PCR-RFLP of the ribosomal DNA internal transcribed spacers (ITS) provides markers for the A and B genomes in<Emphasis Type="Italic"> Musa</Emphasis> L.

Musa acuminata Colla (AA genomes) and Musa balbisiana Colla (BB genomes) are the diploid ancestors of modern bananas that are mostly diploid or triploid cultivars with various combinations of the A and B genomes, including AA, AAA, BB, AAB and ABB. The objective of this study was to identify molecular markers that will facilitate discrimination of the A and B genomes, based on restriction-site variations in the internal transcribed spacers (ITS) of the nuclear ribosomal RNA genes. The ITS regions of seven M. acuminata and five M. balbisiana accessions were each amplified by PCR using specific primers. All accessions produced a 700-bp fragment that is equivalent in size to the ITS of most plants. This fragment was then digested with ten restriction enzymes (AluI, CfoI, DdeI, HaeIII, HinfI, HpaII, MspI, RsaI, Sau3AI and TaqI) and fractionated in 2% agarose gels, stained with ethidium bromide and visualized under UV light. The RsaI digest revealed a single 530-bp fragment unique to the A genome and two fragments of 350-bp and 180-bp that were specific to the B genome. A further 56 accessions representing AA, AAA, AAB, AB and ABB cultivars, and synthetic hybrids, were amplified and screened with RsaI. All accessions with an exclusively A genome showed only the 530-bp fragment, while accessions having only the B-genome lacked the 530-bp fragment but had the 350-bp and 180-bp fragments. Interspecific cultivars possessed all three fragments. The staining intensity of the B-genome markers increased with the number of B-genome complements. These markers can be used to determine the genome constitution of Musa accessions and hybrids at the nursery stage, and, therefore, greatly facilitate genome classification in Musa breeding.Communicated by H.F. Linskens     

Analysis of polymorphic microsatellites within the bovine and ovine prion protein (PRNP) genes

Twenty-four microsatellite sites with at least three repeats were found in the bovine prion protein gene (PRNP) and 23 in the ovine PRNP gene. Eight microsatellite sites were polymorphic in cattle and six in sheep with up to 10 alleles per site. In many cases allelic DNA fragments had variants in microsatellite sites and in flanking regions. Distances between microsatellite sites in eight genes from cattle and sheep occurred on average every 0.9 kb. The numerous polymorphic microsatellite sites will improve analysis of phylogenetic origin of different PRNP alleles and trait association studies for bovine spongiform encephalopathy (BSE) and scrapie.     

Preliminary linkage map of the turkey (Meleagris gallopavo) based on microsatellite markers

The turkey is an agriculturally important species for which, until now, there is no published genetic linkage map based on microsatellite markers--still the markers most used in the chicken and other farm animals. In order to increase the number of markers on a turkey genetic linkage map we decided to map new microsatellite sequences obtained from a GT-enriched turkey genomic library. In different chicken populations more than 35-55% of microsatellites are polymorphic. In the turkey populations tested here, 43% of all turkey primers tested were found to be polymorphic, in both commercial and wild type turkeys. Twenty linkage groups (including the Z chromosome) containing 74 markers have been established, along with 37 other unassigned markers. This map will lay the foundations for further genetic mapping and the identification of genes and quantitative trait loci in this economically important species. Genome comparisons, based on genetic maps, with related species such as the chicken would then also be possible. All primer information, polymerase chain reaction (PCR) conditions, allele sizes and genetic linkage maps can be viewed at http://roslin.thearkdb.org/. The DNA is also available on request through the Roslin Institute.     

A cyclophilin gene marker confirming geographical differentiation of Norway spruce populations and indicating viability response on excess soil-born salinity

A newly identified cyclophilin-encoding cDNA clone was used to design a codominant inherited EST-PCR marker in Norway spruce. The study of the current minor polymorphism revealed a geographically structured differentiation pattern across 17 test populations, showing a slight clinal variation south-north through Europe. Based on the frequency of alleles, isolation-by-distance analysis and the Ewens-Watterson test, we conclude that a selectively neutral random-drift mutation recently occurred within the Alpine population group, thus being responsible for the genetic variation detected. Analysis of tolerant and susceptible subsets of two adjacent Bavarian spruce populations affected by soil-born NaCl pollution, revealed that the cyclophilin marker locus also confirms biased genotype frequencies. Considering an unlinked PCR marker of a ribosomal protein-encoding EST clone, deviations between pooled tolerant subsets and pooled sensitive subsets were proven to be more significant for two-locus homozygous genotypes than for each locus alone. We suggest that both loci are linked to adaptive genomic regions. Their potential to test the feasibility of marker-assisted selection of both NaCl-tolerant and drought resistant tree populations is discussed.     

