Bioinformatic and Molecular Analysis of Satellite Repeat Diversity in Vaccinium Genomes.

Bioinformatic and molecular characterization of satellite repeats was performed to understand the impact of their diversification on Vaccinium genome evolution. Satellite repeat diversity was evaluated in four cultivated and wild species, including the diploid species Vaccinium myrtillus and Vaccinium uliginosum, as well as the tetraploid species Vaccinium corymbosum and Vaccinium arctostaphylos. We comparatively characterized six satellite repeat families using in total 76 clones with 180 monomers. We observed that the monomer units of VaccSat1, VaccSat2, VaccSat5, and VaccSat6 showed a higher order repeat (HOR) structure, likely originating from the organization of two adjacent subunits with differing similarity, length and size. Moreover, VaccSat1, VaccSat3, VaccSat6, and VaccSat7 were found to have sequence similarity to parts of transposable elements. We detected satellite-typical tandem organization for VaccSat1 and VaccSat2 in long arrays, while VaccSat5 and VaccSat6 distributed in multiple sites over all chromosomes of tetraploid V. corymbosum, presumably in long arrays. In contrast, very short arrays of VaccSat3 and VaccSat7 are dispersedly distributed over all chromosomes in the same species, likely as internal parts of transposable elements. We provide a comprehensive overview on satellite species specificity in Vaccinium, which are potentially useful as molecular markers to address the taxonomic complexity of the genus, and provide information for genome studies of this genus.

Adding color to a century-old enigma: multi-color chromosome identification unravels the autotriploid nature of saffron (Crocus sativus) as a hybrid of wild Crocus cartwrightianus cytotypes.

Saffron crocus (Crocus sativus) is the source of the most expensive spice of the world, produced from manually harvested stigmas, thus serving as a cash crop for rural communities. However, despite its economic importance, its genome and chromosomes are poorly studied. C. sativus is a sterile triploid species harboring eight chromosome triplets, and propagated only as a clonal lineage by corms. Saffron's evolutionary origin, parental species and allo- or autotriploidy has been a matter of discussion for almost a century. We performed a survey sequencing of the saffron genome and selected cytogenetic landmark sequences consisting of major tandem repeats, which we used as probes in comparative multicolor fluorescent in situ hybridization (FISH). We tagged 92 chromosomal positions and resolved the chromosomal composition of saffron triplets. By comparative FISH of six Crocus species from 11 accessions, we demonstrate that C. sativus is an autotriploid hybrid derived from heterogeneous Crocus cartwrightianus cytotypes. The FISH reference karyotype of saffron is crucial for integrating genome sequencing data with chromosomes and for investigating the relationship among Crocus species. We provide an evolutionary model of the saffron emergence; the knowledge of the parental origin offers a route towards the resynthesis of C. sativus from C. cartwrightianus to broaden saffron's gene pool.

A cohort study of sciatic pain and measures of internal spinal load in professional drivers.

In a prospective cohort study of 537 male professional drivers, the occurrence of sciatic pain showed stronger associations with measures of internal lumbar load expressed in terms of daily compressive dose, S(ed) (MPa), and risk factor, R (non-dimensional), according to ISO/WD 2631-5 (2013), than with measures of daily vibration exposure calculated as either 8-h energy-equivalent frequency-weighted acceleration (ms(-2) r.m.s.) or vibration dose value (ms(-1.75)) according to the EU Directive on mechanical vibration (2002). Herniated lumbar disc, previous lumbar trauma and physical work load were also powerful predictors of the occurrence of sciatic pain over time. Psychosocial work environment was poorly associated with sciatic pain. The boundary values of risk factor (R) for low and high probabilities of adverse health effects on the lumbar spine, as proposed by international standard ISO/WD 2631-5 (2013), tend to underestimate the health risk in professional drivers. PRACTITIONER SUMMARY: In a prospective cohort study of professional drivers, measures of internal spinal load were better predictors of the occurrence of sciatic pain than the measures of daily vibration exposure established by the EU Directive (2002). Herniated lumbar disc, lumbar trauma and physical work load were also associated with sciatic pain.

Measures of internal lumbar load in professional drivers - the use of a whole-body finite-element model for the evaluation of adverse health effects of multi-axis vibration.

