Rosa Arroyo-García

Analysis of Differential Messenger RNA Expression Between Bovine Blastocysts Produced in Different Culture Systems: Implications for Blastocyst Quality

Abstract Using reverse transcriptase-amplified fragment length polymorphism (RT-AFLP) analysis of differential mRNA expression and semiquantitative reverse transcriptase-polymerase chain reaction, we compared mRNA expression in bovine blastocysts from 4 sources, known to differ in quality in terms of their ability to withstand cryopreservation: 1) in vitro culture in synthetic oviduct fluid of in vitro-matured (IVM)/in vitro fertilized (IVF) zygotes; 2) in vitro culture in TCM-199 supplemented with granulosa cells (coculture) of IVM/IVF zygotes; 3) in vivo culture in the ewe oviduct of IVM/IVF zygotes; or 4) superovulation, artificial insemination, and nonsurgical embryo recovery. Total mRNA was isolated from pools of blastocysts and reverse transcription was performed. Triplicate reactions from each sample were displayed, and only consistent banding variations were recorded. Using AFLP-differential display assay, we found that cDNA banding patterns are highly conserved between the 4 groups of blastocysts studied; however, there was a difference of 7% in bands either missing or expressed across the groups. Fifty bands were reamplified, and a sequence comparison search revealed similarity of 14 isolated fragments to ribosomal and mitochondrial genes, 16 matched to described cDNA, and 20 corresponded to unknown sequences that may represent novel genes. The study of 7 differentially expressed mRNAs known to be involved in developmental process in the embryo suggests roles for apoptosis, oxidative stress, gap junctions, and differentiation in the determination of embryo quality. The aberrant transcription patterns detected in in vitro-produced bovine embryos compared with those produced in vivo may explain their reduced quality in terms of viability after cryopreservation.

Genetic and epigenetic stability of cryopreserved and cold-stored hops (Humulus lupulus L.)

Conventional cold storage and cryopreservation methods for hops (Humulus lupulus L.) are available but, to our knowledge, the genetic and epigenetic stability of the recovered plants have not been tested. This study analyzed 51 accessions of hop using the molecular techniques, Random Amplified DNA Polymorphism (RAPD) and Amplified Fragment Length Polymorphism (AFLP), revealing no genetic variation among greenhouse-grown controls and cold stored or cryopreserved plants. Epigenetic stability was evaluated using Methylation Sensitive Amplified Polymorphism (MSAP). Over 36% of the loci were polymorphic when the cold and cryo-treated plants were compared to greenhouse plants. The main changes were demethylation events and they were common to the cryopreserved and cold stored plants indicating the possible effect of the in vitro establishment process, an essential step in both protocols. Protocol-specific methylation patterns were also detected indicating that both methods produced epigenetic changes in plants following cold storage and cryopreservation.

AFLP evaluation of genetic similarity among laurel populations (Laurus L.)

Two species have traditionally been considered within the genus Laurus: L. nobilis L. and L. azorica (Seub.) Franco. The first is characterized by the presence of glabrous twig leaves and is located in the Mediterranean region. It can be found as cultivated or naturalized, and has been reported in Spain, France, Italy and Greece. L. azorica is characterized by the presence of densely tomentose to hirsute twig leaves and has been described in the Azores, Madeira and the Canary Islands. We have found that some natural populations of Laurus in Northern Spain, which are considered to belong to L. nobilis, have hirsute young buds, with wide variation in hair number and density, in contradiction with taxonomical descriptions reported for this species. In order to evaluate the genetic similarity between these Laurus populations and the two reported species, we have analyzed 14 populations of L. nobilis and L. azorica from different geographical areas, including the Iberian peninsula, the Canary and Madeira Islands, France and Italy, using amplified fragment length polymorphisms (AFLPs). UPGMA clustering and principal component analysis (PCA) of the AFLP data revealed a low genetic similarity between the Iberian populations, including populations from Northern Spain, and the rest of the populations analyzed from France and Italy. Moreover, laurel accessions from the Iberian peninsula showed higher genetic similarity to those from the Canary Islands and Madeira, originally identified as L. azorica, than to samples from populations along the Mediterranean area, morphologically classified as L. nobilis.

