Distinct glycoforms of human alpha1-acid glycoprotein have comparable synthesis rates: a [13C]valine-labelling study in healthy humans

Various alpha1-acid glycoprotein (AGP) glycoforms are present in plasma differing in extent of branching and/or fucosylation of their 5 N-linked glycans, as well as in concentration. It is assumed that hepatic synthesis determines the relative occurrence of the AGP-glycoforms in plasma, but experimental evidence is lacking. In this study, we have investigated the contribution of fractional synthesis rates to the plasma concentration of AGP-glycoforms that differed in relative occurrence in healthy human plasma. During a [13C]valine infusion, AGP was isolated from the plasma of healthy volunteers. Four AGP-glycoforms, differing strongly in plasma concentration were obtained by sequential affinity chromatography over concanavalin-A- and Aleuria aurantia -agarose columns. The incorporation of the [13C]valine tracer into the AGP-glycoforms was measured by gas chromatography combustion isotope ratio mass spectrometry. The mean fractional synthesis rates of the four AGP-glycoforms did not differ significantly between each other as well between individuals. The results indicated a renewal of about 15%/day of the plasma pools of each of the AGP-glycoforms. This is in support to the assumption that the differences in plasma concentration of the AGP-glycoforms are a reflection of the state of the hepatic glycosylation process.     

Agronomic assessment and 15N recovery of urea amended with the urease inhibitor nBTPT (N-(n-butyl) thiophosphoric triamide) for temperate grassland

Three field experiments were undertaken concurrently at one site to evaluate a range of surface-applied nBTPT-amended urea products (0.01, 0.05, 0.1, 0.25 and 0.5% nBTPT w/w) on NH₃ volatilization, grass yield and ¹⁵N recovery in the plant-soil system. Each experiment was repeated on five separate occasions over the 1992 growing season to cover a range of weather conditions. Total NH₃ loss from unamended-urea ranged from 5.5% in early May to 20.8% in June. The inhibitor was highly effective in reducing ammonia volatilization and delaying the time at which maximum rate of NH₃ loss occurred. Over all time periods the % inhibition was 50.4, 82.8, 89.0, 96.5 and 97.0% at the 0.01, 0.05, 0.1, 0.25 and 0.5% nBTPT levels respectively. There was no significant difference in the overall % inhibition in ammonia loss at different times suggesting that the effectiveness of the inhibitor was not dependent on climatic conditions.Over all times incorporation of nBTPT at the 0.05% level increased dry-matter yield by 9% compared to urea alone and increased the shoot recovery of N from 66.7% to 80.9%. Nitrogen saved from volatilization was taken up by the plant, however, the subsequent translation into dry-matter yield appeared to be adversely affected at the high inhibitor rates.There was no significant effect of inhibitor on ¹⁵N recovery in soil at any depth down to 15 cms. nBTPT significantly increased (p < 0.001) the % N derived from fertilizer (% N dff) in the shoot compared to unamended-urea and increased (p < 0.01) the shoot recovery of ¹⁵N from 32% up to 39%. Total ¹⁵N recovery in the soil-plant system was increased by up to 17% by amending urea with nBTPT. This urease inhibitor has been shown to improve the efficiency of urea however, its potential for the European market will be dependent on economic factors.Faculty of Agriculture and Food Science, The Queen's University of Belfast     

