39 results on '"Zuriaga, E."'
Search Results
2. Pomological and chemical diversity in a pomegranate (Punica granatum L.) germplasm collection from Southern European countries
- Author
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Bartual, J., primary, Zuriaga, E., additional, Koka, T., additional, Navarro, M.J., additional, Ortiz, M., additional, and Badenes, M.L., additional
- Published
- 2022
- Full Text
- View/download PDF
3. The Implications of AFLP Data for the Systematics of the Wild Species of Solanum Section Basarthrum
- Author
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Prohens, J., Anderson, G. J., Blanca, J. M., Cañizares, J., Zuriaga, E., and Nuez, F.
- Published
- 2006
4. Morphological characterization of the IVIA persimmon (Diospyros kaki Thunb.) germplasm collection by multivariate analysis
- Author
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Martínez-Calvo, J., Naval, M., Zuriaga, E., Llácer, G., and Badenes, M. L.
- Published
- 2013
- Full Text
- View/download PDF
5. Self-(In)compatibility Systems: Target Traits for Crop-Production, Plant Breeding, and Biotechnology
- Author
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Muñoz-Sanz, J.V., Zuriaga, E., Cruz-García, F., McClure, B., and Romero, Carlos
- Subjects
fungi ,food and beverages - Abstract
Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S-determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition.
- Published
- 2020
6. Self-(In)compatibility Systems: Target Traits for Crop-Production, Plant Breeding, and Biotechnology
- Author
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Muñoz-Sanz, Juan Vicente, Zuriaga, E., Cruz-García, F., McClure, B., Romero, Carlos, Muñoz-Sanz, Juan Vicente, Zuriaga, E., Cruz-García, F., McClure, B., and Romero, Carlos
- Abstract
Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S-determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition.
- Published
- 2020
7. Cost-effective and time-efficient molecular assisted selection for ppv resistance in apricot based on ParPMC2 allele-specific PCR
- Author
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Polo-Oltra, Á., Romero, C., Badenes, María Luisa, Zuriaga, E., Polo-Oltra, Á., Romero, C., Badenes, María Luisa, and Zuriaga, E.
- Abstract
Plum pox virus (PPV) is the most important limiting factor for apricot (Prunus armeniaca L.) production worldwide, and development of resistant cultivars has been proven to be the best solution in the long-term. However, just like in other woody species, apricot breeding is highly time and space demanding, and this is particularly true for PPV resistance phenotyping. Therefore, marker-assisted selection (MAS) may be very helpful to speed up breeding programs. Tightly linked ParPMC1 and ParPMC2, meprin and TRAF-C homology (MATH)-domain-containing genes have been proposed as host susceptibility genes required for PPV infection. Contribution of additional genes to PPV resistance cannot be discarded, but all available studies undoubtedly show a strong correlation between ParPMC2-resistant alleles (ParPMC2res) and PPV resistance. The ParPMC2res allele was shown to carry a 5-bp deletion (ParPMC2-del) within the second exon that has been characterized as a molecular marker suitable for MAS (PMC2). Based on this finding, we propose here a method for PPV resistance selection in apricot by combining high-throughput DNA extraction of 384 samples in 2 working days and the allele-specific genotyping of PMC2 on agarose gel. Moreover, the PMC2 genotype has been determined by PCR or by using whole-genome sequences (WGS) in 175 apricot accessions. These results were complemented with phenotypic and/or genotypic data available in the literature to reach a total of 325 apricot accessions. As a whole, we conclude that this is a time-efficient, cost-effective and straightforward method for PPV resistance screening that can be highly useful for apricot breeding programs.
- Published
- 2020
8. Polyphenol content in apricot fruits
- Author
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Gómez-Martínez, H., primary, Bermejo, A., additional, Zuriaga, E., additional, and Badenes, M.L., additional
- Published
- 2021
- Full Text
- View/download PDF
9. Nutraceutical characterization of apricot fruits of the IVIA’s collection
- Author
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Gómez-Martínez, H., primary, Bermejo, A., additional, Badenes, M.L., additional, and Zuriaga, E., additional
- Published
- 2020
- Full Text
- View/download PDF
10. Genetic diversity among pomegranate germplasm assessed by microsatellite markers
- Author
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Zuriaga, E., primary, Bartual, J., additional, Pintova, J., additional, and Badenes, M.L., additional
- Published
- 2019
- Full Text
- View/download PDF
11. Analysis of genetic diversity among a set of accessions from the IVIA’s persimmon collection
- Author
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Gil-Muñoz, F., primary, Zuriaga, E., additional, Badenes, M.L., additional, and Naval, M.M., additional
- Published
- 2018
- Full Text
- View/download PDF
12. AFLP ANALYSIS OF MUTATIONS INDUCED BY GAMMA IRRADIATION IN 'ROJO BRILLANTE' PERSIMMON
- Author
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Naval, M.M., primary, Zuriaga, E., additional, and Badenes, M.L., additional
- Published
- 2013
- Full Text
- View/download PDF
13. GENETIC TOOLS FOR SELECTING RESISTANCE TO SHARKA DISEASE IN APRICOT
- Author
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Soriano, J.M., primary, Domigo, M.L., additional, Zuriaga, E., additional, Romero, C., additional, Llacer, G., additional, and Badenes, M.L., additional
- Published
- 2012
- Full Text
- View/download PDF
14. Morphological characterization of the IVIA persimmon (Diospyros kaki Thunb.) germplasm collection by multivariate analysis
- Author
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Martínez-Calvo, J., primary, Naval, M., additional, Zuriaga, E., additional, Llácer, G., additional, and Badenes, M. L., additional
- Published
- 2012
- Full Text
- View/download PDF
15. THE STUDY OF MOLECULAR DIVERSITY IN NATURAL POPULATIONS OF WILD AND WEEDY TOMATOES AND ITS IMPLICATIONS IN TOMATO BREEDING
- Author
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Nuez, F., primary, Díez, M.J., additional, Prohens, J., additional, Blanca, J.M., additional, Sifres, A., additional, Picó, B., additional, Cordero, L., additional, and Zuriaga, E., additional
- Published
- 2008
- Full Text
- View/download PDF
16. The study of molecular diversity in natural populations of wild and weedy tomatoes and its implications in tomato breeding
- Author
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Nuez, F., Díez, M. J., Jaime Prohens, Bianca, J. M., Sifres, A., Picó, B., Cordero, L., and Zuriaga, E.
