ALBERT

Beschreibung: ChIP-seq provides a wealth of information on the approximate location of DNA-binding proteins genome-wide. It is known that the targeted motifs in most cases can be found at the peak centers. A high resolution...

Beschreibung: Background Hypoxia inducible factor-1 alpha (HIF-1α) is a transcription factor that plays an important role in neo-vascularisation, embryonic pancreas beta-cell mass development, and beta cell protection. Recently a non synonymous single nucleotide polymorphism (g.C45035T SNP, rs11549465) of HIF-1α gene, resulting in the p.P582S amino acid change has been shown to be associated with type 2 diabetes (T2DM) in a Japanese population. Our aim was to replicate these findings on a Caucasian (Hungarian) population, as well as to study whether this genetic effect is restricted to T2DM or can be expanded to diabetes in general. Methods A large Caucasian sample (N = 890) was recruited including 370 T2DM, 166 T1DM and 354 healthy subjects. Genotyping was validated by two independent methods: a restriction fragment analysis (RFLP) and a real time PCR using TaqMan probes. An overestimation of heterozygotes by RFLP was observed as a consequence of a nearby SNP (rs34005929). Therefore genotyping results of the justified TaqMan system were accepted. The measured genotype distribution corresponded to Hardy-Weinberg equilibrium (P = 0.740) Results As the TT genotype was extremely rare in the population (0.6% in clinical sample and 2.5% in controls), the genotypes were grouped as T absent (CC) and T present (CT and TT). Genotype-wise analysis showed a significant increase of T present group in controls (24.0%) as compared to patients (16.8%, P = 0.008). This genetic effect was demonstrated in the separated samples of type 1 (15.1%, P = 0.020), and also in type 2 (17.6%, P = 0.032) diabetes. Allele-wise analysis gave identical results showing a higher frequency of the T allele in the control sample (13.3%) than in the clinical sample (8.7%, P = 0.002) with similar results in type 1 (7.8%, P = 0.010) and type 2 (9.1%, P = 0.011) diabetes. The odds ratio for diabetes (either type 1 or 2) was 1.56 in the presence of the C allele. Conclusion We confirmed the protective effect of a rare genetic variant of HIF-1α gene against type 2 diabetes in a Caucasian sample. Moreover we demonstrated a genetic contribution of the same polymorphism in type 1 diabetes as well, supporting a possible overlap in pathomechanism for T2DM and a T1DM.

Beschreibung: Type IV collagen is the main component of the basement membrane that gives strength to the blood–gas barrier (BGB). In mammals, the formation of a mature BGB occurs primarily after birth during alveologenesis ...

Beschreibung: Background: In plant genomes, NB-LRR based resistance (R) genes tend to occur in clusters of variable size in a relatively small number of genomic regions. R-gene sequences mostly differentiate by accumulating point mutations and gene conversion events. Potato and tomato chromosome 4 harbours a syntenic R-gene locus (known as the R2 locus in potato) that has mainly been examined in central American/Mexican wild potato species on the basis of its contribution to resistance to late blight, caused by the oomycete pathogen Phytophthora infestans. Evidence to date indicates the occurrence of a fast evolutionary mode characterized by gene conversion events at the locus in these genotypes. Results: A physical map of the R2 locus was developed for three Solanum tuberosum genotypes and used to identify the tomato syntenic sequence. Functional annotation of the locus revealed the presence of numerous resistance gene homologs (RGHs) belonging to the R2 gene family (R2GHs) organized into a total of 4 discrete physical clusters, three of which were conserved across S. tuberosum and tomato. Phylogenetic analysis showed clear orthology/paralogy relationships between S. tuberosum R2GHs but not in R2GHs cloned from Solanum wild species. This study confirmed that, in contrast to the wild species R2GHs, which have evolved through extensive sequence exchanges between paralogs, gene conversion was not a major force for differentiation in S. tuberosum R2GHs, and orthology/paralogy relationships have been maintained via a slow accumulation of point mutations in these genotypes. Conclusions: S. tuberosum and Solanum lycopersicum R2GHs evolved mostly through duplication and deletion events, followed by gradual accumulation of mutations. Conversely, widespread gene conversion is the major evolutionary force that has shaped the locus in Mexican wild potato species. We conclude that different selective forces shaped the evolution of the R2 locus in these lineages and that co-evolution with a pathogen steered selection on different evolutionary paths.

