Characterization of microsatellites and development of chromosome specific STMS markers in bread wheat

Microsatellites, or simple sequence repeats (SSRs), have become the markers of choice for genetic studies with many crop species including wheat. Currently an international effort is underway to enrich the repertoire of available sequence tagged microsatellite site (STMS) markers in wheat. As a part of this effort, we have sequenced 43 clones obtained from a microsatellite-enriched wheat genomic library; 34 clones contained 41 different microsatellites. These microsatellites (mono-, di-, tri- nucleotide repeats) were classified as 19 simple perfect, 18 simple imperfect and 4 compound imperfect types. Dinucleotide repeats were the most abundant (70%). Primer pairs for only 16 microsatellites could be designed, since the flanking sequences of the others were either too short or were otherwise not suitable for designing the microsatellite specific primers. Microsatellite loci of the expected size and polymorphism were successfully amplified from 15 of these 16 primer pairs using three wheat varieties. 14 loci detected by 12 out of the 15 functional primer pairs were assigned to 11 specific chromosomes. An erratum to this article is available at .     

Determinants of variations in coronary revascularization practices

Background:

The ratio of percutaneous coronary interventions to coronary artery bypass graft surgeries (PCI:CABG ratio) varies considerably across hospitals. We conducted a comprehensive study to identify clinical and nonclinical factors associated with variations in the ratio across 17 cardiac centres in the province of Ontario.

Methods:

In this retrospective cohort study, we selected a population-based sample of 8972 patients who underwent an index cardiac catheterization between April 2006 and March 2007 at any of 17 hospitals that perform invasive cardiac procedures in the province. We classified the hospitals into four groups by PCI:CABG ratio (low [< 2.0], low–medium [2.0–2.7], medium–high [2.8–3.2] and high [> 3.2]). We explored the relative contribution of patient, physician and hospital factors to variations in the likelihood of patients receiving PCI or CABG surgery within 90 days after the index catheterization.

Results:

The mean PCI:CABG ratio was 2.7 overall. We observed a threefold variation in the ratios across the four hospital ratio groups, from a mean of 1.6 in the lowest ratio group to a mean of 4.6 in the highest ratio group. Patients with single-vessel disease usually received PCI (88.4%–99.0%) and those with left main artery disease usually underwent CABG (80.8%–94.2%), regardless of the hospital’s procedure ratio. Variation in the management of patients with non-emergent multivessel disease accounted for most of the variation in the ratios across hospitals. The mode of revascularization largely reflected the recommendation of the physician performing the diagnostic catheterization and was also influenced by the revascularization “culture” at the treating hospital.

Interpretation:

The physician performing the diagnostic catheterization and the treating hospital were strong independent predictors of the mode of revascularization. Opportunities exist to improve transparency and consistency around the decision-making process for coronary revascularization, most notably among patients with non-emergent multivessel disease.Large inter-regional and inter-hospital variations exist in the ratio of percutaneous coronary intervention (PCI) procedures to coronary artery bypass graft (CABG) surgeries performed in many countries, but the reasons for these variations are uncertain.^1–3 Bypass surgery was the first method of coronary revascularization to be developed.⁴ The less-invasive alternative of PCI was developed initially to treat single-vessel disease. However, advances in PCI technology (e.g., bare-metal stents and, later, drug-eluting stents) combined with increased operator experience have led to its use for a broader list of indications, including multivessel disease and acute coronary syndromes.^5–7In Ontario, Canada’s most populous province, the overall PCI:CABG ratio has steadily increased, from 1.6 in 2001 to 2.7 in 2006 (unpublished data available from the authors upon request); similar increases have been observed in other jurisdictions.^1,2,8 Although the change in ratio has been driven in part by expanded use of urgent PCI for acute myocardial infarction (MI), increased use of PCI in patients with multivessel disease has likely also been a contributing factor. This application of PCI is more controversial, because several studies, including the recent randomized SYNTAX (Synergy Between Percutaneous Coronary Intervention with TAXUS and Cardiac Surgery) trial, have shown that long-term outcomes of certain patients with multivessel disease were better with CABG surgery than with PCI.^9–13In addition to an overall increase in the PCI:CABG ratio, the amount of variation in the ratio across cardiac centres in Ontario has also steadily increased over time, with more than a threefold regional variation observed in 2006 (unpublished data available from the authors upon request). This degree of variation has raised concerns among some policy-makers and clinicians as to why such striking variations exist in Ontario’s universal health care system. To address this issue, we conducted a comprehensive study to identify clinical and nonclinical factors associated with variations in the PCI:CABG ratio across the province’s 17 cardiac centres.     