The present study aimed to (1) employ the method for evaluation of vibration containing multiple shocks according to ISO/CD 2631-5:2014 (Model 1) and DIN SPEC 45697:2012 in a cohort of 537 professional drivers, (2) deliver the results for a re-analysis of epidemiological data obtained in the VIBRISKS study, (3) clarify the extent to which vibration acceleration and individual variables influence risk values, such as the daily compressive dose S(ed) and the risk factor R, and (4) compare the results with in vivo measurements and those obtained in previous studies with similar models. The risk factor R was influenced by the acceleration, lifetime exposure duration, sitting posture, age at the start of exposure and body mass/body mass index in order of decreasing effect. Age and annual and daily exposure duration had only a marginal effect. The daily compressive dose S(ed) and the risk factor R showed weak linear association with the daily vibration exposure A(8) and the vibration dose value VDV. The study revealed high shear forces in the lumbar spine. PRACTITIONER SUMMARY: In a re-analysis of an epidemiological study of professional drivers, a software tool available with standards DIN SPEC 45697:2012 and ISO/CD 2631­5:2014 Model 1 was used to calculate the risk to the lumbar spine in terms of daily compressive dose S(ed) and risk factor R. The tool was found to be suitable for risk assessment in a large cohort.

Relationships of low back outcomes to internal spinal load: a prospective cohort study of professional drivers.

PURPOSE: To investigate the relationships between low back symptoms and alternative measures of external dose and internal spinal dose in professional drivers exposed to whole body vibration (WBV). METHODS: The occurrence of low back symptoms was investigated in a cohort of 537 drivers over a 2-year follow-up period. Low back pain (LBP), individual characteristics, and work-related risk factors were investigated with a structured questionnaire. Exposure to WBV was evaluated by means of measures of external dose (daily vibration exposure in terms of either equivalent continuous acceleration over an 8-h period (A(8)) or vibration dose value according to the EU Directive on mechanical vibration) and measures of internal lumbar load (daily compressive dose S ed and risk factor R according to ISO/CD 2631-5 2014). RESULTS: In the drivers' cohort, the cumulative incidence of 12-month low back outcomes was 16.8% for LBP, 9.3% for chronic LBP, and 21.8% for sciatic pain. The measures of internal spinal load were better predictors of the occurrence of low back symptoms than the measures of daily vibration exposure. A twofold increase in the risk estimates for low back outcomes was found in the upper quartile of the R factor (0.41-0.72 units) compared to the lower one (0.07-0.19 units). CONCLUSIONS: In this prospective cohort study, measures of internal spinal dose performed better than measures of daily vibration exposure (external dose) for the prediction of low back outcomes in professional drivers. The ISO boundary values of the risk factor R for low and high probabilities of adverse health effects on the lumbar spine tend to underestimate the health risk in professional drivers.

The diversification and activity of hAT transposons in Musa genomes.

Sequencing of plant genomes often identified the hAT superfamily as the largest group of DNA transposons. Nevertheless, detailed information on the diversity, abundance and chromosomal localization of plant hAT families are rare. By in silico analyses of the reference genome assembly and bacterial artificial chromosome (BAC) sequences, respectively, we performed the classification and molecular characterization of hAT transposon families in Musa acuminata. Musa hAT transposons are organized in three families designated MuhAT I, MuhAT II and MuhAT III. In total, 70 complete autonomous elements of the MuhAT I and MuhAT II families were detected, while no autonomous MuhAT III transposons were found. Based on the terminal inverted repeat (TIR)-specific sequence information of the autonomous transposons, 1722 MuhAT I- and MuhAT II-specific miniature inverted-repeat transposable elements (MuhMITEs) were identified. Autonomous MuhAT I and MuhAT II elements are only moderately abundant in the sections of the genus Musa, while the corresponding MITEs exhibit an amplification in Musa genomes. By fluorescent in situ hybridization (FISH), autonomous MuhAT transposons as well as MuhMITEs were localized in subtelomeric, most likely gene-rich regions of M. acuminata chromosomes. A comparison of homoeologous regions of M. acuminata and Musa balbisiana BACs revealed the species-specific mobility of MuhMITEs. In particular, the activity of MuhMITEs II showing transduplications of genomic sequences might indicate the presence of active MuhAT transposons, thus suggesting a potential role of MuhMITEs as modulators of genome evolution of Musa.

