Somatic embryogenesis from seeds in a broad range of Vitis vinifera L. varieties: rescue of true-to-type virus-free plants.

BACKGROUND: Somatic embryogenesis is the preferred method for cell to plant regeneration in Vitis vinifera L. However, low frequencies of plant embryo conversion are commonly found. In a previous work we obtained from cut-seeds of a grapevine infected with the Grapevine leafroll associated viruses 1 and 3 (GLRaV-1 and GLRaV-3), high rates of direct regeneration, embryo plant conversion and sanitation. The aim of this study is to evaluate the usefulness of this procedure for regeneration of other grapevine varieties which include some infected with one to three common grapevine viruses (GLRaV-3, Grapevine fanleaf virus (GFLV) and Grapevine fleck virus (GFkV)). As grapevine is highly heterozygous, it was necessary to select from among the virus-free plants those that regenerated from mother tissues around the embryo, (true-to-type). RESULTS: Somatic embryogenesis and plant regeneration were achieved in a first experiment, using cut-seeds from the 14 grapevine varieties Airén, Cabernet Franc, Cabernet Sauvignon, Mencía, Merlot, Monastrell, Petit Verdot, Pinot Blanc (infected by GFLV and GFkV), Pinot Gris, Pinot Meunier, Pinot Noir, Syrah, Tempranillo (infected by GFLV), and Verdil. All regenerated plants were confirmed to be free of GFkV whereas at least 68% sanitation was obtained for GFLV. The SSR profiles of the virus-free plants showed, in both varieties, around 10% regeneration from mother tissue (the same genetic make-up as the mother plant). In a second experiment, this procedure was used to sanitize the varieties Cabernet Franc, Godello, Merlot and Valencí Blanc infected by GLRaV-3, GFkV and/or GFLV. CONCLUSIONS: Cut-seeds can be used as explants for embryogenesis induction and plant conversion in a broad range of grapevine varieties. The high regeneration rates obtained with this procedure facilitate the posterior selection of true-to-type virus-free plants. A sanitation rate of 100% was obtained for GFkV as this virus is not seed-transmitted. However, the presence of GLRaV-3 and GFLV in some of the regenerated plants showed that both viruses are seed-transmitted. The regeneration of true-to-type virus-free plants from all infected varieties indicates that this methodology may represent an alternative procedure for virus cleaning in grapevine.

Modeling the Accuracy of Three Detection Methods of Grapevine leafroll-associated virus 3 During the Dormant Period Using a Bayesian Approach.

Grapevine leafroll-associated virus 3 (GLRaV-3) has a worldwide distribution and is the most economically important virus that causes grapevine leafroll disease. Reliable, sensitive, and specific methods are required for the detection of the pathogen in order to assure the production of healthy plant material and control of the disease. Although different serological and nucleic acid-based methods have been developed for the detection of GLRaV-3, diagnostic parameters have not been established, and there is no gold standard method. Therefore, the main aim of this work was to determine the sensitivity, specificity, and likelihood ratios of three commonly used methods, including one serological test (double-antibody sandwich enzyme-linked immunosorbent assay [DAS-ELISA]) and two nucleic acid-based techniques (spot and conventional real-time reverse transcription-polymerase chain reaction [RT-PCR]). Latent class models using a Bayesian approach have been applied to determine diagnostic test parameters and to facilitate decision-making regarding diagnostic test selection. Statistical analysis has been based on the results of a total of 281 samples, which were collected during the dormant period from three different populations. The best-fit model out of the 49 implemented models revealed that DAS-ELISA was the most specific method (value = 0.99) and provided the highest degree of confidence in positive results. Conversely, conventional real-time RT-PCR was the most sensitive method (value = 0.98) and produced the highest degree of confidence in negative results. Furthermore, the estimation of likelihood ratios showed that in populations with low GLRaV-3 prevalence the most appropriate method could be DAS-ELISA, while conventional real-time RT-PCR could be the most appropriate method in medium or high prevalence populations. Combining both techniques significantly increases detection accuracy. The flexibility and power of Bayesian latent class models open new possibilities for the evaluation of diagnostic tests for plant viruses.