A whole‐genome,radiation hybrid mapping resource of hexaploid wheat

Generating a contiguous, ordered reference sequence of a complex genome such as hexaploid wheat (2n = 6x = 42; approximately 17 GB) is a challenging task due to its large, highly repetitive, and allopolyploid genome. In wheat, ordering of whole‐genome or hierarchical shotgun sequencing contigs is primarily based on recombination and comparative genomics‐based approaches. However, comparative genomics approaches are limited to syntenic inference and recombination is suppressed within the pericentromeric regions of wheat chromosomes, thus, precise ordering of physical maps and sequenced contigs across the whole‐genome using these approaches is nearly impossible. We developed a whole‐genome radiation hybrid (WGRH) resource and tested it by genotyping a set of 115 randomly selected lines on a high‐density single nucleotide polymorphism (SNP) array. At the whole‐genome level, 26 299 SNP markers were mapped on the RH panel and provided an average mapping resolution of approximately 248 Kb/cR₁₅₀₀ with a total map length of 6866 cR₁₅₀₀. The 7296 unique mapping bins provided a five‐ to eight‐fold higher resolution than genetic maps used in similar studies. Most strikingly, the RH map had uniform bin resolution across the entire chromosome(s), including pericentromeric regions. Our research provides a valuable and low‐cost resource for anchoring and ordering sequenced BAC and next generation sequencing (NGS) contigs. The WGRH developed for reference wheat line Chinese Spring (CS‐WGRH), will be useful for anchoring and ordering sequenced BAC and NGS based contigs for assembling a high‐quality, reference sequence of hexaploid wheat. Additionally, this study provides an excellent model for developing similar resources for other polyploid species.     

Purification of the Ah receptor from C57BL/6J mouse liver

The photoaffinity ligand for the Ah receptor, [125I]-2-azido-3-iodo-7,8-dibromodibenzo-p-dioxin, previously has been shown to selectively label two peptides in the cytosol fraction of C57BL/6J mouse liver: a 95-kDa peptide, the ligand binding moiety of the Ah receptor, and a 70-kDa proteolytic fragment formed from the larger peptide (Poland, A., Glover, E., Ebetino, F. H., and Kende, A.S. (1986) J. Biol. Chem. 261, 6352-6365). These two peptides were partially purified to an approximately 20,000-fold enrichment with a 15-20% yield by the following scheme: 1) photoaffinity labeling of the 35-55% ammonium sulfate fraction of liver cytosol; 2) chromatography on polyethyleneimine-Sepharose coupled at low charge density and heparin/Mn2+ precipitation of the dilute column eluate; 3) DEAE-Sepharose chromatography to remove heparin; 4) chromatography on heparin-Sepharose; 5) preparative sodium dodecyl sulfate-polyacrylamide slab gel electrophoresis followed by electroelution of the protein and ion pair extraction to remove sodium dodecyl sulfate; and 6) high performance liquid chromatography on a reverse-phase C-4 column. Following initial chromatography on polyethyleneimine Sepharose, it was found that substantial subsequent purification could only be achieved under denaturing conditions.     

Genotyping-by-sequencing to remap QTL for type II Fusarium head blight and leaf rust resistance in a wheat–tall wheatgrass introgression recombinant inbred population

Fusarium graminearum Schwabe (Fusarium head blight, FHB) and Puccinia triticina Eriks (leaf rust) are two major fungal pathogens posing a continuous threat to the wheat crop; consequently, identifying resistance genes from various sources is always of importance to wheat breeders. We identified tightly linked single nucleotide polymorphism (SNP) markers for the FHB resistance quantitative trait locus (QTL) Qfhs.pur-7EL and the leaf rust resistance locus Lr19 using genotyping-by-sequencing (GBS) in a wheat–tall wheatgrass introgression-derived recombinant inbred line (RIL) population. One thousand and seven hundred high-confidence SNPs were used to conduct the linkage and QTL analysis. Qfhs.pur-7EL was mapped to a 2.9 cM region containing four markers within a 43.6 cM segment of wheatgrass chromosome 7el₂ that was translocated onto wheat chromosome 7DL. Lr19 from 7el₁ was mapped to a 1.21 cM region containing two markers in the same area, in repulsion. Five lines were identified with the resistance-associated SNP alleles for Qfhs.pur-7EL and Lr19 in coupling. Two SNP markers in the Qfhs.pur-7EL region were converted into PCR-based KASP markers. Investigation of the genetic characteristics of the parental lines of this RIL population indicated that they are translocation lines in two different wheat cultivar genetic backgrounds instead of 7E–7D substitution lines in Thatcher wheat background, as previously reported in the literature.     

