The complex polyploid genome architecture of sugarcane.

Sugarcane, the world's most harvested crop by tonnage, has shaped global history, trade and geopolitics, and is currently responsible for 80% of sugar production worldwide1. While traditional sugarcane breeding methods have effectively generated cultivars adapted to new environments and pathogens, sugar yield improvements have recently plateaued2. The cessation of yield gains may be due to limited genetic diversity within breeding populations, long breeding cycles and the complexity of its genome, the latter preventing breeders from taking advantage of the recent explosion of whole-genome sequencing that has benefited many other crops. Thus, modern sugarcane hybrids are the last remaining major crop without a reference-quality genome. Here we take a major step towards advancing sugarcane biotechnology by generating a polyploid reference genome for R570, a typical modern cultivar derived from interspecific hybridization between the domesticated species (Saccharum officinarum) and the wild species (Saccharum spontaneum). In contrast to the existing single haplotype ('monoploid') representation of R570, our 8.7 billion base assembly contains a complete representation of unique DNA sequences across the approximately 12 chromosome copies in this polyploid genome. Using this highly contiguous genome assembly, we filled a previously unsized gap within an R570 physical genetic map to describe the likely causal genes underlying the single-copy Bru1 brown rust resistance locus. This polyploid genome assembly with fine-grain descriptions of genome architecture and molecular targets for biotechnology will help accelerate molecular and transgenic breeding and adaptation of sugarcane to future environmental conditions.

Association of gene expression with syringyl to guaiacyl ratio in sugarcane lignin.

KEY MESSAGE: A transcriptome analysis reveals the transcripts and alleles differentially expressed in sugarcane genotypes with contrasting lignin composition. Sugarcane bagasse is a highly abundant resource that may be used as a feedstock for the production of biofuels and bioproducts in order to meet increasing demands for renewable replacements for fossil carbon. However, lignin imparts rigidity to the cell wall that impedes the efficient breakdown of the biomass into fermentable sugars. Altering the ratio of the lignin units, syringyl (S) and guaiacyl (G), which comprise the native lignin polymer in sugarcane, may facilitate the processing of bagasse. This study aimed to identify genes and markers associated with S/G ratio in order to accelerate the development of sugarcane bioenergy varieties with modified lignin composition. The transcriptome sequences of 12 sugarcane genotypes that contrasted for S/G ratio were compared and there were 2019 transcripts identified as differentially expressed (DE) between the high and low S/G ratio groups. These included transcripts encoding possible monolignol biosynthetic pathway enzymes, transporters, dirigent proteins and transcriptional and post-translational regulators. Furthermore, the frequencies of single nucleotide polymorphisms (SNPs) were compared between the low and high S/G ratio groups to identify specific alleles expressed with the phenotype. There were 2063 SNP loci across 787 unique transcripts that showed group-specific expression. Overall, the DE transcripts and SNP alleles identified in this study may be valuable for breeding sugarcane varieties with altered S/G ratio that may provide desirable bioenergy traits.

A novel highly differentially expressed gene in wheat endosperm associated with bread quality.

Analysis of gene expression in developing wheat seeds was used to identify a gene, wheat bread making (wbm), with highly differential expression (~1000 fold) in the starchy endosperm of genotypes varying in bread making quality. Several alleles differing in the 5'-upstream region (promoter) of this gene were identified, with one present only in genotypes with high levels of wbm expression. RNA-Seq analysis revealed low or no wbm expression in most genotypes but high expression (0.2-0.4% of total gene expression) in genotypes that had good bread loaf volume. The wbm gene is predicted to encode a mature protein of 48 amino acids (including four cysteine residues) not previously identified in association with wheat quality, possibly because of its small size and low frequency in the wheat gene pool. Genotypes with high wbm expression all had good bread making quality but not always good physical dough qualities. The predicted protein was sulphur rich suggesting the possibility of a contribution to bread loaf volume by supporting the crossing linking of proteins in gluten. Improved understanding of the molecular basis of differences in bread making quality may allow more rapid development of high performing genotypes with acceptable end-use properties and facilitate increased wheat production.

Betaine aldehyde dehydrogenase in plants.

