Proximal placement of lateral thigh skin markers reduces soft tissue artefact during normal gait using the Conventional Gait Model

A primary source of measurement error in gait analysis is soft-tissue artefact. Hip and knee angle measurements, regularly used in clinical decision-making, are particularly prone to pervasive soft tissue on the femur. However, despite several studies of thigh marker artefact it remains unclear how lateral thigh marker height affects results using variants of the Conventional Gait Model. We compared Vicon Plug-in Gait hip and knee angle estimates during gait using a proximal and distal thigh marker placement for ten healthy subjects. Knee axes were estimated by optimizing thigh rotation offsets to minimize knee varus-valgus range during gait. Relative to the distal marker, the proximal marker produced 37% less varus-valgus range and 50% less hip rotation range (p < 0.001), suggesting that it produced less soft-tissue artefact in knee axis estimates. The thigh markers also produced different secondary effects on the knee centre estimate. Using whole gait cycle optimization, the distal marker showed greater minimum and maximum knee flexion (by 6° and 2° respectively) resulting in a 4° reduction in range. Mid-stance optimization reduced distal marker knee flexion by 5° throughout, but proximal marker results were negligibly affected. Based on an analysis of the Plug-in Gait knee axis definition, we show that the proximal marker reduced sensitivity to soft-tissue artefact by decreasing collinearity between the points defining the femoral frontal plane and reducing anteroposterior movement between the knee and thigh markers. This study suggests that a proximal thigh marker may be preferable when performing gait analysis using the Plug-in Gait model.     

Biodiversity assessment using markers for ecologically important traits

Most studies of genetic variation within species to date are based on random markers. However, how well this correlates with quantitative variation is contentious. Yet, functional, or'ecotypic' variation in quantitative traits determines the ecological niche of a species, its future evolutionary potential, and, for livestock, crops and their wild relatives, their usefulness as a genetic resource for breeding. But nowadays we can also assess genetic diversity using markers directly targeted at specific genes or gene families. Such gene-targeted, multilocus profiles of markers can contribute to ex-situ management of genetic resources, ecological studies of diversity, and conservation of endangered species.     

In situ comparison of A-mode ultrasound tracking system and skin-mounted markers for measuring kinematics of the lower extremity

Skin-mounted marker based motion capture systems are widely used in measuring the movement of human joints. Kinematic measurements associated with skin-mounted markers are subject to soft tissue artifacts (STA), since the markers follow skin movement, thus generating errors when used to represent motions of underlying bone segments. We present a novel ultrasound tracking system that is capable of directly measuring tibial and femoral bone surfaces during dynamic motions, and subsequently measuring six-degree-of-freedom (6-DOF) tibiofemoral kinematics. The aim of this study is to quantitatively compare the accuracy of tibiofemoral kinematics estimated by the ultrasound tracking system and by a conventional skin-mounted marker based motion capture system in a cadaveric experimental scenario. Two typical tibiofemoral joint models (spherical and hinge models) were used to derive relevant kinematic outcomes. Intra-cortical bone pins equipped with optical markers were inserted in the tibial and femoral bones to serve as a reference to provide ground truth kinematics. The ultrasound tracking system resulted in lower kinematic errors than the skin-mounted markers (the ultrasound tracking system: maximum root-mean-square (RMS) error 3.44° for rotations and 4.88 mm for translations, skin-mounted markers with the spherical joint model: 6.32° and 6.26 mm, the hinge model: 6.38° and 6.52 mm). Our proposed ultrasound tracking system has the potential of measuring direct bone kinematics, thereby mitigating the influence and propagation of STA. Consequently, this technique could be considered as an alternative method for measuring 6-DOF tibiofemoral kinematics, which may be adopted in gait analysis and clinical practice.     

Genetic diversity assessment of summer squash landraces using molecular markers

Plant identification, classification, and genotyping within a germplasm collection are essential elements for establishing a breeding program that enhances the probability of plants with desirable characteristics in the market place. In this study, random amplified polymorphic DNA (RAPD) was used as a molecular tool to assess the diversity and relationship among 20 summer squash (Curcubita pepo L.) landraces traditionally used to treat hypertension and prostate hyperplasia. A total of 10 RAPD primers produced 65 reproducible bands of which 46 (70.77 %) were polymorphic, indicating a large number of genotypes within the summer squash lines. Cluster analysis divided the summer squash germplasm into two groups, one including one landrace and a second containing 19 landraces that could be divided into five sub-groups. Results of this study indicate the potential of RAPD markers for the identification and assessment of genetic variations among squash landraces and provide a number of choices for developing a successful breeding program to improve summer squash.     

