Transcriptome analysis of Neotyphodium and Epichloë grass endophytes

Large-scale gene discovery has been performed for the grass fungal endophytes Neotyphodium coenophialum, Neotyphodium lolii, and Epichloë festucae. The resulting sequences have been annotated by comparison with public DNA and protein sequence databases and using intermediate gene ontology annotation tools. Endophyte sequences have also been analysed for the presence of simple sequence repeat and single nucleotide polymorphism molecular genetic markers. Sequences and annotation are maintained within a MySQL database that may be queried using a custom web interface. Two cDNA-based microarrays have been generated from this genome resource. They permit the interrogation of 3806 Neotyphodium genes (NchipTM microarray), and 4195 Neotyphodium and 920 Epichloë genes (EndoChipTM microarray), respectively. These microarrays provide tools for high-throughput transcriptome analysis, including genome-specific gene expression studies, profiling of novel endophyte genes, and investigation of the host grass–symbiont interaction. Comparative transcriptome analysis in Neotyphodium and Epichloë was performed

Temporal expression of G-protein-coupled receptor 54 (GPR54), gonadotropin-releasing hormones (GnRH), and dopamine receptor D2 (drd2) in pubertal female grey mullet, Mugil cephalus.

The G-protein-coupled receptor 54 (muGPR54) cDNA was cloned from the brain of the grey mullet, and its expression level, as well as those of the gonadotropin-releasing hormones (GnRH1, GnRH2, GnRH3) and dopamine receptor D2 (drd2), in the brain, pituitary and ovary of pubertal fish (early, intermediate, advanced) were determined by real-time quantitative RT-PCR (QPCR). The muGPR54 cDNA has an open reading frame of 1140 bp with a predicted 380 amino acid peptide, containing seven putative transmembrane domains and putative N-glycosylation and protein kinase C phosphorylation sites. QPCR results showed that the early stage of puberty in grey mullet is characterized by significantly high levels of expression of GPR54, GnRH and drd2 in the brain relative to the intermediate and advanced stages, except for GnRH1 that increased at the advanced stage of puberty. In the pituitary, drd2 expression declined significantly at the advanced stage relative to levels at the intermediate stage. Ovarian expression of GPR54 significantly increased from the intermediate stage of puberty relative to the early stage while that of GnRH1 acutely increased at the advanced stage of puberty. The ovarian expression of drd2 decreased as puberty progressed, but the changes were not significant. The results suggest the possible role of GPR54 and GnRH in positively regulating pubertal development in grey mullet and the dopaminergic inhibition of reproductive function mediated by drd2.

Measurement of genetic and environmental variation in barley (Hordeum vulgare) grain hardness

In this study, we assessed a broad range of barley breeding lines and commercial varieties by three hardness methods (two particle size methods and one crush resistance method (SKCS—Single-Kernel Characterization System), grown at multiple sites to see if there was variation in barley hardness and if that variation was genetic or environmentally controlled. We also developed near-infrared reflectance (NIR) calibrations for these three hardness methods to ascertain if NIR technology was suitable for rapid screening of breeding lines or specific populations. In addition, we used this data to identify genetic regions that may be associated with hardness. There were significant (p<0.05) genetic effects for the three hardness methods. There were also environmental effects, possibly linked to the effect of protein on hardness, i.e. increasing protein resulted in harder grain. Heritability values were calculated at >85% for all methods. The NIR calibrations, with R2 values of >90%, had Standard Error of Prediction values of 0.90, 72 and 4.0, respectively, for the three hardness methods. These equations were used to predict hardness values of a mapping population which resulted in genetic markers being identified on all chromosomes but chromosomes 2H, 3H, 5H, 6H and 7H had markers with significant LOD scores. The two regions on 5H were on the distal end of both the long and short arms. The region that showed significant LOD score was on the long arm. However, the region on the short arm associated with the hardness (hordoindoline) genes did not have significant LOD scores. The results indicate that barley hardness is influenced by both genotype and environment and that the trait is heritable, which would allow breeders to develop very hard or soft varieties if required. In addition, NIR was shown to be a reliable tool for screening for hardness. While the data set used in this study has a relatively low variation in hardness, the tools developed could be applied to breeding populations that have large variation in barley grain hardness.