17. Descriptive Analysis of Carrier and Affected Hereditary Fructose Intolerance in Women during Pregnancy.
- Author
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Zuriaga E, Santander S, Lomba L, Izquierdo-García E, and Luesma MJ
- Abstract
(1) Background: Hereditary fructose intolerance (HFI) is a rare autosomal recessive metabolic disorder resulting from aldolase B deficiency, requiring a fructose, sorbitol and sucrose (FSS)-free diet. Limited information exists on the relationship between pregnancy outcomes and HFI. This study aims to analyze pregnancy-related factors in a cohort of thirty Spanish women, with twenty-three being carriers and seven being HFI-affected (45 pregnancies). (2) Methods: A descriptive, cross-sectional and retrospective study utilized an anonymous questionnaire. (3) Results: Findings encompassed physical and emotional states, nutritional habits, pathology development and baby information. Notable results include improved physical and emotional states compared to the general population, with conventional analyses mostly within normal ranges. Persistent issues after pregnancy included hepatic steatosis, liver adenomas and hemangiomas. Carrier mothers' babies exhibited higher weight than those of patient mothers, while the weights of carrier children born with HFI were similar to disease-affected children. (4) Conclusions: Pregnant women with HFI did not significantly differ in physical and emotional states, except for nausea, vomiting, and cravings. Post-pregnancy, HFI patients and carriers exhibited persistent hepatic issues. Significantly, babies born to HFI-affected mothers had lower weights. This study sheds light on pregnancy outcomes in HFI, emphasizing potential complications and the need for ongoing monitoring and care.
- Published
- 2024
- Full Text
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18. Advances in Cryopreservatives: Exploring Safer Alternatives.
- Author
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Lomba L, García CB, Benito L, Sangüesa E, Santander S, and Zuriaga E
- Subjects
- Cryopreservation, Cell Line, Sulfoxides, Cryoprotective Agents, Ionic Liquids toxicity
- Abstract
Cryopreservation of cells, tissues, and organs is widely used in the biomedical and research world. There are different cryopreservatives that are used for this process; however, many of them, such as DMSO, are used despite the problems they present, mainly due to the toxicity it presents to certain types of samples. The aim of this Review is to highlight the different types of substances used in the cryopreservation process. It has been shown that some of these substances are well-known, as in the case of the families of alcohols, sugars, sulfoxides, etc. However, in recent years, other compounds have appeared, such as ionic liquids, deep eutectic solvents, or certain polymers, which open the door to new cryopreservation methods and are also less toxic to frozen samples.
- Published
- 2024
- Full Text
- View/download PDF
19. Physiological Changes and Transcriptomic Analysis throughout On-Tree Fruit Ripening Process in Persimmon ( Diospyros kaki L.).
- Author
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Dorta T, Gil-Muñoz F, Carrasco F, Zuriaga E, Ríos G, and Blasco M
- Abstract
The involvement of effectors and transcriptional regulators in persimmon fruit maturation has been mostly approached by the literature under postharvest conditions. In order to elucidate the participation of these genes in the on-tree fruit maturation development, we have collected samples from seven persimmon germplasm accessions at different developmental stages until physiological maturation. This study has focused on the expression analysis of 13 genes involved in ethylene biosynthesis and response pathways, as well as the evolution of important agronomical traits such as skin colour, weight, and firmness. Results revealed different gene expression patterns, with genes up- and down-regulated during fruit development progression. A principal component analysis was performed to correlate gene expression with agronomical traits. The decreasing expression of the ethylene biosynthetic genes DkACO1 , DkACO2, and DkACS2 , in concordance with other sensing ( DkERS1 ) and transduction genes ( DkERF18 ), provides a molecular mechanism for the previously described high production of ethylene in immature detached fruits. On the other side, DkERF8 and DkERF16 are postulated to induce fruit softening and skin colour change during natural persimmon fruit ripening via DkXTH9 and DkPSY activation, respectively. This study provides valuable information for a better understanding of the ethylene signalling pathway and its regulation during on-tree fruit ripening in persimmon.
- Published
- 2023
- Full Text
- View/download PDF
20. Single-Bud Expression Analysis of Bud Dormancy Factors in Peach.
- Author
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Puertes A, Polat H, Ramón-Núñez LA, González M, Ancillo G, Zuriaga E, and Ríos G
- Abstract
Transcriptomic and gene expression analysis have greatly facilitated the identification and characterization of transcriptional regulatory factors and effectors involved in dormancy progression and other physiological processes orchestrated during bud development in peach and other temperate fruit species. Gene expression measurements are most usually based on average values from several or many individual buds. We have performed single-bud gene analysis in flower buds of peach across dormancy release using amplicons from the master regulatory DORMANCY-ASSOCIATED MADS-BOX ( DAM ) factors, several jasmonic acid biosynthetic genes, other genes related to flowering development, cell growth resumption, and abiotic stress tolerance. This analysis provides a close view on gene-specific, single-bud variability throughout the developmental shift from dormant to dormancy-released stages, contributing to the characterization of putative co-expression modules and other regulatory aspects in this particular tissue.
- Published
- 2023
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21. Sphingomyelin-Rich Lipid Extract Collar for Canine Atopic Dermatitis.
