Inheritance of chloroplast DNA in two full-sib Vitis populations

The mode of transmission of chloroplasts in 2 grape populations was determined using restriction fragment length polymorphisms of chloroplast DNA (cpDNA) to trace the origin of plastids in the progeny. The populations examined were formed by crossing 2 complex interspecific hybrids: NY 62.136.2 x Yates and Cayuga White x Aurore. Analysis of the restriction banding patterns of total DNA of the 4 parents probed with cpDNA of grape and petunia revealed a high level of polymorphism (63 %) between parents ot the first cross and a low level of polymorphism (15 %) between the parents of the second cross. The restriction banding patterns of the 4 parents were unique, indicating that there were 4 distinct chloroplast genotypes. Analysis of the restriction banding patterns of total DNA of the progeny probed with cpDNA showed that all progeny from both crosses exhibited the banding pattern of the maternal parent. Thus, the mode of plastid transmission in these populations of grape was strictly maternal

Characterization of RAPD markers in Vitis

A study was initiated to investigate the possibility of using RAPD markers in related populations of Vitis. We also sought to design primers that could amplify translation initiation sites (Kozak sequence) as a mean to maximize the production of RAPD markers from single copy DNA sequences in the genome. RAPD bands were labeled and used as probes on blots with either genomic DNA or RAPD products from cvs Aurore, Cayuga White, Horizon and Illinois 547-1. Reamplification of excised RAPD products produced either several bands of smaller size, a single band of smaller size or a single band of the same size as the original band. Among 16 probes hybridized to genomic DNA blots, three probes, including one from the Kozak primer amplification, hybridized to 1-2 bands, 5 probes hybridized to 3-8 bands and 8, including two from a Kozak primer reaction, to more than 10 bands on the genomic DNA blots. Twelve RAPD bands were also probed on RAPD blots derived from the RAPD reaction that produced each probe. Three of those probes hybridized to 1-2 bands, 8 hybridized to 3-8 and one hybridized to more than 10 bands indicating the presence of probe sequences in more than one RAPD band as amplified with the same primers. This result and the observations on reamplification of RAPD bands support the hypothesis that some of the longer RAPD fragments harbor internal priming sites that are either not amplified unless the reaction mixture is saturated with longer other primers indicating amplification from the same sequence but different sized repetitive DNA. RAPD reactions were also run with 16 primers on parental DNA of 2 crosses used in genetic mapping (Cayuga White x Aurore and Horizon x Illinois 547-1). These reactions rated 140 bands; 100 bands were shared by both populations, including 47 polymorphic bands. Ten polymorphic bands in Cayuga White x Aurore and 22 in Horizon x Illinois 547-1 were population specific. The RAPD analysis as well as hybridization of RAPD markers to the genomic blots suggest that linkage analysis could be used in related segregating populations with carefully chosen markers. Tagging single copy regions with Kozak-sequence-derived primers may be possible, but the low number of probes tested and lack of DNA sequence information prevents any definite conclusions

Genetic Analysis of Restriction Fragment Length Polymorphisms in Vitis

The parents and progeny from two crosses (Cayuga White x Aurore and NY62.136.2 x Yates) were examined for the presence of DNA restriction fragment length polymorphisms (RFLPs). Seventeen independent DNA sequences were used in the analysis, 15 obtained from a grape Pstl genomic library and two heterologous probes obtained from other laboratories. Most of the low copy cloned sequences hybridized to more than two restriction fragments, possibly reflecting the polyploid nature of the Vitis genome. Nine of the probes detected RFLPs between parents. Analysis of the progenies (F1generation) revealed segregation for nine distinct polymorphisms generated by seven of the probes.Thus, a relatively high level of polymorphism among parents, as well as heterozygosity within each parent, was evident. Most RFLPs gave segregation ratios close to the 1: 1 ratio predicted for a locus heterozygous in one parent. However four differences between parental phenotypes did not segregated in the progeny, and in three instances fragments present in both parents segregated in the progeny. These peculiar results may be explained by accounting for heterozygosity or homozygosity, respectively, for the DNA segment that generates the polymorphism. We conclude that RFLP studies can be performed on the first filial generation in woody perennials such as Vitis that have a relatively high level of heterozygosity in the genom

Analysis of the relationship between grapevine cultivars, sports and clones via DNA fingerprinting

