Restriction site-associated DNA Sequencing (RAD-Seq) is an economical and efficient method for SNP discovery and genotyping. As with other sequencing-by-synthesis methods, RAD-Seq produces stochastic count data and requires sensitive analysis to develop or genotype markers accurately. We show that there are several sources of bias specific to RAD-Seq that are not explicitly addressed by current genotyping tools, namely restriction fragment bias, restriction site heterozygosity and PCR GC content bias. We explore the performance of existing analysis tools given these biases and discuss approaches to limiting or handling biases in RAD-Seq data. While these biases need to be taken seriously, we believe RAD loci affected by them can be excluded or processed with relative ease in most cases and that most RAD loci will be accurately genotyped by existing tools.
SummaryPectins are a family of complex cell-wall polysaccharides, the biosynthesis of which remains poorly understood. We identified dwarf mutants with reduced cell adhesion at a novel locus, QUASIMODO2 (QUA2). qua2-1 showed a 50% reduction in homogalacturonan (HG) content compared with the wild type, without affecting other cell-wall polysaccharides. The remaining HG in qua2-1 showed an unaltered degree of methylesterification. Positional cloning and GFP fusions showed that QUA2, consistent with a role in HG synthesis, encodes a Golgi-localized protein. In contrast to QUA1, another Golgi-localized protein required for HG-synthesis, QUA2 does not show sequence similarity to glycosyltransferases, but instead contains a putative methyltransferase (MT) domain. The Arabidopsis genome encodes 29 QUA2-related proteins. Interestingly, the transcript profiles of QUA1 and QUA2 are correlated and other pairs of QUA1 and QUA2 homologues with correlated transcript profiles can be identified. Together, the results lead to the hypothesis that QUA2 is a pectin MT, and that polymerization and methylation of homogalacturonan are interdependent reactions.
Caviar-producing sturgeons belonging to the genus Acipenser are considered to be one of the most endangered species groups in the world. Continued overfishing in spite of increasing legislation, zero catch quotas and extensive aquaculture production have led to the collapse of wild stocks across Europe and Asia. The evolutionary relationships among Adriatic, Russian, Persian and Siberian sturgeons are complex because of past introgression events and remain poorly understood. Conservation management, traceability and enforcement suffer a lack of appropriate DNA markers for the genetic identification of sturgeon at the species, population and individual level. This study employed RAD sequencing to discover and characterize single nucleotide polymorphism (SNP) DNA markers for use in sturgeon conservation in these four tetraploid species over three biological levels, using a single sequencing lane. Four population meta-samples and eight individual samples from one family were barcoded separately before sequencing. Analysis of 14.4 Gb of paired-end RAD data focused on the identification of SNPs in the paired-end contig, with subsequent in silico and empirical validation of candidate markers. Thousands of putatively informative markers were identified including, for the first time, SNPs that show population-wide differentiation between Russian and Persian sturgeons, representing an important advance in our ability to manage these cryptic species. The results highlight the challenges of genotyping-by-sequencing in polyploid taxa, while establishing the potential genetic resources for developing a new range of caviar traceability and enforcement tools.
SummaryDespite the fact that several hundred glycosyltransferases have been identified from sequencing of plant genomes, the biological functions of only a handful have been established to date. A Poplar glycosyltransferase 64 (GT64) family member that is differentially expressed during the cell division and elongation phases of cambial growth was identified from previously generated transcript profiling of cambium tissues. The predicted Poplar GT64 protein has a closely related Arabidopsis homolog ECTOPICALLY PARTING CELLS (EPC1). Mutation of the EPC1 gene, one of three Arabidopsis GT64 family members, results in plants with a dramatically reduced growth habit, defects in vascular formation and reduced cell-cell adhesion properties in hypocotyl and cotyledon tissues. Secondary growth is enhanced in epc1 hypocotyl tissues and it is proposed that this results from the abnormal cell-cell adhesion within the cortical parenchyma cell layers. Loss of cellcell contacts within cotyledon and leaf tissues is also proposed to account for vascular patterning defects and the fragile nature of epc1 tissues. The EPC1 protein thus plays a critical role during plant development in maintaining the integrity of organs via cell-cell adhesion, thereby providing mechanical strength and facilitating the movement of metabolites throughout the plant.
We studied DQA1, DQB1, and DPB1 alleles in 31 Finnish families with celiac disease (CD). All healthy first-degree relatives underwent clinical investigation, including in most cases biopsy, to establish whether clinically silent CD was present. Our results indicate that all patients, having either full clinical CD or its silent form, had the susceptibility alleles DQA1*0501 and DQB1*0201. The different clinical outcomes of CD were therefore not directly determined by the DQ alleles. The frequency of DPB1*0101 was also higher in CD patients, but the association appeared secondary to those of DQA1*0501 and DQB1*0201 (DQ2). The primary association of CD with the DQA1*0501 and DQB1*0201 alleles, rather than with HLA haplotypes, was confirmed in multiplex families.
Despite the importance of secondary growth in plants, relatively few genes regulating this process have been identified to date. By using data from detailed transcript profiling of the poplar wood‐forming tissues, 150 genes that are differentially expressed within the zone of secondary growth were identified. In order to determine the possible function of these poplar genes, potential Arabidopsis thaliana orthologs were identified and gene knockout lines analysed. Three selection filters were used to identify the most likely orthologous genes using poplar and Arabidopsis sequence comparisons, expression profiling in secondary thickened Arabidopsis hypocotyls and global expression analysis of Arabidopsis tissues. Three genes encoding AtCSLA2 (At5g22740), the AtGUT1 GT47 glycosyltransferase (At1g27440) and a protein with no proposed function AtUNKA (At4g27435) were selected for further detailed analysis of their role in secondary growth in Arabidopsis. The presented genome‐based approach using both poplar and Arabidopsis systems provides powerful means towards assigning biological functions to enzymes with poorly understood biochemical activity, such as AtCSLA2 and AtGUT1, as well as for proteins with no known function.
Jejunal biopsy specimens from 25 patients with IgA deficiency (IgAd) were studied immunohistochemically to find markers of inflammation. Five of the 25 patients had coeliac disease (CD): they were on a gluten free diet and had normal jejunal morphology. Only two of 15 specimens from control subjects had CD25+ cells in the surface epithelium, while this was seen in 19 out of 20 specimens from IgAd patients (p<0.0001). A significant increase of CD25+ cells was also noted in the lamina propria of IgAd patients. The median percentage of crypt cells in mitosis (Ki67+ cells) was higher in the specimens from IgAd patients (26%) than in those from controls (13%, p<0.001). The densities of yS T cell receptor positive cells in the surface epithelium and lamina propria did not differ in the specimens from IgAd patients and those of controls nor was the expression of HLA class II antigens augmented in the surface epithelium. These findings were similar for the IgAd patients whether or not the patient had DQB 0201 allele, a genetic marker which is strongly associated with CD. The inadequacy of the local immunoglobulins in patients with IgAd may lead to increased T cell activation, which is accompanied by the appearance of intraepithelial CD25+ cells and with an increase in the mitotic rate in the crypts. (Gut 1995; 37: 519-523)
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