Varietal identification and assessment of genetic relationships in Hippeastrum using RAPD markers

The National Botanical Research Institute (NBRI) in Lucknow, India, maintains germplasm of Hippeastrum, a beautiful summer blooming ornamental. Germplasm collections comprise NBRI hybrids developed through selective breeding, hybrids with unknown parentage, local species, and Dutch hybrids for research purposes. Considering the importance of protecting plant breeders’ rights for commercial exploitation of hybrids, a PCR-based technique (random amplified polymorphic DNA—RAPD) was used to correctly identify known and unknown hybrids and to determine cultivar relatedness. RAPD profiles were used very successfully to trace and confirm the parentage of all the hybrids tested and to determine clear molecular relationships among varieties.     

Ground reaction: intrinsic and extrinsic variability assessment and related method for artefact treatment

Ground reaction (GR) components measured by a dynamometric platform represent the dynamic interaction of the moving human body with the ground and depend on the subject-platform relative position and orientation. The observed variability among the GR measurements of the walking trials of an individual is either due to variability in the motor performance (intrinsic variability) or due to changes in the direction of walking and in the position and orientation of the striking foot relative to the platform (extrinsic variability). A method, based on the median operator, is presented here which lets us quantify the two components of variability. The application of the method to a large data set of normal subjects evidenced changes in progression direction/foot orientation (95th percentile value is 6.9 degrees ), which can dramatically change the patterns of GR components. This result warns about improper analysis of ground reaction measurement.An algorithm for restoring GR measurements affected by artefact was derived from the above method. This tool can be of valuable aid in clinical practice where patients' conditions suggest to not insist on repetition of trials even if the required number of correct foot placements has not been achieved. The artefact correction algorithm has been applied to a large data set artificially corrupted to evaluate its robustness.     

Foot kinematics during walking measured using bone and surface mounted markers

The aim was to compare kinematic data from an experimental foot model comprising four segments ((i) heel, (ii) navicular/cuboid (iii) medial forefoot, (iv) lateral forefoot), to the kinematics of the individual bones comprising each segment. The foot model was represented using two different marker attachment protocols: (a) markers attached directly to the skin; (b) markers attached to rigid plates mounted on the skin. Bone data were collected for the tibia, talus, calcaneus, navicular, cuboid, medial cuneiform and first and fifth metatarsals (n=6). Based on the mean differences between the three data sets during stance, the differences between any two of the three kinematic protocols (i.e. bone vs skin, bone vs plate, skin vs plate) were >3 degrees in only 35% of the data and >5 degrees in only 3.5% of the data. However, the maximum difference between any two of the three protocols during stance was >3 degrees in 100% of the data, >5 degrees in 73% of the data and >8 degrees in 23% of the data. Differences were greatest for motion of the combined navicular/cuboid relative to the calcaneus and the medial forefoot segment relative to the navicular/cuboid. The differences between the data from the skin and plate protocols were consistently smaller than differences between either protocol and the kinematic data for each bone comprising the segment. The pattern of differences between skin and plate protocols and the actual bone motion showed no systematic pattern. It is unlikely that one rigid body foot model and marker attachment approach is always preferable over another.     

Toward a more robust assessment of intraspecies diversity, using fewer genetic markers

Phylogenetic sequence analysis of single or multiple genes has dominated the study and census of the genetic diversity among closely related bacteria. It remains unclear, however, how the results based on a few genes in the genome correlate with whole-genome-based relatedness and what genes (if any) best reflect whole-genome-level relatedness and hence should be preferentially used to economize on cost and to improve accuracy. We show here that phylogenies of closely related organisms based on the average nucleotide identity (ANI) of their shared genes correspond accurately to phylogenies based on state-of-the-art analysis of their whole-genome sequences. We use ANI to evaluate the phylogenetic robustness of every gene in the genome and show that almost all core genes, regardless of their functions and positions in the genome, offer robust phylogenetic reconstruction among strains that show 80 to 95% ANI (16S rRNA identity, >98.5%). Lack of elapsed time and, to a lesser extent, horizontal transfer and recombination make the selection of genes more critical for applications that target the intraspecies level, i.e., strains that show >95% ANI according to current standards. A much more accurate phylogeny for the Escherichia coli group was obtained based on just three best-performing genes according to our analysis compared to the concatenated alignment of eight genes that are commonly employed for phylogenetic purposes in this group. Our results are reproducible within the Salmonella, Burkholderia, and Shewanella groups and therefore are expected to have general applicability for microevolution studies, including metagenomic surveys.     

Genetic diversity assessment in greek Medicago truncatula genotypes using microsatellite markers

In this study we examined the genetic diversity and geographic scale of genotype distribution within the model legume species Medicago truncatula widely distributed in pasture and marginal agricultural lands in Greece and other Mediterranean countries. Thirty one Medicago truncatula and Medicago littorialis accessions were chosen on the basis of their geographical distributions and studied using 9 polymorphic simple sequence repeats (SSR) markers. The number of alleles per locus varied between 3 and 7. A total of 42 alleles were detected with a mean value of 4.66 alleles per locus. Geographic origin was not related with genotypic similarity among accessions. However, there were instances of close genetic relatedness between accessions from neighboring locations in a geographic compartment. In conclusion, the presented data revealed extensive M. truncatula genotype dispersal in Greece pointing to the significance of preserving local genetic resources in their natural environment.     