Genetic Variation and Evolutionary Analysis of Eggplant Mottled Dwarf Virus Isolates from Spain.

The genetic variation and population structure of gene N (nucleocapsid) and part of gene L (replicase) from 13 eggplant mottle dwarf virus (EMDV) isolates from Spain were evaluated and compared with sequences of EMDV isolates from other countries retrieved from GenBank. Phylogenetic inference of part of gene L showed three main clades, one containing an EMDV isolate from Australia and the other two containing isolates from Iran and Europe, as well as four subclades. EMDV isolates from Spain were genetically very similar and grouped in a subclade together with one isolate from Germany and one from the UK. No new recombination events were detected in addition to one recombination previously reported, suggesting that recombination is rare for EMDV. The comparison of synonymous and non-synonymous rates showed that negative selection played an important role, and only two codons were under positive selection. Genetic differentiation (Fst test), phylogenetic and nucleotide diversity analyses suggest a unique introduction of EMDV to Spain and low gene flow with other countries. In contrast, Greece and Italy showed diverse populations with high gene flow between both.

Changes in Body Reserves, Non-Esterified Fatty Acids, and Leptin during the Reproductive Lifespan of the Rabbit Female.

The aim of this work is to study changes in body weight, perirenal fat thickness (PFT), and non-esterified fatty acid (NEFA) and leptin concentrations throughout the reproductive life of the rabbit female and their correlations when a semi-intensive reproductive rhythm is applied. A total of 46 lactating females were used. Body weight, PFT, and NEFA and leptin concentration were recorded at 12 weeks of age, at first mating and delivery, and at second, third, and fourth mating, 12th d of gestation, and delivery. The highest body weight was detected on the 12th d of any gestation, around 4280 g, and the lowest weight was at delivery, around 4030 g. PFT increased until third mating. NEFA and leptin concentration showed a cyclical pattern throughout the reproductive lifespan of the females. NEFAs presented the highest concentration at delivery within each reproductive cycle and levels decreased over the course of the deliveries (0.423 mmol/L at first delivery, 0.406 mmol/L at second delivery, 0.371 mmol/L at third delivery, and 0.309 mmol/L at fourth delivery). Similar NEFA concentrations at mating and on the 12th d of gestation were obtained. Leptin showed the highest concentrations at mating within each reproductive cycle. Leptin decreased between mating and delivery in all reproductive cycles and it was close to 1 ng/mL HE. Low or null correlations were shown between body weight, PFT, and NEFA and leptin concentration at mating, 12th d of gestation, and delivery. In conclusion, females are able to maintain a semi-intensive reproductive rhythm across four parities weighing around 4 kg from first mating. Females had an increased perirenal fat thickness until third delivery, and their NEFA concentration was maximum at delivery and leptin concentration was maximum at mating. Body weight, PFT, and NEFA and leptin concentration should be measured during critical moments of reproductive life in order to determine body condition and energy mobilization, due to their low or null correlations.

Correlated Response on Growth Traits and Their Variabilities to Selection for Ovulation Rate in Rabbits Using Genetic Trends and a Cryopreserved Control Population.

The aim of this work was to estimate correlated responses in growth traits and their variabilities in an experiment of selection for ovulation rate during 10 generations in rabbits. Individual weight at 28 days old (IW28, kg) and at 63 days old (IW63, kg) was analyzed, as well as individual growth rate (IGR = IW63 - IW28, kg). The variability of each growth trait was calculated as the absolute value of the difference between the individual value and the mean value of their litter. Data were analyzed using Bayesian methodology. The estimated heritabilities of IW28, IW63 and IGR were low, whereas negligible heritabilities were obtained for growth variability traits. The common litter effect was high for all growth traits, around 30% of the phenotypic variance, whereas low maternal effect for all growth traits was obtained. Low genetic correlations between ovulation rate and growth traits were found, and also between ovulation rate and the variability of growth traits. Therefore, genetic trends methods did not show correlated responses in growth traits. A similar result was also obtained using a cryopreserved control population.