Use of methylation filtration and C(0)t fractionation for analysis of genome composition and comparative genomics in bread wheat

We investigated the compositional and structural differences in sequences derived from different fractions of wheat genomic DNA obtained using methylation filtration and C(0)t fractionation. Comparative analysis of these sequences revealed large compositional and structural variations in terms of GC content, different structural elements including repeat sequences (e.g., transposable elements and simple sequence repeats), protein coding genes, and non-coding RNA genes. A correlation between methylation status [determined on the basis of selective inclusion/exclusion in methylation-filtered (MF) library] of different repeat elements and expression level was observed. The expression levels were determined by comparing MF sequences with expressed sequence tags (ESTs) available in the public domain. Only a limited overlap among MF, high C(0)t (HC), and ESTs was observed, suggesting that these sequences may largely either represent the low-copy non-transcribed sequences or include genes with low expression levels. Thus, these results indicated a need to study MF and HC sequences along with ESTs to fully appreciate complexity of wheat gene space.     

Development of large-scale AFLP markers in jute

Amplified fragment length polymorphism (AFLP) analysis was conducted to investigate the level of polymorphism in four jute genotypes including two genotypes (JRC 321 and CMU 010) of Corchorus capsularis (the white jute) and two genotypes (JRO 524 and PPO4) of Corchorus olitorius (the tossa jute). Out of 1024 primer combinations that were tried, as many as 281 combinations of selective primers (13 EcoRI and 64 MseI) were selected, which produced a total of 9092 amplicons, including 752 (8.3%) polymorphic bands in C. capsularis and a total of 8856 amplicons including 1477 (16.7%) polymorphic bands in C. olitorius. The average number of bands/primer combination was 32.3 for C. capsularis and 31.5 for C. olitorius. For C. capsularis, highest polymorphism of 56.6% was shown by primer combination E35M50, while for C. olitorius highest polymorphism of 50% was shown by E41M91. In C. olitorius, 30–50% polymorphism was observed with 27 primer combinations, but in C. capsularis only 3 primer combinations gave this level of polymorphism. Similarly, in C. capsularis <10% polymorphism was detected by 115 primer combinations while in C. olitorius, <10% polymorphism was shown by only 56 primer combinations. These results indicate a higher level of polymorphism in C. olitorius relative to that in C. capsularis. The occurrence of such a large number of polymorphic AFLP markers will facilitate preparation of molecular maps and QTL analysis in jute.     

Orthology between genomes of Brachypodium, wheat and rice

Background

Differential expression of perforin (PRF1), a gene with a pivotal role in immune surveillance, can be attributed to differential methylation of CpG sites in its promoter region. A reproducible method for quantitative and CpG site-specific determination of perforin methylation is required for molecular epidemiologic studies of chronic diseases with immune dysfunction.

Findings

We developed a pyrosequencing based method to quantify site-specific methylation levels in 32 out of 34 CpG sites in the PRF1 promoter, and also compared methylation pattern in DNAs extracted from whole blood drawn into PAXgene blood DNA tubes (whole blood DNA) or DNA extracted from peripheral blood mononuclear cells (PBMC DNA) from the same normal subjects. Sodium bisulfite treatment of DNA and touchdown PCR were highly reproducible (coefficient of variation 1.63 to 2.18%) to preserve methylation information. Application of optimized pyrosequencing protocol to whole blood DNA revealed that methylation level varied along the promoter in normal subjects with extremely high methylation (mean 86%; range 82–92%) in the distal enhancer region (CpG sites 1–10), a variable methylation (range 49%–83%) in the methylation sensitive region (CpG sites 11–17), and a progressively declining methylation level (range 12%–80%) in the proximal promoter region (CpG sites 18–32) of PRF1. This pattern of methylation remained the same between whole blood and PBMC DNAs, but the absolute values of methylation in 30 out of 32 CpG sites differed significantly, with higher values for all CpG sites in the whole blood DNA.