- Author
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Segarra S, Sanmiguel D, Zuriaga E, Leclerc S, Cabañas J, Seigneuric E, Miquel A, Vázquez A, and Ferrer L
- Abstract
The management of canine atopic dermatitis (CAD) is complex, and it needs to be multimodal, combining topical and systemic therapies. Given that the currently available options are not always totally effective and might have some associated adverse effects, novel alternatives are needed. For this reason, a new collar for CAD was developed with 2.5% of a sphingomyelin-rich lipid extract (LE) with proven benefits for skin health. The release of the active ingredient when incorporated into the collar was tested in vitro, showing an adequate kinetic profile. Then, the efficacy and safety of the collar were assessed in 12 client-owned dogs with CAD in a pilot study. After eight weeks, the dogs experienced significant clinical improvements on the Canine Atopic Dermatitis Extent and Severity Index (CADESI)-4, Pruritus Index for Canine Atopic Dermatitis (PCAD) and Pruritus Visual Analogue Scale (PVAS) scores, without any adverse effects. Additionally, further in vitro studies were performed, indicating that this LE collar should be compatible with antiparasitic collars (with deltamethrin or imidacloprid/flumethrin) if worn simultaneously. Given the observed benefits of this LE collar, combining it with other CAD therapies could potentially allow for drug sparing, reduction in adverse effects, enhanced owner compliance, and reduced treatment costs.
- Published
- 2023
- Full Text
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22. Editorial: Towards the Identification of Useful Genes for Prunus Breeding.
- Author
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Zuriaga E, Tao R, and Romero C
- Abstract
Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
- Published
- 2022
- Full Text
- View/download PDF
23. Characterization of the Spanish Pomegranate Germplasm Collection Maintained at the Agricultural Experiment Station of Elche to Identify Promising Breeding Materials.
- Author
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Zuriaga E, Pintová J, Bartual J, and Badenes ML
- Abstract
Pomegranates were one of the first domesticated fruit crops, and their long history resulted in the development of local cultivars all over the world. Spain is one of the main producers and exporters of this crop in the Mediterranean Basin, but in order to maintain the competitiveness of this crop, new varieties should be developed. For this purpose, the pomegranate germplasm collection hold at the Agricultural Experiment Station of Elche, a public institution dependent on the Valencian regional government, is an interesting tool. However, the detailed characterization of any germplasm collection is a fundamental requirement to be able to make the most of these resources, allowing to identify putative promising accessions and to optimize the design of the future crosses. In this work, the genetic diversity of 94 accessions of this collection was analyzed using 19 microsatellite markers. As a result, 85 different genotypes were identified. These genetic profiles could be useful for varietal identification. Despite this genetic diversity, no clear substructure was observed, except for the ornamental accessions, that could be related to the vegetative propagation of the species. Additionally, the morphological characterization of this collection has made it possible to identify some materials that may be of interest as a source of traits for breeding. Results presented here pave the way for further genetic analyses, allowing the selection of parents to obtain segregating populations, as well as their descendants by the use of molecular assisted selection.
- Published
- 2022
- Full Text
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24. Regulatory circuits involving bud dormancy factor PpeDAM6.
- Author
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Lloret A, Quesada-Traver C, Conejero A, Arbona V, Gómez-Mena C, Petri C, Sánchez-Navarro JA, Zuriaga E, Leida C, Badenes ML, and Ríos G
- Abstract
DORMANCY-ASSOCIATED MADS-BOX (DAM) genes have recently emerged as key potential regulators of the dormancy cycle and climate adaptation in perennial species. Particularly, PpeDAM6 has been proposed to act as a major repressor of bud dormancy release and bud break in peach (Prunus persica). PpeDAM6 expression is downregulated concomitantly with the perception of a given genotype-dependent accumulation of winter chilling time, and the coincident enrichment in H3K27me3 chromatin modification at a specific genomic region. We have identified three peach BASIC PENTACYSTEINE PROTEINs (PpeBPCs) interacting with two GA-repeat motifs present in this H3K27me3-enriched region. Moreover, PpeBPC1 represses PpeDAM6 promoter activity by transient expression experiments. On the other hand, the heterologous overexpression of PpeDAM6 in European plum (Prunus domestica) alters plant vegetative growth, resulting in dwarf plants tending toward shoot meristem collapse. These alterations in vegetative growth of transgenic lines associate with impaired hormone homeostasis due to the modulation of genes involved in jasmonic acid, cytokinin, abscisic acid, and gibberellin pathways, and the downregulation of shoot meristem factors, specifically in transgenic leaf and apical tissues. The expression of many of these genes is also modified in flower buds of peach concomitantly with PpeDAM6 downregulation, which suggests a role of hormone homeostasis mechanisms in PpeDAM6-dependent maintenance of floral bud dormancy and growth repression., (© 2021. The Author(s).)
- Published
- 2021
- Full Text
- View/download PDF
25. Insights of Phenolic Pathway in Fruits: Transcriptional and Metabolic Profiling in Apricot ( Prunus armeniaca ).
- Author
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Gómez-Martínez H, Gil-Muñoz F, Bermejo A, Zuriaga E, and Badenes ML
- Subjects
- Alcohol Oxidoreductases genetics, Alcohol Oxidoreductases metabolism, Oxidoreductases genetics, Oxidoreductases metabolism, Phenylalanine Ammonia-Lyase genetics, Phenylalanine Ammonia-Lyase metabolism, Plant Proteins genetics, Plant Proteins metabolism, Polyphenols genetics, Prunus genetics, Metabolome, Polyphenols biosynthesis, Prunus metabolism, Transcriptome
- Abstract
There is an increasing interest in polyphenols, plant secondary metabolites, in terms of fruit quality and diet, mainly due to their antioxidant effect. However, the identification of key gene enzymes and their roles in the phenylpropanoid pathway in temperate fruits species remains uncertain. Apricot ( Prunus armeniaca ) is a Mediterranean fruit with high diversity and fruit quality properties, being an excellent source of polyphenol compounds. For a better understanding of the phenolic pathway in these fruits, we selected a set of accessions with genetic-based differences in phenolic compounds accumulation. HPLC analysis of the main phenolic compounds and transcriptional analysis of the genes involved in key steps of the polyphenol network were carried out. Phenylalanine ammonia-lyase (PAL), dihydroflavonol-4-reductase (DFR) and flavonol synthase (FLS) were the key enzymes selected. Orthologous of the genes involved in transcription of these enzymes were identified in apricot: ParPAL1 , ParPAL2 , ParDFR , ParFLS1 and ParFLS2 . Transcriptional data of the genes involved in those critical points and their relationships with the polyphenol compounds were analyzed. Higher expression of ParDFR and ParPAL2 has been associated with red-blushed accessions. Differences in expression between paralogues could be related to the presence of a BOXCOREDCPAL cis-acting element related to the genes involved in anthocyanin synthesis ParFLS2 , ParDFR and ParPAL2 .
