Karyotype variation is indicative of subgenomic and ecotypic differentiation in switchgrass.

BACKGROUND: Karyotypes can provide information about taxonomic relationships, genetic aberrations, and the evolutionary origins of species. However, differentiation of the tiny chromosomes of switchgrass (Panicum virgatum L.) and creation of a standard karyotype for this bioenergy crop has not been accomplished due to lack of distinguishing features and polyploidy. RESULTS: A cytogenetic study was conducted on a dihaploid individual (2n = 2X = 18) of switchgrass to establish a chromosome karyotype. Size differences, condensation patterns, and arm-length ratios were used as identifying features and fluorescence in-situ hybridization (FISH) assigned 5S and 45S rDNA loci to chromosomes 7 and 2 respectively. Both a maize CentC and a native switchgrass centromeric repeat (PviCentC) that shared 73% sequence identity demonstrated a strong signal on chromosome 3. However, only the PviCentC probe labeled the centromeres of all chromosomes. Unexpected PviCentC and 5S rDNA hybidization patterns were consistent with severe reduction or total deletion of these repeats in one subgenome. These patterns were maintained in tetraploid and octoploid individuals. The 45S rDNA repeat produced the expected number of loci in dihaploid, tetraploid and octoploid individuals. Differences observed at the 5S rDNA loci between the upland and lowland ecotypes of switchgrass provided a basis for distinguishing these subpopulations. CONCLUSION: Collectively, these results provide a quantitative karyotype of switchgrass chromosomes. FISH analyses indicate genetic divergence between subgenomes and allow for the classification of switchgrass plants belonging to divergent genetic pools. Furthermore, the karyotype structure and cytogenetic analysis of switchgrass provides a framework for future genetic and genomic studies.

Phenotypic Examination of Camelina sativa (L.) Crantz Accessions from the USDA-ARS National Genetics Resource Program.

Camelina sativa (L.) Crntz. is a hardy self-pollinated oilseed plant that belongs to the Brassicaceae family; widely grown throughout the northern hemisphere until the 1940s for production of vegetable oil but was later displaced by higher-yielding rapeseed and sunflower crops. However, interest in camelina as an alternative oil source has been renewed due to its high oil content that is rich in polyunsaturated fatty acids, antioxidants as well as its ability to grow on marginal lands with minimal requirements. For this reason, our group decided to screen the existing (2011) National Genetic Resources Program (NGRP) center collection of camelina for its genetic diversity and provide a phenotypic evaluation of the cultivars available. Properties evaluated include seed and oil traits, developmental and mature morphologies, as well as chromosome content. Selectable marker genes were also evaluated for potential use in biotech manipulation. Data is provided in a raw uncompiled format to allow other researchers to analyze the unbiased information for their own studies. Our evaluation has determined that the NGRP collection has a wide range of genetic potential for both breeding and biotechnological manipulation purposes. Accessions were identified within the NGRP collection that appear to have desirable seed harvest weight (5.06 g/plant) and oil content (44.1%). Other cultivars were identified as having fatty acid characteristics that may be suitable for meal and/or food use, such as low (<2%) erucic acid content, which is often considered for healthy consumption and ranged from a high of 4.79% to a low of 1.83%. Descriptive statistics are provided for a breadth of traits from 41 accessions, as well as raw data, and key seed traits are further explored. Data presented is available for public use.

Emergence and spread of azithromycin-resistant Neisseria gonorrhoeae in Scotland.