Comparative molecular cytogenetic analyses of a major tandemly repeated DNA family and retrotransposon sequences in cultivated jute Corchorus species (Malvaceae).

BACKGROUND AND AIMS: The cultivated jute species Corchorus olitorius and Corchorus capsularis are important fibre crops. The analysis of repetitive DNA sequences, comprising a major part of plant genomes, has not been carried out in jute but is useful to investigate the long-range organization of chromosomes. The aim of this study was the identification of repetitive DNA sequences to facilitate comparative molecular and cytogenetic studies of two jute cultivars and to develop a fluorescent in situ hybridization (FISH) karyotype for chromosome identification. METHODS: A plasmid library was generated from C. olitorius and C. capsularis with genomic restriction fragments of 100-500 bp, which was complemented by targeted cloning of satellite DNA by PCR. The diversity of the repetitive DNA families was analysed comparatively. The genomic abundance and chromosomal localization of different repeat classes were investigated by Southern analysis and FISH, respectively. The cytosine methylation of satellite arrays was studied by immunolabelling. KEY RESULTS: Major satellite repeats and retrotransposons have been identified from C. olitorius and C. capsularis. The satellite family CoSat I forms two undermethylated species-specific subfamilies, while the long terminal repeat (LTR) retrotransposons CoRetro I and CoRetro II show similarity to the Metaviridea of plant retroelements. FISH karyotypes were developed by multicolour FISH using these repetitive DNA sequences in combination with 5S and 18S-5·8S-25S rRNA genes which enable the unequivocal chromosome discrimination in both jute species. CONCLUSIONS: The analysis of the structure and diversity of the repeated DNA is crucial for genome sequence annotation. The reference karyotypes will be useful for breeding of jute and provide the basis for karyotyping homeologous chromosomes of wild jute species to reveal the genetic and evolutionary relationship between cultivated and wild Corchorus species.

Survey of sugar beet (Beta vulgaris L.) hAT transposons and MITE-like hATpin derivatives.

Genome-wide analyses of repetitive DNA suggest a significant impact particularly of transposable elements on genome size and evolution of virtually all eukaryotic organisms. In this study, we analyzed the abundance and diversity of the hAT transposon superfamily of the sugar beet (B. vulgaris) genome, using molecular, bioinformatic and cytogenetic approaches. We identified 81 transposase-coding sequences, three of which are part of structurally intact but nonfunctional hAT transposons (BvhAT), in a B. vulgaris BAC library as well as in whole genome sequencing-derived data sets. Additionally, 116 complete and 497 truncated non-autonomous BvhAT derivatives lacking the transposase gene were in silico-detected. The 116 complete derivatives were subdivided into four BvhATpin groups each characterized by a distinct terminal inverted repeat motif. Both BvhAT and BvhATpin transposons are specific for species of the genus Beta and closely related species, showing a localization on B. vulgaris chromosomes predominantely in euchromatic regions. The lack of any BvhAT transposase function together with the high degree of degeneration observed for the BvhAT and the BvhATpin genomic fraction contrasts with the abundance and activity of autonomous and non-autonomous hAT transposons revealed in other plant species. This indicates a possible genus-specific structural and functional repression of the hAT transposon superfamily during Beta diversification and evolution.

Seat-to-head transfer function of seated men--determination with single and three axis excitations at different magnitudes.

Most research has investigated the seat-to-head transmissibility during single-axis excitations. Associations between head accelerations and discomfort or effects on vision were reported. Possible differences between the seat-to-head transmissibility determined during different vibration magnitudes with a variable number of excitation axes have not been systematically examined. An experimental study was performed with 8 male subjects sitting on a rigid seat with hands on a support. They were exposed to random whole-body vibration (E1=0.45 ms(-2), E2=0.90 ms(-2), and E3=1.80 ms(-2)) to single- and three-axis vibration. All translational and rotational seat-to-head transmissibilities were calculated. The effects of the factors vibration magnitude and number of axes on the peak modulus and frequency of the seat-to-head transmissibilities were tested. In general the head motions follow constant pattern. These pattern of head motions comprise a combination of rotational and translational shares of transmissions, i.e. the curves show a dependence on the factors 'vibration magnitude' and 'number of vibration axes'. Mechanical properties of the soft tissue, relative motions of body parts, and muscle reactions were supposed to cause the nonlinearities of the head. Future research should consider effects of multi-axis vibration, if conclusions shall be drawn for the evaluation of possible health effects and model validations.