- Published
- 2021
- Full Text
- View/download PDF
26. Self-(In)compatibility Systems: Target Traits for Crop-Production, Plant Breeding, and Biotechnology.
- Author
-
Muñoz-Sanz JV, Zuriaga E, Cruz-García F, McClure B, and Romero C
- Abstract
Self-incompatibility (SI) mechanisms prevent self-fertilization in flowering plants based on specific discrimination between self- and non-self pollen. Since this trait promotes outcrossing and avoids inbreeding it is a widespread mechanism of controlling sexual plant reproduction. Growers and breeders have effectively exploited SI as a tool for manipulating domesticated crops for thousands of years. However, only within the past thirty years have studies begun to elucidate the underlying molecular features of SI. The specific S -determinants and some modifier factors controlling SI have been identified in the sporophytic system exhibited by Brassica species and in the two very distinct gametophytic systems present in Papaveraceae on one side and in Solanaceae, Rosaceae, and Plantaginaceae on the other. Molecular level studies have enabled SI to SC transitions (and vice versa ) to be intentionally manipulated using marker assisted breeding and targeted approaches based on transgene integration, silencing, and more recently CRISPR knock-out of SI-related factors. These scientific advances have, in turn, provided a solid basis to implement new crop production and plant breeding practices. Applications of self-(in)compatibility include widely differing objectives such as crop yield and quality improvement, marker-assisted breeding through SI genotyping, and development of hybrids for overcoming intra- and interspecific reproductive barriers. Here, we review scientific progress as well as patented applications of SI, and also highlight future prospects including further elucidation of SI systems, deepening our understanding of SI-environment relationships, and new perspectives on plant self/non-self recognition., (Copyright © 2020 Muñoz-Sanz, Zuriaga, Cruz-García, McClure and Romero.)
- Published
- 2020
- Full Text
- View/download PDF
27. QSAR modelling for predicting the toxic effects of traditional and derived biomass solvents on a Danio rerio biomodel.
- Author
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Zuriaga E, Giner B, Valero MS, Gómez M, García CB, and Lomba L
- Subjects
- Animals, Lethal Dose 50, Regression Analysis, Solvents chemistry, Zebrafish, Biomass, Ecotoxicology, Green Chemistry Technology methods, Quantitative Structure-Activity Relationship, Solvents toxicity
- Abstract
The increasing interest in the development of ecofriendly solvents has led to the synthesis of benign alternative chemicals with minimized environmental impacts. These kinds of chemicals are known as Green solvents. In this work, we selected three families of solvents (furfural, lactate and levulinate families) derived from biomass that are structurally related. Most of the previous ecotoxicological studies of these solvents have focused on invertebrate models such as bacteria, algae and crustaceans. To complete this information, in this work, the acute toxicity of these solvents was studied in Danio rerio (D. rerio). Sublethal and lethal effects were also observed, and the LC
50 was obtained. The LC50 values ranged from 13.21 to 12073 mg L-1 , with furfural being the most toxic compound and tetrahydrofurfuryl alcohol the least toxic. Furthermore, the results indicated that a frequent sublethal effect was heart oedema or malformation, even in some cases at concentrations lower than the LC50 . A QSAR analysis was also performed to model the toxicological effect towards D. rerio for the studied solvents obtained from biomass and traditional solvents. A total of 15 molecular descriptors of the solvents were obtained using Gaussian 03 software. Finally, we also used the physicochemical property Log P, calculated from ACD/LogP, for QSAR modelling. Multivariable regression analysis showed that the minimum set of independent variables that leads to the best regression is Log P, the energy of the lowest unoccupied molecular orbital (ELUMO) and the heat capacity (CV). The proposed model was validated using several internal and external methods., (Copyright © 2019. Published by Elsevier Ltd.)- Published
- 2019
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28. Acute and subacute effects of drugs in embryos of Danio rerio. QSAR grouping and modelling.
- Author
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Lomba L, Ribate MP, Zuriaga E, García CB, and Giner B
- Subjects
- 1-Octanol chemistry, Animals, Drug-Related Side Effects and Adverse Reactions, Pharmaceutical Preparations chemistry, Quantitative Structure-Activity Relationship, Toxicity Tests, Water chemistry, Embryo, Nonmammalian drug effects, Models, Theoretical, Water Pollutants, Chemical chemistry, Water Pollutants, Chemical toxicity, Zebrafish embryology
- Abstract
The final fate of many drugs is release into the natural aquatic environment. It is necessary to assess the toxicity caused by this situation and the associated concerns for human beings. Zebrafish (Danio rerio) is a common biomodel used to assess toxicity in aquatic environments. The zebrafish embryo toxicity test was selected to evaluate the acute toxicity of several drugs (diphenhydramine, gentamicin, tobramycin, enalapril and lidocaine) due to the lack of such information. Lethal and sublethal effects were detected, and the LC
50 values of the drugs ranged from 11.0 mg/L to 422·102 mg/L. For all of the drugs tested, these values were higher than the concentrations found in the natural environment. Therefore, there was a low environmental toxicological risk. Nevertheless, teratogenic effects were also recorded when embryos of zebrafish were exposed to caffeine (control drug), diphenhydramine and lidocaine at lower concentrations than the respective LC50 values. Quantitative structure-activity relationship analysis was also performed to analyse these drugs and other chemicals with pharmaceutical uses as well as previous toxicological data in this vertebrate after 48 h of exposure. It is estimated that the partition coefficient, log P, is the main physicochemical property related to the ecotoxicological data and can be used for the development of a mathematical model., (Copyright © 2019 Elsevier Inc. All rights reserved.)- Published
- 2019
- Full Text
- View/download PDF
29. Exploring the usefulness of key green physicochemical properties: Quantitative structure-activity relationship for solvents from biomass.