Plant betaine aldehyde dehydrogenases (BADHs) have been the target of substantial research, especially during the last 20 years. Initial characterisation of BADH as an enzyme involved in the production of glycine betaine (GB) has led to detailed studies of the role of BADH in the response of plants to abiotic stress in vivo, and the potential for transgenic expression of BADH to improve abiotic stress tolerance. These studies have, in turn, yielded significant information regarding BADH and GB function. Recent research has identified the potential for BADH as an antibiotic-free marker for selection of transgenic plants, and a major role for BADH in 2-acetyl-1-pyrroline-based fragrance associated with jasmine and basmati style aromatic rice varieties.

Robust allele-specific polymerase chain reaction markers developed for single nucleotide polymorphisms in expressed barley sequences.

Many methods have been developed to assay for single nucleotide polymorphisms (SNPs), but generally these depend on access to specialised equipment. Allele-specific polymerase chain reaction (AS-PCR) is a method that does not require specialised equipment (other than a thermocycler), but there is a common perception that AS-PCR markers can be unreliable. We have utilised a three primer AS-PCR method comprising of two flanking-primers combined with an internal allele-specific primer. We show here that this method produces a high proportion of robust markers (from candidate allele specific primers). Forty-nine inter-varietal SNP sites in 31 barley (Hordeum vulgare L.) genes were targeted for the development of AS-PCR assays. The SNP sites were found by aligning barley expressed sequence tags from public databases. The targeted genes correspond to cDNAs that have been used as restriction fragment length polymorphic probes for linkage mapping in barley. Two approaches were adopted in developing the markers. In the first approach, designed to maximise the successful development of markers to a SNP site, markers were developed for 18 sites from 19 targeted (95% success rate). With the second approach, designed to maximise the number of markers developed per primer synthesised, markers were developed for 18 SNP sites from 30 that were targeted (a 60% success rate). The robustness of markers was assessed from the range of annealing temperatures over which the PCR assay was allele-specific. The results indicate that this form of AS-PCR is highly successful for the development of robust SNP markers.

Single nucleotide polymorphism, haplotype diversity and recombination in the Isa gene of barley.

The Isa gene from barley--an intronless gene expressed in maternal tissues of the seed--has a likely role in defence against pathogens. The protein product--bi-functional alpha-amylase/subtilisin inhibitor--inhibits the seed's own amylase in addition to the bacterial protease subtilisin and fungal xylanase. Sixteen barley genotypes were targeted to amplify and sequence the Isa gene region to detect sequence polymorphisms, since little is known about genetic diversity at this locus. A total of 80 single nucleotide polymorphisms (SNPs) and 23 indels were detected in 2,164 bp of sequence containing the Isa transcript, promoter and 3' non-transcribed region (overall one SNP per 27 bp and one indel per 94 bp), with eight sequence-based haplotypes distinguishable amongst the 16 varieties. Sequencing a polymorphic region in the promoter in an additional 27 barley genotypes increased the number of sequence-based haplotypes discovered to 11. However there is low haplotype diversity amongst the cultivated barley varieties sampled, with most varieties represented by a single haplotype. There was minor amino acid diversity in the protein, with five out of ten SNP sites in the coding region predicted to produce amino acid substitutions. SNP analysis indicated a history of recombination events--a minimum of seven based on the initial eight haplotypes from the whole sequenced region. Most of the recombination events occurred in the highly polymorphic regions, the 3' non-transcribed region and sequences flanking a microsatellite in the Isa promoter.

Single nucleotide polymorphisms in cytochrome P450 genes from barley.

