Doubled haploidy and induced mutation

Doubled haploid (DH) systems have many attractive features for inducing and fixing mutations. Doubled haploidy provides the fastest route to homozygosity with the greatest fidelity. The ability to fix mutations via doubled haploidy is a key factor, especially as induced mutations␣are predominantly recessive and cannot normally be detected until the M₂ generation at the earliest. The DH systems themselves provide an opportunity to target haploid as well as doubled haploid cells for mutation treatment and capture the mutation in a homozygous, pure line.     

Identification of cultivars and accessions of Lolium, Festuca and Festulolium hybrids through the detection of simple sequence repeat polymorphism

Molecular diversity and genetic affinity in the Lolium/Festuca grass complex have been assessed using simple sequence repeat (SSR) marker technology. The genotypic set was derived from three accessions of perennial ryegrass, two cultivars of Italian ryegrass, two cultivars of meadow fescue, two cultivars of tall fescue and 10 accessions from different intergeneric hybrid (Festulolium) combinations. The majority of the genomic DNA‐derived SSR primer pairs from perennial ryegrass (LPSSR) and Italian ryegrass (LMSSR) produced clear, simple and distinctive amplification products from the majority of the genotypes. The efficiency of cross‐specific amplification for LPSSR markers varied from 38% in meadow fescue to 93% in two cultivars of Festulolium and from 57% in meadow fescue to 87% in Italian ryegrass for LMSSR markers. Of 40 amplified markers, 14 (35%) produced species‐difference alleles in the relation to cultivars used in the present study. Thirty‐five LPSSR locus‐derived alleles were found to be specific to Lolium species, four to meadow fescue and six to tall fescue. For LMSSR alleles, eight were specific to Lolium species and five were only associated with Italian ryegrass, and null alleles were detected for meadow fescue in all instances. These species‐difference markers could clearly identify different accessions of Festulolium. Cluster analysis separated the individual taxa and showed grouping of intergeneric hybrids based on genomic composition. The data distinguished between the species and reflected the known pedigree of the cultivars and the differences between the species. The dendrogram also distinguished between the Festulolium accessions and clearly demonstrated the relations between Festulolium hybrids and their parent species.     

A high-throughput DNA extraction method for barley seed

A non-destructive, quick DNA extraction method for barley seed is described. The method is simple and consists of drilling out a sample from the seed, adding sodium hydroxide, heating in a microwave oven and neutralizing with Tris-HCl. The seed DNA extract can be used directly for PCR with extra cycles added to the PCR programme compared to PCR programmes used for leaf extracts. This protocol was developed in particular for a micro satellite marker genetically linked to barley yellow mosaic virus resistance, but it can be applied toother markers of interest for barley breeding. The quick seed extraction protocol makes it possible to handle thousands of samples per day. Extraction of DNA from seed also facilitates transfer of plant material compared to the long-distance transfer of leaf samples. This revised version was published online in July 2006 with corrections to the Cover Date.     

Effects of serotonin, adrenaline and other vasoactive drugs on the branchial blood vessels of the Antarctic fish Pagothenia borchgrevinki

Myography and an isolated, perfused gill arch preparation were used to investigate the actions of drugs on the gill vasculature of an Antarctic teleost fish, Pagothenia borchgrevinki. Serotonin produced a dose-dependent vasoconstriction of the afferant branchial arteries, the gill arch and the efferent branchial arteries, and was the most potent vasoconstrictor tested. Acetylcholine, at concentrations exceeding 1 × 10^-6M, vasoconstricted the gill arch, but had a negligible effect on the branchial arteries. The responses to adrenaline, with and without the -adrenergic blocking drug, sotalol and the effects of the -adrenergic agonist drug isoprenaline indicated the presence of both -adrenergic and -adrenergic receptors in the gill vasculature, with the vasoconstrictory action of the former predominating in the efferent vasculature. Angiotensin II was without effect in either preparation. The results are a further demonstration of the dominance of vasoconstrictory responses in the control of gill cular resistance in P. borchgrevinki: which has been associated previously with the sub-zero temperatures at which the animals are found.     

Microbial aggregation of sand in a maritime dune succession

Aggregation of sand by microorganisms was assessed in a dune succession ranging from unstable foredunes to stable fixed dunes. The microorganisms isolated from aggregates were fungi, bacteria, actinomycetes and algae, including cyanobacteria. Aggregation increased as the dunes became stabilized and the higher plant succession developed. The number and weight of aggregates were interrelated and most aggregates were in the > 1 <1.4 and > 2 mm ranges. The smaller aggregates were colonized by fewer fungal species than the larger ones. The general aspects of microbial aggregation in sand dunes are discussed.     

Retained surgical swabs in 13 dogs

To report the signalment, history, clinical signs, diagnostic and surgical findings and outcome in dogs and cats with retained swabs, medical records of dogs and cats that were referred to four referral centres were reviewed. Cases in which a retained surgical swab was identified during surgery at the referral hospital were selected for further study. The signalment, history, clinical signs, diagnostic and surgical findings and outcome in these cases were reviewed. Thirteen dogs with retained surgical swabs were included in the study. Bodyweight ranged from 9 to 45 kg. The initial surgery was a 'non-routine' abdominal procedure in seven dogs, ovariohysterectomy in five dogs and perineal hernia repair in one dog. Time from initial surgery to presentation at the referral centre ranged from four days to seven years. Treatment involved major surgery in eight of the 13 dogs. Outcome was excellent in 12 of 13 cases, one dog was euthanased.     

