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Effect of Oxytocin Treatment on Artificial Insemination with Frozen-Thawed Semen in Murciano-Granadina Goats

  • Viudes-De-Castro, María P.
  • Salvador, I.
  • Marco-Jiménez, Francisco
  • Gómez, Ernesto A.
  • Silvestre, Miguel A.
The site where the semen is deposited appears to be one of the most important factors affecting pregnancy of inseminated goats. In Murciano-Granadina (MG) goats, post-cervical insemination is achieved in a limited number of females. An effective way to increase fertility rate could be by increasing post-cervical inseminations. Effect of exogenous oxytocin application to facilitate the cervical penetration and its effect on kidding rate and prolificacy in MG goats were investigated. Oestrus was synchronized using progesterone-impregnated sponges for 11 days. Females were randomly divided into three groups (n = 190) and received either an i.v. injection of 100 or 200 IU of oxytocin or saline solution 15 min before being inseminated. Data on semen deposition depth were recorded for each animal using a catheter scaled in centimetres (up to 4 cm). Depth of semen deposition was affected by the oxytocin treatment (p < 0.05). Oxytocin enhanced cervical passage only with the dose of 200 IU compared with the control group, increasing the deposition depth (2.9 cm vs 1.9 cm). No significant effect of oxytocin treatment on kidding rate and prolificacy was detected. Depth of semen deposition affected kidding rate (p < 0.01). In conclusion, oxytocin treatment improved the depth of semen deposition in AI of MG goats, but kidding rate and prolificacy was not affected. More studies must be conducted to assess the minimal effective dose required for sufficient cervical dilation, and to determine the effects of such doses of oxytocin on uterine motility, sperm transport and fertility in goats.
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The Complete Genome Sequence of Citrus Vein Enation Virus (CVEV) Obtained through Deep Sequencing of Small RNAs, Acta Horticulturae

  • Vives, María C.
  • Velázquez, Karelia
  • Pina, José A.
  • Moreno, Pedro
  • Guerri, José
  • Navarro, Luis
Citrus vein enation (VE), a graft-transmissible disease naturally spread by several aphid species in a persistent mode, has been reported in many citrus growing areas. It causes vein enations on leaves and woody galls on trunk and branches of sensitive citrus species such as Mexican lime, rough lemon and Citrus volkameriana. The disease is currently diagnosed by biological indexing on sensitive indicator plants, an expensive and time-consuming method. In order to identify its causal agent and develop specific and reliable molecular detection methods, we analyzed small RNAs (sRNAs) from VE-infected Etrog citron plants by deep sequencing using the Illumina Solexa platform. Assembly of VE-associated sRNAs yielded several contigs that showed sequence homology with Pea enation mosaic virus 1 (PEMV-1), the type species of genus Enamovirus, family Luteoviridae. The gaps between adjacent contigs were filled by RT-PCR amplification, cloning and sequencing in order to obtain the complete genome sequence of a new virus, Citrus vein enation virus (CVEV). The CVEV genomic RNA has 5,983 nt organized in five open reading frames, resembling that of PEMV-1. Phylogenetic comparison of amino acid signatures in RNA-dependent RNA polymerases of the family Luteoviridae clearly grouped CVEV with PEMV-1. Therefore, we propose that CVEV should be included in the genus Enamovirus. A rapid and specific detection procedure was developed based on RT-PCR with CVEV-specific primers.
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Development of a full-genome cDNA clone of Citrus leaf blotch virus and infection of citrus plants

  • Vives, María C.
  • Martin, Susana
  • Ambrós, Silvia
  • Renovell, Agueda
  • Navarro, Luis
  • Pina, José A.
  • Moreno, Pedro
  • Guerri, José
Citrus leaf blotch virus (CLBV), a member of the family Flexiviridae, has a similar to 9-kb single-stranded, positive-sense genomic RNA encapsidated by a 41-kDa coat protein. CLBV isolates are associated with symptom production in citrus including leaf blotching of Dweet tangor and stem pitting in Etrog citron (Dweet mottle disease), and some isolates are associated with bud union crease on trifoliate rootstocks, but Koch's postulates for this virus were not fulfilled. A full-genome cDNA of CLBV isolate SRA-153, which induces bud union crease, was placed under the T7 promoter (clone T7-CLBV), or between the 35S promoter and the Nos-t terminator, with or without a ribozyme sequence downstream of the CLBV sequence (clones 35SRbz-CLBV and 35S-CLBV). RNA transcripts from T7-CLBV failed to infect Etrog citron and Nicotiana occidentalis and N. benthamiana plants, whereas agro-inoculation with binary vectors carrying 35SRbz-CLBV or 35S-CLBV, and the p19 silencing suppressor, caused systemic infection and production of normal CLBV virions. Virus accumulation was similar in citron plants directly agro-infiltrated, or mechanically inoculated with wild-type or 35SRbz-CLBV-derived virions from Nicotiana, and the three sources incited the symptoms characteristic of Dweet mottle disease, but not bud union crease. Our results show that (1) virions derived from an infectious clone show the same replication, movement and pathogenicity characteristics as the wild-type CLBV; (2) CLBV is the causal agent of Dweet mottle disease but not of the bud union crease syndrome; and (3) for the first time an RNA virus could be successfully agro-inoculated on citrus plants. This infectious clone may become a useful viral vector for citrus genomic studies.
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Characterization of two kinds of subgenomic RNAs produced by citrus leaf blotch virus

