Genomics

Genome-wide analysis of the Hsf gene family in Brassica oleracea and a comparative analysis of the Hsf gene family in B. oleracea , B. rapa and B. napus Abstract The global climate change-induced abiotic and biotic stresses are predicted to affect crop-growing seasons and crop yield. Heat stress transcription factors (Hsfs) have been suggested to play a significant role in various stress responses. They are an integral part of the signal transduction pathways that operate in response to environmental stresses. Brassica oleracea is one of the agronomical important crop species which consists of cabbage, cauliflower, broccoli, Brussels sprout, kohlrabi and kale. The identification and roles of Hsfs in this important Brassica species are unknown. The availability of whole genome sequence of B. oleracea provides us an opportunity for performing in silico analysis of Hsf genes in B. oleracea. Thirty-five putative genes encoding Hsf proteins were identified and classified into A, B and C classes. Their evolution, physical location, gene structure, domain structure and tissue-specific expression patterns were investigated. Further, a comparative analysis of the Hsf gene family in B. oleracea, B. rapa and B. napus highlighted the role of hybridisation and allopolyploidy in the evolution of the largest known Hsf gene family in B. napus. The presence of orthologous gene clusters, found in Brassica species, but not in A. thaliana, suggested that polyploidisation has resulted in the formation of new Brassica-specific orthologous gene clusters. Gene duplication analysis indicated that the evolution of the Hsf gene family was under strong purifying selection in these Brassica species. High-level synteny was observed within the B. napus genome. Conservation of physical location, the similarity of structure and similar expression profiles between the B. napus Hsf genes and the corresponding genes from B. oleracea and B. rapa suggest a high functional similarity between these genes. This study paves the way for further investigation of Hsf genes in improving stress tolerance in B. oleracea. The genes thus identified may be useful for developing crop varieties resilient to the global climate change.

The RNA-binding protein FXR1 modulates prostate cancer progression by regulating FBXO4 Abstract This paper is to characterize the expression status of Fragile X Mental Retardation, Autosomal Homolog 1 (FXR1) in prostate cancer cells and understand its mechanistic involvement in the tumor biology of prostate cancer. The relative expression of FXR1 in prostate cancer cells was determined by real-time polymerase chain reaction and Western blotting. Cell proliferation in FXR1-deficient cells was evaluated by cell counting and MTT assays. The migrative and invasive capacities were measured by transwell assay. The potential regulatory effect of FXR1 on FBXO4 was interrogated using luciferase reporter assay. The direct bind of FXR1 with FBXO4 transcripts was analyzed by RNA immunoprecipitation and RNA pull-down assay. We observed aberrant overexpression of FXR1 in prostate cancer cells at both transcript and protein levels. FXR1 deficiency was associated with inhibited cell proliferation/viability and compromised migration/invasion in prostate cancer cells. Mechanistically, FXR1 negatively regulated FBXO4 transcripts via direct association with its 3′UTR and promoted mRNA degradation. FBXO4 knockdown predominantly rescued the tumor-suppressive phenotype in FXR1-deficient cells. We uncovered the oncogenic role of FXR1 in prostate cancer cells and further demonstrated its dependence on FBXO4. Our data highlight the importance of FXR1-FBXO4 signaling in prostate cancer.

Cryptochrome deletion in p53 mutant mice enhances apoptotic and anti-tumorigenic responses to UV damage at the transcriptome level Abstract Previous studies have demonstrated that deletion of cryptochrome (Cry) genes protects p53−/− mutant mice from the early onset of cancer and extends their median life-span by about 1.5-fold. Subsequent in vitro studies had revealed that deletion of Crys enhances apoptosis in response to UV damage through activation of p73 and inactivation of GSK3β. However, it was not known at the transcriptome-wide level how deletion of Crys delays the onset of cancer in p53−/− mutant mice. In this study, the RNA-seq approach was taken to uncover the differentially expressed genes (DEGs) and pathways following UV-induced DNA damage in p53−/− and p53−/−Cry1−/−Cry2−/− mouse skin fibroblasts. Gene set enrichment analysis with the DEGs demonstrated enrichment in immune surveillance-associated genes regulated by IFN-γ and genes involved in TNFα signaling via NF-κB. Furthermore, protein network analysis enabled identification of DEGs p21, Sirt1, and Jun as key players, along with their interacting partners. It was also observed that the DEGs contained a high ratio of non-coding transcripts. Collectively, the present study suggests new genes in NF-κB regulation and IFN-γ response, as well as non-coding RNAs, may contribute to delaying the onset of cancer in p53−/−Cry1−/−Cry2−/− mice and increasing the life-span of these animals compared to p53−/− mice.