Isolation and characterization of novel microsatellite markers and their application for diversity assessment in cultivated groundnut (Arachis hypogaea)

Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is the fourth most important oilseed crop in the world, grown mainly in tropical, subtropical and warm temperate climates. Due to its origin through a single and recent polyploidization event, followed by successive selection during breeding efforts, cultivated groundnut has a limited genetic background. In such species, microsatellite or simple sequence repeat (SSR) markers are very informative and useful for breeding applications. The low level of polymorphism in cultivated germplasm, however, warrants a need of larger number of polymorphic microsatellite markers for cultivated groundnut. Results: A microsatellite- enriched library was constructed from the genotype TMV2. Sequencing of 720 putative SSR-positive clones from a total of 3,072 provided 490 SSRs. 71.2% of these SSRs were perfect type, 13.1% were imperfect and 15.7% were compound. Among these SSRs, the GT/CA repeat motifs were the most common (37.6%) followed by GA/CT repeat motifs (25.9%). The primer pairs could be designed for a total of 170 SSRs and were optimized initially on two genotypes. 104 (61.2%) primer pairs yielded scorable amplicon and 46 (44.2%) primers showed polymorphism among 32 cultivated groundnut genotypes. The polymorphic SSR markers detected 2 to 5 alleles with an average of 2.44 per locus. The polymorphic information content (PIC) value for these markers varied from 0.12 to 0.75 with an average of 0.46. Based on 112 alleles obtained by 46 markers, a phenogram was constructed to understand the relationships among the 32 genotypes. Majority of the genotypes representing subspecies hypogaea were grouped together in one cluster, while the genotypes belonging to subspecies fastigiata were grouped mainly under two clusters. Conclusion. Newly developed set of 104 markers extends the repertoire of SSR markers for cultivated groundnut. These markers showed a good level of PIC value in cultivated germplasm and therefore would be very useful for germplasm analysis, linkage mapping, diversity studies and phylogenetic relationships in cultivated groundnut as well as related Arachis species.

Heterologous signals allow efficient targeting of a nuclear-encoded fusion protein to plastids and endoplasmic reticulum in diverse plant species

Approximately 30% of plant nuclear genes appear to encode proteins targeted to the plastids or endoplasmic reticulum (ER). The signals that direct proteins into these compartments are diverse in sequence, but, on the basis of a limited number of tests in heterologous systems, they appear to be functionally conserved across species. To further test the generality of this conclusion, we tested the ability of two plastid transit peptides and an ER signal peptide to target green fluorescent protein (GFP) in 12 crops, including three monocots (barley, sugarcane, wheat) and nine dicots (Arabidopsis, broccoli, cabbage, carrot, cauliflower, lettuce, radish, tobacco, turnip). In all species, transient assays following microprojectile bombardment or vacuum infiltration using Agrobacterium showed that the plastid transit peptides from tomato DCL (defective chloroplast and leaves) and tobacco RbcS [ribulose bisphosphate carboxylase (Rubisco) small subunit] genes were effective in targeting GFP to the leaf plastids. GFP engineered as a fusion to the N-terminal ER signal peptide from Arabidopsis basic chitinase and a C-terminal HDEL signal for protein retention in the ER was accumulated in the ER of all species. The results in tobacco were confirmed in stably transformed cells. These signal sequences should be useful to direct proteins to the plastid stroma or ER lumen in diverse plant species of biotechnological interest for the accumulation of particular recombinant proteins or for the modification of particular metabolic streams.

An assessment of the genetic relationship between sweet and grain sorghums, within Sorghum bicolor ssp. bicolor (L.) Moench, using AFLP markers

Compared to grain sorghums, sweet sorghums typically have lower grain yield and thick, tall stalks which accumulate high levels of sugar (sucrose, fructose and glucose). Unlike commercial grain sorghum (S. bicolor ssp. bicolor) cultivars, which are usually F1 hybrids, commercial sweet sorghums were selected as wild accessions or have undergone limited plant breeding. Although all sweet sorghums are classified within S. bicolor ssp. bicolor, their genetic relationship with grain sorghums is yet to be investigated. Ninety-five genotypes, including 31 sweet sorghums and 64 grain sorghums, representing all five races within the subspecies bicolor, were screened with 277 polymorphic amplified fragment length polymorphism (AFLP) markers. Cluster analysis separated older sweet sorghum accessions (collected in mid 1800s) from those developed and released during the early to mid 1900s. These groups were emphasised in a principle component analysis of the results such that sweet sorghum lines were largely distinguished from the others, particularly by a group of markers located on sorghum chromosomes SBI-08 and SBI-10. Other studies have shown that QTL and ESTs for sugar-related traits, as well as for height and anthesis, map to SBI-10. Although the clusters obtained did not group clearly on the basis of racial classification, the sweet sorghum lines often cluster with grain sorghums of similar racial origin thus suggesting that sweet sorghum is of polyphyletic origin within S. bicolor ssp. bicolor.