  • Vives, María C.
  • Galipienso, Luis
  • Navarro, Luis
  • Moreno, Pedro
  • Guerri, José
Citrus leaf blotch virus (CLBV) has a single-stranded, positive-sense, genomic RNA (gRNA) organized in three ORFs, which encode a polyprotein involved in replication (RID), a potential movement protein (MP), and coat protein (CP). Northern blot hybridization of total, virion, or double-stranded RNA with probes of different gRNA regions revealed that CLBV produces two 3'-coterminal and two 5'-coterminal subgenomic RNAs (sgRNAs). The 3'-coterminal sgRNAs contain the MP (3'MP sgRNA) and CP (3'CP sgRNA) genes and untranslated regions (UTRs) of 123 and 284 nt, respectively, at their 5' end. These sgRNAs start with a hexanucleotide which is also present at the 5' terminus of the gRNA. The 5'-coterminal sgRNAs; have 6795 and 5798 nt, colinear with the gRNA, and contain ORF1 and most MP gene (5'RPMP sgRNA) and most ORF1 (5'RP sgRNA), respectively. Their 3' termini map 35 and 40 nt upstream of the transcription initiation of the 3'CP and 3'MP sgRNAs, respectively, next to a potential promoter element. Our results suggest that, as in alphaviruses, CLBV internal genes are expressed via 3'-coterminal sgRNAs transcribed from the minus gRNA strand, The 5'-coterminal sgRNAs may result from early termination of the gRNA during the plus-strand synthesis, (C) 2002 Elsevier Science (USA).
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The nucleotide sequence and genomic organization of Citrus leaf blotch virus: Candidate type species for a new virus genus

  • Vives, María C.
  • Galipienso, Luis
  • Navarro, Luis
  • Moreno, Pedro
  • Guerri, José
The complete nucleotide sequence of Citrus leaf blotch virus (CLBV) was determined. CLBV genomic RNA (gRNA) has 8747 nt, excluding the 3'-terminal poly(A) tail, and contains three open reading frames (ORFs) and untranslated regions (UTR) of 73 and 541 nucleotides at the 5' and 3' termini, respectively. ORF1 potentially encodes a 227.4-kDa polypeptide, which has methyltransferase, papain-like protease, helicase, and RNA-dependent RNA polymerase motifs. ORF2 encodes a 40.2-kDa polypeptide containing a motif characteristic of cell-to-cell movement proteins. The 40.7-kDa polypeptide encoded by ORF3 was identified as the coat protein. The genome organization of CLBV resembles that of viruses in the genus Trichovirus, but they differ in various aspects: (i) in trichoviruses ORF2 overlaps ORFs 1 and 3, whereas in CLBV, ORFs 2 and 3 are separated and ORFs 1 and 2 overlap in one nucleotide; (ii) CLBV gRNA and CP are larger than those of trichoviruses; and (iii) the CLBV 3' UTR is larger than that of trichoviruses. Phylogenetic comparisons based on CP amino acid signatures clearly separates CLBV from trichoviruses. Also contrasting with trichoviruses, CLBV could not be transmitted to Chenopodium quince Willd. Considering these singularities, we propose that CLBV should be included in a new virus genus. (C) 2001 Academic Press.
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Low genetic variation between isolates of Citrus leaf blotch virus from different host species and of different geographical origins