Computational detection and experimental validation of segmental duplications and associated copy number variations in water buffalo ( Bubalus bubalis ) Abstract Duplicated sequences are an important source of gene evolution and structural variation within mammalian genomes. Using a read depth approach based on next-generation sequencing, we performed a genome-wide analysis of segmental duplications (SDs) and associated copy number variations (CNVs) in the water buffalo (Bubalus bubalis). By aligning short reads of Olimpia (the reference water buffalo) to the UMD3.1 cattle genome, we identified 1,038 segmental duplications comprising 44.6 Mb (equivalent to ~1.73% of the cattle genome) of the autosomal and X chromosomal sequence in the buffalo genome. We experimentally validated 70.3% (71/101) of these duplications using fluorescent in situ hybridization. We also detected a total of 1,344 CNV regions across 14 additional water buffaloes, amounting to 59.8 Mb of variable sequence or the equivalent of 2.2% of the cattle genome. The CNV regions overlap 1,245 genes that are significantly enriched for specific biological functions including immune response, oxygen transport, sensory system and signal transduction. Additionally, we performed array Comparative Genomic Hybridization (aCGH) experiments using the 14 water buffaloes as test samples and Olimpia as the reference. Using a linear regression model, a high Pearson correlation (r = 0.781) was observed between the log2 ratios between copy number estimates and the log2 ratios of aCGH probes. We further designed Quantitative PCR assays to confirm CNV regions within or near annotated genes and found 74.2% agreement with our CNV predictions. These results confirm sub-chromosome-scale structural rearrangements present in the cattle and water buffalo. The information on genome variation that will be of value for evolutionary and phenotypic studies, and may be useful for selective breeding of both species.

Clinical manifestations and AR gene mutations in Kennedy's disease Abstract Kennedy's disease, resulted from the expansion of a CAG repeat in exon 1 of androgen receptor (AR) gene, is a motor neuron degenerative disease in the brainstem and spinal cord with the slow development of facial, bulbar, and limb muscle degeneration. To investigate the clinical manifestations and gene mutations in Han Chinese patients with Kennedy's disease. The clinical manifestations of 5 male Han Chinese patients including 2 probands and their relatives from 2 families and 1 sporadic case were retrospectively studied. The CAG repeats in the first exon of AR were screened in 5 Han Chinese people including 2 probands and their healthy relatives from 2 families and 1 sporadic case by polymerase chain reaction (PCR) and direct sequencing. The average age at onset of Kennedy's disease was 48.20 ± 8.70 (mean ± SD) years and the average duration was 7.60 ± 5.32 years. All the patients showed slow onset and progressive weakness, wasting, and fasciculations of the whole body. Four patients demonstrated decreased fertility and 1 patient showed mild gynecomastia. Serum creatine kinase and testosterone levels were elevated mildly in 2 and 1 patients, respectively. The electromyogram showed neurogenic abnormalities. Muscle magnetic resonance demonstrated reduced muscle volume and fatty infiltration. Three different enlarged CAG domains were discovered in the 2 families and 1 sporadic patient with Kennedy's disease, and the CAG repeat number was 48, 43, and 44, respectively. The clinical manifestations of Kennedy's disease in Han Chinese middle-aged men were progressive weakness and atrophy in the bulbar and spinal muscles, occasionally demonstrating incomplete androgen insensitivity syndrome. These patients were also characterized with enlarged CAG repeat number in the first exon of AR, indicating that CAG number could be used in the diagnosis of Han Chinese patients with Kennedy's disease.

Discovery and profiling of small RNAs from Puccinia triticina by deep sequencing and identification of their potential targets in wheat Abstract Cross-kingdom RNAi is a well-documented phenomenon where sRNAs generated by host and pathogens may govern resistance or susceptible phenotypes during host-pathogen interaction. With the first example of the direct involvement of fungal generated sRNAs in virulence of plant pathogenic fungi Botrytis cinerea and recently from Puccinia striiformis f. sp. tritici, we attempted to identify sRNAs in Puccinia triticina (P. triticina). Four sRNA libraries were prepared and sequenced using Illumina sequencing technology and a total of ~ 1–1.28 million potential sRNAs and two microRNA-like small RNA (mil-RNAs) candidates were identified. Computational prediction of targets using a common set of sRNAs and P. triticina mil-RNAs (pt-mil-RNAs) within P. triticina and wheat revealed the majority of the targets as repetitive elements in P. triticina whereas in wheat, the target genes were identified to be involved in many biological processes including defense-related pathways. We found 9 receptor-like kinases (RLKs) and 14 target genes of each related to reactive oxygen species (ROS) pathway and transcription factors respectively, including significant numbers of target genes from various other categories. Expression analysis of twenty selected sRNAs, targeting host genes pertaining to ROS related, disease resistance, metabolic processes, transporter, apoptotic inhibitor, and transcription factors along with two pt-mil-RNAs by qRT-PCR showed distinct patterns of expression of the sRNAs in urediniospore-specific libraries. In this study, for the first time, we report identification of novel sRNAs identified in P. triticina including two pt-mil-RNAs that may play an important role in biotrophic growth and pathogenicity.