An asparaginyl endopeptidase mediates in vivo protein backbone cyclization

Proteases can catalyze both peptide bond cleavage and formation, yet the hydrolysis reaction dominates in nature. This presents an interesting challenge for the biosynthesis of backbone cyclized (circular) proteins, which are encoded as part of precursor proteins and require post-translational peptide bond formation to reach their mature form. The largest family of circular proteins are the plant-produced cyclotides; extremely stable proteins with applications as bioengineering scaffolds. Little is known about the mechanism by which they are cyclized in vivo but a highly conserved Asn (occasionally Asp) residue at the C terminus of the cyclotide domain suggests that an enzyme with specificity for Asn (asparaginyl endopeptidase; AEP) is involved in the process. Nicotiana benthamiana does not endogenously produce circular proteins but when cDNA encoding the precursor of the cyclotide kalata B1 was transiently expressed in the plants they produced the cyclotide, together with linear forms not commonly observed in cyclotide-containing plants. Observation of these species over time showed that in vivo asparaginyl bond hydrolysis is necessary for cyclization. When AEP activity was suppressed, either by decreasing AEP gene expression or using a specific inhibitor, the amount of cyclic cyclotide in the plants was reduced compared with controls and was accompanied by the accumulation of extended linear species. These results suggest that an AEP is responsible for catalyzing both peptide bond cleavage and ligation of cyclotides in a single processing event.

Large cryptic internal sequence repeats in protein structures from Homo sapiens

Amino acid sequences are known to constantly mutate and diverge unless there is a limiting condition that makes such a change deleterious. However, closer examination of the sequence and structure reveals that a few large, cryptic repeats are nevertheless sequentially conserved. This leads to the question of why only certain repeats are conserved at the sequence level. It would be interesting to find out if these sequences maintain their conservation at the three-dimensional structure level. They can play an active role in protein and nucleotide stability, thus not only ensring proper functioning but also potentiating malfunction and disease. Therefore, insights into any aspect of the repeats - be it structure, function or evolution - would prove to be of some importance. This study aims to address the relationship between protein sequence and its three-dimensional structure, by examining if large cryptic sequence repeats have the same structure.

Allostery in tRNA Synthetases Elucidated from MD Simulations and Protein Structure Networks

tRNA synthetases (aaRS) are enzymes crucial in the translation of genetic code. The enzyme accylates the acceptor stem of tRNA by the congnate amino acid bound at the active site, when the anti-codon is recognized by the anti-codon site of aaRS. In a typical aaRS, the distance between the anti-codon region and the amino accylation site is approximately 70 Å. We have investigated this allosteric phenomenon at molecular level by MD simulations followed by the analysis of protein structure networks (PSN) of non-covalent interactions. Specifically, we have generated conformational ensembles by performing MD simulations on different liganded states of methionyl tRNA synthetase (MetRS) from Escherichia coli and tryptophenyl tRNA synthetase (TrpRS) from Human. The correlated residues during the MD simulations are identified by cross correlation maps. We have identified the amino acids connecting the correlated residues by the shortest path between the two selected members of the PSN. The frequencies of paths have been evaluated from the MD snapshots[1]. The conformational populations in different liganded states of the protein have been beautifully captured in terms of network parameters such as hubs, cliques and communities[2]. These parameters have been associated with the rigidity and plasticity of the protein conformations and can be associated with free energy landscape. A comparison of allosteric communication in MetRS and TrpRS [3] elucidated in this study highlights diverse means adopted by different enzymes to perform a similar function. The computational method described for these two enzymes can be applied to the investigation of allostery in other systems.