- Author
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Zuriaga E, Giner B, Ribate MP, García CB, and Lomba L
- Subjects
- Aliivibrio fischeri physiology, Animals, Aquatic Organisms metabolism, Daphnia metabolism, Ecotoxicology, Biomass, Chemical Phenomena, Quantitative Structure-Activity Relationship, Solvents chemistry
- Abstract
In recent decades there has been a growing interest in the development of new solvents from biomass. Some of these new solvents have been classified as green because of their renewable and sustainable source. However, characterization from the ecotoxicological and physicochemical points of view is needed to categorize them as green solvents. We have selected several key physicochemical properties that can reflect environmental features (density, boiling point, critical aggregation concentration, and log p) and explored their usefulness for preliminarily assessing the green character of the studied solvents. Specifically, we have studied several solvents from biomass: lactate family (methyl, ethyl, and butyl lactate), furfural family (furfural, 5-methylfurfural, furfuryl alcohol, and tetrahydrofurfuryl alcohol), and levulinate family (methyl, ethyl, and butyl levulinate). To fill the gaps and complete some toxicity data for the environment, we have measured the ecotoxicity using 2 of the most common and versatile biomodels, bacteria Vibrio fischeri and crustacean Daphnia magna, for furfural- and lactate-derived compounds. The results indicate that solvents from biomass can be categorized as green because their toxicity for the environment is low. Finally, a quantitative structure-activity relationship (QSAR) study was performed with the selected key properties and the ecotoxicological information. Despite the different structure of the chemicals under study, good correlations were found for the studied organisms. It seems that log p and critical aggregation concentration reflect the greatest part of the ecotoxic behavior, whereas density and boiling point cannot reflect toxicity signals. However, these properties are rather useful for assessing the final environmental fate of the studied chemicals. Environ Toxicol Chem 2018;37:1014-1023. © 2017 SETAC., (© 2017 SETAC.)
- Published
- 2018
- Full Text
- View/download PDF
30. Resistance to Plum Pox Virus (PPV) in apricot (Prunus armeniaca L.) is associated with down-regulation of two MATHd genes.
- Author
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Zuriaga E, Romero C, Blanca JM, and Badenes ML
- Subjects
- Genomics, Plant Proteins metabolism, Prunus armeniaca metabolism, Prunus armeniaca virology, Disease Resistance, Down-Regulation, Plant Diseases virology, Plant Proteins genetics, Plum Pox Virus physiology, Prunus armeniaca genetics, Transcriptome
- Abstract
Background: Plum pox virus (PPV), causing Sharka disease, is one of the main limiting factors for Prunus production worldwide. In apricot (Prunus armeniaca L.) the major PPV resistance locus (PPVres), comprising ~ 196 kb, has been mapped to the upper part of linkage group 1. Within the PPVres, 68 genomic variants linked in coupling to PPV resistance were identified within 23 predicted transcripts according to peach genome annotation. Taking into account the predicted functions inferred from sequence homology, some members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing genes were pointed as PPV resistance candidate genes., Results: Here, we have characterized the global apricot transcriptome response to PPV-D infection identifying six PPVres locus genes (ParP-1 to ParP-6) differentially expressed in resistant/susceptible cultivars. Two of them (ParP-3 and ParP-4), that encode MATHd proteins, appear clearly down-regulated in resistant cultivars, as confirmed by qRT-PCR. Concurrently, variant calling was performed using whole-genome sequencing data of 24 apricot cultivars (10 PPV-resistant and 14 PPV-susceptible) and 2 wild relatives (PPV-susceptible). ParP-3 and ParP-4, named as Prunus armeniaca PPVres MATHd-containing genes (ParPMC), are the only 2 genes having allelic variants linked in coupling to PPV resistance. ParPMC1 has 1 nsSNP, while ParPMC2 has 15 variants, including a 5-bp deletion within the second exon that produces a frameshift mutation. ParPMC1 and ParPMC2 are adjacent and highly homologous (87.5% identity) suggesting they are paralogs originated from a tandem duplication. Cultivars carrying the ParPMC2 resistant (mutated) allele show lack of expression in both ParPMC2 and especially ParPMC1., Conclusions: Accordingly, we hypothesize that ParPMC2 is a pseudogene that mediates down-regulation of its functional paralog ParPMC1 by silencing. As a whole, results strongly support ParPMC1 and/or ParPMC2 as host susceptibility genes required for PPV infection which silencing may confer PPV resistance trait. This finding may facilitate resistance breeding by marker-assisted selection and pave the way for gene edition approaches in Prunus.
- Published
- 2018
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31. A disulfide bond A-like oxidoreductase is a strong candidate gene for self-incompatibility in apricot (Prunus armeniaca) pollen.
- Author
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Muñoz-Sanz JV, Zuriaga E, Badenes ML, and Romero C
- Subjects
- Disulfides, Genetic Loci genetics, Genotype, High-Throughput Nucleotide Sequencing, Loss of Function Mutation, Oxidoreductases genetics, Plant Proteins genetics, Plant Proteins metabolism, Pollen genetics, Pollen physiology, Prunus armeniaca genetics, Prunus armeniaca physiology, Sequence Analysis, DNA, Oxidoreductases metabolism, Pollen enzymology, Prunus armeniaca enzymology, Self-Incompatibility in Flowering Plants genetics
- Abstract
S-RNase based gametophytic self-incompatibility (SI) is a widespread prezygotic reproductive barrier in flowering plants. In the Solanaceae, Plantaginaceae and Rosaceae gametophytic SI is controlled by the pistil-specific S-RNases and the pollen S-locus F-box proteins but non-S-specific factors, namely modifiers, are also required. In apricot, Prunus armeniaca (Rosaceae), we previously mapped two pollen-part mutations that confer self-compatibility in cultivars Canino and Katy at the distal end of chromosome 3 (M-locus) unlinked to the S-locus. Here, we used high-resolution mapping to identify the M-locus with an ~134 kb segment containing ParM-1-16 genes. Gene expression analysis identified four genes preferentially expressed in anthers as modifier gene candidates, ParM-6, -7, -9 and -14. Variant calling of WGS Illumina data from Canino, Katy, and 10 self-incompatible cultivars detected a 358 bp miniature inverted-repeat transposable element (MITE) insertion in ParM-7 shared only by self-compatible apricots, supporting ParM-7 as strong candidate gene required for SI. ParM-7 encodes a disulfide bond A-like oxidoreductase protein, which we named ParMDO. The MITE insertion truncates the ParMDO ORF and produces a loss of SI function, suggesting that pollen rejection in Prunus is dependent on redox regulation. Based on phylogentic analyses we also suggest that ParMDO may have originated from a tandem duplication followed by subfunctionalization and pollen-specific expression., (© The Author 2017. Published by Oxford University Press on behalf of the Society for Experimental Biology.)