OBJECTIVES: The aim of this study was to analyse the trend in azithromycin susceptibility (AzDS) of Neisseria gonorrhoeae in Scotland between April 2004 and December 2007, and to characterize isolates exhibiting decreased AzDS or high-level azithromycin resistance (AzHLR). METHODS: Antibiotic susceptibility testing and N. gonorrhoeae multiantigen sequence typing (NG-MAST) were performed on all gonococcal isolates received by the Scottish Bacterial Sexually Transmitted Infections Reference Laboratory (SBSTIRL) during the study period. RESULTS: AzHLR isolates were observed for the first time in 2004 and increased from 0.3% to 3.9% in 2007. AzDS declined from 2.1% to 1.3% in the same period. Taken together, AzDS and AzHLR isolates accounted for 5.2% of the gonococcal infections in Scotland in 2007. NG-MAST revealed that only a small number of sequence types (STs) contained AzHLR and AzDS isolates; these STs also included azithromycin-susceptible isolates. Most STs containing AzHLR isolates were genetically related on the basis of their por and tbpB alleles; however, demographic data suggested that they formed discrete sexual networks. CONCLUSIONS: AzHLR strains of N. gonorrhoeae are increasing in Scotland. A 1 g dose of azithromycin should not be considered as an alternative antibiotic therapy for gonococcal infections. The use of azithromycin to treat chlamydia in patients co-infected with N. gonorrhoeae results in a level of azithromycin in vivo that is sublethal for N. gonorrhoeae, which may lead to resistance.

Rectal chlamydial infection among men who have sex with men: partner notification as a means of nucleic acid amplification test validation.

Nucleic acid amplification tests for the diagnosis of rectal chlamydial infection are not yet licenced. However, the detection of urethral Chlamydia trachomatis in sexual contacts of men who have sex with men (MSM) with rectal chlamydiae might support the validity of such tests. This was a retrospective study of the outcome of partner notification among MSM with rectal chlamydia who consecutively attended a sexually transmitted diseases clinic. During the study period, 5213 MSM attended the Department as new or 'return new' patients. There were 243 (5%) episodes of rectal chlamydial infection. In 87 men, at least one sexual partner was identified and attended the clinic. Overall, 34 (39%) of the 87 evaluable sexual contacts had urethral chlamydial infection. The finding of urethral C. trachomatis infection in a sizeable proportion of sexual contacts of men with rectal chlamydiae lends further support to the validation of the test system for the diagnosis of rectal chlamydia.

Evaluation of the fluorescent treponemal antibody absorption test for detection of antibodies (immunoglobulins G and M) to Treponema pallidum in serologic diagnosis of syphilis.

We compared the fluorescent treponemal antibody-absorption (FTA-ABS) (immunoglobulin (Ig)G + IgM) assay with the (micro-) Treponema pallidum haemagglutination assay (TPHA), the T. pallidum particle agglutination assay (TPPA), the Murex syphilis ICE (ICE) enzyme-linked immunosorbent assay (ELISA), the Diesse Enzywell TP (TP) (ELISA) using 122 serum samples and the Western blot (WB) assay using 42 serum samples whose results were inharmonious with other tests. Additionally, the Captia syphilis-M (IgM) (ELISA) were performed. All sera had already been examined by the rapid plasma reagin (RPR) card test, a non-treponemal test and the TPHA, a treponemal test using routine screening tests. Agreements of the FTA-ABS with the TPHA test, the TPPA test, the ICE test and the TP test were 97.5%, 95.9%, 98.3% and 98.3%, respectively. The results suggest that the FTA-ABS test is a useful confirmatory test, but can be inadequate as a confirmatory test for serologic diagnosis of syphilis by giving equivocal and false-negative results even rarely.

Extended surveillance of gonorrhoea in Scotland 2003.

In 2003, a national surveillance of demographic, behavioural, clinical and laboratory data on gonorrhoea at genitourinary (GU) medicine clinics in Scotland was undertaken. The data-set represented 77% of all gonorrhoea cases. Findings were compared with data reported from England and Wales. Young women (16-19 years) and young men (20-24. years) represented the greatest proportion of heterosexual infections in Scotland (36 and 30%, respectively) and in England and Wales (37 and 32%, respectively). In Scotland (relative to England and Wales), men who have sex with men (MSM) accounted for more of the total gonorrhoea; there were more heterosexuals aged 45+ years; fewer belonged to ethnic minorities; fewer had had gonorrhoea previously; more heterosexual men had a sexual partner abroad; ciprofloxacin resistance was higher. During the year, first-line therapy changed from ciprofloxacin to a third-generation cephalosporin. Extended surveillance for gonorrhoea is vital in guiding appropriately targeted interventions as the epidemiology of gonorrhoea may differ in neighbouring countries.

From the Lab to the Farm: An Industrial Perspective of Plant Beneficial Microorganisms.