Construction of a SNP and SSR linkage map in autotetraploid blueberry using genotyping by sequencing

The construction of the first genetic map in autotetraploid blueberry has been made possible by the development of new SNP markers developed using genotyping by sequencing in a mapping population created from a cross between two key highbush blueberry cultivars, Draper × Jewel (Vaccinium corymbosum). The novel SNP markers were supplemented with existing SSR markers to enable the alignment of parental maps.  In total, 1794 single nucleotide polymorphic (SNP) markers and 233 simple sequence repeat (SSR) markers exhibited segregation patterns consistent with a random chromosomal segregation model for meiosis in an autotetraploid. Of these, 700 SNPs and 85 SSRs were utilized for construction of the ‘Draper’ genetic map, and 450 SNPs and 86 SSRs for the ‘Jewel’ map.  The ‘Draper’ map comprises 12  linkage groups (LG), associated with the haploid chromosome number for blueberry, and totals 1621 cM while the ‘Jewel’ map comprises 20 linkage groups totalling 1610 cM. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents. Tentative alignments of the two parental maps have been made on the basis of shared SSR alleles and linkages to double-simplex markers segregating in both parents.     

Finding fathers: spatio-temporal analysis of paternity assignment in grey seals (Halichoerus grypus)

Molecular studies of pinniped breeding systems exhibit a broad range of agreement and disagreement with observational indices of male breeding success. Grey seal studies have reported considerable discrepancies between genetic and behavioural paternity measures that have been interpreted as evidence of previously unidentified male strategies and/or tactics. Therefore, these studies have the power to fundamentally alter our perceptions of mating systems. However, other pinniped studies exhibit no such disagreements, and one possible explanation for disparities may be sampling biases in space and time. Therefore, it is essential that potential sampling biases are examined to evaluate the likelihood of previously unidentified male strategies. We examined paternities assigned at the North Rona grey seal colony between 1999 and 2002 in relation to concurrent detailed behavioural and locational data for males and females. We found that (i) for females observed in sexual interaction(s) during their oestrus period, it was highly probable that one of the interacting males fathered their next pup; (ii) over 80% of assigned paternities agreed with observations of the in-colony behaviour and spatio-temporal proximity of the males and females involved; and (iii) a minority of females exhibit mate choice and seek sires outside their local male's home range, although evidence suggests that these females mate on the colony rather than at sea. In conclusion, nearly all paternities assigned agreed with expectation based upon detailed knowledge of the spatio-temporal patterns of individuals during the breeding season. We found little evidence of unidentified male strategies at North Rona, Scotland, whereas further examination of mechanisms of female choice may be productive.     

Application of Genotyping-by-Sequencing on Semiconductor Sequencing Platforms: A Comparison of Genetic and Reference-Based Marker Ordering in Barley

The rapid development of next-generation sequencing platforms has enabled the use of sequencing for routine genotyping across a range of genetics studies and breeding applications. Genotyping-by-sequencing (GBS), a low-cost, reduced representation sequencing method, is becoming a common approach for whole-genome marker profiling in many species. With quickly developing sequencing technologies, adapting current GBS methodologies to new platforms will leverage these advancements for future studies. To test new semiconductor sequencing platforms for GBS, we genotyped a barley recombinant inbred line (RIL) population. Based on a previous GBS approach, we designed bar code and adapter sets for the Ion Torrent platforms. Four sets of 24-plex libraries were constructed consisting of 94 RILs and the two parents and sequenced on two Ion platforms. In parallel, a 96-plex library of the same RILs was sequenced on the Illumina HiSeq 2000. We applied two different computational pipelines to analyze sequencing data; the reference-independent TASSEL pipeline and a reference-based pipeline using SAMtools. Sequence contigs positioned on the integrated physical and genetic map were used for read mapping and variant calling. We found high agreement in genotype calls between the different platforms and high concordance between genetic and reference-based marker order. There was, however, paucity in the number of SNP that were jointly discovered by the different pipelines indicating a strong effect of alignment and filtering parameters on SNP discovery. We show the utility of the current barley genome assembly as a framework for developing very low-cost genetic maps, facilitating high resolution genetic mapping and negating the need for developing de novo genetic maps for future studies in barley. Through demonstration of GBS on semiconductor sequencing platforms, we conclude that the GBS approach is amenable to a range of platforms and can easily be modified as new sequencing technologies, analysis tools and genomic resources develop.