Beschreibung: Background: Activation of numerous protective mechanisms during cold acclimation is important for the acquisition of freezing tolerance in perennial ryegrass (Lolium perenne L.). To elucidate the molecular mechanisms of cold acclimation in two genotypes (‘Veyo’ and ‘Falster’) of perennial ryegrass from distinct geographical origins, we performed transcriptome profiling during cold acclimation using RNA-Seq. Methods: We cold-acclimated plants from both genotypes in controlled conditions for a period of 17 days and isolated Total RNA at various time points for high throughput sequencing using Illumina technology. RNA-seq reads were aligned to genotype specific references to identify transcripts with significant changes in expression during cold acclimation. Results: The genes induced were involved in protective mechanisms such as cell response to abiotic stimulus, signal transduction, redox homeostasis, plasma membrane and cell wall modifications, and carbohydrate metabolism in both genotypes. ‘Falster’ genotype, adapted to cold climates, showed a stronger transcriptional differentiation during cold acclimation, and more differentially expressed transcripts related to stress, signal transduction, response to abiotic stimulus, and metabolic processes compared to ‘Veyo’. ‘Falster’ genotype also showed an induction of more transcripts with sequence homology to fructosyltransferase genes (FTs) and a higher fold induction of fructan in response to low-temperature stress. The circadian rhythm network was perturbed in the ‘Veyo’ genotype adapted to warmer climates. Conclusion: In this study, the differentially expressed genes during cold acclimation, potentially involved in numerous protective mechanisms, were identified in two genotypes of perennial ryegrass from distinct geographical origins. The observation that the circadian rhythm network was perturbed in ‘Veyo’ during cold acclimation may point to a low adaptability of ‘Veyo’ to low temperature stresses. This study also revealed the transcriptional mechanisms underlying carbon allocation towards fructan biosynthesis in perennial ryegrass.

Beschreibung: Background: Wheat is the leading source of vegetable protein in the human diet, and metabolites are crucial for both plant development and human nutrition. The recent advances in metabolomics provided an opportunity to perform an untargeted metabolite analysis in this important crop. Results: Wheat was characterised at the metabolite level during cold acclimation and transition from the vegetative to the generative phase. The relationship between these changes and chromosome 5A and the maintained vegetative phase (mvp) mutation was also investigated. Samples were taken from the shoots and crowns during four developmental stages: plants grown at 20/17°C, after cold treatment but still during the vegetative phase, at the double ridge and during spikelet formation. The levels of 47 compounds were identified by gas chromatography-mass spectrometry, of which 38 were annotated. The cold treatment, in general, increased the concentrations of osmolites but not in all lines and not equally in the shoots and crowns. The accumulation of proline was not associated with the vernalisation process or with frost tolerance. The mvp mutation and chromosome 5A substitutions altered the amounts of several metabolites compared to those of the Tm and CS, respectively, during each developmental stage. The Ch5A substitution resulted in more substantial changes at the metabolite level than did the Tsp5A substitution. While Ch5A mainly influenced the sugar concentrations, Tsp5A altered the level of tricarboxylic acid cycle intermediates during the vegetative/generative transition. A much higher trehalose, proline, glutamine, asparagine, and unidentified m/z 186 content was detected in crowns than in shoots that may contribute to the frost tolerance of crowns. Conclusions: Substantial influences of chromosome 5A and the mvp mutation on metabolism during four different developmental stages were demonstrated. The distinct and overlapping accumulation patterns of metabolites suggest the complex genetic regulation of metabolism in the shoots and crowns.

