Development of a minimal KASP marker panel for distinguishing genotypes in apple collections

Accurate identification of named accessions in germplasm collections is extremely important, especially for vegetatively propagated crops which are expensive to maintain.  Thus, an inexpensive, reliable, and rapid genotyping method is essential because it avoids the need for laborious and time-consuming morphological comparisons.  Single Nucleotide Polymorphism (SNP) marker panels containing large numbers of SNPs have been developed for many crop species, but such panels are much too large for basic cultivar identification.  Here, we have identified a minimum set of SNP markers sufficient to distinguish apple cultivars held in the English and Welsh national collections providing a cheaper and automatable alternative to the markers currently used by the community.  We show that SNP genotyping with a small set of well selected markers is equally efficient as microsatellites for the identification of apple cultivars and has the added advantage of automation and reduced cost when screening large numbers of sample

Pedigree reconstruction for triploid apple cultivars using single nucleotide polymorphism array data

Societal Impact Statement Many economically, culturally, and historically important apple cultivars are triploids, which have three copies of each chromosome instead of the more typical two copies in diploids. Despite their prevalence and importance, there have been conflicting reports regarding their origin and their ability to beget diploids. New genetic analysis methodologies outlined in this study have clarified the genetic origin of triploid apple cultivars and suggest that triploidy has been a dead end in historic apple pedigrees. The specific results of this study have resolved the pedigrees of many cultivars, including the famous English cultivar Cox's Orange Pippin and the oldest known US cultivar Roxbury Russet. Summary In apple (Malus × domestica), most cultivars are diploid, though a sizeable number are triploids, which tend to be stronger growing, more robust, and bear larger fruit. However, triploidy is also associated with strongly reduced fertility. Some recorded pedigrees for historical apple cultivars include triploids as parents of diploids, despite this reputation of poor fertility. This information, coupled with some initiatives using triploids in breeding efforts, result in confusion about how possible or common it is for triploids to be parents of diploid offspring. To date, no studies have systematically evaluated and identified pedigrees of triploid apple cultivars to resolve these contradictions. Here, we describe a method to make triploid genotype calls using Illumina Infinium single nucleotide polymorphism (SNP) array data through a novel Python script: ploidyClassifier. SNP data for 219 unique triploids was compared alongside 2498 unique diploid apple accessions to conduct pedigree reconstruction. Unreduced gamete-donating parents were identified for over half of the triploid accessions. From those, reduced gamete-donating parents were identified for nearly half. Full or partial pedigrees for many classic triploids were uncovered, including that of the oldest known American cultivar, ‘Roxbury Russet’. All tested pedigrees from literature that listed triploids as parents of diploids were deemed false, including that of the well-known ‘Cox's Orange Pippin’, whose previously unreported second parent was also identified here as ‘Rosemary Russet’. These results together suggest that historic triploids are mostly or solely the product of diploid parentage and that triploidy has been a dead end in historic apple pedigrees

Delayed chilling appears to counteract flowering advances of apricot in southern UK

Temperatures are rising across the globe, and the UK is no exception. Spring phenology of perennial fruit crops is to a large extent determined by temperature during effective chilling (endo-dormancy) and heat accumulation (eco-dormancy) periods. We used the apricot flowering records of the UK National Fruit Collections (NFC) to determine the influence of temperature trends over recent decades (1960 to 2014) on apricot (Prunus armeniaca L.) flowering time. Using Partial Least Squares (PLS) regression, we determined the respective periods for calculating chill and heat accumulation. Results suggested intervals between September 27th and February 26th and between December 31st and April 12th as the effective chilling and warming periods, respectively. Flowering time was correlated with temperature during both periods, with warming during chilling corresponding to flowering delays by 4.82 d°C-1, while warming during heat accumulation was associated with bloom advances by 9.85 d°C-1. Heat accumulation started after accumulating 62.7 ± 5.6 Chill Portions, and flowering occurred after a further 3744 ± 1538 Growing Degree Hours (above a base temperature of 4°C, with optimal growth at 26°C). When examining the time series, the increase in temperature during the chilling period did not appear to decrease overall chill accumulation during the chilling period but to delay the onset of chill accumulation and the completion of the the average chill accumulation necessary to start heat accumulation. The resulting delay in heat responsiveness appeared to weaken the phenology-advancing effect of spring warming. These processes may explain why apricot flowering time remained relatively unchanged despite significant temperature increases. A consequence of this may be a reduction of frost risk for early flowering crops such as apricot in the UK

