Implementation of two high through-put techniques in a novel application: detecting point mutations in large EMS mutated plant populations

Background  

The establishment of mutant populations together with the strategies for targeted mutation detection has been applied successfully to a large number of organisms including many species in the plant kingdom. Considerable efforts have been invested into research on tomato as a model for berry-fruit plants. With the progress of the tomato sequencing project, reverse genetics becomes an obvious and achievable goal.     

The serological BHV1 status of dams determines the precolostral status of their calves

Precolostral calves and their dams were serologically investigated for the presence of antibodies against Bovine Herpesvirus 1 in diagnostic tests with a very high sensitivity and specificity. Although the syndesmo-chorial type of placenta of ruminants does not transfer gamma globulins, a large number of calves had antibodies, in most cases in a very low concentration. Significant correlations were found between the serological status of the dam, the status of the calf, and the titre of antibodies. Oral intake of maternal blood by the calf at birth or transmission or leakage of maternal antibodies during pregnancy might be possible causes of precolostrally positive calves. From the results it is concluded that to reduce the risk of obtaining BHV1-positive calves, BHV1-negative dams should be selected for breeding purposes.     

Dispersal of Dictyocaulus viviparus larvae from bovine faeces in Ireland

An involvement of Pilobolus species fungus in the dispersal of Dictyocaulus viviparus third stage larvae from dung to surrounding herbage under Irish conditions was investigated. The presence of Pilobolus kleinii on artificial dung pats containing first stage larvae of D viviparus was associated with a 19-fold increase (P less than 0.05) in numbers of third stage larvae recovered from the surrounding herbage. A subjective examination of natural dung pats showed that the presence of Pilobolus species was significantly correlated with hours of bright sunshine (r = -0.5, P less than 0.01), total rainfall (r = 0.41, P less than 0.05) and the height of herbage surrounding the pats (r = 0.31, P less than 0.001). A multiple regression analysis showed that meteorological parameters and the height of surrounding herbage accounted for 38 per cent of the variation in growth of Pilobolus species on dung pats. The incidence of extensive damage to natural dung pats within five days of deposition, caused by biotic factors, another possible cause of D viviparus third stage larvae dispersal, varied from 0 to 92 per cent of the pats depending on their degree of dryness.     

Effect of Leptin on In Vivo Goat Embryo Production

The aim of this study was to evaluate the effect of leptin administration during superovulation on in vivo goat embryo production. Ten mature does were superovulated with 133 mg follicle‐stimulating hormone (FSH) i.m. in six descending doses at 12‐h intervals. The goats received 4.8 μg/kg human recombinant leptin s.c. (leptin group, n = 5) or phosphate‐buffered saline (PBS) (control group, n = 5) with the first and second FSH doses. The does were mated and subjected to embryo collection by transcervical technique 6 days later. The total number of cells per embryo and the number of cells with fragmented DNA were assessed in selected blastocysts by combining Hoechst 33342 and terminal dUTP nick‐end labelling (TUNEL) staining. Plasma concentrations of oestradiol (E₂) and progesterone (P₄) were determined by electrochemiluminescence from the day of FSH treatment, on the day of superovulatory oestrus and on the day before embryo collection. Compared with the control group, the does that received leptin had a higher number of transferable embryos (p < 0.005), fewer embryos classified as degenerated (p < 0.001) and fewer TUNEL‐positive cells/blastocyst (p < 0.001). The number of transferable embryos was positively correlated with E₂ concentrations on day of oestrus (r = 0.562; p < 0.01) and P₄ concentrations on the day of embryo collection (r = 0.912; p < 0.001). We concluded that in vivo leptin administration during FSH treatment improved embryo quality and affected ovarian steroidogenesis in superovulated goats.     

