Development and preliminary evaluation of a 90?K Axiom? SNP array for the allo-octoploid cultivated strawberry Fragaria × ananassa

Background

A high-throughput genotyping platform is needed to enable marker-assisted breeding in the allo-octoploid cultivated strawberry Fragaria × ananassa. Short-read sequences from one diploid and 19 octoploid accessions were aligned to the diploid Fragaria vesca ‘Hawaii 4’ reference genome to identify single nucleotide polymorphisms (SNPs) and indels for incorporation into a 90 K Affymetrix® Axiom® array. We report the development and preliminary evaluation of this array.

Results

About 36 million sequence variants were identified in a 19 member, octoploid germplasm panel. Strategies and filtering pipelines were developed to identify and incorporate markers of several types: di-allelic SNPs (66.6%), multi-allelic SNPs (1.8%), indels (10.1%), and ploidy-reducing “haploSNPs” (11.7%). The remaining SNPs included those discovered in the diploid progenitor F. iinumae (3.9%), and speculative “codon-based” SNPs (5.9%). In genotyping 306 octoploid accessions, SNPs were assigned to six classes with Affymetrix’s “SNPolisher” R package. The highest quality classes, PolyHigh Resolution (PHR), No Minor Homozygote (NMH), and Off-Target Variant (OTV) comprised 25%, 38%, and 1% of array markers, respectively. These markers were suitable for genetic studies as demonstrated in the full-sib family ‘Holiday’ × ‘Korona’ with the generation of a genetic linkage map consisting of 6,594 PHR SNPs evenly distributed across 28 chromosomes with an average density of approximately one marker per 0.5 cM, thus exceeding our goal of one marker per cM.

Conclusions

The Affymetrix IStraw90 Axiom array is the first high-throughput genotyping platform for cultivated strawberry and is commercially available to the worldwide scientific community. The array’s high success rate is likely driven by the presence of naturally occurring variation in ploidy level within the nominally octoploid genome, and by effectiveness of the employed array design and ploidy-reducing strategies. This array enables genetic analyses including generation of high-density linkage maps, identification of quantitative trait loci for economically important traits, and genome-wide association studies, thus providing a basis for marker-assisted breeding in this high value crop.

Electronic supplementary material

The online version of this article (doi:10.1186/s12864-015-1310-1) contains supplementary material, which is available to authorized users.     

Regulation of matrix metalloproteinase (MMP) activity by the low-density lipoprotein receptor-related protein (LRP). A new function for an "old friend"

Matrix metalloproteinases (MMPs) are essential contributors to a microenvironment that promotes tumour progression. During the two last decades, inhibition of MMPs has become the focus of considerable interest for cancer therapy, and numerous synthetic metalloproteinase inhibitors have been developed by the pharmaceutical industry. However, clinical trials have shown disappointing efficacy or unexpected toxicity and new targets are thus eagerly awaited. The identification of endocytic clearance of several MMPs by the low-density lipoprotein receptor-related protein (LRP) might provide insight into novel strategies for controlling MMP level during malignant processes. This review attempts to summarize recent aspects on the cellular and molecular basis of LRP-mediated endocytic disposal of MMPs.     

Collagen and elastin cross-linking: a mechanism of constrictive remodeling after arterial injury

Constrictive remodeling after arterial injury is related to collagen accumulation. Cross-linking has been shown to induce a scar process in cutaneous wound healing and is increased after arterial injury. We therefore evaluated the effect of cross-linking inhibition on qualitative and quantitative changes in collagen, elastin, and arterial remodeling after balloon injury in the atherosclerotic rabbit model. Atherosclerotic-like lesions were induced in femoral arteries of 28 New Zealand White rabbits by a combination of air desiccation and a high-cholesterol diet. After 1 mo, balloon angioplasty was performed in both femoral arteries. Fourteen rabbits were fed beta-aminopropionitrile (beta-APN, 100 mg/kg) and compared with 14 untreated animals. The remodeling index, i.e., the ratio of external elastic lamina at the lesion site to external elastic lamina at the reference site, was determined 4 wk after angioplasty for both groups. Pyridinoline was significantly decreased in arteries from beta-APN-treated animals compared with controls, confirming inhibition of collagen cross-linking: 0.30 (SD 0.03) and 0.52 (SD 0.02) mmol/mol hydroxyproline, respectively (P = 0.002). Scanning and transmission electron microscopy showed a profound disorganization of collagen fibers in arteries from beta-APN-treated animals. The remodeling index was significantly higher in beta-APN-treated than in control animals [1.1 (SD 0.3) vs. 0.8 (SD 0.3), P = 0.03], indicating favorable remodeling. Restenosis decreased by 33% in beta-APN-treated animals: 32% (SD 16) vs. 48% (SD 24) (P = 0.02). Neointimal collagen density was significantly lower in beta-APN-treated animals than in controls: 23.0% (SD 3.8) vs. 29.4% (SD 4.0) (P = 0.004). These findings suggest that collagen and elastin cross-linking plays a role in the healing process via constrictive remodeling and restenosis after balloon injury in the atherosclerotic rabbit model.     