Any successful strategy aimed at enhancing crop productivity with microbial products ultimately relies on the ability to scale at regional to global levels. Microorganisms that show promise in the lab may lack key characteristics for widespread adoption in sustainable and productive agricultural systems. This paper provides an overview of critical considerations involved with taking a strain from discovery to the farmer's field. In addition, we review some of the most effective microbial products on the market today, explore the reasons for their success and outline some of the major challenges involved in industrial production and commercialization of beneficial strains for widespread agricultural application. General processes associated with commercializing viable microbial products are discussed in two broad categories, biofertility inoculants and biocontrol products. Specifically, we address what farmers desire in potential microbial products, how mode of action informs decisions on product applications, the influence of variation in laboratory and field study data, challenges with scaling for mass production, and the importance of consistent efficacy, product stability and quality. In order to make a significant impact on global sustainable agriculture, the implementation of plant beneficial microorganisms will require a more seamless transition between laboratory and farm application. Early attention to the challenges presented here will improve the likelihood of developing effective microbial products to improve crop yields, decrease disease severity, and help to feed an increasingly hungry planet.

Sensitivity of microscopy for the rapid diagnosis of gonorrhoea in men and women and the role of gonorrhoea serovars.

Auditing the sensitivity of microscopic diagnosis of gonorrhoea is recommended by the current guidelines. A retrospective study was performed of 596 cases of positive cultures for Neisseria gonorrhoeae in modified New York City culture (MNYC) media diagnosed from 1995 to 1999. The sensitivity of the cervical slides in women was 51% while in men who have sex with men (MSM) the sensitivity of urethral and rectal slides were 89% and 54% respectively. The sensitivity of urethral slides in heterosexual men was 84%. Neisseria serovar 1B02 among MSM and serovar 1B31 among women were mostly undiagnosed with microscopy. Serovars 1A05, 1A21, B08 among heterosexual men were exclusively associated with negative microscopy. Microscopy is important in the rapid detection and treatment of gonorrhoea. Infections with certain serovars are less likely to be detected by microscopy; making them more likely to spread within the community. Culture from different ano-genital sites is essential to maximize detection of gonorrhoea in all patients.

Brachypodium sylvaticum, a model for perennial grasses: transformation and inbred line development.

Perennial species offer significant advantages as crops including reduced soil erosion, lower energy inputs after the first year, deeper root systems that access more soil moisture, and decreased fertilizer inputs due to the remobilization of nutrients at the end of the growing season. These advantages are particularly relevant for emerging biomass crops and it is projected that perennial grasses will be among the most important dedicated biomass crops. The advantages offered by perennial crops could also prove favorable for incorporation into annual grain crops like wheat, rice, sorghum and barley, especially under the dryer and more variable climate conditions projected for many grain-producing regions. Thus, it would be useful to have a perennial model system to test biotechnological approaches to crop improvement and for fundamental research. The perennial grass Brachypodiumsylvaticum is a candidate for such a model because it is diploid, has a small genome, is self-fertile, has a modest stature, and short generation time. Its close relationship to the annual model Brachypodiumdistachyon will facilitate comparative studies and allow researchers to leverage the resources developed for B. distachyon. Here we report on the development of two keystone resources that are essential for a model plant: high-efficiency transformation and inbred lines. Using Agrobacterium tumefaciens-mediated transformation we achieved an average transformation efficiency of 67%. We also surveyed the genetic diversity of 19 accessions from the National Plant Germplasm System using SSR markers and created 15 inbred lines.

Complete chloroplast genome sequences of Mongolia medicine Artemisia frigida and phylogenetic relationships with other plants.