Protective role of zinc in nickel induced hepatotoxicity in rats

This study was planned to determine the protective role of zinc, if any, in attenuating the toxicity induced by nickel sulfate in rat liver. Female Sprague Dawley (SD) rats received either nickel alone in the dose of 800 mg/l in drinking water, zinc alone in the dose of 227 mg/l in drinking water, and nickel plus zinc or drinking water alone for a total duration of eight weeks. The effects of different treatments were studied on various parameters in rat liver which include antioxidant enzymes, levels of nickel and zinc and histoarchitecture at the light microscopic level. Further, the activities of hepatic marker enzymes AST and ALT were also studied in rat serum. Nickel treatment to the normal control animals, resulted in a significant increase in lipid peroxidation and enzyme activities of catalase and glutathione-S-transferase. On the contrary, nickel treatment to normal rats caused a significant inhibition in the levels of reduced glutathione. Superoxide dismutase activity was found to be decreased which however was not significant. Interestingly, when Zn was supplemented to nickel treated rats, the activities of catalase, and glutathione-S-transferase and the levels of GSH and lipid peroxidation came back to within normal limits. Activities of serum AST and ALT were increased significantly following nickel treatment to normal rats. Simultaneous zinc administration to nickel treated rats tended to restore the altered levels of AST and ALT. Normal control and zinc treated animals revealed normal histology of liver. On the other hand, nickel treated animals showed alterations in normal hepatic histoarchitecture which comprise of vacuolization of the hepatocytes and dilatation of sinusoids as well as increase in the number of bi-nucleated cells. Administration of zinc to nickel treated rats resulted in marked improvement in the structure of hepatocytes, thus emphasizing the protective potential of zinc in restoring the altered hepatic histoarchitecture. The nickel administration to normal rats indicated increased concentrations of nickel and decreased concentrations of zinc. However, zinc effectively brought the altered levels of nickel and zinc to within normal range. The study concludes that zinc has the potential in alleviating the toxic effects of nickel in rat liver because of its property to induce metallothionein (S-rich protein) as a free radical scavenger, or its indirect action in reducing the levels of oxygen reactive species.     

Understanding muscle markers: aggregation and construct validity

Musculoskeletal markers are frequently used to reconstruct past lifestyles and activity patterns. Yet, the reliability of muscle marker measurements has been called into question because they allegedly fail to correlate with cross-sectional properties and exercise patterns, and are confounded by body size. In this study, the principle of aggregation was used to sum muscle markers over 7 insertion sites (4 humeral, 2 radial, and 1 ulnar) and examine the effects on them of body size, age, sex, and cross-sectional properties. Analyses were made of a sample of 91 (66 males, 25 females) Native British Columbians (3500-1500 years BP) and 18th century Quebec prisoners. Muscle markers were measured using three-point observer rating scales; size was measured by standard methods; age and sex were determined through pelvic, cranial, and dental morphology; and cross-sectional properties were calculated from radiographs. Whereas any single muscle marker component failed to correlate with age, size, sex, or cross-sections, aggregate muscle marker correlated with: age, r = 0.49; size, r = 0.38; sex, r = 0.40; and, cross-sections, r = 0.38; P < 0.001. Older individuals had greater muscle markers, as did larger individuals, males, and those with more robust cross-sections. Based on partial correlations and regression analyses, age was the best overall predictor of aggregate muscle marker.     