Relationship between Prenatal Characteristics and Body Condition and Endocrine Profile in Rabbits.

This study evaluated the relationship between prenatal characteristics and body condition and endocrine profile. A total of 25 non-lactating multiparous females were used. Body condition, measured as body weight and perirenal fat thickness, non-esterified fatty acids (NEFA), leptin, progesterone and 17ß-estradiol were recorded at mating and 12 d of gestation. Ovulation rate, number of foetuses, ovary and foetal weight, length and weight of uterine horn, available space per foetus and maternal and foetal placental morphometry were recorded at 12 d of gestation. Ovulation rate showed a positive linear relationship with number of foetuses, ovary weight and NEFA. A negative linear relationship between ovulation rate and perirenal fat thickness and leptin was obtained. Ovulation rate was maximum when body weight and 17ß-estradiol were 4.4 kg and 22.7 pg/mL, respectively. Foetal weight showed a positive relationship with perirenal fat thickness and a negative relationship with leptin. An increase in progesterone and NEFA concentration was related to a positive linear increase in number of foetuses and in uterine horn weight. Space available per foetus was affected both by the number of vessels that reach the implantation site and by position of the foetus in the uterine horn. In conclusion, body condition during mating and early gestation should be maintained within an optimal range to ensure the best prenatal characteristics. While 17ß-estradiol, NEFA and leptin affected the ovulation rate, progesterone and NEFA affected foetal development. The number of vessels that reach the implantation site determines early foetal survival.

Persistent Southern Tomato Virus (STV) Interacts with Cucumber Mosaic and/or Pepino Mosaic Virus in Mixed- Infections Modifying Plant Symptoms, Viral Titer and Small RNA Accumulation.

Southern tomato virus (STV) is a persistent virus that was, at the beginning, associated with some tomato fruit disorders. Subsequent studies showed that the virus did not induce apparent symptoms in single infections. Accordingly, the reported symptoms could be induced by the interaction of STV with other viruses, which frequently infect tomato. Here, we studied the effect of STV in co- and triple-infections with Cucumber mosaic virus (CMV) and Pepino mosaic virus (PepMV). Our results showed complex interactions among these viruses. Co-infections leaded to a synergism between STV and CMV or PepMV: STV increased CMV titer and plant symptoms at early infection stages, whereas PepMV only exacerbated the plant symptoms. CMV and PepMV co-infection showed an antagonistic interaction with a strong decrease of CMV titer and a modification of the plant symptoms with respect to the single infections. However, the presence of STV in a triple-infection abolished this antagonism, restoring the CMV titer and plant symptoms. The siRNAs analysis showed a total of 78 miRNAs, with 47 corresponding to novel miRNAs in tomato, which were expressed differentially in the plants that were infected with these viruses with respect to the control mock-inoculated plants. These miRNAs were involved in the regulation of important functions and their number and expression level varied, depending on the virus combination. The number of vsiRNAs in STV single-infected tomato plants was very small, but STV vsiRNAs increased with the presence of CMV and PepMV. Additionally, the rates of CMV and PepMV vsiRNAs varied depending on the virus combination. The frequencies of vsiRNAs in the viral genomes were not uniform, but they were not influenced by other viruses.

Ovarian Transcriptomic Analysis Reveals Differential Expression Genes Associated with Cell Death Process after Selection for Ovulation Rate in Rabbits.

Litter size is an essential trait in rabbit meat production but with low heritability. A selection experiment for ovulation rate has been performed for 10 generations to improve litter size in rabbits. The selected line increased two ova more than the control line but nevertheless a negative correlation was observed with prenatal survival. A transcriptomic study was performed, using microarrays, in ovarian tissue from females belonging to the selected line and the control line. Our results showed 1357 differential expressed genes and nineteen potential biomarkers associated with prenatal mortality, which could explain differences between litter size in rabbits. Cell death was the most relevant process.