Beschreibung: Background: Cultivated peanut or groundnut (Arachis hypogaea L.) is an important oilseed crop with an allotetraploid genome (AABB, 2n = 4x = 40). Both the low level of genetic variation within the cultivated gene pool and its polyploid nature limit the utilization of molecular markers to explore genome structure and facilitate genetic improvement. Nevertheless, a wealth of genetic diversity exists in diploid Arachis species (2n = 2x = 20), which represent a valuable gene pool for cultivated peanut improvement. Interspecific populations have been used widely for genetic mapping in diploid species of Arachis. However, an intraspecific mapping strategy was essential to detect chromosomal rearrangements among species that could be obscured by mapping in interspecific populations. To develop intraspecific reference linkage maps and gain insights into karyotypic evolution within the genus, we comparatively mapped the A- and B-genome diploid species using intraspecific F2 populations. Exploring genome organization among diploid peanut species by comparative mapping will enhance our understanding of the cultivated tetraploid peanut genome. Moreover, new sources of molecular markers that are highly transferable between species and developed from expressed genes will be required to construct saturated genetic maps for peanut. Results: A total of 2,138 EST-SSR (expressed sequence tag-simple sequence repeat) markers were developed by mining a tetraploid peanut EST assembly including 101,132 unigenes (37,916 contigs and 63,216 singletons) derived from 70,771 long-read (Sanger) and 270,957 short-read (454) sequences. A set of 97 SSR markers were also developed by mining 9,517 genomic survey sequences of Arachis. An SSR-based intraspecific linkage map was constructed using an F2 population derived from a cross between K 9484 (PI 298639) and GKBSPSc 30081 (PI 468327) in the B - genome species A. batizocoi. A high degree of macrosynteny was observed when comparing the homoeologous linkage groups between A (A. duranensis) and B (A. batizocoi) genomes. Comparison of the A - and B - genome genetic linkage maps also showed a total of five inversions and one major reciprocal translocation between two pairs of chromosomes under our current mapping resolution. Conclusions: Our findings will contribute to understanding tetraploid peanut genome origin and evolution and eventually promote its genetic improvement. The newly developed EST-SSR markers will enrich current molecular marker resources in peanut.

Beschreibung: Background: White clover (Trifolium repens L.) is an allotetraploid species possessing two highly collinear ancestral sub-genomes. The apparent existence of highly similar homeolog copies for the majority of genes in white clover is problematic for the development of genome-based resources in the species. This is especially true for the development of genetic markers based on single nucleotide polymorphisms (SNPs), since it is difficult to distinguish between homeolog-specific and allelic variants. Robust methods for categorising single nucleotide variants as allelic or homeolog-specific in large transcript datasets are required. We illustrate one potential approach in this study. Results: We used 454-pyrosequencing sequencing to generate ~760,000 transcript sequences from an 8th generation white clover inbred line. These were assembled and partially annotated to yield a reference transcript set comprising 71,545 sequences. We subsequently performed Illumina sequencing on three further white clover samples, generating 14 million transcript reads from a mixed sample comprising 24 divergent white clover genotypes, and 50 million reads on two further eighth generation white clover inbred lines. Mapping these reads to the reference transcript set allowed us to develop a significant SNP resource for white clover, and to partition the SNPs from the inbred lines into categories reflecting allelic or homeolog-specific variation. The potential for using haplotype reconstruction and progenitor genome comparison to assign haplotypes to specific ancestral sub-genomes of white clover is demonstrated for sequences corresponding to genes encoding dehydration responsive element binding protein and acyl-coA oxidase. Conclusions: In total, 208,854 independent SNPs in 31,715 reference sequences were discovered, approximately three quarters of which were categorised as representing allelic or homeolog-specific variation using two inbred lines. This represents a significant resource for white clover genomics and genetics studies. We discuss the potential to extend the analysis to identify a "core set" of ancestrally derived homeolog specific variants in white clover.

Beschreibung: Background: Nicotiana benthamiana is a widely used model plant species for research on plant-pathogen interactions as well as other areas of plant science. It can be easily transformed or agroinfiltrated, therefore it is commonly used in studies requiring protein localization, interaction, or plant-based systems for protein expression and purification. To discover and characterize the miRNAs and their cleaved target mRNAs in N. benthamiana, we sequenced small RNA transcriptomes and degradomes of two N. benthamiana accessions and validated them by Northern blots. Results: We used a comprehensive molecular approach to detect and to experimentally validate N. benthamiana miRNAs and their target mRNAs from various tissues. We identified 40 conserved miRNA families and 18 novel microRNA candidates and validated their target mRNAs with a genomic scale approach. The accumulation of thirteen novel miRNAs was confirmed by Northern blot analysis. The conserved and novel miRNA targets were found to be involved in various biological processes including transcription, RNA binding, DNA modification, signal transduction, stress response and metabolic process. Among the novel miRNA targets we found the mRNA of REPRESSOR OF SILENCING (ROS1). Regulation of ROS1 by a miRNA provides a new regulatory layer to reinforce transcriptional gene silencing by a post-transcriptional repression of ROS1 activity. Conclusions: The identified conserved and novel miRNAs along with their target mRNAs also provides a tissue specific atlas of known and new miRNA expression and their cleaved target mRNAs of N. benthamiana. Thus this study will serve as a valuable resource to the plant research community that will be beneficial well into the future.