Conical tomography of freeze-fracture replicas: a method for the study of integral membrane proteins inserted in phospholipid bilayers

We have used conical tomography to study the structure of integral proteins in their phospholipid bilayer environments. Complete conical series were collected from replicas of the water channel aquaporin-0 (AQP0), a 6.6 nm side tetramer with a molecular weight of approximately 120 kDa that was purified and reconstituted in liposomes. The replicas were tilted at 38 degrees , 50 degrees or 55 degrees and rotated by 2.5 degrees , 4 degrees , or 5 degrees increments until completing 360 degrees turns. The elliptical paths of between 6 and 12 freeze-fracture particles aligned the images to a common coordinate system. Using the weighted back projection algorithm, small volumes of the replicas were independently reconstructed to reconstitute the field. Using the Fourier Shell Correlation computed from reconstructions of even and odd projections of the series, we estimated a resolution of 2-3 nm, a value that was close to the thickness of the replica (approximately 1.5 nm). The 3D reconstructions exhibited isotropic resolution along the x-y plane, which simplified the analysis of particles oriented randomly in the membrane plane. In contrast to reconstructions from single particles imaged using random conical tilt [J. Mol. Biol. 325 (2003) 210], the reconstructions using conical tomography allowed the size and shape of individual particles representing the AQP0 channel to be identified without averaging or imposing symmetry. In conclusion, the reconstruction of freeze-fracture replicas with electron tomography has provided a novel experimental approach for the study of integral proteins inserted in phospholipid bilayers.     

Low density lipoprotein receptor-related protein mediates endocytic clearance of pro-MMP-2.TIMP-2 complex through a thrombospondin-independent mechanism

The low density lipoprotein receptor-related protein (LRP) mediates the endocytic clearance of various proteinases and proteinase.inhibitor complexes, including thrombospondin (TSP)-dependent endocytosis of matrix metalloproteinase (MMP)-2 (or gelatinase A), a key effector of extracellular matrix remodeling and cancer progression. However, the zymogen of MMP-2 (pro-MMP-2) mostly occurs in tissues as a complex with the tissue inhibitor of MMPs (TIMP-2). Here we show that clearance of the pro-MMP-2.TIMP-2 complex is also mediated by LRP, because addition of receptor-associated protein (RAP), a natural LRP ligand antagonist, inhibited endocytosis and lysosomal degradation of (125)I-pro-MMP-2.TIMP-2. Both TIMP-2 and the pro-MMP-2 collagen-binding domain independently competed for endocytosis of (125)I-pro-MMP-2.TIMP-2 complex. Surface plasmon resonance studies indicated that pro-MMP-2, TIMP-2, and pro-MMP-2.TIMP-2 directly interact with LRP in the absence of TSP. LRP-mediated endocytic clearance of (125)I-pro-MMP-2 was inhibited by anti-TSP antibodies and accelerated upon complexing with TSP-1, but these treatments had no effect on (125)I-pro-MMP-2.TIMP-2 uptake. This implies that mechanisms of clearance by LRP of pro-MMP-2 and pro-MMP-2.TIMP-2 complex are different. Interestingly, RAP did not inhibit binding of (125)I-pro-MMP-2.TIMP-2 to the cell surface. We conclude that clearance of pro-MMP-2.TIMP-2 complex is a TSP-independent two-step process, involving (i) initial binding to the cell membrane in a RAP-insensitive manner and (ii) subsequent LRP-dependent (RAP-sensitive) internalization and degradation.     