BACKGROUND: Artemisia frigida Willd. is an important Mongolian traditional medicinal plant with pharmacological functions of stanch and detumescence. However, there is little sequence and genomic information available for Artemisia frigida, which makes phylogenetic identification, evolutionary studies, and genetic improvement of its value very difficult. We report the complete chloroplast genome sequence of Artemisia frigida based on 454 pyrosequencing. METHODOLOGY/PRINCIPAL FINDINGS: The complete chloroplast genome of Artemisia frigida is 151,076 bp including a large single copy (LSC) region of 82,740 bp, a small single copy (SSC) region of 18,394 bp and a pair of inverted repeats (IRs) of 24,971 bp. The genome contains 114 unique genes and 18 duplicated genes. The chloroplast genome of Artemisia frigida contains a small 3.4 kb inversion within a large 23 kb inversion in the LSC region, a unique feature in Asteraceae. The gene order in the SSC region of Artemisia frigida is inverted compared with the other 6 Asteraceae species with the chloroplast genomes sequenced. This inversion is likely caused by an intramolecular recombination event only occurred in Artemisia frigida. The existence of rich SSR loci in the Artemisia frigida chloroplast genome provides a rare opportunity to study population genetics of this Mongolian medicinal plant. Phylogenetic analysis demonstrates a sister relationship between Artemisia frigida and four other species in Asteraceae, including Ageratina adenophora, Helianthus annuus, Guizotia abyssinica and Lactuca sativa, based on 61 protein-coding sequences. Furthermore, Artemisia frigida was placed in the tribe Anthemideae in the subfamily Asteroideae (Asteraceae) based on ndhF and trnL-F sequence comparisons. CONCLUSION: The chloroplast genome sequence of Artemisia frigida was assembled and analyzed in this study, representing the first plastid genome sequenced in the Anthemideae tribe. This complete chloroplast genome sequence will be useful for molecular ecology and molecular phylogeny studies within Artemisia species and also within the Asteraceae family.

Comparative analysis of tandem repeats from hundreds of species reveals unique insights into centromere evolution.

BACKGROUND: Centromeres are essential for chromosome segregation, yet their DNA sequences evolve rapidly. In most animals and plants that have been studied, centromeres contain megabase-scale arrays of tandem repeats. Despite their importance, very little is known about the degree to which centromere tandem repeats share common properties between different species across different phyla. We used bioinformatic methods to identify high-copy tandem repeats from 282 species using publicly available genomic sequence and our own data. RESULTS: Our methods are compatible with all current sequencing technologies. Long Pacific Biosciences sequence reads allowed us to find tandem repeat monomers up to 1,419 bp. We assumed that the most abundant tandem repeat is the centromere DNA, which was true for most species whose centromeres have been previously characterized, suggesting this is a general property of genomes. High-copy centromere tandem repeats were found in almost all animal and plant genomes, but repeat monomers were highly variable in sequence composition and length. Furthermore, phylogenetic analysis of sequence homology showed little evidence of sequence conservation beyond approximately 50 million years of divergence. We find that despite an overall lack of sequence conservation, centromere tandem repeats from diverse species showed similar modes of evolution. CONCLUSIONS: While centromere position in most eukaryotes is epigenetically determined, our results indicate that tandem repeats are highly prevalent at centromeres of both animal and plant genomes. This suggests a functional role for such repeats, perhaps in promoting concerted evolution of centromere DNA across chromosomes.

Chloroplast genome variation in upland and lowland switchgrass.

Switchgrass (Panicum virgatum L.) exists at multiple ploidies and two phenotypically distinct ecotypes. To facilitate interploidal comparisons and to understand the extent of sequence variation within existing breeding pools, two complete switchgrass chloroplast genomes were sequenced from individuals representative of the upland and lowland ecotypes. The results demonstrated a very high degree of conservation in gene content and order with other sequenced plastid genomes. The lowland ecotype reference sequence (Kanlow Lin1) was 139,677 base pairs while the upland sequence (Summer Lin2) was 139,619 base pairs. Alignments between the lowland reference sequence and short-read sequence data from existing sequence datasets identified as either upland or lowland confirmed known polymorphisms and indicated the presence of other differences. Insertions and deletions principally occurred near stretches of homopolymer simple sequence repeats in intergenic regions while most Single Nucleotide Polymorphisms (SNPs) occurred in intergenic regions and introns within the single copy portions of the genome. The polymorphism rate between upland and lowland switchgrass ecotypes was found to be similar to rates reported between chloroplast genomes of indica and japonica subspecies of rice which were believed to have diverged 0.2-0.4 million years ago.