Plant cytochrome P450s are known to be essential in a number of economically important pathways of plant metabolism but there are also many P450s of unknown function accumulating in expressed sequence tag (EST) and genomic databases. To detect trait associations that could assist in the assignment of gene function and provide markers for breeders selecting for commercially important traits, detection of polymorphisms in identified P450 genes is desirable. Polymorphisms in EST sequences provide so-called perfect markers for the associated genes. The International Triticeae EST Cooperative data base of 24,344 ESTs was searched for sequences exhibiting homology to P450 genes representing the nine known clans of plant P450s. Seventy five P450 ESTs were identified of which 24 had best matches in Genbank to P450 genes of known function and 51 to P450s of unknown function. Sequence information from PCR products amplified from the genomic template DNA of 11 barley varieties was obtained using primers designed from six barley P450 ESTs and one durum wheat P450 EST. Single nucleotide polymorphisms (SNPs) between barley varieties were identified using five of the seven PCR products. A maximum of five SNPs and three haplotypes among the 11 barley lines were detected in products from any one primer pair. SNPs in three PCR products led to changes between barley varieties in at least one restriction site enabling genotyping and mapping without the expense of a specialist SNP detection system. The overall frequency of SNPs across the 11 barley varieties was 1 every 131 bases.

Evaluating the potential of SSR flanking regions for examining taxonomic relationships in the Vitaceae.

Three EST-derived microsatellite loci from Vitis vinifera were amplified and sequenced across eight species of Vitaceae from four different genera. Phylogenetic analysis of the microsatellite's flanking regions produced informative results in congruence with previous studies. Generic relationships were respected and the data produced sufficient inter-specific variation to distinguish between Cayratia acris and Cayratia saponaria, two very closely related species. Overall, the sequence alignments showed that priming sites were conserved, whereas microsatellite repeats were present in most cases but structurally variable. The sequence data provided information on the evolutionary patterns of various microsatellite repeats and their correlation to evolutionary relationships among taxa.

Microsatellite markers from sugarcane (Saccharum spp.) ESTs cross transferable to erianthus and sorghum.

Analysis of a sugarcane (Saccharum spp.) EST (expressed sequence tag) library of 8678 sequences revealed approximately 250 microsatellite or simple sequence repeats (SSRs) sequences. A diversity of dinucleotide and trinucleotide SSR repeat motifs were present although most were of the (CGG)(n) trinucleotide motif. Primer sets were designed for 35 sequences and tested on five sugarcane genotypes. Twenty-one primer pairs produced a PCR product and 17 pairs were polymorphic. Primer pairs that produced polymorphisms were mainly located in the coding sequence with only a single pair located within the 5' untranslated region. No primer pairs producing a polymorphic product were found in the 3' untranslated region. The level of polymorphism (PIC value) in cultivars detected by these SSRs was low in sugarcane (0.23). However, a subset of these markers showed a significantly higher level of polymorphism when applied to progenitor and related genera (Erianthus sp. and Sorghum sp.). By contrast, SSRs isolated from sugarcane genomic libraries amplify more readily, show high levels of polymorphism within sugarcane with a higher PIC value (0.72) but do not transfer to related species or genera well.

Microsatellite variation and assessment of genetic structure in tea tree (Melaleuca alternifolia-Myrtaceae).

Analysis of five microsatellite loci in 500 Melaleuca alternifolia individuals produced 98 alleles that were useful for population genetic studies. Considerable levels of observed heterozygosity were recorded (HO = 0.724), with approximately 90% of the variability being detected within populations. A low level of selfing (14%) was suggested to be the principal cause of excess homozygosity in a number of populations (overall FIS = 0.073). This study showed low levels of inbreeding in certain populations as well as a significant isolation-by-distance model. Only two groups of populations (Queensland and New South Wales) constituted different genetic provenances as a result of geographical isolation. The M. alternifolia data suggest that microsatellite loci did not always arise by a stepwise mutation process but that larger jumps in allele size may be involved in their evolution.

A pharmacokinetic study of midazolam in dogs: nasal drop vs. atomizer administration.

PURPOSE: The purpose of this investigation was to compare the pharmacokinetics of midazolam following intravenous, intranasal drop, and nasal-atomizer administration in beagle dogs. METHODS: Six animals weighing 9-13 kg were used in a repeated-measure design, group assignment based on route of drug administration. Midazolam (1.5 mg/kg) was administered with the delivery route based on group assignment. Blood samples were obtained at baseline and at 1, 3, 5, 7, 10, 15, 20, 30, and 45 min after administration. Cerebrospinal fluid samples (CSF) were obtained at 5 and 10 min after administration. Plasma and CSF concentrations of midazolam were determined by electron-capture gas-liquid chromatography. RESULTS: Comparison between groups and over time demonstrated that both nasal routes resulted in significantly higher CSF concentrations relative to corresponding plasma levels, and that nasal-atomizer administration produced significantly higher CSF concentrations compared to the drop approach.