Leaf rust ( Puccinia triticina ) mediated RNAi in wheat ( Triticum aestivum L.) prompting host susceptibility Abstract Significance of microRNAs in regulating gene expression in higher eukaryotes as well as in pathogens like fungi to suppress host defense is a well-established phenomenon. The present study focuses on leaf rust fungi Puccinia triticina (Pathotype 77-5) mediated RNAi to make wheat (Triticum aestivum L.) more susceptible. To reach such conclusions, we first confirmed the presence of argonaute (AGO) and dicer-like protein (DCL) family sequences in Puccinia. Bioinformatic tools were applied to retrieve the sequences from Puccinia genome followed by cloning and sequencing from P. triticina pathotype 77-5 cDNA to obtain the specific sequences. Their homologs were searched in other 14 Puccinia races to relate them with pathogenesis. Further, precursor sequences for three miRNA-like RNA molecules (milRs) were cloned from P. triticina cDNA. Their target genes like MAP kinase were successfully predicted and validated through degradome mapping and qRT-PCR. Gradual increase in milR2 (milR and milR*) expression over progressive time point of infection and positive expression for all the milRs within 77-5 urediniospores confirmed a complete host- independent RNAi activity by P. triticina.

Proteomic and transcriptomic analysis to unravel the influence of high temperature on banana fruit during postharvest storage Abstract Banana, an important food, incurs significant economic losses due to high storage temperature. Integrative analysis of proteome and transcriptome profiles of the banana peel stored at 20 °C (control) and 30 °C (HT) was used to investigate the molecular mechanism in response to high temperature stress. Critical proteins and genes relating to the response of banana fruit to HT stress were evaluated using partial least squares-discriminant analysis (PLS-DA) and orthogonal signal correction partial least squares-discriminant analysis (OPLS-DA). HT stress influenced proteins/genes related to chlorophyll metabolism, fruit firmness, signal transduction, energy metabolism, and stress response and defense. Together with scanning electron microscopy (SEM) and real time quantitative PCR (RT-qPCR) results, it can be concluded that HT stress resulted in stay-green ripening of banana fruit. Additionally, HT stress accelerated firmness loss and senescence of banana peel, might mainly through regulating hormone signaling pathway, stress protective ability, and energy metabolism in the banana peel. Our study provided a clearer understanding of regulatory mechanisms of HT treatment on banana fruit and potential genetic resources for the improvement of high temperature-tolerant characteristics in banana fruit.

Transcriptomics analysis of propiconazole-treated Cochliobolus sativus reveals new putative azole targets in the plant pathogen Abstract Cochliobolus sativus (anamorph: Bipolaris sorokiniana) is a filamentous fungus from the class Dothideomycetes. It is a pathogen of cereals including wheat and barley, and causes foliar spot blotch, root rot, black point on grains, head blight, leaf blight, and seedling blight diseases. Annual yields of these economically important cereals are severely reduced due to this pathogen attack. Evolution of fungicide resistant pathogen strains, availability of a limited number of potent antifungal compounds, and their efficacy are the acute issues in field management of phytopathogenic fungi. Propiconazole is a widely used azole fungicide to control the disease in fields. The known targets of azoles are the demethylase enzymes involved in ergosterol biosynthesis. Nonetheless, azoles have multiple modes of action, some of which have not been explored yet. Identifying the off-target effects of fungicides by dissecting gene expression profiles in response to them can provide insights into their modes of action and possible mechanisms of fungicide resistance. Moreover it can also reveal additional targets for development of new fungicides. Hence, we analyzed the global gene expression profile of C. sativus on exposure to sub-lethal doses of propiconazole in a time series. The gene expression patterns were confirmed using quantitative reverse transcriptase PCR (qRT-PCR). This study revealed overexpression of target genes from the sterol biosynthesis pathway supporting the reported mode of resistance against azoles. In addition, some new potential targets have also been identified, which could be explored to develop new fungicides and plant protection strategies.