- Published
- 2017
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32. Self-(in)compatibility in apricot germplasm is controlled by two major loci, S and M.
- Author
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Muñoz-Sanz JV, Zuriaga E, López I, Badenes ML, and Romero C
- Subjects
- Genotype, Mutation, Phylogeography, Pollen genetics, Prunus armeniaca physiology, Prunus armeniaca genetics, Self-Incompatibility in Flowering Plants genetics
- Abstract
Background: Apricot (Prunus armeniaca L.) exhibits a gametophytic self-incompatibility (GSI) system and it is mostly considered as a self-incompatible species though numerous self-compatible exceptions occur. These are mainly linked to the mutated S
C -haplotype carrying an insertion in the S-locus F-box gene that leads to a truncated protein. However, two S-locus unlinked pollen-part mutations (PPMs) termed m and m' have also been reported to confer self-compatibility (SC) in the apricot cultivars 'Canino' and 'Katy', respectively. This work was aimed to explore whether other additional mutations might explain SC in apricot as well., Results: A set of 67 cultivars/accessions with different geographic origins were analyzed by PCR-screening of the S- and M-loci genotypes, contrasting results with the available phenotype data. Up to 20 S-alleles, including 3 new ones, were detected and sequence analysis revealed interesting synonymies and homonymies in particular with S-alleles found in Chinese cultivars. Haplotype analysis performed by genotyping and determining linkage-phases of 7 SSR markers, showed that the m and m' PPMs are linked to the same m0- haplotype. Results indicate that m0 -haplotype is tightly associated with SC in apricot germplasm being quite frequent in Europe and North-America. However, its prevalence is lower than that for SC in terms of frequency and geographic distribution. Structures of 34 additional M-haplotypes were inferred and analyzed to depict phylogenetic relationships and M1-2 was found to be the closest haplotype to m0. Genotyping results showed that four cultivars classified as self-compatible do not have neither the SC - nor the m0 -haplotype., Conclusions: According to apricot germplasm S-genotyping, a loss of genetic diversity affecting the S-locus has been produced probably due to crop dissemination. Genotyping and phenotyping data support that self-(in)compatibility in apricot relies mainly on the S- but also on the M-locus. Regarding this latter, we have shown that the m0 -haplotype associated with SC is shared by 'Canino', 'Katy' and many other cultivars. Its origin is still unknown but phylogenetic analysis supports that m0 arose later in time than SC from a widely distributed M-haplotype. Lastly, other mutants putatively carrying new mutations conferring SC have also been identified deserving future research.- Published
- 2017
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33. Can pharmacogenetics help patients under chronic treatment with coumarin anticoagulants?
- Author
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López I, Sangüesa E, Vancraenendonck Y, Zuriaga E, Ribate MP, and García CB
- Subjects
- Anticoagulants administration & dosage, Coumarins administration & dosage, Dose-Response Relationship, Drug, Genotype, Humans, Anticoagulants therapeutic use, Coumarins therapeutic use, Pharmacogenetics, Vitamin K antagonists & inhibitors
- Abstract
Vitamin K antagonists are highly effective antithrombotic drugs. However, appropriate dosing is difficult to establish owing to its narrow therapeutic window as well as widespread inter- and intra-individual variability in dosage. Compared with dosing solely based on clinical information, pharmacogenetics can help improve the therapy with coumarins by decreasing the time to reach a stable dose and reducing the risk of bleeding. Most of the studies about genotyping of patients using vitamin K antagonists have focused on predicting the stable dose. Two genes have been shown to have the most influence on dosing: VKORC1 and CYP2C9. Furthermore, genotyping of more genes, such as CYP4F2 and APOE, is also being included in some dosing algorithms. The role of genotype beyond the initial dose-titration phase is less clear. Thus, a proven genetically determined risk of unstable dose or bleeding could help with the selection of patients who require more frequent monitoring of dose. On the other hand, patients who have a genetically determined stable dose could self-monitor their international normalized ratio (INR), making the therapy less expensive and more convenient.
- Published
- 2016
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34. Acute toxicological effects on the earthworm Eisenia fetida of 18 common pharmaceuticals in artificial soil.
- Author
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Pino MR, Val J, Mainar AM, Zuriaga E, Español C, and Langa E
- Subjects
- Animals, Oligochaeta, Pharmaceutical Preparations chemistry, Soil chemistry, Soil Pollutants toxicity, Toxicity Tests, Acute
- Abstract
Following soil applications of recycled water and biosolids, pharmaceutical residues can eventually enter the terrestrial environment. In vitro and in vivo assays have largely focused on the acute ecotoxicity of these compounds in aquatic systems. However, studies on the ecotoxicological effects of pharmaceuticals in soil biota are especially scarce. The aim of this study was to investigate the acute toxicity of 18 pharmaceuticals (4 NSAIDs, 5 blood lipid-lowering agents, 6 β-blockers and 3 antibiotics) that are usually found in the environment by using an Eisenia fetida bioassay. In addition, the presence of these pharmaceuticals in artificial soil was verified at the end of the test. Our results indicate that seven of the studied drugs cause acute adverse effects in E. fetida, in particular, the NSAIDs and the blood lipid-lowering agents. Ibuprofen (LC50=64.80 mg/kg) caused the highest acute toxicity for all tested compounds, followed by diclofenac (LC50=90.49 mg/kg) and simvastatin (LC50=92.70 mg/kg). Other tested pharmaceuticals from NSAIDs and blood lipid-lowering families have toxicity effects, from a LC50=140.87 mg/kg for gemfibrozil to 795.07 mg/kg for lovastatin. Atorvastatin, bezafibrate, β-blockers and antibiotics showed no detectable lethality in E. fetida. The four NSAIDs showed evidence of modification of their original chemical structure after 14 days so the detected toxicity may be due to the original product as well as their degradation products. The three blood lipid-lowering agents seem to be more stable in soil. From an environmental perspective, the lethal concentrations of the tested drugs are much greater than those reported in wastewater and biosolids, therefore acute toxic effects may be improbable. However, little is known about the accumulation of these substances in soils after regular applications, so accumulative and chronic effects cannot be excluded. Moreover, more studies are needed to determine the role of the degradation products of these pharmaceuticals on terrestrial toxicity., (Copyright © 2015 Elsevier B.V. All rights reserved.)