Examining the Role of Collective Action in an Informal Seed System: A Case Study from the Central Valleys of Oaxaca, Mexico

This paper explores social arrangements associated with seed transactions among small-scale maize farmers in the Central Valleys of Oaxaca, Mexico, where no formal seed supply system exists. We test the hypothesis that individual farmers have strong incentives to participate in some form of collective action to ensure their access to seed. Six communities were studied, three of them in detail, using in-depth, semistructured interviews with key informants; focus group discussions; and a tracer study that followed seed flows among farmers. Farmers mostly saved seed and only occasionally acquired seed from outside sources. We found no evidence of a specialized social organization based on collective action to mediate seed flows. Seed transactions are infrequent, bilateral, and ad hoc, although trust is an important component, as it ensures reliable information about the seed is provided. Implications of these findings are discussed, especially for genetic diversity if the current supply system breaks down.     

Discovery and optimization of a potent and selective triazolopyridinone series of c-Met inhibitors

Deregulation of the receptor tyrosine kinase c-Met has been implicated in several human cancers and is an attractive target for small molecule drug discovery. Herein, we report the discovery of a structurally diverse series of carbon-linked quinoline triazolopyridinones, which demonstrates nanomolar inhibition of c-Met kinase activity. This novel series of inhibitors exhibits favorable pharmacokinetics as well as potent inhibition of HGF-mediated c-Met phosphorylation in a mouse liver pharmacodynamic model.     

Polymorphisms in the mitochondrial ribosome recycling factor EF-G2mt/MEF2 compromise cell respiratory function and increase atorvastatin toxicity

Mitochondrial translation, essential for synthesis of the electron transport chain complexes in the mitochondria, is governed by nuclear encoded genes. Polymorphisms within these genes are increasingly being implicated in disease and may also trigger adverse drug reactions. Statins, a class of HMG-CoA reductase inhibitors used to treat hypercholesterolemia, are among the most widely prescribed drugs in the world. However, a significant proportion of users suffer side effects of varying severity that commonly affect skeletal muscle. The mitochondria are one of the molecular targets of statins, and these drugs have been known to uncover otherwise silent mitochondrial mutations. Based on yeast genetic studies, we identify the mitochondrial translation factor MEF2 as a mediator of atorvastatin toxicity. The human ortholog of MEF2 is the Elongation Factor Gene (EF-G) 2, which has previously been shown to play a specific role in mitochondrial ribosome recycling. Using small interfering RNA (siRNA) silencing of expression in human cell lines, we demonstrate that the EF-G2mt gene is required for cell growth on galactose medium, signifying an essential role for this gene in aerobic respiration. Furthermore, EF-G2mt silenced cell lines have increased susceptibility to cell death in the presence of atorvastatin. Using yeast as a model, conserved amino acid variants, which arise from non-synonymous single nucleotide polymorphisms (SNPs) in the EF-G2mt gene, were generated in the yeast MEF2 gene. Although these mutations do not produce an obvious growth phenotype, three mutations reveal an atorvastatin-sensitive phenotype and further analysis uncovers a decreased respiratory capacity. These findings constitute the first reported phenotype associated with SNPs in the EF-G2mt gene and implicate the human EF-G2mt gene as a pharmacogenetic candidate gene for statin toxicity in humans.     

Post-translational tyrosine nitration of eosinophil granule toxins mediated by eosinophil peroxidase

Nitration of tyrosine residues has been observed during various acute and chronic inflammatory diseases. However, the mechanism of tyrosine nitration and the nature of the proteins that become tyrosine nitrated during inflammation remain unclear. Here we show that eosinophils but not other cell types including neutrophils contain nitrotyrosine-positive proteins in specific granules. Furthermore, we demonstrate that the human eosinophil toxins, eosinophil peroxidase (EPO), major basic protein, eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), and the respective murine toxins, are post-translationally modified by nitration at tyrosine residues during cell maturation. High resolution affinity-mass spectrometry identified specific single nitration sites at Tyr349 in EPO and Tyr33 in both ECP and EDN. ECP and EDN crystal structures revealed and EPO structure modeling suggested that the nitrated tyrosine residues in the toxins are surface exposed. Studies in EPO(-/-), gp91phox(-/-), and NOS(-/-) mice revealed that tyrosine nitration of these toxins is mediated by EPO in the presence of hydrogen peroxide and minute amounts of NOx. Tyrosine nitration of eosinophil granule toxins occurs during maturation of eosinophils, independent of inflammation. These results provide evidence that post-translational tyrosine nitration is unique to eosinophils.