Analysis of genes encoding seed storage proteins (SSPs) in chickpea ( Cicer arietinum L.) reveals co-expressing transcription factors and a seed-specific promoter Abstract Improvement of the quality and quantity of chickpea seed protein can be greatly facilitated by an understanding of the genic organization and the genetic architecture of the genes encoding seed storage proteins (SSPs). The aim of this study was to provide a comprehensive analysis of the chickpea SSP genes, putative co-expressing transcription factors (TFs), and to identify a seed-specific SSP gene promoter. A genome-wide identification of SSP genes in chickpea led to the identification of 21 non-redundant SSP encoding genes located on 6 chromosomes. Phylogenetic analysis grouped SSP genes into 3 subgroups where members within the same clade demonstrated similar motif composition and intron-exon organization. Tandem duplications were identified to be the major contributors to the expansion of the SSP gene family in chickpea. Co-expression analysis revealed 14 TFs having expression profiles similar to the SSP genes that included members of important TF families that are known to regulate seed development. Expression analysis of SSP genes and TFs revealed significantly higher expression in late stages of seed development as well as in high seed protein content (HPC) genotypes. In silico analysis of the promoter regions of the SSP encoding genes revealed several seed-specific cis-regulatory elements such as RY repeats, ACGT motifs, CAANTG, and GCN4. A candidate promoter was analyzed for seed specificity by generating stable transgenics in Arabidopsis. Overall, this study provides a useful resource to explore the regulatory networks involved in SSP synthesis and/or accumulation for utilization in developing nutritionally improved chickpea genotypes. Complementary gene interaction and xenia effect controls the seed coat colour in interspecific cross between Trifolium alexandrinum and T. apertum Abstract Trifolium alexandrinum (Egyptian clover) is a widely cultivated winter annual fodder. Present work deals with inheritance of the seed coat colour in segregating progenies of the interspecific cross between T. alexandrinumand T. apertum. Although, both the parent species possessed yellow seed coat, the F1 seeds were black coloured in the reciprocal cross (T. apertum × T. alexandrinum). Seeds borne on individual F2 plants and the advancing generations segregated in yellow and black seed coat colour, which confirmed xenia effect. F2 seeds collected from individual F1 plants exhibited nine black and seven yellow segregation ratio. The segregation of the seed coat colour recorded from F3 to F5 generations revealed that yellow seed coat was true breeding (i.e. non-segregating) in this interspecific cross (including the reciprocal crosses). However, the black seeded progenies were either true breeding or segregated in nine black: seven yellow ratio or three black: one yellow ratio suggesting a complementary gene interaction or duplicate recessive epistasis. It indicated that the seed coat colour is controlled by complementary gene interaction along with xenia effect in interspecific crosses between T. alexandrinum and T. apertum. Occurrence of the complementary genes across the species could suggest T. apertum to be the progenitor of T. alexandrinum. Inheritance of seed coat colour in reference to its importance in Egyptian clover breeding is also discussed. TaEPFL1 , an EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) secreted peptide gene, is required for stamen development in wheat Abstract Members of the EPIDERMAL PATTERNING FACTOR-LIKE (EPFL) family play diverse roles in plant growth and development, including the guidance of inflorescence architecture and pedicel length. In this work, we identified and characterized the EFPL gene TaEPFL1 from the wheat pistillody mutant HTS-1. Sequence alignment and phylogenetic analysis indicated that TaEPFL1 belongs to the EPFL1 gene. Quantitative real-time RT-PCR analysis showed that the TaEPFL1 gene is expressed at an abnormally high level in pistillody stamens compared with that in pistils and stamens. Heterologous expression of the TaEPFL1 gene in Arabidopsis caused shortened filaments and pedicels and might reduce the level of AtACO2 gene expression. These results suggest that TaEPFL1 plays an important role in the development of stamen and that overexpression of TaEPFL1results in abnormal stamens. We deduced that the overexpression of the TaEPFL1 gene may contribute to the homeotic transformation of stamens into pistils or pistil-like structures in wheat. These data offer insights into the molecular mechanism of pistillody mutation in wheat. Genome-wide identification and expression analysis of the AAAP family in Medicago truncatula Abstract The amino acid/auxin permease (AAAP) gene family plays an important role in the long-distance amino acid transport pathway and takes part in various stages of plant growth and development. However, little is known about the AAAP gene family in Medicago truncatula. Here, we identified 86 putative MtAAAP family members using genome sequence information. Based on phylogenetic analysis, these MtAAAP genes were categorized into eight distinct subfamilies. The MtAAAP genes were mapped on 8 chromosomes and duplication events appeared widely, with 19 and 21 pairs of MtAAAP genes showing segment and tandem duplication events, respectively. Ratio of Ka/Ks indicated that duplicated genes underwent purifying selection. Analysis of RNA-seq data showed that MtAAAP genes exhibited specific expression patterns among different tissues and abiotic stress, indicating that MtAAAP members were involved in plant developmental regulation and stress responses. Expression patterns of 16 MtAAAP genes under abiotic stress were verified by qRT-PCR. The present study provides a foundation for the functional analysis of MtAAAPs in developmental regulation and stress responses. Genetic structure