Comparative molecular cytogenetics of major repetitive sequence families of three Dendrobium species (Orchidaceae) from Bangladesh.

BACKGROUND AND AIMS: Dendrobium species show tremendous morphological diversity and have broad geographical distribution. As repetitive sequence analysis is a useful tool to investigate the evolution of chromosomes and genomes, the aim of the present study was the characterization of repetitive sequences from Dendrobium moschatum for comparative molecular and cytogenetic studies in the related species Dendrobium aphyllum, Dendrobium aggregatum and representatives from other orchid genera. METHODS: In order to isolate highly repetitive sequences, a c(0)t-1 DNA plasmid library was established. Repeats were sequenced and used as probes for Southern hybridization. Sequence divergence was analysed using bioinformatic tools. Repetitive sequences were localized along orchid chromosomes by fluorescence in situ hybridization (FISH). KEY RESULTS: Characterization of the c(0)t-1 library resulted in the detection of repetitive sequences including the (GA)(n) dinucleotide DmoO11, numerous Arabidopsis-like telomeric repeats and the highly amplified dispersed repeat DmoF14. The DmoF14 repeat is conserved in six Dendrobium species but diversified in representative species of three other orchid genera. FISH analyses showed the genome-wide distribution of DmoF14 in D. moschatum, D. aphyllum and D. aggregatum. Hybridization with the telomeric repeats demonstrated Arabidopsis-like telomeres at the chromosome ends of Dendrobium species. However, FISH using the telomeric probe revealed two pairs of chromosomes with strong intercalary signals in D. aphyllum. FISH showed the terminal position of 5S and 18S-5.8S-25S rRNA genes and a characteristic number of rDNA sites in the three Dendrobium species. CONCLUSIONS: The repeated sequences isolated from D. moschatum c(0)t-1 DNA constitute major DNA families of the D. moschatum, D. aphyllum and D. aggregatum genomes with DmoF14 representing an ancient component of orchid genomes. Large intercalary telomere-like arrays suggest chromosomal rearrangements in D. aphyllum while the number and localization of rRNA genes as well as the species-specific distribution pattern of an abundant microsatellite reflect the genomic diversity of the three Dendrobium species.

Diversity of a complex centromeric satellite and molecular characterization of dispersed sequence families in sugar beet (Beta vulgaris).

BACKGROUND AND AIMS: The aim of this work was the identification and molecular characterization of novel sugar beet (Beta vulgaris) repetitive sequences to unravel the impact of repetitive DNA on size and evolution of Beta genomes via amplification and diversification. METHODS: Genomic DNA and a pool of B. vulgaris repetitive sequences were separately used as probes for a screening of high-density filters from a B. vulgaris plasmid library. Novel repetitive motifs were identified by sequencing and further used as probes for Southern analyses in the genus Beta. Chromosomal localization of the repeats was analysed by fluorescent in situ hybridization on chromosomes of B. vulgaris and two other species of the section Beta. KEY RESULTS: Two dispersed repetitive families pDvul1 and pDvul2 and the tandemly arranged repeat family pRv1 were isolated from a sugar beet plasmid library. The dispersed repetitive families pDvul1 and pDvul2 were identified in all four sections of the genus Beta. The members of the pDvul1 and pDvul2 family are scattered over all B. vulgaris chromosomes, although amplified to a different extent. The pRv1 satellite repeat is exclusively present in species of the section Beta. The centromeric satellite pBV1 by structural variations of the monomer and interspersion of pRv1 units forms complex satellite structures, which are amplified in different degrees on the centromeres of 12 chromosomes of the three species of the Beta section. CONCLUSIONS: The complexity of the pBV1 satellite family observed in the section Beta of the genus Beta and, in particular, the strong amplification of the pBV1/pRv1 satellite in the domesticated B. vulgaris indicates the dynamics of centromeric satellite evolution during species radiation within the genus. The dispersed repeat families pDvul1 and pDvul2 might represent derivatives of transposable elements.

