Automated tetraploid genotype calling by hierarchical clustering

Key message

New software to make tetraploid genotype calls from SNP array data was developed, which uses hierarchical clustering and multiple F1 populations to calibrate the relationship between signal intensity and allele dosage.

Abstract

SNP arrays are transforming breeding and genetics research for autotetraploids. To fully utilize these arrays, the relationship between signal intensity and allele dosage must be calibrated for each marker. We developed an improved computational method to automate this process, which is provided as the R package ClusterCall. In the training phase of the algorithm, hierarchical clustering within an F1 population is used to group samples with similar intensity values, and allele dosages are assigned to clusters based on expected segregation ratios. In the prediction phase, multiple F1 populations and the prediction set are clustered together, and the genotype for each cluster is the mode of the training set samples. A concordance metric, defined as the proportion of training set samples equal to the mode, can be used to eliminate unreliable markers and compare different algorithms. Across three potato families genotyped with an 8K SNP array, ClusterCall scored 5729 markers with at least 0.95 concordance (94.6% of its total), compared to 5325 with the software fitTetra (82.5% of its total). The three families were used to predict genotypes for 5218 SNPs in the SolCAP diversity panel, compared with 3521 SNPs in a previous study in which genotypes were called manually. One of the additional markers produced a significant association for vine maturity near a well-known causal locus on chromosome 5. In conclusion, when multiple F1 populations are available, ClusterCall is an efficient method for accurate, autotetraploid genotype calling that enables the use of SNP data for research and plant breeding.

     

Mitochondrial genome mutations and neuronal dysfunction of induced pluripotent stem cells derived from patients with Alzheimer's disease

ObjectivesPatient‐derived induced pluripotent stem cells (iPSCs) are materials that can be used for autologous stem cell therapy. We screened mtDNA mutations in iPSCs and iPSC‐derived neuronal cells from patients with Alzheimer''s disease (AD). Also, we investigated whether the mutations could affect mitochondrial function and deposition of β‐amyloid (Aβ) in differentiated neuronal cells.Materials and MethodsmtDNA mutations were measured and compared among iPSCs and iPSC‐derived neuronal cells. The selected iPSCs carrying mtDNA mutations were subcloned, and then their growth rate and neuronal differentiation pattern were analyzed. The differentiated cells were measured for mitochondrial respiration and membrane potential, as well as deposition of Aβ.ResultsMost iPSCs from subjects with AD harbored ≥1 mtDNA mutations, and the number of mutations was significantly higher than that from umbilical cord blood. About 35% and 40% of mutations in iPSCs were shared with isogenic iPSCs and their differentiated neuronal precursor cells, respectively, with similar or different heteroplasmy. Furthermore, the mutations in clonal iPSCs were stable during extended culture and neuronal differentiation. Finally, mtDNA mutations could induce a growth advantage with higher viability and proliferation, lower mitochondrial respiration and membrane potential, as well as increased Aβ deposition.ConclusionThis study demonstrates that mtDNA mutations in patients with AD could lead to mitochondrial dysfunction and accelerated Aβ deposition. Therefore, early screening for mtDNA mutations in iPSC lines would be essential for developing autologous cell therapy or drug screening for patients with AD.

mtDNA mutations were found in induced pluripotent stem cells derived from patients with Alzheimer''s disease. The mutations could induce growth advantage due to their high viability and proliferation. Differentiated neuronal cells with mtDNA mutations exhibited mitochondrial and neuronal dysfunction, as well as increased Aβ deposition.     

Nano-patterned layers of a grafted coumarinic chromophore.

We report on the grafting of coumarin chromophores on flat silicon surfaces and in regions of nanometric dimensions drawn on silicon surfaces. The coumarin derivative was grafted by using the quaternization of a tertiary amine group of the chromophore with a ((chloromethyl)phenylethyl)-dimethylchlorosilane (CMPDCS) grafted on silicon. Complete characterization of the grafted layer was performed as a function of reaction time by X-ray photoelectron spectroscopy, X-ray reflectometry, atomic force microscopy, fluorescence spectroscopy and laser-scanning confocal microscopy. The results indicate that about one chromophore molecule is grafted every second CMPDCS molecule, resulting in a surface density of coumarin of slightly more than one coumarin per nm2. A broadening of the distribution of the fluorescence lifetimes was observed, suggesting that the grafted molecules experience a larger distribution of environments in the grafted layer than in solution. Since this reaction is fully compatible with silicon processing technology, the grafting could also be performed in nano-regions of size as small as 250 nm defined by combining electron-beam lithography with silanization. In such nano-sized regions the distribution of fluorescence lifetimes was narrower, suggesting a possible influence of the confinement on the organization of the molecules.     