Epigenetic changes detected in micropropagated hop plants

Micropropagation is a widely used technique in hops (Humulus lupulus L.). However, to the best of our knowledge, the genetic and epigenetic stability of the microplants has never been tested before. In the present study, two hop accessions were established in vitro and micropropagated for 2 years. The genetic and epigenetic stability of the in vitro plants was analyzed with several molecular techniques: random amplified DNA polymorphism (RAPD), retrotransposon microsatellite amplified polymorphism (REMAP), and methylation-sensitive amplification polymorphism (MSAP). No genetic variation among control and treated plants was found, even after 12 cycles of micropropagation. Epigenetic variation was detected, first, when field and in vitro samples were compared. Nearly a 30% of the detected fragments presented the same pattern of alterations in all the vitroplants. Second, lower levels of epigenetic variation were detected among plants from the different subcultures. Part of this detected variation seemed to be accumulated along the 12 sequential subcultures tested.

A small XY chromosomal region explains sex determination in wild dioecious V. vinifera and the reversal to hermaphroditism in domesticated grapevines

BackgroundIn Vitis vinifera L., domestication induced a dramatic change in flower morphology: the wild sylvestris subspecies is dioecious while hermaphroditism is largely predominant in the domesticated subsp. V. v. vinifera. The characterisation of polymorphisms in genes underlying the sex-determining chromosomal region may help clarify the history of domestication in grapevine and the evolution of sex chromosomes in plants. In the genus Vitis, sex determination is putatively controlled by one major locus with three alleles, male M, hermaphrodite H and female F, with an allelic dominance M > H > F. Previous genetic studies located the sex locus on chromosome 2. We used DNA polymorphisms of geographically diverse V. vinifera genotypes to confirm the position of this locus, to characterise the genetic diversity and traces of selection in candidate genes, and to explore the origin of hermaphroditism.ResultsIn V. v. sylvestris, a sex-determining region of 154.8 kb, also present in other Vitis species, spans less than 1% of chromosome 2. It displays haplotype diversity, linkage disequilibrium and differentiation that typically correspond to a small XY sex-determining region with XY males and XX females. In male alleles, traces of purifying selection were found for a trehalose phosphatase, an exostosin and a WRKY transcription factor, with strikingly low polymorphism levels between distant geographic regions. Both diversity and network analysis revealed that H alleles are more closely related to M than to F alleles.ConclusionsHermaphrodite alleles appear to derive from male alleles of wild grapevines, with successive recombination events allowing import of diversity from the X into the Y chromosomal region and slowing down the expansion of the region into a full heteromorphic chromosome. Our data are consistent with multiple domestication events and show traces of introgression from other Asian Vitis species into the cultivated grapevine gene pool.

Anthocyanin composition of several wild grape accessions.

This study investigated the anthocyanin composition of 21 mostly Spanish wild grapevine accessions preserved at El Encín Germoplasm Bank and selected in consideration of observed ampelographic differences and molecular characterization. Sampling was carried out in 2006, 2007, and 2008. After extraction from grape skins, total anthocyanins was determined by spectrophotometry and the anthocyanin fingerprint of grapes, based on 15 anthocyanin variables, was determined by HPLC. Total anthocyanin concentration was similar to that found in winegrape cultivars. The accessions studied showed considerable variability in their anthocyanin fingerprints and it was possible to distinguish several groups, similar to previous reports on the anthocyanin fingerprint of winegrapes. The anthocyanin composition of wild grapevine accessions was similar to that of cultivated grapes. Nevertheless, the presence of wild accessions with anthocyanin fingerprints uncommon or nonexistent in Spanish cultivated varieties suggests that the genetic variability related to anthocyanins in Spanish wild grapevine populations may be higher than that of cultivated varieties commonly considered of Spanish origin.

The influence of European and American wild germplasm in hop (Humulus lupulus L.) cultivars

Microsatellite variation at the nuclear and chloroplast genomes was evaluated for wild European and wild American hops, in order to assess the genetic diversity and origin of cultivated hops. Seven nuclear loci and 32 chloroplast loci were used in the analysis of 182 hop accessions including wild European (68), wild American (48), and cultivars (66). A total of 116 alleles were identified using 7 nuclear microsatellites showing different averages of polymorphism and distribution in the wild American and European accessions and cultivars. Two main groups were established as revealed by several statistical analyses; one including European wild accessions and cultivars and a second group consisting of American wild accessions. Three polymorphic chloroplast microsatellite loci were detected, six alleles were scored which defined a total of five haplotypes that were exclusive or presented different distribution between American and European wild accessions. A major influence of the wild European haplotypes was detected among hop cultivars. To the best of our knowledge, this is the first work reporting the use of chloroplast microsatellites in hops.