In vitro propagation of Vitis vinifera L. cv. 'Monastrell'

Background: A protocol for the micropropagation of the grape (Vitis vinifera L.) cultivar 'Monastrell' was developed. Initial plant material was obtained from the sanitary selection of grapevine plants performed by real-time RT-PCR to confirm the absence of Grapevine fanleaf virus, Arabis mosaic virus, Grapevine leafroll-associated virus 1, Grapevine leafroll-associated virus 3, and Grapevine fleck virus. Results: The effects of the salt composition (comparing Lloyd and McCown woody plant medium and Murashige and Skoog medium 1/2 macronutrients) and the growth regulator benzylaminopurine (BAP), at 0 and 8.9 µM, on plant propagation were evaluated using nodes as explants. The most efficient procedure consisted of bud induction in the medium with Lloyd and McCown woody plant salts and 8.9 µM BAP for 30 d along with elongation in cytokinin-free medium for 60 d, which gave 22 nodes/explant (174 plants/initial plant). A second cycle of propagation in a medium without BAP for another 60 d could give approximately 10,000 nodes, which can be obtained after an additional 2 months of culture. All plants acclimatized after the second cycle of multiplication were successfully transferred to soil. Conclusion: We developed an optimal protocol for V. vinifera cv. 'Monastrell' micropropagation, the first described for this cultivar.

Rabbit oviductal glycoprotein 1 gene: genomic organization polymorphism analysis and mRNA expression.

The OVGP1 is an oviductal glycoprotein that has positive effects on fertilization and early embryo development. We have amplified and sequenced the rabbit OVGP1 gene, which spans 13 kb and it is formed by 11 exons and 10 introns. To find polymorphisms, a region encompassing the promoter to intron 1 has been sequenced in 22 rabbits of the H, V, R, and A Spanish lines. We have identified five SNPs and one triallelic microsatellite in the promoter region, and three SNPs and one dinucleotide INDEL in intron 1. The 10 polymorphic sites cosegregate forming two haplotypes. The allelic frequencies of the microsatellite have been analyzed in 98 rabbits belonging to the four lines and the three alleles were found in all the lines. The relative quantification of the OVGP1 mRNA in liver, kidneys, lungs, skeletal muscle, ovary, uterus, and oviduct reveals that the OVGP1 expression in the oviduct is 5,500 higher than in the uterus or ovary, whereas a low level of basal expression is detected in non-reproductive tissues. We have also compared the mRNA expression between samples of oviducts taken from non-mated rabbit and samples of oviducts at different stages of the early pregnancy, but no significant differences were found.

TIMP-1 as candidate gene for embryo survival in two divergent lines selected for uterine capacity in rabbits.

Selection on uterine capacity has been used in animal breeding as a way to improve the litter size. A divergent selection experiment for uterine capacity was performed in rabbits during ten generations. After the first generations of selection, large differences in number of implanted embryos were obtained between high and low lines. The major part of the differences between lines was due to embryo survival. A segregation analysis suggested the presence of a major gene affecting the reproductive traits. The objective of this work was to test the TIMP-1 gene as a candidate gene for embryo survival in rabbits since it stands up as a target for the investigation of reproductive problems in humans. We have analyzed the parental generation of a F2 cross which consists of 8 and 14 animals from the high and low uterine capacity lines, respectively. The rabbit TIMP-1 gene structure and sequence has been determined, including the proximal promoter region. Despite of the absence of polymorphism between lines in the screened regions (CDS, proximal promoter, exon 1, intron 1, and exon 2), a real-time RT-PCR quantification of the TIMP-1 mRNA in oviduct has shown significant differences between high and low lines at 62 hr of gestation, just when rabbit embryos are located in the oviduct, postulating TIMP-1 as an interesting candidate gene to be involved in the phenotypic differences between the two rabbit lines.