otorgan un alto interés comercial. En concreto el “Tomate Valenciano d’El Perelló” es cada vez más apreciado en las superficies comerciales. Este tomate presenta diferentes rasgos agronómicos y de calidad que lo hacen atractivo para el agricultor y par a el consumidor. No obstante, a veces presenta problemas de uniformidad de producción así como de otras características importantes. Por otra parte, esta variedad presenta una elevada susceptibilidad al virus del mosaico del tomate ( ToMV ). Así, una forma muy interesante de potenciar este cultivo es obtener variedades de tomate valenciano más uniforme así como resistentes a ToMV . En este trabajo se lleva a cabo un programa de selección así como un programa de mejora genética mediante el Mé todo de Retrocruzamientos con el que se pretende por una parte aumentar la uniformidad de producción así como la presencia de rasgos morfológicos característicos de esta variedad, a la vez que se introgresa el gen de resistencia a ToMV ( Tm2 2 ). La selecci ón de los individuos portadores de la resistencia se realiza mediante la Selección Asistida por Marcadores y se comprueba mediante inoculación y análisis ELISA. En estos momentos se dispone de la cuarta generación de autofecundación en el programa de selec ción si como del segundo Retrocruzamiento (RC2). Al mismo tiempo se est á llevando a cabo el tercer Retrocruzamiento (RCBorràs Palomares, D. (2016). Mejora genética de la "Tomaca Valenciana d'El Perelló" para la resistencia al virus del mosaico del tomate (Tomato mosaic virus, ToMV). http://hdl.handle.net/10251/67874TFG