Allelic variation in the VvMYBA1 and VvMYBA2 domestication genes in natural grapevine populations (Vitis vinifera subsp. sylvestris)

Grape skin color is among the most important qualitative traits on which selection is based in wine and table grape breeding programmes. Skin color is determined by the quantity and composition of anthocyanins. In prior work on cultivated forms, it was shown that polymorphisms in the grape transcription factor family VvMYBA are responsible for anthocyanin content variation in the berries of cultivated grapevine (Vitis vinifera subsp. vinifera). Wild grapevine (V. vinifera subsp. sylvestris) is the ancestor of the cultivated V. vinifera subsp. sativa and has black-colored berries. The purpose of this study was to determine how the VvmybA1 and VvmybA2 polymorphisms emerged and affected the genetic diversity of wild grapevines in the Mediterranean basin by examining samples from the Iberian Peninsula, Italian Peninsula and Caucasian region. Our observations provide evidence that variation in the two transcriptional regulators generated a novel allele series via length polymorphisms in VvmybA1 and a point mutation in VvmybA2, which is lacking in cultivated grapevine. Further, correlation was detected between allele composition and anthocyanin contents. According to polymorphisms in both VvMYBA genes at the color locus, we were able to identify several haplotypes. The most ancestral haplotype (HapN) was found in wild grapevine in the western Mediterranean region and corresponded to wine grape cultivars, whereas recent haplotypes were detected in eastern regions. These eastern zones showed the most diverse haplotypes, which appeared in table cultivars where intense breeding practices may have replaced the original haplotype diversity. These findings provide information about the evolution of grapes since their domestication and have direct implications for wine quality.

Evaluation of Microsatellite Detection Using Autoradiography and Capillary Electrophoresis in Hops

Two sequence tagged sites (STS) detection techniques were used in the identification of 25 hop cultivars. In cultivar identification, capillary electrophoresis is more efficient than autoradiography. First, it was more sensitive in detection of fragments that allowed observation of the instability in the locus 7a82 due to the small size differences of its alleles. Additionally, the fluorescent detection of fragments was a nonsubjective technique suitable of automation that increased the flexibility of work because no radioactive labeling or long vertical polyacrylamide electrophoresis was needed. Only two cultivars, Hersbrucker and Hallertauer, were indistinguishable, but further molecular analysis revealed that both were included in a separate cluster within the European hop cultivars. The statistical analysis of fluorescence and radioactive detection data including allelic frequencies, heterozygosity, and probability of identity are displayed in this study, which corroborates the following advantages of the capillary electrophoresis technique: high sensitivity, amenable to automation, and its nonsubjectivity. Therefore, fluorescent-labeled primer microsatellite detection by capillary electrophoresis is a powerful tool in cultivar identification for hop breeders, merchants, and brewers.

Spontaneous variation regarding grape berry skin color: A comprehensive study of berry development by means of biochemical and molecular markers

Understanding grape berry development and the metabolism of different classes of compounds responsible for traits like berrys color is imperative to control and improve quality aspects of grapes. A colorimetric, biochemical and molecular characterization allowed the comprehensive description of the pigment-related characteristics of nine berry skin color somatic variants, belonging to four different varieties. Although the observed berry skin color variability was not fully explained by MybA locus, the phenolic profiles allowed inferring about specific interferences among the biosynthetic pathways. Data were consistent concerning that grapes showing cyanidin-3-O-glucoside as the major anthocyanin and flavonols with two substituent groups in the lateral B-ring are generally originated by a white ancestor. After retro-mutation, these grapes seem to keep the dysfunction on flavonoid hydroxylases enzymes, which negatively affect the synthesis of both flavonols and anthocyanins with three substituent groups in the lateral B-ring. Overall, the obtained results indicate that the color differences observed between somatic variants are not solely the result of the total amount of compounds synthesized, but rather reflect a different dynamics of the phenolic pathway among the different color variants of the same variety. CHEMICAL COMPOUNDS Gallic acid (PubChem CID: 370); Caftaric acid (PubChem CID: 6,440,397); Catechin (PubChem CID: 73,160); Epigallocatechin gallate (PubChem CID: 65,064); Quercetin-3-O-galactoside (PubChem CID: 5,281,643); Quercetin-3-O-glucoside (PubChem CID: 25,203,368); Malvidin-3-O-glucoside (PubChem CID: 443,652); Peonidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,849); Malvidin-3-O-p-coumaroylglucoside (PubChem CID: 44,256,988); Resveratrol-3-O-glucoside (PubChem CID: 25,579,167).