Structural Polymorphism of the Nucleosomal DNA and Implications for Protein Binding

A nucleosome forms a basic unit of the chromosome structure. A biologically relevant question is how much of the nucleosomal conformational space is accessible to protein-free DNA, and what proportion of the nucleosomal conformations are induced by bound histones. To investigate this, we have analysed high resolution xray crystal structure datasets of DNA in protein-free as well as protein-bound forms, and compared the dinucleotide step parameters for the two datasets with those for high resolution nucleosome structures. Our analysis shows that most of the dinucleotide step parameter values for the nucleosome structures lie within the range accessible to protein-free DNA, indirectly indicating that the histone core plays more of a stabilizing role. The nucleosome structures are observed to assume smooth and nearly planar curvature, implying that ‘normal’ B-DNA like parameters can give rise to a curved geometry at the gross structural level. Different nucleosome

A high-density consensus map of barley linking DArT markers to SSR, RFLP and STS loci and agricultural traits

Background: Molecular marker technologies are undergoing a transition from largely serial assays measuring DNA fragment sizes to hybridization-based technologies with high multiplexing levels. Diversity Arrays Technology (DArT) is a hybridization-based technology that is increasingly being adopted by barley researchers. There is a need to integrate the information generated by DArT with previous data produced with gel-based marker technologies. The goal of this study was to build a high-density consensus linkage map from the combined datasets of ten populations, most of which were simultaneously typed with DArT and Simple Sequence Repeat (SSR), Restriction Enzyme Fragment Polymorphism (RFLP) and/or Sequence Tagged Site (STS) markers. Results: The consensus map, built using a combination of JoinMap 3.0 software and several purpose-built perl scripts, comprised 2,935 loci (2,085 DArT, 850 other loci) and spanned 1,161 cM. It contained a total of 1,629 'bins' (unique loci), with an average inter-bin distance of 0.7 ± 1.0 cM (median = 0.3 cM). More than 98% of the map could be covered with a single DArT assay. The arrangement of loci was very similar to, and almost as optimal as, the arrangement of loci in component maps built for individual populations. The locus order of a synthetic map derived from merging the component maps without considering the segregation data was only slightly inferior. The distribution of loci along chromosomes indicated centromeric suppression of recombination in all chromosomes except 5H. DArT markers appeared to have a moderate tendency toward hypomethylated, gene-rich regions in distal chromosome areas. On the average, 14 ± 9 DArT loci were identified within 5 cM on either side of SSR, RFLP or STS loci previously identified as linked to agricultural traits. Conclusion: Our barley consensus map provides a framework for transferring genetic information between different marker systems and for deploying DArT markers in molecular breeding schemes. The study also highlights the need for improved software for building consensus maps from high-density segregation data of multiple populations.

Characterisation of chicken Campylobacter jejuni isolates using resolution optimised single nucleotide polymorphisms and binary gene markers

The principal objective of this study was to determine if Campylobacter jejuni genotyping methods based upon resolution optimised sets of single nucleotide polymorphisms (SNPs) and binary genetic markers were capable of identifying epidemiologically linked clusters of chicken-derived isolates. Eighty-eight C. jejuni isolates of known flaA RFLP type were included in the study. They encompassed three groups of ten isolates that were obtained at the same time and place and possessed the same flaA type. These were regarded as being epidemiologically linked. Twenty-six unlinked C. jejuni flaA type I isolates were included to test the ability of SNP and binary typing to resolve isolates that were not resolved by flaA RFLP. The remaining isolates were of different flaA types. All isolates were typed by real-time PCR interrogation of the resolution optimised sets of SNPs and binary markers. According to each typing method, the three epidemiologically linked clusters were three different clones that were well resolved from the other isolates. The 26 unlinked C. jejuni flaA type I isolates were resolved into 14 SNP-binary types, indicating that flaA typing can be unreliable for revealing epidemiological linkage. Comparison of the data with data from a fully typed set of isolates associated with human infection revealed that abundant lineages in the chicken isolates that were also found in the human isolates belonged to clonal complex (CC) -21 and CC-353, with the usually rare C-353 member ST-524 being especially abundant in the chicken collection. The chicken isolates selected to be diverse according to flaA were also diverse according to SNP and binary typing. It was observed that CC-48 was absent in the chicken isolates, despite being very common in Australian human infection isolates, indicating that this may be a major cause of human disease that is not chicken associated.