of Kho population from north-western Pakistan based on mtDNA control region sequences Abstract The Kho population speaking Khowar language reside since long ago in Chitral District of North-western Pakistan. So far, no report is available about their genetic structure and relationship with surrounding population groups. We partially sequenced the mitochondrial DNA control region from 16 unrelated Kho male and female individuals of different ages. The D-Loop region sequences of Kho were aligned and compared with the revised Cambridge Reference Sequence (rCRS). The genetic data of Kho was compared with surrounding north-western Pakistani population groups including Pathan, Kashmiri, and Hazara. Comparison with rCRS identified overall 49 different haplotypes for Kho samples. Among these 21 haplotypes were shared by more than one Kho individuals. The genetic diversity and power of discrimination observed for Kho group were 0.215 and 0.202 respectively indicating the Kho tribe as a least differentiated group among north-western Pakistani populations. The haplogroup mapping, phylogenetic and haplotype network analysis revealed the nearby maternal ancestral relationship between Kho and Kashmiri populations. The haplogroups analysis demonstrates the western Eurasian ancestral origin of Kho samples. However, the appearance of a few South Asian haplogroups with low frequency speculate the Kho tribe as an admixed population of western Eurasian and South Asian genetic components. Comparative mitogenomic and phylogenetic analysis of Apalone spinifera and Apalone ferox (Testudines: Trionychidae) Abstract The soft-shell turtles Apalone spinifera (AS) and Apalone ferox (AF) are two important economic species. AF is found in the Yellow River of China, and is a confirmed member of the Trionychidae family. However, the classification of AS was in dispute. Mitochondrial genomes (mitogenomes) have been widely used for species identification, as well as population and phylogenetic analysis. In order to understand the phylogenetic and mitogenomic features of AS and AF, the complete mitogenomes were sequenced, annotated and analyzed in this study. The complete mitogenomes of AS and AF are 16,817 bp and 16,756 bp in length, respectively. Both mitogenomes contain 37 genes, seven short intergenic spacers and two long intergenic spacers. Comparative analysis showed that there are 1,137 variation sites (6.79%) between the two mitogenomes. AS and AF mitogenomes both show a usage preference in terms of nucleotides, codons and amino acids. In addition, the non-synonymous substitution rate/synonymous substitution rate indicates that all protein-coding genes (PCGs) have undergone a strong purifying selection. Phylogenetic trees constructed by 13 PCGs show a clear phylogenetic relationship of the soft-shell turtles and suggest that AS is a sister species to AF of the genus Apalone. The data could be useful for further research of species identification, population analysis and the mitogenomic features of soft-shell turtles. Genetic diversity and demography of the critically endangered Roberts' false brook salamander ( Pseudoeurycea robertsi ) in Central Mexico Abstract Land use changes are threatening the maintenance of biodiversity. Genetic diversity is one of the main indicators of biological diversity and is highly important as it shapes the capability of populations to respond to environmental changes. We studied eleven populations of Pseudoeurycea robertsi, a micro-endemic and critically endangered species from the Nevado de Toluca Volcano, a mountain that is part of the Trans-Mexican Volcanic Belt, Mexico. We sequenced the mitochondrial cytochrome b gene from 71 individuals and genotyped 9 microsatellites from 150 individuals. Our results based on the cytochrome b showed two divergent lineages, with moderate levels of genetic diversity and a recently historical demographic expansion. Microsatellite-based results indicated low levels of heterozygosity for all populations and few alleles per locus, as compared with other mole salamander species. We identified two genetically differentiated subpopulations with a significant level of genetic structure. These results provide fundamental data for the development of management plans and conservation efforts for this critically endangered species. Adaptive evolution of the ACSL gene family in Carnivora Abstract Carnivores exhibit various fat contents and energy reserves to adapt to their environments. However, the molecular mechanisms underlying lipid metabolic differences among carnivores have not been well explored. Long-chain acyl-CoA synthetases (ACSLs) catalyze the initial step in lipid metabolism by activating fatty acids (FAs), and they drive acyl-CoAs toward anabolic lipid synthesis or catabolic β-oxidation. We identified the sequences of the genes of the ACSL family (ACSL1, ACSL3, ACSL4, ACSL5 and ACSL6) in the sable (Martes zibellina) via transcriptome sequencing. The ACSL gene sequences of 13 other carnivores were obtained from NCBI. Phylogenetic results showed that unlike the widely accepted carnivore phylogeny, Canidae and Felidae tend to group together based on ACSL4 and ACSL6. The evolutionary analyses identified a series of positively selected amino acid residues in ACSL1, ACSL4 and ACSL5. Two radical amino acid substitutions detected in sable suggested potential insights into the molecular mechanism underlying the relatively low fat content in this animal. This is the first study to investigate the molecular mechanisms underlying the adaptive evolution of fat metabolism in carnivores. Overall, the ACSL genes were under different evolutionary forces in carnivores, and some genes have undergone adaptive evolution in lipid metabolism. Characterization and phylogeny of bitter taste receptor genes (Tas2r) in Squamata Abstract The perception of bitter taste is linked to the detection of toxins. Therefore, it facilitates avoiding the consumption of potential toxins in the diet. At