Investigating the in vivo calcium transport path to developing potato tuber using 45Ca: a new concept in potato tuber calcium nutrition

Calcium is believed to be transported with water in the xylem. Consistent with this proposal, low‐transpiring organs such as potato Solanum tuberosum tubers are known to suffer from calcium deficiency. Although roots on tubers and stolons have been shown to supply water to tubers, there is no direct evidence for the calcium transport pathway to tubers. Both a xylem and a phloem transport pathway have been suggested. We investigated in vivo calcium transport to developing potato, cv. Dark Red Norland and cv. Russet Burbank, tubers using ⁴⁵Ca in a controlled environment facility. Whole plant split pot experiments allowed the placement of ⁴⁵Ca either in the main (basal) root or the tuber and stolon areas of the pot. The results showed that ⁴⁵Ca was transported to the shoot with the transpiration stream from both areas but was not re‐translocated to tubers or the main (basal) root system even 57 days after ⁴⁵Ca application. Radioactivity could only be detected in the tuber when ⁴⁵Ca was fed to the stolon and tuber area. When ⁴⁵Ca was fed to specific tubers, radioactivity was detected in the aerial shoot; however, no activity was detected in other tubers or the main (basal) roots. In another set of experiments, roots on a stolon near a tuber were precisely fed ⁴⁵Ca and Safranin O. The radioactive signal exactly overlapped the water transport pathway in the tuber marked with Safranin O dye, suggesting that water and calcium can be simultaneously transported from stolon roots to the tuber. No transport of ⁴⁵Ca across the tuber periderm was detected 8 days after ⁴⁵Ca was applied to the tuber periderm. This indicated that no significant transport of calcium occurs from the soil across the periderm. Our results provide evidence that: (1) calcium is not re‐translocated via the phloem from the aerial shoot tubers and main (basal) roots; (2) the main root system does not supply calcium to the tuber; (3) calcium is not transported across the periderm to the interior tuber tissue; (4) calcium is transported to the tuber via the xylem along with water, and the roots on the stolon associated with the tuber supply water and calcium to the developing tuber; and (5) transpirational demand is a significant determinant of calcium distribution within the plant.     

Calcium and zinc complexes of pyrroglutamate analogs detected by electrospray ionization mass spectrometry

The complexation of calcium and zinc cations by pyrroglutamate analogs has been studied in the gas phase by means of electrospray ionization mass spectrometry (ESI–MS). Complexes were obtained from the solutions of calcium perchlorate and zinc perchlorate in acetonitrile. The complexes with calcium are singly and doubly charged with various stoichiometries while zinc complexes are singly charged except for one ligand. Solvation with acetonitrile and presence of perchlorate counter-ions are observed when the complexes are in the gas phase. The complexes formed with both metals are mainly L₂M and LM species. All tested compounds are better complexing agents for calcium than for zinc.     

Root zone calcium modulates the response of potato plants to heat stress

Potato plant growth and development are known to be severely impacted by heat stress. Here plants grown in a chemically inert medium of 1 : 1 quartzite : perlite (v : v) were subjected to either 35/25°C (stress) or 20/15°C (control) day/night air temperatures and four concentrations of root zone calcium (5, 25, 125 and 600 µ M Ca) for 3 weeks. We report for the first time that potato plant growth under heat stress can persist at specific levels of Ca²⁺ in the root zone and that the Ca²⁺ level required for growth under heat stress exceeds that required for growth under normal temperatures. We also provide strong, initial evidence that the ability of high Ca²⁺ levels to mitigate heat stress effects results from shifts in meristematic activity. Total foliar mass and leaf area were essentially unaffected by Ca²⁺ level under control temperatures. Under heat stress, leaf area was reduced to about 5% of the control at 5 and 25 µ M Ca but to only 70% of the control at 125 and 600 µ M Ca. Likewise, total foliar mass was reduced under heat stress to about 30% of the control at 5 and 25 µ M Ca but total foliar mass was greater under heat stress than control conditions at 125 and 600 µ M Ca. This increase at higher Ca²⁺ concentrations was due primarily to axillary shoot growth. Anatomical studies of leaves grown under heat stress show that cell expansion was impaired by heat stress and this impairment was overcome by increasing root zone calcium levels. These results provide insight into the mechanism by which root zone Ca²⁺ may modulate plant response to heat stress.