Standardisation of a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole in Fasciola hepatica

A sheep trial was performed to standardise a coproantigen reduction test (CRT) protocol for the diagnosis of resistance to triclabendazole (TCBZ) in Fasciola hepatica). The CRT employs the BIO K201 Fasciola coproantigen ELISA (Bio-X Diagnostics, Jemelle, Belgium) to test for the presence of F. hepatica coproantigens in a faecal sample. If it is coproantigen-positive, the CRT protocol recommends that faecal samples are re-tested for coproantigens at 14 days post-treatment (dpt), with negative testing at this point indicating TCBZ success. Initial work aimed to confirm the sensitivity of the BIO K201 ELISA for Fasciola infection and investigate whether coproantigens represent a robust reduction marker of TCBZ efficacy. Thirty-eight, indoor-reared sheep were artificially infected with F. hepatica isolates known to be susceptible (Cullompton) and resistant (Sligo) to TCBZ action, respectively. Treatment was administered at 12 weeks post-infection (wpi), with 2 sheep groups, infected with each isolate, culled at 2 and 4 weeks post-treatment (wpt), respectively. Necropsy was performed to confirm treatment efficacy. Individual faecal samples were collected twice-weekly throughout the trial period. Additional work focused on the effect of temperature on faecal sample collection and storage. Faecal samples collected from sheep positive for F. hepatica infection were sub-sampled and left at room temperature. Individual sub-samples were tested by ELISA on consecutive days and these readings compared to the original test result on the day of collection. In addition, ELISA values were compared between faecal sub-samples prepared on the day of sampling and post storage at -20°C. Also, an immunocytochemical study was performed to determine the tissue site of origin of the coproantigen protein in the fluke. Results showed that the BIO K201 ELISA was sensitive for Fasciola coproantigens, with coproantigens detectable from 5 wpi onwards. The suitability of coproantigens as a diagnostic marker of TCBZ efficacy was supported by the absence and presence of coproantigens in TCBZ-treated Cullompton (TCBZ-susceptible) and Sligo (TCBZ-resistant) F. hepatica infections at 2 and 4 wpt, respectively. Study results suggest that low to moderate temperature has little, if any, impact on coproantigen stability in faecal samples, but that higher temperatures may have. Immunolabelling for the coproantigen showed that it was specific to the gastrodermal cells of both adult and juvenile flukes.     

Effects of high-intensity exercise on plasma catecholamines in the thoroughbred horse.

In Study 1, a single speed test of 6 to 12 m/sec was performed for 2 mins at an incline of 5 degrees on a high-speed treadmill (single-step test). Only one speed was performed per session and blood samples were taken before and after the test. In Study 2 horses cantered for 1 min at increasing speeds of 6 to 13 m/sec on an incline of 3 degrees (multiple-step test). Blood samples were taken before exercise, throughout the test and during recovery. In the single-step test plasma concentrations of adrenaline and noradrenaline both increased at speeds of 9 m/sec, as did blood lactate. Mean concentrations of adrenaline and noradrenaline at the end of the 12 m/sec test were 153 and 148 nmol/litre, respectively. Plasma concentrations were similar over all speeds although there was a tendency for the increase in noradrenaline to be greater than that of adrenaline at the lower speeds. The multiple-step test resulted in smaller increases in both adrenaline and noradrenaline. Although again closely correlated, increases in adrenaline were 20-30% greater than those for noradrenaline. In both exercise models, changes in plasma adrenaline and noradrenaline values with exercise showed an exponential relationship to plasma lactate. A plasma half-life of less than 30 secs was indicated during recovery from the multiple-step test. Changes in adrenaline and noradrenaline were much greater than previously recorded in man and emphasise the importance of catecholamines in mediating the physiological response of the horse to exercise.     

Effects of supplemental alfalfa hay on feed intake and digestion by Holstein steers consuming high-quality bermudagrass or orchardgrass hay

Effects of level and frequency of supplementation with alfalfa (A) on feed intake and digestion by steers fed bermudagrass (B) or orchardgrass (O) were determined in two Latin square experiments. In Exp. 1, six Holstein steers (224 kg) were fed B (2.25% N; 71.4% NDF) or O (2.52% N; 64.3% NDF) with 0, 15 or 30% (DM) A (2.70% N; 44.0% NDF). Total DMI was 2.43, 2.72 and 2.85% BW for B and 2.98, 3.00 and 2.87% BW for O with 0, 15 and 30% A, respectively. Total DMI was affected by forage (P less than .05), A level (linear; P less than .06) and a forage x A level (linear) interaction (P less than .05). Digestible OM intake increased .42 (15%) and .67 kg (24%) with feeding of 15 and 30% A, respectively, for B, but for O, only dietary inclusion of 30% A elevated digestible OM intake (.14 kg and 4% increases). In Exp. 2, five Holstein steers (165 kg) were fed B (1.81% N; 78.6% NDF) alone or with A (2.76% N; 52.8% NDF). Morning meals consisted of ad libitum B (OA), .3% BW of A daily (.3A), .6% BW of A every 2nd d (.6A), .9% BW of A every 3rd d (.9A) or 1.2% BW of A every 4th d (1.2A). All steers received B in the afternoon ad libitum, and B was given in the morning when A was not fed. Total DMI was 2.31, 2.12, 2.12, 2.26 and 2.29% BW for OA, .3A, .6A, .9A and 1.2A, respectively (SE .049). Grass characteristics affected response in feed intake to legume supplementation. Frequency of dietary legume addition may alter feed intake.