Aluminium tolerance in wheat: correlating hydroponic evaluations with field and soil performances*

The objective of this study was to correlate root length of wheat (Triticum aestivum L.) genotypes grown in Al-containing, acidic hydroponic solutions, with root weights from acid-soil experiments and field scores from Brazilian acid-field trials. A total of 43 wheat genotypes, primarily from Brazil, were evaluated by growing seedlings for 4 days in hydroponic solutions containing 0.0–4.0 mg/l Al. The root growth rate of all the genotypes was reduced with the addition of Al to the solution and the Al-sensitive and Al-tolerant wheat genotypes were clearly identified. Genotypes with intermediate Al-tolerance levels showed variable root lengths in response to Al stress. Correlations between root length or a root tolerance index (RTI) in the Al solutions versus acid-soil experiments and acid-field trials were highly significant (r = 0.71–0.85, P < 0.01). The most significant correlation was observed among seedlings grown in 1 mg/1 Al. This study presents evidence that this short duration and simple screening technique provides a highly significant correlation with previous acid-soil Al-tolerance evaluations. Furthermore, the data obtained suggest that hydroponic screening of wheat seedlings for Al tolerance may be used in breeding programmes or in screening germplasm collections.     

Estimating the genetic relationship of hybrid bromegrass to smooth bromegrass and meadow bromegrass using RAPD markers

The two most widely grown bromegrass species in North America are smooth bromegrass, a hay type grass, and meadow bromegrass, a pasture type grass. Hybrid populations between these two species have been developed through hybridization and recurrent selection. The objective of this study was to determine the genetic relatedness of the hybrid bromegrass population S‐9073M to its parental populations using random amplified polymorphic DNA (RAPD) markers and to determine genetic variation within and between populations. Individuals from each of the three populations were genotyped at 43 polymorphic RAPD loci. One of the RAPD fragments was meadow bromegrass‐specific. Cluster analysis showed three groups representing the two parental populations and the hybrid population. An analysis of molecular variance (AMOVA), showed that the hybrid population had the highest within‐population variation, followed by meadow bromegrass and smooth bromegrass. The interpopulation genetic distance (phi‐statistic =Φ_st) was highest between meadow and smooth bromegrass and lowest between smooth and hybrid bromegrass. The hybrid population was genetically intermediate to smooth bromegrass and meadow bromegrass, but closer to smooth bromegrass, possibly reflecting the selection criteria used in the development of this hybrid.     

Allelic changes in bread wheat cultivars were associated with long-term wheat trait improvements

Genetic impacts under selective breeding of agricultural crops have been frequently investigated with molecular tools, but inadequate attention has been paid to assess genetic changes under long-term genetic improvement of plant traits. Here we analyzed allelic changes with respect to wheat trait improvement in 78 Canadian hard red spring wheat cultivars released from 1845 to 2004 and screened with 370 mapped SSR markers. The improvements in quality, maturity, yield, disease, stem rust, leaf rust, sawfly resistance, and agronomy were considered. A total of 154 (out of 370) loci with significant allelic changes across 21 chromosomes were detected in the 78 wheat cultivars separated into improved versus non-improved groups for eight traits. The number of significant loci for improving a trait ranged from four for quality to 68 for yield and averaged 35. Many more loci with significant allelic reduction for improving a trait were detected than those with significant allelic increase. Selection for early maturity introduced more alleles, but improving the other traits purged more alleles. Significantly lower numbers of unique alleles were found in the cultivars with improved traits. The distributions of unique allele counts also varied greatly across the 21 chromosomes with respect to trait improvement. Significant SSR variation between two cultivar groups was observed for improvement in seven traits, but not in stem rust. The proportional SSR variation residing between two groups ranged from 0.014 to 0.118. The proportional SSR variations within the improved cultivar groups consistently were much lower than those within the non-improved groups. These findings clearly demonstrate the association between allelic changes and wheat trait improvements and are useful for understanding the genetic modification of the wheat genome by long-term wheat breeding.     

Reduction of particulate air pollution lowers the risk of heritable mutations in mice

Urban and industrial air pollution can cause elevated heritable mutation rates in birds and rodents. The relative importance of airborne particulate matter versus gas-phase substances in causing these genetic effects under ambient conditions has been unclear. Here we show that high-efficiency particulate-air (HEPA) filtration of ambient air significantly reduced heritable mutation rates at repetitive DNA loci in laboratory mice housed outdoors near a major highway and two integrated steel mills. These findings implicate exposure to airborne particulate matter as a principal factor contributing to elevated mutation rates in sentinel mice and add to accumulating evidence that air pollution may pose genetic risks to humans and wildlife.