Towards a joint international database: alignment of SSR marker data for European collections of cherry germplasm

The objective of our study was the alignment of microsatellite or simple sequence repeat (SSR) marker data across germplasm collections of cherry within Europe. Through the European Co-operative program for Plant Genetic Resources ECPGR, a number of European germplasm col-lections had previously been analysed using standard sets of SSR loci. However, until now these datasets remained unaligned. We used a combination of standard reference genotypes and ad-hoc selections to compile a central dataset representing as many alleles as possible from national datasets produced in France, Great Britain, Germany, Italy, Sweden and Switzerland. Through the comparison of alleles called in data from replicated samples we were able to create a series of alignment factors, supported across 448 different allele calls, that allowed us to align a dataset of 2241 SSR profiles from six countries. The proportion of allele comparisons that were either in agreement with the alignment factor or confounded by null alleles ranged from 67% to 100% and this was further improved by the inclusion of a series of allele-specific adjustments. The aligned dataset allowed us to identify groups of previously unknown matching accessions and to identify and resolve a number of errors in the prior datasets. The combined and aligned dataset represents a significant step forward in the co-ordinated management of field collections of cherry in Europe

Genetic analysis of a major international collection of cultivated apple varieties reveals previously unknown historic heteroploid and inbred relationships

Domesticated apple (Malus x domestica Borkh.) is a major global crop and the genetic diversity held within the pool of cultivated varieties is important for the development of future cultivars. The aim of this study was to investigate the diversity held within the domesticated form, through the analysis of a major international germplasm collection of cultivated varieties, the UK National Fruit Collection, consisting of over 2,000 selections of named cultivars and seedling varieties. We utilised Diversity Array Technology (DArT) markers to assess the genetic diversity within the collection. Clustering attempts, using the software STRUCTURE revealed that the accessions formed a complex and historically admixed group for which clear clustering was challenging. Comparison of accessions using the Jaccard similarity coefficient allowed us to identify clonal and duplicate material as well as revealing pairs and groups that appeared more closely related than a standard parent-offspring or full-sibling relations. From further investigation, we were able to propose a number of new pedigrees, which revealed that some historically important cultivars were more closely related than previously documented and that some of them were partially inbred. We were also able to elucidate a number of parent-offspring relationships that had resulted in a number of important polyploid cultivars. This included reuniting polyploid cultivars that in some cases dated as far back as the 18th century, with diploid parents that potentially date back as far as the 13th century

Imaging and Tissue Biomarkers of Choline Metabolism in Diffuse Adult Glioma: 18F-Fluoromethylcholine PET/CT, Magnetic Resonance Spectroscopy, and Choline Kinase α

The cellular and molecular basis of choline uptake on PET imaging and MRS-visible choline containing compounds is not well understood. Choline kinase alpha (ChoKa) is an enzyme that phosphorylates choline, an essential step in membrane synthesis. We investigate choline metabolism through 18F-fluoromethylcholine (18F-FMC) PET, MRS and tissue ChoKa in human glioma. 14 patients with suspected diffuse glioma underwent multimodal 3T MRI and dynamic 18F FMC PET/CT prior to surgery. Co-registered PET and MRI data were used to target biopsies to regions of high and low choline signal, and immunohistochemistry for ChoKa expression was performed. 18F-FMC/PET differentiated WHO grade IV from grade II and III tumours, whereas MRS differentiated grade III/IV from grade II tumours. Tumoural 18F-FMC/PET uptake was higher than in normal-appearing white matter across all grades and markedly elevated within regions of contrast enhancement. 18F-FMC/PET correlated weakly with MRS Cho ratios. ChoKa expression on IHC was negative or weak in all but one GBM sample, and did not correlate with tumour grade or imaging choline markers. MRS and 18F-FMC/PET provide complimentary information on glioma choline metabolism. Tracer uptake is, however, potentially confounded by blood-brain barrier permeability. ChoKa overexpression does not appear to be a common feature in diffuse glioma

Reliability of dynamic contrast-enhanced magnetic resonance imaging data in primary brain tumours: a comparison of Tofts and shutter speed models