the molecular level, bitter taste is mediated by taste 2 receptors (Tas2rs). Studies on Tas2r have made major advances in recent years. However, little is known about Tas2rs in Squamata, the second largest order of extant vertebrates. To explore the repertoire and phylogenetic relationships among Tas2r genes in Squamata, we identified and characterized Tas2rs from genome assemblies of 15 Squamata species. We observed considerable Tas2r contraction and expansion in the suborders Serpentes and Lacertilia, respectively. Phylogenetic and reconciliation analysis suggested that lineage-specific gene gains and losses could have led to the Tas2r contraction and expansion in Squamata. Different Tas2r repertoires in Serpents and Lacertilia also reflect their oral anatomical features and taste behaviors. Our findings offer novel perspectives into the study of taste and dietary protection in Squamata species. Correction to: Genome-wide analysis of the MYB-CC gene family of maize In the original publication of the article, the incorrect version of Fig. 1 was mistakenly used. The correct version of the figure is provided in this correction. AFLP markers show low levels of clonal propagation and high genotypic diversity in the rare, southernmost populations of Linnaea borealis L. (Caprifoliaceae) in the Western Alps Abstract In plants, clonal propagation is a common reproductive strategy in parallel to sexual reproduction. It has both advantages and drawbacks, and the potential complete loss of sexual reproduction causes serious conservation concerns, especially because population maintenance then only relies on adult survival and low genetic diversity leads to decreased adaptive potential. We investigated the rare, southernmost populations of the mostly circumboreal twinflower Linnaea borealis, located in the Western Alps. Based on 105 AFLP markers and 118 leaf samples, including replicates, we estimated the genetic similarity threshold above which samples belong to a single clone. Although the species is known for extensive clonal propagation, we observed high genotypic diversity within the seven studied populations and almost all samples were genetically distinct. Nevertheless, some clonal samples were detected in two populations, separated by up to 180 m. We found a strong genetic differentiation among populations (overall Fst = 0.38), which was congruent with the previously documented high plastid diversity in the region. We therefore hypothesize that Alpine populations are relicts of the Quaternary glacial periods, when the species probably survived at these lower latitudes before colonizing Northern Europe. Regarding conservation, our results suggest that most extant plants result from sexual reproduction and that populations are not highly threatened. Nevertheless, since clones can be very long-lived and almost no seedlings were observed in recent years, events of sexual reproduction may be ancient. The current reproductive dynamics should therefore be studied to estimate e.g. pollinators activity, proportions of flowering plants, and seed set. Identification of placental genes linked to selective intrauterine growth restriction (IUGR) in dichorionic twin pregnancies: gene expression profiling study Abstract A linkage of dichorionic (DC) twin pregnancies with selective intrauterine growth restriction (IUGR) to alterations in placental gene expression is unclear. The aim of the study was to identify placental genes related to hypoxia, adipogenesis and human growth which may contribute to IUGR development. The study group (IUGR/AGA) comprised dichorionic (DC) twin pregnancies, where the weight of the twins differed by > 15%; in addition, one twin was small for gestational age (< 10th percentile-SGA) (IUGR) while the other was appropriate for gestational age (> 10th percentile-AGA). In the control group (AGA/AGA), both fetuses were AGA and their weights differed by < 15%. In the first step (selection), placental expression of 260 genes was analysed by commercial PCR profiler array or qPCR primer assay between six pairs of IUGR/AGA twins. In the second stage (verification), the expression of 20 genes with fold change (FC) > 1.5 selected from the first stage was investigated for 75 DC pregnancies: 23 IUGR/AGA vs. 52 AGA/AGA. The expression of Angiopoetin 2, Leptin and Kruppel-like factor 4 was significantly higher, and Glis Family Zinc Finger 3 was lower, in placentas of SGA fetuses (FC = 3.3; 4.4; 1.6; and − 1.8, respectively; p < 0.05). The dysregulation of gene expression related to angiogenesis and growth factors in placentas of twins born from IUGR/AGA pregnancies suggest that these alternations might represent biological fetal adaptation to the uteral condition. Moreover, DC twin pregnancies may be a good model to identify the differences in placental gene expression between SGA and AGA fetuses. Personalized medicine: going to the dogs? Abstract Interindividual variation in drug response occurs in canine patients just as it does in human patients. Although canine pharmacogenetics still lags behind human pharmacogenetics, significant life-saving discoveries in the field have been made over the last 20 years, but much remains to be done. This article summarizes the available published data about the presence and impact of genetic polymorphisms on canine drug transporters, drug-metabolizing enzymes, drug receptors/targets, and plasma protein binding while comparing them to their human counterparts when applicable. In addition, precision medicine in cancer treatment as an application of canine pharmacogenetics and pertinent considerations for canine pharmacogenetics testing is reviewed. The field is poised to transition from single pharmacogene-based studies, pharmacogenetics, to pharmacogenomic-based studies to enhance our understanding of interindividual variation of drug response in dogs. Advances made in the field of canine pharmacogenetics will not only improve the health and well-being of dogs and dog breeds, but