  • Vives, María C.
  • Rubio, Luis
  • Galipienso, Luis
  • Navarro, Luis
  • Moreno, Pedro
  • Guerri, José
The population structure and genetic diversity of Citrus leaf blotch virus (CLBV) were estimated by single-strand conformation polymorphism and nucleotide sequence analyses of two genomic regions located within the replicase (R) and the coat protein (C) genes. Analysis of 30 cDNA clones of each genomic region from two CLBV isolates showed that both isolates contained a predominant haplotype and others closely related. Analysis of 37 CLBV Spanish field isolates showed low genetic diversity (0.0041 and 0.0018 for genomic regions R and C, respectively). Comparison of 14 CLBV isolates from Spain, Japan, USA, France and Australia showed genetic diversities of 0.0318 (R) and 0.0209 (C), respectively. No correlation was found between genetic distance and geographical origin or host species of the isolates. The ratio between nonsynonymous and synonymous substitutions was the lowest found in a plant virus, indicating a strong negative selective pressure in both genomic regions.
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Genetic Variation and Possible Mechanisms Driving the Evolution of Worldwide Fig mosaic virus Isolates

  • Walia, Jeewan Jyot
  • Willemsen, Anouk
  • Elci, Eminur
  • Caglayan, Kadriye
  • Falk, Bryce W.
  • Rubio, Luis
Fig mosaic virus (FMV) is a multipartite negative-sense RNA virus infecting fig trees worldwide. FMV is transmitted by vegetative propagation and grafting of plant materials, and by the eriophyid mite Aceria ficus. In this work, the genetic variation and evolutionary mechanisms shaping FMV populations were characterized. Nucleotide sequences from four genomic regions (each within the genomic RNAs 1, 2, 3, and 4) from FMV isolates from different countries were determined and analyzed. FMV genetic variation was low, as is seen for many other plant viruses. Phylogenetic analysis showed some geographically distant FMV isolates which clustered together, suggesting long-distance migration. The extent of migration was limited, although varied, between countries, such that FMV populations of different countries were genetically differentiated. Analysis using several recombination algorithms suggests that genomes of some FMV isolates originated by reassortment of genomic RNAs from different genetically similar isolates. Comparison between nonsynonymous and synonymous substitutions showed selection acting on some amino acids; however, most evolved neutrally. This and neutrality tests together with the limited gene flow suggest that genetic drift plays an important role in shaping FMV populations.
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Usefulness of thermography for plant water stress detection in citrus and persimmon trees

  • Ballester, Carlos
  • Jimenez-Bello, M. A.
  • Castel, Juan R.
  • Intrigliolo, Diego S.
The feasibility of using canopy temperature (T-c) measured with a hand-operated infrared thermographic camera as a water stress indicator was evaluated in the field during two seasons on citrus and persimmon trees subjected to different levels of deficit irrigation. In both species, which differ in leaf anatomy and stomatal response to environmental conditions, T-c, was compared with midday stem water potential (Psi(s)) measurements. In persimmon trees, leaf stomatal conductance (g(s)) was also measured. In 2009, images were taken from the sunlit and shady sides of the canopies. Based on the results obtained, during the second experimental season images were taken from the sunlit side of the trees and also from above the canopy. In persimmon, trees under deficit irrigation had lower Psi(s) and g(s) what resulted in a clear increase in T-c regardless of the position from where the pictures were taken. The maximum T-c difference between deficit-irrigated and control trees observed was of 4.4 degrees C, which occurred when the stressed trees had Psi(s) values 1.1 MPa lower than the control ones. In persimmon trees, T-c was the most sensitive indicator of plant water status particularly due to the lower tree-to-tree variability as compared to Psi(s) and g(s). On the other hand, in citrus trees T-c was not always affected by plant water stress. Only in the second experimental season, when air vapour pressure deficit values were below 2.7 kPa and images were also taken from above the canopies, deficit-irrigated trees had higher T-c than the control ones, this difference being at most 1.7 degrees C. Overall, the results show that hand-operated thermographic cameras can be used to detect plant water stress in both fruit tree species. Nevertheless, the use of T-c measurements to detect plant water stress appears to be more precise in persimmon than in orange citrus. This might be because persimmon trees have larger leaf size which determines higher canopy resistance allowing for higher increases in canopy temperature in response to water stress via stomatal closure. (C) 2012 Elsevier B.V. All rights reserved.
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Sequencing of diverse mandarin, pummelo and orange genomes reveals complex history of admixture during citrus domestication

