Rosa Peiró

Identification of Single-Nucleotide Polymorphism in the Progesterone Receptor Gene and Its Association With Reproductive Traits in Rabbits

A total of 598 F2 does from a cross between the high and low lines selected divergently for uterine capacity during 10 generations were used in a candidate gene analysis. The presence of major genes affecting the number of implanted embryos and uterine capacity has been suggested in lines divergently selected for uterine capacity. Uterine capacity is a main component of litter size. The progesterone receptor gene was tested as a candidate gene to determine whether polymorphisms explain differences in litter size and its components. Fragments of the promoter region and exons 1–8 were amplified and sequenced. One SNP was found in the promoter region, 2464G>A, three SNPs in the 5′-UTR exon 1, and a silence SNP in exon 7. The first four SNPs were segregated in two haplotypes. The allele G found in the promoter region was found in 75% of the high-line parental animals and in 29% of the low-line parental animals. The GG genotype had 0.5 kits and 0.5 implanted embryos more than the AA genotype. At 48 hr of gestation, the difference in early embryo survival and embryonic stage of development was small. However, at 72 hr of gestation, the GG genotype had 0.36 embryos more than the AA genotype and also had a more advanced embryonic stage of development, showing a lower percentage of compacted morulae and a higher percentage of blastocysts. The difference in litter size between the GG and GA genotypes was similar to the difference found between homozygote genotypes; however, differences in implanted embryos, early embryo survival, and embryo development were not detected between the GG and GA genotypes.

Candidate gene analysis for reproductive traits in two lines of rabbits divergently selected for uterine capacity.

The objective of this work was to analyze 3 functional candidate genes for reproduction in 2 lines of rabbits divergently selected by uterine capacity. Both lines were selected for 10 generations. The selection was then relaxed until the 17th generation, when it was compounded by 61 and 63 does of the High and Low lines, respectively. We sequenced the SCGB1A1 gene, which encodes the main protein secreted by the rabbit in the uterus and seems to play an important role in implantation. We found 6 SNP in the promoter region cosegregating in 2 haplotypes in both lines with similar frequency. We also analyzed IGF1 mRNA because of its effects on embryo development, but we did not find any polymorphism between individuals of the 2 lines. The third gene analyzed was the TIMP1, which encodes a protein involved in many biological processes related to reproduction. We determined the sequence of its promoter region and found 1 SNP (g.1423A>G) segregating with different frequencies in both lines (0.60 for allele A in the High line and 0.82 for allele G in the Low line). The association study performed in an F(2) population (n = 598) generated by the cross of the 2 lines of rabbits revealed that the AA genotype had 0.88 embryos more than the GG genotype at 72 h of gestation. The difference increased to 2.23 embryos at implantation, but no difference was found between genotypes at birth. These results suggest that TIMP1 could be a candidate gene for embryo implantation and embryo survival.

Expression of progesterone receptor related to the polymorphism in the PGR gene in the rabbit reproductive tract

The association of the 2464G > A SNP found in the promoter region of the rabbit progesterone receptor gene with progesterone receptor (PR) expression was evaluated by Western blot analysis. This SNP was associated with 2 lines divergently selected for uterine capacity, the high line selected to increase uterine capacity and the low line selected to decrease uterine capacity. Two progesterone isoforms were obtained using a commercial monoclonal antibody: the PR-B isoform described previously in rabbits, and the PR-A isoform, not described previously in rabbits. The GG genotype, the genotype more frequent in the high line, showed less PR-B and PR-A expression than the AA genotype in the oviduct (GG/AA(PR-B) = 0.81 and GG/AA(PR-A) = 0.73) and uterus (around 0.70 in both isoforms). The GA genotype showed similar PR-A expression in both tissues and also similar PR-B expression in the oviduct to the GG genotype. Conversely, the GG genotype showed less PR-B expression than the GA genotype in the uterus (GG/GA(PR-B) = 0.79). Similar expression of both PR isoforms was found in the uterus at d 2 and 3 of gestation; meanwhile, an increase of both isoforms was observed in the oviduct. Similar PR-A expression was observed in the ampulla and isthmus; meanwhile, the PR-B expression in the isthmus was double that in the ampulla.

Investigation of the oviductal glycoprotein 1 (OVGP1) gene associated with embryo survival and development in the rabbit.

An association study was performed in rabbits between early embryo survival and development, and the nonconservative SNP 12944C>G located in exon 11 and the triallellic microsatellite [(GT)(15)T(G)(5), (GT)(14)T(G)(5), and (GT)(11)T(G)(7))] located in the promoter region of the oviductal glycoprotein 1 (OVGP1) gene. We analyzed an F(2) cross of 2 lines of rabbits divergently selected for uterine capacity. A total of 172 and 159 females were slaughtered at 48 and 72 h of gestation, respectively, to determine whether OVGP1 influences ovulation rate, fertilization rate, early embryo survival, and embryonic stage of development. The results of the SNP indicated that all genotypes showed similar early embryo survival and a similar embryonic stage of development at 48 h of gestation. However, at 72 h of gestation, the GG genotype showed greater early embryo survival than the CC genotype (0.56 embryos) and their embryos presented less embryonic development. Analysis of the microsatellite was performed to ascertain the presence or absence of the allele (GT)(14)T(G)(5). At both stages of gestation, the (GT)(14)T(G)(5)/(GT)(14)T(G)(5) genotype showed greater early embryo survival (0.94 and 1.54 embryos at 48 and 72 h of gestation, respectively) and less embryonic development than the homozygous genotypes without the allele (GT)(14)T(G)(5).