The influence of meperidine and nitrous oxide on respiratory depression in rats.

Narcotic sedation is commonly accomplished with nitrous oxide (N2O) coadministration. Concerns regarding respiratory morbidity and mortality with drug combinations have been reported in the literature, particularly in patients not receiving supplemental oxygen (O2). The purpose of this investigation was to determine the effect of meperidine alone and in combination with N2O on respiration in laboratory rats by evaluating cardiovascular and arterial blood gas data. Fifty-four Sprague-Dawley rats were assigned to one of six groups (nine per group). Groups were allocated based upon the dosage of meperidine administered (0, 4.0, or 8.0 mg/kg intraperitoneally [i.p.]) and exposure to N2O (50% with oxygen) or O2 (100%). Following meperidine administration, animals were placed into a sealed chamber through which flowed either N2O or O2. Arterial blood was obtained, at baseline and at 15-min intervals, from a femoral artery catheter and pH, O2, CO2 (mm Hg), and oxygen saturation (%) were determined. Plasma samples were analyzed using a System 1306 pH/blood gas analyzer. Group comparisons demonstrated that: (a) N2O coadministration, in animals pretreated with meperidine, did not result in increased arterial CO2 levels, and (b) as expected, arterial O2 levels in all groups increased significantly from preexposure baseline values (P < 0.05). This investigation demonstrated that the coadministration of N2O to meperidine-sedated animals did not enhance respiratory depression.

Talon cusp with associated adjacent supernumerary tooth.

Talon cusp may occur with other dental anomalies. A case is reported in which talon cusp is associated with a supernumerary tooth, suggesting genetic inheritance as a causative factor.

Dental management of children with asthma.

Asthma affects about 1 in 10 children. The condition is characterized by acute respiratory distress brought on by environmental factors. The condition is treated with medications aimed to reduce reaction to stimulants by the airway. Dental management involves attention to the status of the patient and awareness of stimulants of the reactive airway. Clinical recommendations are provided.

The influence of midazolam and nitrous oxide on respiratory depression in laboratory rats.

Midazolam in combination with nitrous oxide (N2O) is a commonly used sedative approach for pediatric dental patients. Respiratory morbidity and mortality have been reported with midazolam administration, particularly when used in combination with other drugs in the absence of supplemental oxygen. Thus, the purpose of this investigation was to determine the effect of midazolam alone and in combination with N2O on respiration in laboratory rats by measuring arterial blood gas levels. Sixty-four Sprague-Dawley rats, weighing 250-320 g, were assigned to one of eight groups (eight per group). Groups were allocated based upon the dosage of midazolam administered (0, 1.0, 2.0 or 4.0 mg/kg i.p.) and exposure to N2O/02 (50%/50%) or room air. Arterial blood was obtained from a femoral artery catheter and pH, O2, CO2 (mm Hg), and oxygen saturation (%) were determined. Samples were analyzed using a System 1306 pH/Blood Gas Analyzer. Baseline arterial blood gasses were obtained for each animal and at 15-min intervals following midazolam administration throughout the 45-min experiment. Following midazolam administration, animals were placed into a sealed chamber through which flowed either N2O or room air. Group comparisons demonstrated that: 1) arterial CO2 levels increased in midazolam-exposed animals breathing room air, but not in those exposed to N2O (P < 0.05), and 2) as expected, N2O/O2-exposed animals showed an increase in arterial O2 and a %saturation that was not observed in room air groups (P < 0.01). This investigation demonstrated that coadministration of N2O/O to midazolam-exposed animals did not result in hypercarbia, an early indicator of respiratory depression.

Supernumerary and congenitally absent teeth: a literature review.

This review article examines supernumerary teeth as to prevalence, location, clinical and radiographic appearance, etiology, complications and management of supernumerary teeth; congenital absence of teeth as to prevalence and location, clinical features and complications, etiology, treatment considerations, and coexistance of supernumeraries and congenital absence of teeth.