  • Wu, G. Albert
  • Prochnik, Simon
  • Jenkins, Jerry
  • Salse, Jerome
  • Hellsten, Uffe
  • Murat, Florent
  • Perrier, Xavier
  • Ruiz, Manuel
  • Scalabrin, Simone
  • Terol, Javier
  • Takita, Marco Aurelio
  • Labadie, Karine
  • Poulain, Julie
  • Couloux, Arnaud
  • Jabbari, Kamel
  • Cattonaro, Federica
  • Del Fabbro, Cristian
  • Pinosio, Sara
  • Zuccolo, Andrea
  • Chapman, Jarrod
  • Grimwood, Jane
  • Tadeo, Francisco R.
  • Estornell, Leandro H.
  • Munoz-Sanz, Juan V.
  • Ibanez, Victoria
  • Herrero-Ortega, Amparo
  • Aleza, Pablo
  • Perez-Perez, Julian
  • Ramon, Daniel
  • Brunel, Dominique
  • Luro, Francois
  • Chen, Chunxian
  • Farmerie, William G.
  • Desany, Brian
  • Kodira, Chinnappa
  • Mohiuddin, Mohammed
  • Harkins, Tim
  • Fredrikson, Karin
  • Burns, Paul
  • Lomsadze, Alexandre
  • Borodovsky, Mark
  • Reforgiato, Giuseppe
  • Freitas-Astua, Juliana
  • Quetier, Francis
  • Navarro, Luis
  • Roose, Mikeal
  • Wincker, Patrick
  • Schmutz, Jeremy
  • Morgante, Michele
  • Machado, Marcos Antonio
  • Talón, Manuel
  • Jaillon, Olivier
  • Ollitrault, Patrick
  • Gmitter, Frederick
  • Rokhsar, Daniel
Cultivated citrus are selections from, or hybrids of, wild progenitor species whose identities and contributions to citrus domestication remain controversial. Here we sequence and compare citrus genomes-a high-quality reference haploid clementine genome and mandarin, pummelo, sweet-orange and sour-orange genomes- and show that cultivated types derive from two progenitor species. Although cultivated pummelos represent selections from one progenitor species, Citrus maxima, cultivated mandarins are introgressions of C. maxima into the ancestral mandarin species Citrus reticulata. The most widely cultivated citrus, sweet orange, is the offspring of previously admixed individuals, but sour orange is an F1 hybrid of pure C. maxima and C. reticulata parents, thus implying that wild mandarins were part of the early breeding germplasm. A Chinese wild 'mandarin' diverges substantially from C. reticulata, thus suggesting the possibility of other unrecognized wild citrus species. Understanding citrus phylogeny through genome analysis clarifies taxonomic relationships and facilitates sequence-directed genetic improvement.
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Is Pre-Veraison Irrigation Cut-Off more Convenient than Post-Veraison Water Stress as a Strategy to Improve Grape Composition in Vitis vinifera 'Tempranillo' in Spain?, Acta Horticulturae

  • Yeves, A.
  • Perez, D.
  • Risco, D.
  • Intrigliolo, Diego S.
  • Castel, Juan R.
Irrigation of grapevines to meet the full seasonal water needs (ETc) usually negatively affects wine composition. In addition, our previous results showed that under Mediterranean climate replacing approximately 50% of full ETc ensures a substantial yield increase, compared with rain-fed conditions, with no detrimental effects on grape composition. The question then is whether it would be better to concentrate the water applications during the pre-veraison or during the postveraison period. A field experiment was carried out in a 'Tempranillo' vineyard during 2007 and 2008 where different irrigation strategies were applied at two crop levels (Normal and High). Rain-fed (NI) vines were compared with vines that were constantly irrigated at 75% of ETc (Control). Irrigation to the Control commenced when midday stem water potential (Psi(stem)) values reached -0.7 MPa. In addition, a spring water shortage strategy (ED) was applied by withholding irrigation until. stem Psi(stem) surpassed -1.0 MPa. After that, 75% of ETc was applied. Finally, a LD treatment was irrigated as per the Control up to veraison, and thereafter water application was reduced to approximately 2550% of ETc, trying to avoid. stem surpass -1.4 MPa. Results indicated that all irrigation regimes increased vine yield up to 50% with respect to the NI and no differences in yield among the irrigated treatments occurred. However, there were large differences in berry composition among the different irrigation strategies. The ED was more effective than LD in reducing berry growth leading to more concentrated berries in terms of anthocyanins. The LD impaired berry sugar accumulation due to the detrimental effect of water stress on leaf photosynthetic rate. Thus, in our conditions of scarce water resources, applying moderate water deficits before veraison and irrigating without considerable restriction afterwards, appears as the most convenient irrigation strategy. Crop level did not affect grape composition.
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