Analysis of the oviductal glycoprotein 1 polymorphisms and their effects on components of litter size in rabbits

The objective of this work was to study the effect of the oviductal glycoprotein 1 (OVGP1) genotype and mRNA expression on litter size and other fertility measures, as OVGP1 has positive effects on fertilization and early embryo development. We have analysed an F(2) cross of two lines of rabbits divergently selected for uterine capacity. The OVGP1 mRNA expression was analysed in both lines, but no differences were observed between them. The promoter region and mRNA were sequenced in the F(0) generation, and 17 polymorphic sites were found to co-segregate in three haplotypes (A, B and C). An association study was performed between several reproductive traits and a triallelic microsatellite identified in the promoter region as well as a non-synonymous SNP located in exon 11 [g.12944C>G (p.Arg468Gly)]. The alleles g.12944G and g.325(GT)(14)T(G)(5) of the B haplotype have a positive effect on the total number of kits born, number born alive, number of implanted embryos and foetal and prenatal embryo survival.

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.

Evaluation of conditions for in vitro storage of commercial and minor grapevine (Vitis vinifera L.) cultivars

ABSTRACT In vitro culture represents a tool for the ex situ conservation of a high number of sanitised plants in a reduced space. However, the culture media and/or other growing conditions need to be optimised to minimising plant growth and storage cost. Growth on MW medium was evaluated in the commercial cultivars ‘Airén’, ‘Bobal’, ‘Chardonnay’, ‘Garnacha Blanca’, ‘Moscatel de Alejandría’, ‘Moscatel de Grano Menudo’, ‘Pedro Ximénez’, ‘Pinot Blanc’, ‘Pinot Gris’, ‘Sauvignon Blanc’, and ‘Tempranillo’; the minor cultivars ‘Chelva’, ‘Valencí Negre’, ‘Valencí Blanc’, and ‘Verdil’; and the endangered cv. ‘Esclafacherre’. Different growth rates were observed among cultivars: those with faster growth need to be subcultured every 1.5–2.0 months; those with the slowest growth every 3.5–4.0 months. The effect of halving the sucrose in MW reduced the growth of the cultivars that grew faster without compromising survival. When IBA was removed from MW, growth was also reduced in some cultivars. Therefore, small modifications of the MW composition are adequate for grapevine in vitro storage under standard incubation conditions. This is an advantage with respect to the change of temperature used in other work to achieve growth reduction, and allows the use of the same chamber for different in vitro culture procedures.

Morphological characterization of the cucumber ( Cucumis sativus L.) collection of the COMAV’s Genebank

The cucumber (Cucumis sativus L.) is an important crop worldwide. In the present study the morphological characterization of 206 cucumber accessions, 195 from Spain and 11 outgroups from other countries, was carried out. One hundred and seventy-eight of them came from the COMAV’s Genebank, 116 collected by the COMAV and the others 62 maintained at this institution as safety duplicates of the BGHZ collection. Seventeen more accessions supplied by BGHZ were included in the present research. Five plants per accession were characterized, with 17 qualitative and nine quantitative descriptors, eight of them referred to plant traits and 18 related to the fruit. Fruit descriptors were evaluated in at least 25 fruits per accession. The accessions were classified in five groups: ‘White’, ‘Short’, ‘French’, ‘Long’ and ‘Very long’, based on the morphology of their fruits and their similarity to commercial types. Principal Component Analysis showed that, with few exceptions, the accessions grouped to the previously established groups. Variability found among and within groups displayed the potential of these plant materials in breeding programs for different traits. The morphological characterization allowed the selection of the 67.2% of the collection, eliminating the most similar accessions.

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

BackgroundSomatic 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).ResultsSomatic 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.ConclusionsCut-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.

A set of PCR-based markers for management of a library of Solanum lycopersicoides introgression lines

Summary A collection of introgression lines (ILs) of Solanum lycopersicoides Dunal in the genetic background of cultivated tomato (S. lycopersicum L.) is a valuable tool for tomato breeding. Efficient management of the collection requires the use of molecular markers. The objective of this work was to identify polymerase chain reaction (PCR)-based markers that were polymorphic between the parents of the ILs, namely ‘VF36’ and ‘LA2951’. In total, 81 primer pairs were tested on genomic DNA from both parents. Genomic DNA of the inter-specific hybrid between the two parents, from which the IL collection had been derived, was also tested. The markers used were either cleaved amplified polymorphic sequence (CAPS) markers or were derived from a set of conserved orthologous genes. In both cases, the markers had been mapped in tomato and described in the SOL Genomics Network. Forty-seven of the markers tested produced a single PCR product in ‘VF36’ and ‘LA2951’. Eleven markers revealed polymorphisms as differences in band-sizes between the two parents. At least one restriction enzyme that generated polymorphism was identified in 29 of the remaining 36 markers. Among other applications, some of these markers have been used to identify plants carrying a target DNA fragment among segregating generations, or to delimit the length of an introgressed sequence.