Conclusion

This reproducible, site-specific and quantitative method for methylation determination of PRF1 based on pyrosequencing without cloning is well suited for large-scale molecular epidemiologic studies of diseases with immune dysfunction. PBMC DNA may be better suited than whole blood DNA for examining methylation levels in genes associated with immune function.     

Differential Recognition and Hydrolysis of Host Carbohydrate Antigens by Streptococcus pneumoniae Family 98 Glycoside Hydrolases

The presence of a fucose utilization operon in the Streptococcus pneumoniae genome and its established importance in virulence indicates a reliance of this bacterium on the harvesting of host fucose-containing glycans. The identities of these glycans, however, and how they are harvested is presently unknown. The biochemical and high resolution x-ray crystallographic analysis of two family 98 glycoside hydrolases (GH98s) from distinctive forms of the fucose utilization operon that originate from different S. pneumoniae strains reveal that one enzyme, the predominant type among pneumococcal isolates, has a unique endo-β-galactosidase activity on the Lewis^Y antigen. Altered active site topography in the other species of GH98 enzyme tune its endo-β-galactosidase activity to the blood group A and B antigens. Despite their different specificities, these enzymes, and by extension all family 98 glycoside hydrolases, use an inverting catalytic mechanism. Many bacterial and viral pathogens exploit host carbohydrate antigens for adherence as a precursor to colonization or infection. However, this is the first evidence of bacterial endoglycosidase enzymes that are known to play a role in virulence and are specific for distinct host carbohydrate antigens. The strain-specific distribution of two distinct types of GH98 enzymes further suggests that S. pneumoniae strains may specialize to exploit host-specific antigens that vary from host to host, a factor that may feature in whether a strain is capable of colonizing a host or establishing an invasive infection.Streptococcus pneumoniae asymptomatically colonizes the nasopharynx of 10–40% of people, but given the appropriate opportunity, it can become an extremely aggressive pathogen (1–3). This bacterium causes millions of deaths annually (1), is acquiring antibiotic resistance (4), and shows a disturbing and lethal synergy with the Influenza virus (5). The ability of S. pneumoniae to cause invasive disease is increasingly being linked with the capacity of this bacterium to attack and process the glycans present in host tissues (see Ref. 6 for a review). Indeed, large scale screening of pneumococcal virulence factors has revealed a large complement of genes devoted to complex carbohydrate metabolism that contribute to pneumococcal virulence (7–9). Recent elegant studies have focused on showing how a group of three exo-glycosidases sequentially trim complex human N-glycans (10, 11). These enzymes, however, only make up a fraction of the 39 glycosidases predicted to be in the pneumococcal genome (TIGR4 strain); at least 18 of these 39 are required for full virulence of the bacterium (7). Despite the growing appreciation for the role of carbohydrate metabolism in pneumococcal virulence and the possibility of targeting such metabolic pathways with small molecule therapeutic compounds, the bulk of the carbohydrate-active proteins of S. pneumoniae remain unexamined. As such, we presently have a relatively superficial but growing appreciation for the array of host glycans that S. pneumoniae can degrade.Several S. pneumoniae genes whose protein products are dedicated to the harvesting and processing of the sugar fucose are beginning to emerge as an important set of pneumococcal virulence factors (12). Comparative genomic studies of several S. pneumoniae genomes has suggested genetic variability at this locus; however, some components of the operon were observed to be present in all of the studied isolates (13). Through our recent identification and characterization of a novel solute-binding protein present in an alternate pneumococcal fucose utilization operon, we have made the observation that there are two different fucose utilization operons distributed among pneumococcal strains (14). Although the organization and composition of the two operons is different, both pathways are predicted to be initiated by the action of a family 98 glycoside hydrolase that is probably secreted (for a discussion of the sequence classification system of glycoside hydrolases, see Ref. 15). This GH98 is the same as that identified as a virulence factor in the TIGR4 strain (7). Remarkably, the GH98 enzymes from the two different pathways display different modular architectures, and their shared catalytic modules only have modest amino acid sequence identity. Given the placement of these enzymes in a fucose utilization operon, we hypothesized that they have activity on fucose-containing glycans; however, their divergent sequences and different modular arrangements led us to postulate that they would have different glycan substrate specificities.Here we describe the specificity and catalytic mechanism for these two different types of S. pneumoniae GH98 enzymes, one from the TIGR4 strain (Sp4GH98) and the other from the SP3-BS71 strain (Sp3GH98). Both enzymes act as endo-β-1,4-galactosidases on the galactosyl-β-1,4-N-acetylglucosamine linkage found in type 2 carbohydrate blood group antigens, although Sp4GH98 displays specificity for the Lewis^Y antigen, whereas Sp3GH98 is highly selective for the same linkage in the blood group A/B-antigens. The biochemical analysis of these enzymes in combination with the determination of their structures in complex with products and substrates provides molecular level insight to their catalytic mechanism and how they discriminate between their respective substrates. We discuss these results in the context of the recent association of the pneumococcal fucose utilization operon with the virulence of S. pneumoniae (7, 12) and the possible strain-specific dependence of pneumococcal virulence on the carbohydrate antigens presented by different hosts.     