Atmospheric pressure chemical ionization-mass spectrometry method to improve the determination of dansylated polyamines

Determination of polyamine pools is still a step impossible to circumvent in studies aimed at determining the pathophysiological role of natural polyamines. In addition, polyamine measurement in biological fluids and tissues may have clinical relevance, especially in cancer patients. Among the wide panel of analytical methods developed for the quantification of polyamines, high-performance liquid chromatographic (HPLC) separation of polyamines after derivatization with dansyl chloride remains the most commonly used method. In this work, we show that atmospheric pressure chemical ionization-mass spectrometry (MS) can be used to detect and quantify biologically relevant polyamines after dansylation, without chromatographic separation. Positive-ion mass spectra for each dansylated polyamine were generated after optimization by flow injection analysis (FIA). FIA coupled with MS detection by selected ion monitoring greatly increased the sensitivity of the polyamine detection. The method is linear over a wide range of polyamine concentrations and allows detection of quantities as low as 5 fmol. The FIA/MS method is about 50-fold more sensitive than the conventional HPLC/fluorimetry procedure. A good correlation (r>0.98) between these two methods was observed. The FIA/MS method notably reduces the time of analysis per sample to 1.5 min and turns out to be rapid, efficient, cost saving, reproducible, and sufficiently simple to allow its routine application.     

Molecular mapping of a new gene Rrs4 CI 11549 for resistance to barley scald (Rhynchosporium secalis)

Doubled haploid (DH) progeny from a cross between the scald susceptible barley (Hordeum vulgare L.) cultivar Ingrid and the resistant accession CI 11549 (Nigrinudum) was evaluated for resistance in the pathogen Rhynchosporium secalis (Oudem) J.J. Davis. Two linked and incompletely dominant loci confer resistance CI 11549 against isolate 4004. One is an allele at the complex Rrs1 locus on chromosome 3H close to the centromere; the other is located 22 cM distally on the long arm. The latter locus is designated Rrs4. In BC₃-lines into Ingrid from CI 2222 (another Nigrinudum) resistance seems governed by one locus close to the telomeric region of chromosome 7H, probably allelic to Rrs2. In neither case did we find any trace of the recessive gene rh8 reported to be present in Nigrinudum. Various resistance donors of Ethiopian origin designated as Nigrinudum, Jet or Abyssinian were identical to a great extent with respect to markers, but differed in resistance to different isolates of scald or in barley yellow dwarf virus (BYDV) resistance. The implications for their use as differentials in scald tests and screening of germplasm collections are discussed.     

Molecular genecology of temperature response in Lolium perenne: 1. preliminary analysis to reduce false positives

Molecular genecology is the study of geographical clines in frequencies of molecular markers and their relationship to ecological clines in environmental conditions. This study outlines the principles underlying the selection of populations, focusing on avoiding 'false positives'- noncausal correlations between allele frequency and the environment. The principles are illustrated by identifying a set of populations of Lolium perenne for the study of temperature responses. The selected set of populations encompasses a 20 degrees C range in mean January temperature. Their freezing tolerance shows a linear trend with winter temperature, LT50 decreasing by 0.25 degrees C for each 1 degrees C reduction in mean January temperature.     

Molecular genecology of temperature response in Lolium perenne: 2. association of AFLP markers with ecogeography

Improved winter hardiness is an important breeding objective in the forage grass Lolium perenne. This is a complex trait with several components, including the ability to survive and grow at low temperature, to acclimate to cold, tolerate wind, snow cover and ice encasement. Marker-assisted selection has the potential to increase the efficiency of breeding for improved cold tolerance. Here we describe a genecological approach to identifying molecular markers that are associated with adaptation to low winter temperatures. AFLP was used to assess the genetic diversity in 29 wild populations of ryegrass (Lolium perenne) representing a pan-European temperature cline in terms of their geographical origin. A further 18 populations from a temperature cline in Bulgaria were also analysed. In addition, two varieties and five populations representing parents of mapping families currently in use at IGER were included in the analysis. Principal coordinate (PCoA) and cluster analyses of the molecular marker data showed that the Bulgarian altitude cline populations could be distinguished clearly from the other populations. Two regression analyses were carried out; one to identify AFLP markers that correlated in frequency with low mean January temperature of the geographical origin of the population, and another to identify AFLP markers correlating in frequency with the cold tolerance phenotype of the populations, as determined by LT50 values in freezing tests. In the first analysis six AFLP markers showed significant type II trends with mean January temperature, and in the second analysis 28 bands had a significant univariate relationship with the LT50 value of the accessions. In steps 2 and 3 of the stepwise analysis a further 4 and 5 bands, respectively, improved the fit significantly. The results of the two types of regression analysis are discussed in relation to ecogeography and cold tolerance phenotype of the populations.