- Published
- 2015
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35. Genomic analysis reveals MATH gene(s) as candidate(s) for Plum pox virus (PPV) resistance in apricot (Prunus armeniaca L.).
- Author
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Zuriaga E, Soriano JM, Zhebentyayeva T, Romero C, Dardick C, Cañizares J, and Badenes ML
- Subjects
- Arabidopsis genetics, Chromosomes, Artificial, Bacterial genetics, Genetic Association Studies, Genetic Loci genetics, Genotype, Hybridization, Genetic, Plant Diseases genetics, Polymorphism, Single Nucleotide genetics, Recombination, Genetic genetics, Sequence Analysis, DNA, Synteny genetics, Disease Resistance genetics, Genes, Plant genetics, Genomics, Plant Diseases virology, Plum Pox Virus physiology, Prunus genetics, Prunus virology
- Abstract
Sharka disease, caused by Plum pox virus (PPV), is the most important viral disease affecting Prunus species. A major PPV resistance locus (PPVres) has been mapped to the upper part of apricot (Prunus armeniaca) linkage group 1. In this study, a physical map of the PPVres locus in the PPV-resistant cultivar 'Goldrich' was constructed. Bacterial artificial chromosome (BAC) clones belonging to the resistant haplotype contig were sequenced using 454/GS-FLX Titanium technology. Concurrently, the whole genome of seven apricot varieties (three PPV-resistant and four PPV-susceptible) and two PPV-susceptible apricot relatives (P. sibirica var. davidiana and P. mume) were obtained using the Illumina-HiSeq2000 platform. Single nucleotide polymorphisms (SNPs) within the mapped interval, recorded from alignments against the peach genome, allowed us to narrow down the PPVres locus to a region of ∼196 kb. Searches for polymorphisms linked in coupling with the resistance led to the identification of 68 variants within 23 predicted transcripts according to peach genome annotation. Candidate resistance genes were ranked combining data from variant calling and predicted functions inferred from sequence homology. Together, the results suggest that members of a cluster of meprin and TRAF-C homology domain (MATHd)-containing proteins are the most likely candidate genes for PPV resistance in apricot. Interestingly, MATHd proteins are hypothesized to control long-distance movement (LDM) of potyviruses in Arabidopsis, and restriction for LDM is also a major component of PPV resistance in apricot. Although the PPV resistance gene(s) remains to be unambiguously identified, these results pave the way to the determination of the underlying mechanism and to the development of more accurate breeding strategies., (© 2013 BSPP AND JOHN WILEY & SONS LTD.)
- Published
- 2013
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36. An S-locus independent pollen factor confers self-compatibility in 'Katy' apricot.
- Author
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Zuriaga E, Muñoz-Sanz JV, Molina L, Gisbert AD, Badenes ML, and Romero C
- Subjects
- Chromosome Mapping, Genetic Linkage, Genotype, Mutation, Open Reading Frames genetics, Synteny, Genetic Loci genetics, Pollen genetics, Pollen physiology, Prunus genetics, Prunus physiology, Self-Incompatibility in Flowering Plants genetics
- Abstract
Loss of pollen-S function in Prunus self-compatible cultivars has been mostly associated with deletions or insertions in the S-haplotype-specific F-box (SFB) genes. However, self-compatible pollen-part mutants defective for non-S-locus factors have also been found, for instance, in the apricot (Prunus armeniaca) cv. 'Canino'. In the present study, we report the genetic and molecular analysis of another self-compatible apricot cv. termed 'Katy'. S-genotype of 'Katy' was determined as S(1)S(2) and S-RNase PCR-typing of selfing and outcrossing populations from 'Katy' showed that pollen gametes bearing either the S(1)- or the S(2)-haplotype were able to overcome self-incompatibility (SI) barriers. Sequence analyses showed no SNP or indel affecting the SFB(1) and SFB(2) alleles from 'Katy' and, moreover, no evidence of pollen-S duplication was found. As a whole, the obtained results are compatible with the hypothesis that the loss-of-function of a S-locus unlinked factor gametophytically expressed in pollen (M'-locus) leads to SI breakdown in 'Katy'. A mapping strategy based on segregation distortion loci mapped the M'-locus within an interval of 9.4 cM at the distal end of chr.3 corresponding to ∼1.29 Mb in the peach (Prunus persica) genome. Interestingly, pollen-part mutations (PPMs) causing self-compatibility (SC) in the apricot cvs. 'Canino' and 'Katy' are located within an overlapping region of ∼273 Kb in chr.3. No evidence is yet available to discern if they affect the same gene or not, but molecular markers seem to indicate that both cultivars are genetically unrelated suggesting that every PPM may have arisen independently. Further research will be necessary to reveal the precise nature of 'Katy' PPM, but fine-mapping already enables SC marker-assisted selection and paves the way for future positional cloning of the underlying gene.
- Published
- 2013
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37. Physical mapping of a pollen modifier locus controlling self-incompatibility in apricot and synteny analysis within the Rosaceae.
