In Vitro vs In Silico Detected SNPs for the Development of a Genotyping Array: What Can We Learn from a Non-Model Species?

Background: There is considerable interest in the high-throughput discovery and genotyping of single nucleotide polymorphisms (SNPs) to accelerate genetic mapping and enable association studies. This study provides an assessment of EST-derived and resequencing-derived SNP quality in maritime pine (Pinus pinaster Ait.), a conifer characterized by a huge genome size (~23.8 Gb/C). [br/] Methodology/Principal Findings: A 384-SNPs GoldenGate genotyping array was built from i/ 184 SNPs originally detected in a set of 40 re-sequenced candidate genes (in vitro SNPs), chosen on the basis of functionality scores, presence of neighboring polymorphisms, minor allele frequencies and linkage disequilibrium and ii/ 200 SNPs screened from ESTs (in silico SNPs) selected based on the number of ESTs used for SNP detection, the SNP minor allele frequency and the quality of SNP flanking sequences. The global success rate of the assay was 66.9%, and a conversion rate (considering only polymorphic SNPs) of 51% was achieved. In vitro SNPs showed significantly higher genotyping-success and conversion rates than in silico SNPs (+11.5% and +18.5%, respectively). The reproducibility was 100%, and the genotyping error rate very low (0.54%, dropping down to 0.06% when removing four SNPs showing elevated error rates). [br/] Conclusions/Significance: This study demonstrates that ESTs provide a resource for SNP identification in non-model species, which do not require any additional bench work and little bio-informatics analysis. However, the time and cost benefits of in silico SNPs are counterbalanced by a lower conversion rate than in vitro SNPs. This drawback is acceptable for population-based experiments, but could be dramatic in experiments involving samples from narrow genetic backgrounds. In addition, we showed that both the visual inspection of genotyping clusters and the estimation of a per SNP error rate should help identify markers that are not suitable to the GoldenGate technology in species characterized by a large and complex genome

Development and implementation of a highly-multiplexed SNP array for genetic mapping in maritime pine and comparative mapping with loblolly pine

<p>Abstract</p> <p>Background</p> <p>Single nucleotide polymorphisms (SNPs) are the most abundant source of genetic variation among individuals of a species. New genotyping technologies allow examining hundreds to thousands of SNPs in a single reaction for a wide range of applications such as genetic diversity analysis, linkage mapping, fine QTL mapping, association studies, marker-assisted or genome-wide selection. In this paper, we evaluated the potential of highly-multiplexed SNP genotyping for genetic mapping in maritime pine (<it>Pinus pinaster </it>Ait.), the main conifer used for commercial plantation in southwestern Europe.</p> <p>Results</p> <p>We designed a custom GoldenGate assay for 1,536 SNPs detected through the resequencing of gene fragments (707 <it>in vitro </it>SNPs/Indels) and from Sanger-derived Expressed Sequenced Tags assembled into a unigene set (829 <it>in silico </it>SNPs/Indels). Offspring from three-generation outbred (G2) and inbred (F2) pedigrees were genotyped. The success rate of the assay was 63.6% and 74.8% for <it>in silico </it>and <it>in vitro </it>SNPs, respectively. A genotyping error rate of 0.4% was further estimated from segregating data of SNPs belonging to the same gene. Overall, 394 SNPs were available for mapping. A total of 287 SNPs were integrated with previously mapped markers in the G2 parental maps, while 179 SNPs were localized on the map generated from the analysis of the F2 progeny. Based on 98 markers segregating in both pedigrees, we were able to generate a consensus map comprising 357 SNPs from 292 different loci. Finally, the analysis of sequence homology between mapped markers and their orthologs in a <it>Pinus taeda </it>linkage map, made it possible to align the 12 linkage groups of both species.</p> <p>Conclusions</p> <p>Our results show that the GoldenGate assay can be used successfully for high-throughput SNP genotyping in maritime pine, a conifer species that has a genome seven times the size of the human genome. This SNP-array will be extended thanks to recent sequencing effort using new generation sequencing technologies and will include SNPs from comparative orthologous sequences that were identified in the present study, providing a wider collection of anchor points for comparative genomics among the conifers.</p

Head-down tilt bed rest with or without artificial gravity is not associated with motor unit remodeling

© 2020, The Author(s). Purpose: The objective of this study was to assess whether artificial gravity attenuates any long-duration head-down 60 bed rest (HDBR)-induced alterations in motor unit (MU) properties. Methods: Twenty-four healthy participants (16 men; 8 women; 26–54 years) underwent 60-day HDBR with (n = 16) or without (n = 8) 30 min artificial gravity daily induced by whole-body centrifugation. Compound muscle action potential (CMAP), MU number (MUNIX) and MU size (MUSIX) were estimated using the method of Motor Unit Number Index in the Abductor digiti minimi and tibialis anterior muscles 5 days before (BDC-5), and during day 4 (HDT4) and 59 (HDT59) of HDBR. Results: The CMAP, MUNIX, and MUSIX at baseline did not change significantly in either muscle, irrespective of the intervention (p > 0.05). Across groups, there were no significant differences in any variable during HDBR, compared to BDC-5. Conclusion: Sixty days of HDBR with or without artificial gravity does not induce alterations in motor unit number and size in the ADM or TA muscles in healthy individuals