DNA fingerprinting utilizing RAPD polymorphisms was employed to investigate the relationship among 16 grapevine cultivars and sports thought to have arisen from these cultivars. From 53 primers, a total of 464 bands were generated, of which 29 % were common to all genotypes tested. Cluster analysis classified all tested cultivars into two main groups (Vitis vinifera L. and V. x Labruscana Bailey) as expected. No polymorphism was detected among known clones of Chardonnay (Ch. clone 7, Ch. clone 78 and Ch. Geneva clone) or Pinot noir (P. n. clone 29, P. n. Geneva clone and P. n. Pernand). Pinot Meunier, Pinot gris, and Gamay Beaujolais displayed patterns indistinguishable from Pinot noir. Auxerrois and Melon showed unique patterns and may be classified as distinct cultivars. Chardonnay clone 7 shared 84 % of its bands with Pinot noir. There was more than 97 % RAPD amplicon homology between Niagara and two supposed sports, and between Concord and a red-fruited sport. Taking into account the error rate in scoring RAPD bands, the evidence is against the hypothesis that the three sports are distinct cultivars. While RAPD banding patterns could not distinguish between known clones, they were useful for distinguishing between phenotypically similar cultivars and for assessing the origins of cultivars thought to have originated as sports

Virtual Fencing Predictable for Cattle? A Simple Method to Test Whether and How Fast Cattle Can Learn the Association Between Acoustic Signal and Electric Pulse

Virtual fencing (VF) offers promising future prospects for improved grazing management as it has the potential to simplify fencing. VF lines are easily drawn and shifted via GPS coordinates. A VF collar emits an acoustic signal when the animal approaches the VF line. The signal stops immediately when the animal turns around. If the animal continues to move towards the VF line, a short electric pulse is emitted. A teaching and an operating mode are provided by the VF collars. The animals automatically change mode when they respond correctly to 20 consecutive acoustic signals without receiving an electric pulse. A prerequisite for using the technology is the ability of the grazing animal to learn to predict the electric pulse, therefore we used the time until mode change (from teaching to operating) to evaluate the learning ability and speed of 16 Fleckvieh heifers equally divided into two groups. All heifers were naive to VF prior to the study (conducted 05.07-16.07.2021). On the first day, the two groups were equipped with VF collars (® Nofence, AS, Batnfjordsøra Norway) and assigned to two adjacent pastures. On day eight, the collars were deactivated for a short time and then activated to start in teaching mode again to analyze differences in mode change speed when they were naive to the technology or experienced. The animals remained on the same pasture after reactivation of the collars. We investigated the time to reach theoperating mode (Δ) for each consecutive round (days one and eight) and found a significant difference (p \u3c 0.0001). Average Δ was 49.32 ± 0.41 h and 2.31± 0.41 h for round one and two, respectively. The faster mode change speed of the second round suggested successful learning. Given our study results, cattle learned to predict (and avoid) the electric pulse of VF collars

More Than a Fencing System? Testing the Validity of Virtual Fencing Collars for Animal Monitoring on Pasture

High labour requirements for fencing and animal monitoring appear to be general obstacles for the wider use of pastureland for grazing livestock. Virtual fencing (VF) enables a less laborious pasture management. Fence lines can be easily drawn and moved using GPS data. The advantages of VF for reducing the labour inherent to controlled grazing management are obvious. Potential additional animal monitoring opportunities arising from such a VF system that uses real-time GPS data have not yet been studied. Lying is seen as an indicator for assessing comfort or restlessness of cattle. Therefore, we focus on lying behavior in this evaluation. Based on data from conventional GPS collars, lying and standing often cannot be distinguished. The VF collars (® Nofence, AS, Batnfjordsøra Norway) used in this study detect low movement via an integrated accelerometer and then send the same GPS position during this time of low movement, in order to save battery life. We tested whether this battery life save function could be suitable for the detection of lying behavior. To address this question in a two-step-approach, we first compared observational data with IceTag pedometer (IceRobotics Ltd. Edinburgh, Scotland) data. In the second step, the pedometer data were compared to VF collar data via a confusion matrix. With 93% precision, 89% accuracy and 83% recall in this second step, the use of the VF collars can be recommended for a valid measure of lying behavior monitoring on pasture, which would be an added benefit to VF technology

Rotational Stocking with Virtual Fences: Effects on Grassland Diet Digestibility, Livestock Performance and Stress Levels of Heifers

Virtual fencing is an innovative technology for simplified, less laborious dynamic grazing management and remote animal monitoring. The effect of this novel technology on animal welfare is still a matter of debate. Previous research suggests no differences in stress experience responses of cattle between the two fencing systems on continuously stocked pastures. This study investigated differences in diet organic matter digestibility, livestock performance and fecal cortisol metabolite concentrations of heifers on pastures in a rotational grazing system, fenced with a virtual fence (Nofence, Batnfjordsøra Norway) compared to heifers fenced with a traditional electric wire fence. The study was conducted in 8 weeks from July to September 2021 using 32 heifers divided into four groups allocated to the two fencing systems (two replicates). The experimental pasture of each group was subdivided into four paddocks for rotational grazing. Fecal samples were collected the first day and last day on pasture of each rotation period. Grassland herbage samples were taken by hand plucking pre- and post-grazing and analyzed using near infrared reflectance spectroscopy. Heifers were weighed prior to and after each rotation. The results suggest that no differences occur between the two fencing treatments with respect to diet digestibility, livestock performance or stress level, pointing at no trade-off to livestock performance or animal welfare when using virtual fencing