- Author
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Zuriaga E, Molina L, Badenes ML, and Romero C
- Subjects
- Chromosomes, Artificial, Bacterial, Fragaria genetics, Genotype, Malus genetics, Plant Proteins genetics, Pollen genetics, Pollen physiology, Prunus genetics, Prunus physiology, Rosaceae genetics, Rosaceae physiology, Synteny genetics
- Abstract
S-locus products (S-RNase and F-box proteins) are essential for the gametophytic self-incompatibility (GSI) specific recognition in Prunus. However, accumulated genetic evidence suggests that other S-locus unlinked factors are also required for GSI. For instance, GSI breakdown was associated with a pollen-part mutation unlinked to the S-locus in the apricot (Prunus armeniaca L.) cv. 'Canino'. Fine-mapping of this mutated modifier gene (M-locus) and the synteny analysis of the M-locus within the Rosaceae are here reported. A segregation distortion loci mapping strategy, based on a selectively genotyped population, was used to map the M-locus. In addition, a bacterial artificial chromosome (BAC) contig was constructed for this region using overlapping oligonucleotides probes, and BAC-end sequences (BES) were blasted against Rosaceae genomes to perform micro-synteny analysis. The M-locus was mapped to the distal part of chr.3 flanked by two SSR markers within an interval of 1.8 cM corresponding to ~364 Kb in the peach (Prunus persica L. Batsch) genome. In the integrated genetic-physical map of this region, BES were mapped against the peach scaffold_3 and BACs were anchored to the apricot map. Micro-syntenic blocks were detected in apple (Malus × domestica Borkh.) LG17/9 and strawberry (Fragaria vesca L.) FG6 chromosomes. The M-locus fine-scale mapping provides a solid basis for self-compatibility marker-assisted selection and for positional cloning of the underlying gene, a necessary goal to elucidate the pollen rejection mechanism in Prunus. In a wider context, the syntenic regions identified in peach, apple and strawberry might be useful to interpret GSI evolution in Rosaceae.
- Published
- 2012
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38. Narrowing down the apricot Plum pox virus resistance locus and comparative analysis with the peach genome syntenic region.
- Author
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Vera Ruiz EM, Soriano JM, Romero C, Zhebentyayeva T, Terol J, Zuriaga E, Llácer G, Abbott AG, and Badenes ML
- Subjects
- Chromosome Mapping, Chromosome Segregation genetics, Chromosomes, Artificial, Bacterial genetics, Contig Mapping, Genes, Plant genetics, Genetic Association Studies, Genetic Loci genetics, Genetic Markers, Genotype, Microsatellite Repeats genetics, Plant Diseases genetics, Plant Diseases virology, Prunus immunology, Disease Resistance genetics, Genome, Plant genetics, Plant Diseases immunology, Plum Pox Virus immunology, Prunus genetics, Prunus virology, Synteny genetics
- Abstract
Sharka disease, caused by the Plum pox virus (PPV), is one of the main limiting factors for stone fruit crops worldwide. Only a few resistance sources have been found in apricot (Prunus armeniaca L.), and most studies have located a major PPV resistance locus (PPVres) on linkage group 1 (LG1). However, the mapping accuracy was not sufficiently reliable and PPVres was predicted within a low confidence interval. In this study, we have constructed two high-density simple sequence repeat (SSR) improved maps with 0.70 and 0.68 markers/cm, corresponding to LG1 of 'Lito' and 'Goldrich' PPV-resistant cultivars, respectively. Using these maps, and excluding genotype-phenotype incongruent individuals, a new binary trait locus (BTL) analysis for PPV resistance was performed, narrowing down the PPVres support intervals to 7.3 and 5.9 cm in 'Lito' and 'Goldrich', respectively. Subsequently, 71 overlapping oligonucleotides (overgo) probes were hybridized against an apricot bacterial artificial chromosome (BAC) library, identifying 870 single BACs from which 340 were anchored onto a map region of approximately 30-40 cm encompassing PPVres. Partial BAC contigs assigned to the two allelic haplotypes (resistant/susceptible) of the PPVres locus were built by high-information content fingerprinting (HICF). In addition, a total of 300 BAC-derived sequences were obtained, and 257 showed significant homology with the peach genome scaffold_1 corresponding to LG1. According to the peach syntenic genome sequence, PPVres was predicted within a region of 2.16 Mb in which a few candidate resistance genes were identified., (© 2011 The Authors. Molecular Plant Pathology © 2011 BSPP and Blackwell Publishing Ltd.)
- Published
- 2011
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39. AFLP and DNA sequence variation in an Andean domesticate, pepino (Solanum muricatum, Solanaceae): implications for evolution and domestication.
- Author
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Blanca JM, Prohens J, Anderson GJ, Zuriaga E, Cañizares J, and Nuez F
- Abstract
The pepino (Solanum muricatum) is a vegetatively propagated, domesticated native of the Andes, where it grows with wild relatives. We used AFLPs and a 1-kb sequence of the 3-methylcrotonyl-CoA carboxylase gene to study variation of 27 accessions of S. muricatum and 35 collections of 10 species of wild relatives (Solanum section Basarthrum). A total of 298 AFLP fragments and 29 DNA sequence haplotypes were detected. Cluster and principal coordinate analyses and other genetic parameters estimated from both types of markers, show that S. muricatum is closely related to the species from one of the series (Caripensia) of section Basarthrum and that >90% of the variation of the cultigen is also represented in that series. Pepino is highly diverse, either because it is not monophyletic or it has been subjected to regular introgression with wild species, or both. Although a continuous distribution of the genetic variation occurred within the cultivated species, three genetic clusters were recognized. Cluster 1 is mostly centered in Ecuador, cluster 2 in Ecuador and Peru, and cluster 3 in Colombia and Ecuador. Cluster 3 also includes all modern cultivars studied. These results and other evidence suggest that northern Ecuador/southern Colombia is the main center of pepino diversity and the center of origin. The high genetic variation of this cultigen indicates that domestication does not always produce a genetic bottleneck.
- Published
- 2007
- Full Text
- View/download PDF
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