Mov Disord

OBJECTIVE: To determine the rates of brain atrophy progression in vivo in patients with multiple system atrophy (MSA). BACKGROUND: Surrogate biomarkers of disease progression are a major unmet need in MSA. Small-scale longitudinal studies in patients with MSA using magnetic resonance imaging (MRI) to assess progression of brain atrophy have produced inconsistent results. In recent years, novel MRI post-processing methods have been developed enabling reliable quantification of brain atrophy in an automated fashion. METHODS: Serial 3D-T1-weighted MRI assessments (baseline and after 1 year of follow-up) of 43 patients with MSA were analyzed and compared to a cohort of early-stage Parkinson's disease (PD) patients and healthy controls (HC). FreeSurfer's longitudinal analysis stream was used to determine the brain atrophy rates in an observer-independent fashion. RESULTS: Mean ages at baseline were 64.4 +/- 8.3, 60.0 +/- 7.5, and 59.8 +/- 9.2 years in MSA, PD patients and HC, respectively. A mean disease duration at baseline of 4.1 +/- 2.5 years in MSA patients and 2.3 +/- 1.4 years in PD patients was observed. Brain regions chiefly affected by MSA pathology showed progressive atrophy with annual rates of atrophy for the cerebellar cortex, cerebellar white matter, pons, and putamen of -4.24 +/- 6.8%, -8.22 +/- 8.8%, -4.67 +/- 4.9%, and - 4.25 +/- 4.9%, respectively. Similar to HC, atrophy rates in PD patients were minimal with values of -0.41% +/- 1.8%, -1.47% +/- 4.1%, -0.04% +/- 1.8%, and -1.54% +/- 2.2% for cerebellar cortex, cerebellar white matter, pons, and putamen, respectively. CONCLUSIONS: Patients with MSA show significant brain volume loss over 12 months, and cerebellar, pontine, and putaminal volumes were the most sensitive to change in mid-stage disease. (c) 2023 The Authors. Movement Disorders published by Wiley Periodicals LLC on behalf of International Parkinson and Movement Disorder Society

The haemotoxicity of mitomycin in a repeat dose study in the female CD-1 mouse

Mitomycin (MMC), like many antineoplastic drugs, induces a predictable, dose-related, bone marrow depression in man and laboratory animals; this change is generally reversible. However, there is evidence that MMC may also cause a late-stage or residual bone marrow injury. The present study in female CD-1 mice investigated the haematological and bone marrow changes induced by MMC in a repeat dose study lasting 50 days. Control and MMC-treated mice were dosed intraperitoneally on eight occasions over 18 days with vehicle, or MMC at 2.5 mg/kg, autopsied (n = 6–12) at 1, 7, 14, 28, 42 and 50 days after the final dose and haematological changes investigated. Femoral nucleated bone marrow cell counts and levels of apoptosis were also evaluated and clonogenic assays carried out; serum levels of FLT3 ligand (FL) were assessed. At day 1 post-dosing, MMC induced significant reductions in RBC, Hb and haematocrit (HCT) values, and there were decreases in reticulocyte, platelet, and femoral nucleated cell counts (FNCC); neutrophil, lymphocyte and monocyte values were also significantly reduced. On days 7 and 14 post-dosing, all haematological parameters showed evidence of a return towards normal values, but at these times, and at day 28, values for RBC and FNCC remained significantly reduced in comparison with controls. At days 42 and 50 post-dosing, many haematological parameters in MMC-treated mice had returned to control levels; however, there remained evidence of late-stage effects on RBC, Hb and HCT values, and FNCC also continued to be significantly decreased. Results for granulocyte-macrophage colony-forming units and erythroid colonies showed a profound decrease immediately post-dosing, but a return to normal values was evident at day 50. Serum FL concentrations demonstrated very significant increases in the immediate post-dosing period, but a return to normal was seen at day 50 post-dosing; a relatively similar pattern was seen in the number of apoptotic femoral marrow nucleated cells. The histopathological examination of kidney tissues from MMC animals at day 42 and 50 post-dosing showed evidence of hydronephrosis with cortical glomerular/tubular atrophy and degeneration. It is therefore concluded that MMC administered on eight occasions over 18 days to female CD-1 mice at 2.5 mg/kg induced profound changes in haematological and bone marrow parameters in the immediate post-dosing period with a return to normal levels at day 50 post-dosing; however, there was evidence of mild but significant late-stage/residual effects on RBC and FNCC, and on cells of the erythroid lineage in the bone marrow