Purpose To investigate the robustness of pharmacokinetic modelling of DCE-MRI brain tumour data and to ascertain reliable perfusion parameters through a model selection process and a stability test. Methods DCE-MRI data of 14 patients with primary brain tumours were analysed using the Tofts model (TM), the extended Tofts model (ETM), the shutter speed model (SSM) and the extended shutter speed model (ESSM). A no-effect model (NEM) was implemented to assess overfitting of data by the other models. For each lesion, the Akaike Information Criteria (AIC) was used to build a 3D model selection map. The variability of each pharmacokinetic parameter extracted from this map was assessed with a noise propagation procedure, resulting in voxel-wise distributions of the coefficient of variation (CV). Results The model selection map over all patients showed NEM had the best fit in 35.5% of voxels, followed by ETM (32%), TM (28.2%), SSM (4.3%) and ESSM (<0.1%). In analysing the reliability of Ktrans, when considering regions with a CV<20%, ≈25% of voxels were found to be stable across all patients. The remaining 75% of voxels were considered unreliable. Conclusions The majority of studies quantifying DCE-MRI data in brain tumours only consider a single model and whole-tumour statistics for the output parameters. Appropriate model selection, considering tissue biology and its effects on blood brain barrier permeability and exchange conditions, together with an analysis on the reliability and stability of the calculated parameters, is critical in processing robust brain tumour DCE-MRI data

Identification of genetic variants associated with Huntington's disease progression: a genome-wide association study

Background Huntington's disease is caused by a CAG repeat expansion in the huntingtin gene, HTT. Age at onset has been used as a quantitative phenotype in genetic analysis looking for Huntington's disease modifiers, but is hard to define and not always available. Therefore, we aimed to generate a novel measure of disease progression and to identify genetic markers associated with this progression measure. Methods We generated a progression score on the basis of principal component analysis of prospectively acquired longitudinal changes in motor, cognitive, and imaging measures in the 218 indivduals in the TRACK-HD cohort of Huntington's disease gene mutation carriers (data collected 2008–11). We generated a parallel progression score using data from 1773 previously genotyped participants from the European Huntington's Disease Network REGISTRY study of Huntington's disease mutation carriers (data collected 2003–13). We did a genome-wide association analyses in terms of progression for 216 TRACK-HD participants and 1773 REGISTRY participants, then a meta-analysis of these results was undertaken. Findings Longitudinal motor, cognitive, and imaging scores were correlated with each other in TRACK-HD participants, justifying use of a single, cross-domain measure of disease progression in both studies. The TRACK-HD and REGISTRY progression measures were correlated with each other (r=0·674), and with age at onset (TRACK-HD, r=0·315; REGISTRY, r=0·234). The meta-analysis of progression in TRACK-HD and REGISTRY gave a genome-wide significant signal (p=1·12 × 10−10) on chromosome 5 spanning three genes: MSH3, DHFR, and MTRNR2L2. The genes in this locus were associated with progression in TRACK-HD (MSH3 p=2·94 × 10−8 DHFR p=8·37 × 10−7 MTRNR2L2 p=2·15 × 10−9) and to a lesser extent in REGISTRY (MSH3 p=9·36 × 10−4 DHFR p=8·45 × 10−4 MTRNR2L2 p=1·20 × 10−3). The lead single nucleotide polymorphism (SNP) in TRACK-HD (rs557874766) was genome-wide significant in the meta-analysis (p=1·58 × 10−8), and encodes an aminoacid change (Pro67Ala) in MSH3. In TRACK-HD, each copy of the minor allele at this SNP was associated with a 0·4 units per year (95% CI 0·16–0·66) reduction in the rate of change of the Unified Huntington's Disease Rating Scale (UHDRS) Total Motor Score, and a reduction of 0·12 units per year (95% CI 0·06–0·18) in the rate of change of UHDRS Total Functional Capacity score. These associations remained significant after adjusting for age of onset. Interpretation The multidomain progression measure in TRACK-HD was associated with a functional variant that was genome-wide significant in our meta-analysis. The association in only 216 participants implies that the progression measure is a sensitive reflection of disease burden, that the effect size at this locus is large, or both. Knockout of Msh3 reduces somatic expansion in Huntington's disease mouse models, suggesting this mechanism as an area for future therapeutic investigation

Novel transposon tagging and the expression of potentially lethal constructs in Arabidopsis thaliana

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