Compact Saloplastic Poly(Acrylic Acid)/Poly(Allylamine) Complexes: Kinetic Control Over Composition, Microstructure, and Mechanical Properties

Durable compact polyelectrolyte complexes (CoPECs) with controlled porosity and mechanical properties are prepared by ultracentrifugation. Because thestarting materials, poly(allylamine hydrochloride) (PAH) and poly(acrylic acidsodium salt) (PAA), are weak acids/bases, both composition and morphology are controlled by solution pH. In addition, the nonequilibrium nature of polyelectrolyte complexation can be exploited to provide a range of compositions and porosities under the infl uence of polyelectrolyte addition order and speed, and concentration. Confocal microscopy shows these “saloplastic” materials to be highly porous, where pore formation is attributed to a combination of deswelling of the polyelectrolyte matrix and expansion of small inhomogenities by osmotic pressure. The porosity (15–70%) and the pore size ( < 5 μ m to > 70 μ m) of these materials can be tuned by adjusting the PAA to PAH ratio, the salt concentration, and the pH. The modulus of these CoPECs depends on the ratio of the two polyelectrolytes, with stoichiometric complexes being the stiffest due to optimized charge pairing, which correlates with maximized crosslinking density. Mechanical properties, pore sizes, and pore density of these materials make them well suited to three dimensional supports for tissue engineering applications

Functional consequences of seven novel mutations in the CYP11B1 Gene: four mutations associated with nonclassic and three mutations causing classic 11 -Hydroxylase Deficiency

Context: Steroid 11β-hydroxylase (CYP11B1) deficiency (11OHD) is the second most common form of congenital adrenal hyperplasia (CAH). Cases of nonclassic 11OHD are rare compared with the incidence of nonclassic 21-hydroxylase deficiency. Objective: The aim of the study was to analyze the functional consequences of seven novel CYP11B1 mutations (p.M88I, p.W116G, p.P159L, p.A165D, p.K254_A259del, p.R366C, p.T401A) found in three patients with classic 11OHD, two patients with nonclassic 11OHD, and three heterozygous carriers for CYP11B1 mutations. Methods: We conducted functional studies employing a COS7 cell in vitro expression system comparing wild-type (WT) and mutant CYP11B1 activity. Mutants were examined in a computational three-dimensional model of the CYP11B1 protein. Results: All mutations (p.W116G, p.A165D, p.K254_A259del) found in patients with classic 11OHD have absent or very little 11β-hydroxylase activity relative to WT. The mutations detected in patients with nonclassic 11OHD showed partial functional impairment, with one patient being homozygous (p.P159L; 25% of WT) and the other patient compound heterozygous for a novel mild p.M88I (40% of WT) and the known severe p.R383Q mutation. The two mutations detected in heterozygous carriers (p.R366C, p.T401A) also reduced CYP11B1 activity by 23 to 37%, respectively. Conclusion: Functional analysis results allow for the classification of novel CYP11B1 mutations as causative for classic and nonclassic 11OHD, respectively. Four partially inactivating mutations are predicted to result in nonclassic 11OHD. These findings double the number of mild CYP11B1 mutations previously described as associated with mild 11OHD. Our data are important to predict phenotypic expression and provide important information for clinical and genetic counseling i

Effect of mild cortisol cosecretion on body composition and metabolic parameters in patients with primary hyperaldosteronism

Objective To investigate the effects of simultaneous cortisol cosecretion (CCS) on body composition in computed tomography (CT)-imaging and metabolic parameters in patients with primary aldosteronism (PA) with the objective of facilitating early detection. Design Retrospective cohort study. Patients Forty-seven patients with PA and CCS confirmed by 1-mg dexamethasone suppression test (DST) with a cutoff of ≥1.8 µg/dL were compared with PA patients with excluded CCS (non-CCS, n = 47) matched by age and sex. Methods Segmentation of the fat compartments and muscle area at the third lumbar region was performed on non-contrast-enhanced CT images with dedicated segmentation software. Additionally, liver, spleen, pancreas and muscle attenuation were compared between the two groups. Results Mean cortisol after DST was 1.2 µg/dL (33.1 nmol/L) in the non-CCS group and 3.2 µg/dL (88.3 nmol/L) in the CCS group with mild autonomous cortisol excess (MACE). No difference in total, visceral and subcutaneous fat volumes was observed between the CCS and non-CCS group (p = .7, .6 and .8, respectively). However, a multivariable regression analysis revealed a significant correlation between total serum cholesterol and results of serum cortisol after 1-mg DST (p = .026). Classification of the patients based on visible lesion on CT and PA-lateralization via adrenal venous sampling also did not show any significant differences in body composition. Conclusion MACE in PA patients does not translate into body composition changes on CT-imaging. Therefore, early detection of concurrent CCS in PA is currently only attainable through biochemical tests. Further investigation of the long-term clinical adverse effects of MACE in PA is necessary