may provide insight into individual drug efficacy and toxicity in human patients as well. Is population structure in the genetic biobank era irrelevant, a challenge, or an opportunity? Abstract Replicable genetic association signals have consistently been found through genome-wide association studies in recent years. The recent dramatic expansion of study sizes improves power of estimation of effect sizes, genomic prediction, causal inference, and polygenic selection, but it simultaneously increases susceptibility of these methods to bias due to subtle population structure. Standard methods using genetic principal components to correct for structure might not always be appropriate and we use a simulation study to illustrate when correction might be ineffective for avoiding biases. New methods such as trans-ethnic modeling and chromosome painting allow for a richer understanding of the relationship between traits and population structure. We illustrate the arguments using real examples (stroke and educational attainment) and provide a more nuanced understanding of population structure, which is set to be revisited as a critical aspect of future analyses in genetic epidemiology. We also make simple recommendations for how problems can be avoided in the future. Our results have particular importance for the implementation of GWAS meta-analysis, for prediction of traits, and for causal inference. EDAR , LYPLAL1 , PRDM16 , PAX3 , DKK1 , TNFSF12 , CACNA2D3 , and SUPT3H gene variants influence facial morphology in a Eurasian population Abstract In human society, the facial surface is visible and recognizable based on the facial shape variation which represents a set of highly polygenic and correlated complex traits. Understanding the genetic basis underlying facial shape traits has important implications in population genetics, developmental biology, and forensic science. A number of single nucleotide polymorphisms (SNPs) are associated with human facial shape variation, mostly in European populations. To bridge the gap between European and Asian populations in term of the genetic basis of facial shape variation, we examined the effect of these SNPs in a European–Asian admixed Eurasian population which included a total of 612 individuals. The coordinates of 17 facial landmarks were derived from high resolution 3dMD facial images, and 136 Euclidean distances between all pairs of landmarks were quantitatively derived. DNA samples were genotyped using the Illumina Infinium Global Screening Array and imputed using the 1000 Genomes reference panel. Genetic association between 125 previously reported facial shape-associated SNPs and 136 facial shape phenotypes was tested using linear regression. As a result, a total of eight SNPs from different loci demonstrated significant association with one or more facial shape traits after adjusting for multiple testing (significance threshold p < 1.28 × 10−3), together explaining up to 6.47% of sex-, age-, and BMI-adjusted facial phenotype variance. These included EDAR rs3827760, LYPLAL1 rs5781117, PRDM16 rs4648379, PAX3 rs7559271, DKK1 rs1194708, TNFSF12 rs80067372, CACNA2D3 rs56063440, and SUPT3H rs227833. Notably, the EDAR rs3827760 and LYPLAL1 rs5781117 SNPs displayed significant association with eight and seven facial phenotypes, respectively (2.39 × 10−5 < p < 1.28 × 10−3). The majority of these SNPs showed a distinct allele frequency between European and East Asian reference panels from the 1000 Genomes Project. These results showed the details of above eight genes influence facial shape variation in a Eurasian population. The Four Horsemen of the 'Omicsalypse': ontology, replicability, probability and epistemology Abstract Much of modern genomics and the other 'omics' that tag along, assert that the causal bases of biomedical outcomes are genomically enumerable lists whose effects are predictable with 'precision', extensible from samples to all, and enabled by ever-greater hypothesis-free data accumulation. The assertion rests on fundamental, if often implicit assumptions, that (1) the phenomena are based on underlying law-like biological causation, and, therefore, are (2) replicable and (3) even if not deterministic, have specifiable, stable, essentially parametric, probabilities, all of which (4) essentially equates induction with deduction, enabling asymptotically accurate prediction based on past observation. These glowing promises are the four horsemen of a genocentric 'Omicsalypse'. But what if the assumptions are wrong or appropriate only to an extent that is unknowable, even in principle? Might there be better ways to understand complex traits? Variants in KIAA0825 underlie autosomal recessive postaxial polydactyly Abstract Postaxial polydactyly (PAP) is a common limb malformation that often leads to cosmetic and functional complications. Molecular evaluation of polydactyly can serve as a tool to elucidate genetic and signaling pathways that regulate limb development, specifically, the anterior-posterior specification of the limb. To date, only five genes have been identified for nonsyndromic PAP: FAM92A, GLI1, GLI3, IQCE and ZNF141. In this study, two Pakistani multiplex consanguineous families with autosomal recessive nonsyndromic PAP were clinically and molecularly evaluated. From both pedigrees, a DNA sample from an affected member underwent exome sequencing. In each family, we identified a segregating frameshift (c.591dupA [p.(Q198Tfs*21)]) and nonsense variant (c.2173A > T [p.