Validation of QTL for grain weight using MAS-derived pairs of NILs in bread wheat (Triticum aestivum L.)

Near isogenic lines (NILs) are ideal material for a variety of genetic studies including validation of specific QTL. In the present study, eight pairs of NILs for grain weight were developed, seven in the background of Raj3765, and one in the background of K9107. For this purpose, marker-assisted selection (MAS) was used for the transfer of three grain weight QTL (QGw.ccsu-1A.2, QGw.ccsu-1A.3 and QGw.ccsu-1B.1) that were earlier identified in our laboratory. Two genotypes of each of the eight pairs of NILs, differed for QTL alleles (QTL_Hgw derived from the donor parent and the QTL_Lgw derived from the recipient parent). Each pair of NILs involved a solitary QTL except one NIL, which differed for all the three QTL. The difference in thousand grain weight (TGW) in two NILs of an individual pair ranged from 2.8 to 7.5 g, thus validating the effect of the QTL for TGW, although the quantum of difference did not always match the phenotypic variance of the corresponding QTL. As expected, the NILs which involved all the three QTL had the maximum difference of 7.5 g in TGW, and the NILs which involved QTL, QGw.ccsu-1A.2 had minimum average difference of 2.8 g for TGW. The NILs produced during the present study may be used in future for MAS and for fine mapping of TGW QTL.

     

Investigation and assessment of environmental pollution in Gonghu Bay,Taihu Lake,China: A year-long study

Abstract

To understand environmental pollution and assess water quality in Gonghu Bay of Taihu Lake, the eutrophication status and microcystins (MCs) occurrence were monitored monthly from December 2015 to November 2016. Moreover, the pollution of metals and antibiotics was investigated and assessed. In May 2016, the comprehensive trophic level index (∑TLI) at most sampling sites in Gonghu Bay were hypereutrophic, demonstrating that the eutrophication status in Gonghu Bay is still quite intense. The persistent and ubiquitous occurrence of MCs was observed in the water of Gonghu Bay throughout the monitoring period. Moreover, various metal elements and antibiotic compounds were found in the surface water of Gonghu Bay, in which arsenic (As) and sulfamethoxazole (SMX) respectively showed high risks. Overall, the water quality of eastern Gonghu Bay is affected mainly by eutrophication, while the antibiotic pollution level is higher in the southern part of Gonghu Bay. Continuous and long-term monitoring efforts are required to document the tendency of water quality change in Gonghu Bay and other parts of Taihu Lake to improve environmental management works.     

Single-trait,multi-locus and multi-trait GWAS using four different models for yield traits in bread wheat

Molecular Breeding - Rice (Oryza sativa L.) is a saline-alkali-sensitive crop. Saline-alkali environments can seriously affect the growth, development, and yield of rice. The mechanisms of salt...