Mobilization and evolutionary history of miniature inverted-repeat transposable elements (MITEs) in Beta vulgaris L.

We have identified three families of miniature inverted-repeat transposable elements (VulMITEs) in the genome of sugar beet (Beta vulgaris L.), evidently derived from a member of the Vulmar family of mariner transposons. While VulMITEs I are typical stowaway-like MITEs, VulMITEs II and VulMITEs III are rearranged stowaway elements of increased size. The integration of divergent moderately and highly repetitive sequences into VulMITEs II and, in particular in VulMITEs III, respectively, shows that amplification of repetitive DNA by MITEs contribute to the increase of genome size with possible implications for plant genome evolution. Fluorescent in-situ hybridization (FISH), for the first time visualizing stowaway MITE distribution on plant chromosomes, revealed a dispersed localization of VulMITEs along all B. vulgaris chromosomes. Analysis of the flanking sequences identified a dispersed repeat as target site for the integration of the stowaway element VulMITE I. Recent transposition of VulMITE I, which most likely occurred during the domestication of cultivated beets, was concluded from insertional polymorphisms between different B. vulgaris cultivars and species.

Characterization of a protein of the plastid inner envelope having homology to animal inorganic phosphate, chloride and organic-anion transporters.

A protein from Arabidopsis thaliana (L.) Heynh. showing homology to animal proteins of the NaPi-1 family, involved in the transport of inorganic phosphate, chloride, glutamate and sialic acid, has been characterized. This protein, named ANTR2 (for anion transporters) was shown by chloroplast subfractionation to be localized to the plastid inner envelope in both A. thaliana and Spinacia oleracea (L.). Immunolocalization revealed that ANTR2 was expressed in the leaf mesophyll cells as well as in the developing embryo at the upturned-U stage. Five additional homologues of ANTR2 are found in the Arabidopsis genome, of which one was shown by green fluorescent protein (GFP) fusion to be also located in the chloroplast. All ANTR proteins share homology to the animal NaPi-1 family, as well as to other organic-anion transporters that are members of the Anion:Cation Symporter (ACS) family, and share the main features of transporters from this family, including the presence of 12 putative transmembrane domains and of a 7-amino acid motif in the fourth putative transmembrane domain. ANTR2 thus represent a novel protein of the plastid inner envelope that is likely to be involved in anion transport.

Molecular characterization of Vulmar1, a complete mariner transposon of sugar beet and diversity of mariner- and En/Spm-like sequences in the genus Beta.

Transposons of the Tc1-mariner superfamily are widespread in eukaryotic genomes. We have isolated the mariner element Vulmar1 from Beta vulgaris L., which is 3909 bp long and bordered by perfect terminal inverted repeats of 32 bp with homology to terminal inverted repeats of transposons from soybean and rice. According to a characteristic amino acid signature, Vulmar1 can be assigned to the DD39D group of mariner transposons. Vulmar1 is flanked by a 5'-TA-3' target site duplication that is typical for mariner transposons. Southern hybridization revealed that mariner-like copies are highly abundant in Beta species, and sequence analysis of 10 transposase fragments from representative species of the four Beta sections revealed an identity between 34% and 100% after conceptual translation. By fluorescent in situ hybridization, Vulmar1 was detected in distal euchromatin as well as in some intercalary and pericentromeric regions of all B. vulgaris chromosomes. In addition, using PCR, we were able to amplify fragments of the transposase gene of En/Spm-like transposons in the genus Beta. En/Spm-like transposase sequences are highly amplified in four Beta sections and showed a considerable degree of conservation (88.5-100%) at the protein level, while the homology to corresponding regions of En/Spm transposons of other plant species ranges from 49.5% to 62.5%. By fluorescent in situ hybridization, En/Spm-like transposon signals of strong intensity were detected on all chromosomes of B. vulgaris.