Epithiospecifier protein activity in broccoli: The link between terminal alkenyl glucosinolates and sulphoraphane nitrile

The chemical nature of the hydrolysis products from the glucosinolate-myrosinase system depends on the presence or absence of supplementary proteins, such as epithiospecifier proteins (ESPs). ESPs (non-catalytic cofactors of myrosinase) promote the formation of epithionitriles from terminal alkenyl glucosinolates and as recent evidence suggests, simple nitriles at the expense of isothiocyanates. The ratio of ESP activity to myrosinase activity is crucial in determining the proportion of these nitriles produced on hydrolysis. Sulphoraphane, a major isothiocyanate produced in broccoli seedlings, has been found to be a potent inducer of phase 2 detoxification enzymes. However, ESP may also support the formation of the non-inductive sulphoraphane nitrile. Our objective was to monitor changes in ESP activity during the development of broccoli seedlings and link these activity changes with myrosinase activity, the level of terminal alkenyl glucosinolates and sulphoraphane nitrile formed. Here, for the first time, we show ESP activity increases up to day 2 after germination before decreasing again to seed activity levels at day 5. These activity changes paralleled changes in myrosinase activity and terminal alkenyl glucosinolate content. There is a significant relationship between ESP activity and the formation of sulforaphane nitrile in broccoli seedlings. The significance of these findings for the health benefits conferred by eating broccoli seedlings is briefly discussed.

The use of high resolution melting (HRM) to map single nucleotide polymorphism markers linked to a covered smut resistance gene in barley

Using an established genetic map, a single gene conditioning covered smut resistance, Ruh.7H, was mapped to the telomere region of chromosome 7HS in an Alexis/Sloop doubled haploid barley population. The closest marker to Ruh.7H, abg704 was 7.5 cM away. Thirteen loci on the distal end of 7HS with potential to contain single nucleotide polymorphisms (SNPs) were identified by applying a comparative genomics approach using rice sequence data. Of these, one locus produced polymorphic co-dominant bands of different size while two further loci contained SNPs that were identified using the recently developed high resolution melting (HRM) technique. Two of these markers flanked Ruh.7H with the proximal marker located 3.8 cM and the distal marker 2.7 cM away. This is the first report on the application of the HRM technique to SNP detection and to rapid scoring of known cleaved amplified polymorphic sequence (CAPS) markers in plants. This simple, precise post-PCR technique should find widespread use in the fine-mapping of genetic regions of interest in complex cereal and other plant genomes.

DArT markers: Diversity analyses and mapping in Sorghum bicolor

The sequential nature of gel-based marker systems entails low throughput and high costs per assay. Commonly used marker systems such as SSR and SNP are also dependent on sequence information. These limitations result in high cost per data point and significantly limit the capacity of breeding programs to obtain sufficient return on investment to justify the routine use of marker-assisted breeding for many traits and particularly quantitative traits. Diversity Arrays Technology (DArT™) is a cost effective hybridisation-based marker technology that offers a high multiplexing level while being independent of sequence information. This technology offers sorghum breeding programs an alternative approach to whole-genome profiling. We report on the development, application, mapping and utility of DArT™ markers for sorghum germplasm. Results: A genotyping array was developed representing approximately 12,000 genomic clones using PstI+BanII complexity with a subset of clones obtained through the suppression subtractive hybridisation (SSH) method. The genotyping array was used to analyse a diverse set of sorghum genotypes and screening a Recombinant Inbred Lines (RIL) mapping population. Over 500 markers detected variation among 90 accessions used in a diversity analysis. Cluster analysis discriminated well between all 90 genotypes. To confirm that the sorghum DArT markers behave in a Mendelian manner, we constructed a genetic linkage map for a cross between R931945-2-2 and IS 8525 integrating DArT and other marker types. In total, 596 markers could be placed on the integrated linkage map, which spanned 1431.6 cM. The genetic linkage map had an average marker density of 1/2.39 cM, with an average DArT marker density of 1/3.9 cM. Conclusion: We have successfully developed DArT markers for Sorghum bicolor and have demonstrated that DArT provides high quality markers that can be used for diversity analyses and to construct medium-density genetic linkage maps. The high number of DArT markers generated in a single assay not only provides a precise estimate of genetic relationships among genotypes, but also their even distribution over the genome offers real advantages for a range of molecular breeding and genomics applications.