(K725*)]) in KIAA0825 (also known as C5orf36). Although KIAA0825 encodes a protein of unknown function, it has been demonstrated that its murine ortholog is expressed during limb development. Our data contribute to the establishment of a catalog of genes important in limb patterning, which can aid in diagnosis and obtaining a better understanding of the biology of polydactyly. Quality assurance checklist and additional considerations for canine clinical genetic testing laboratories: a follow-up to the published standards and guidelines Abstract There is currently no oversight for canine clinical genetic testing laboratories. We published an initial set of standards and guidelines with the goal of providing a basis for which canine testing laboratories could evaluate their quality assurance programs. To further those standards and guidelines, we have developed a checklist that can be used as a self-evaluation to identify gaps in their programs for continual quality improvement over time. Because there is currently no organization willing to oversee an external proficiency program, the checklist provides the first step toward an internal, self-assessment that can be used periodically to monitor improvements. In addition, we attempt to address concerns from the canine community regarding rare or private mutations, genetic screening using array-based technologies, non-peer reviewed tests that are being offered, and the clinical validity of certain mutations in particular breeds. Through coordination, conversation and hard work, the canine genetic testing community can strive to organize to improve testing and to provide more transparency to consumers and better outcomes for dogs. Prediction of skin color, tanning and freckling from DNA in Polish population: linear regression, random forest and neural network approaches Abstract Predicting phenotypes from DNA has recently become extensively studied field in forensic research and is referred to as Forensic DNA Phenotyping. Systems based on single nucleotide polymorphisms for accurate prediction of iris, hair and skin color in global population, independent of bio-geographical ancestry, have recently been introduced. Here, we analyzed 14 SNPs for distinct skin pigmentation traits in a homogeneous cohort of 222 Polish subjects. We compared three different algorithms: General Linear Model based on logistic regression, Random Forest and Neural Network in 18 developed prediction models. We demonstrate Random Forest to be the most accurate algorithm for 3- and 4-category estimations (total of 58.3% correct calls for skin color prediction, 47.2% for tanning prediction, 50% for freckling prediction). Binomial Logistic Regression was the best approach in 2-category estimations (total of 69.4% correct calls, AUC = 0.673 for tanning prediction; total of 52.8% correct calls, AUC = 0.537 for freckling prediction). Our study confirms the association of rs12913832 (HERC2) with all three skin pigmentation traits, but also variants associated solely with certain pigmentation traits, namely rs6058017 and rs4911414 (ASIP) with skin sensitivity to sun and tanning abilities, rs12203592 (IRF4) with freckling and rs4778241 and rs4778138 (OCA2) with skin color and tanning. Finally, we assessed significant differences in allele frequencies in comparison with CEU data and our study provides a starting point for the development of prediction models for homogeneous populations with less internal differentiation than in the global predictive testing. A review of gene-by-air pollution interactions for cardiovascular disease, risk factors, and biomarkers Abstract Air pollution is recognized as causal factor for cardiovascular disease (CVD) and is associated with multiple CVD risk factors. Substantial research effort has been invested in understanding the linkages between genetic variation and CVD risk, resulting in over 50 CVD-associated genetic loci. More recently, gene–air pollution interaction studies have quantified the contribution of genetic variation to inter-individual heterogeneity in air pollution health risks, and aided in elucidating mechanisms of air pollution exposure health risks. Here, we perform a comprehensive review of gene–air pollution interaction studies for CVD, as well as risk factors and emerging CVD biomarkers. The literature review revealed that most published interaction studies have been candidate gene studies, causing observed interactions to cluster in a few genes related to detoxification (GSTM1and GSTT1), inflammation (IL-6), iron processing (HFE), and microRNA processing (GEMIN4 and DGCR8). There have been a few genome-wide interaction studies with results indicating that interactions extend beyond commonly considered genetic loci. Gene–air pollution interactions are observed for exposure periods ranging from hours to years and a variety of air pollutants including particulate matter, gaseous pollutants, and pollutant sources such as traffic. Though the existing evidence for the existence of relevant gene–air pollution interactions for CVD outcomes is substantial, it could be strengthened by improved replication and meta-analyses as well as functional validation. Mutation signatures in germline mitochondrial genome provide insights into human mitochondrial evolution and disease Abstract Variations in mitochondrial DNA (mtDNA) have been fundamental for understanding human evolution and are causative for a plethora of inherited mitochondrial diseases, but the mutation signatures of germline mtDNA and their value in understanding mitochondrial pathogenicity remain unknown. Here, we carried out a systematic analysis of mutation patterns in germline mtDNA based on 97,566 mtDNA variants from 45,494 full-length sequences and revealed a highly non-stochastic and replication-coupled mutation signature characterized by nucleotide-specific mutation pressure (G > T>A > C) and position-specific selection pressure, suggesting the existence of an intensive mutation–selection interplay in germline mtDNA. We provide evidence that this mutation–selection interplay has strongly shaped the mtDNA sequence during evolution, which not only manifests as an oriented alteration of amino acid compositions of mitochondrial encoded proteins, but also explains the long-lasting mystery of CpG depletion in mitochondrial genome. Finally, we demonstrated that these insights may be integrated to better understand the pathogenicity of disease-implicated mitochondrial variants.