Non-invasive assessment of parasitic nematode species diversity in wild Soay sheep using molecular markers

Considerable effort has been put into detecting and identifying parasitic nematodes in live ruminants, but to date most studies are limited to a small group of nematodes and/or to experimentally infected sheep. In this study, a PCR-based assay using species-specific primer pairs, located in the second internal transcribed spacer ribosomal DNA, was developed to identify nine different species from six different families of parasitic nematodes in a wild, unmanaged and naturally infected population of sheep. Each primer pair was tested for its specificity and sensitivity and it exclusively amplified the species it was designed for and exhibited a high degree of sensitivity. The method was applied to eggs and cultured larvae to identify the parasitic nematodes present in a pooled faecal sample from several host individuals with unknown parasite burden. To test detection reliability, a faecal sample from an individual with known parasite burden (through post-mortem analysis) was also examined. All species present could be correctly identified by PCR, but detecting very low levels and/or early stages of infection proved to be difficult. The method was also tested for its applicability to high through-put screening of faecal samples.     

An improved micropropagation and assessment of genetic stability of micropropagated Salvadora oleoides using RAPD and ISSR markers

An improved micropropagation method has been developed for Salvadora oleoides, a valuable tree species of alkaline and arid regions. Nodal explant obtained from a mature tree (30- to 35-year-old) responded optimally (80.0 %) on BAP (2.0 mg l^?1) and produced (4.56 ± 0.52) shoots. Shoots were further multiplied by subculturing the in vitro excised shoots and transferring them to MS medium containing either BAP (0.0–2.0 mg l^?1) alone or in combination with lower concentrations of an auxin (IAA or NAA 0.05–0.4 mg l^?1). Among all the PGRs combination tested, MS medium supplemented with BAP (0.5 mg l^?1) and IAA (0.1 mg l^?1) formed the maximum number of shoots (68.40 ± 2.74 per culture bottle) with an average height (6.59 ± 0.30 cm), after 6 weeks of culture. Rooting in regenerated shoots was achieved by ex vitro methods and about 92.5 % of shoots were rooted with 5.25 ± 0.64 roots per shoot and an average length of 2.76 ± 0.53 cm after 3 weeks of incubation in the green house. More than (80 %) of hardened plantlets survived in the field conditions. Genetic stability of the discussed protocol was confirmed by two DNA-based fingerprinting techniques i.e. RAPD and ISSR. Of the 10 RAPD primers finally selected, a total of 42 bands (out of 43) were monomorphic and one polymorphic, whereas from 10 ISSR primers selected, all the 43 bands were monomorphic revealing a high level of genetic homogeneity in the regenerated plants and the donor plant. In the present investigation, we achieved significantly more number of shoots during multiplication, which are higher than all previous reports and further evaluated the genetic fidelity of protocol for the first time in S. oleoides, which concludes the clonal (true-to-type) nature of micropropagated plantlets.     

Callus-mediated and direct protocorm-like body formation of Bletilla striata and assessment of clonal fidelity using ISSR markers

Two efficient morphogenetic pathways for micropropagation of Bletilla striata (Thunb.) Reichb. f. have been established through the callus-mediated and direct formation of protocorm-like bodies (PLBs) from protocorms and shoot tips. Green calli were induced from the basal surface of protocorms and the cut-end of shoot tips on Vacin and Went (VW) medium supplemented with 2,4-dichlorophenoxyacetic acid (2,4-D) or α-naphthalene acetic acid (NAA) after 3–5 weeks, with the highest frequency of explants forming callus (48.0 %) from protocorms at 1.0 mg l^?1 2,4-D. The calli obtained from all plant growth regulator (PGR) treatments could proliferate and differentiate PLBs on the PGR-free medium. NAA and 2,4-D significantly enhanced the growth of callus. The fastest growth rate of callus was achieved at the combination of 1.0 mg l^?1 2,4-D and 1.0 mg l^?1 TDZ with 46.2-fold within 3 months. The regeneration of PLBs from callus was significantly improved by 6-benzyladenine (BA), and a mean number of 48.4 PLBs was produced from 100 mg calli at 1.0 mg l^?1 BA within 3 months. BA and thidiazuron (TDZ) promoted the direct formation of PLBs from explants. The highest frequency of direct PLBs formation (76.0 %) and the highest mean number of PLBs per explant (30.2) were observed in protocorms cultured with 0.5 mg l^?1 BA. Assessment of clonal fidelity by inter-simple sequence repeat (ISSR) markers revealed similarity ranges of 99.8–100.0 % between the regenerants and their mother plants and 99.5–100.0 % among the regenerants, which suggested the micropropagation protocols were genetically stable.     

Development and assessment of DArT markers in triticale

Triticale (X Triticosecale Wittm.) is a hybrid derived by crossing wheat (Triticum sp.) and rye (Secale sp.). Till date, only a limited number of simple sequence repeat (SSRs) markers have been used in triticale molecular analyses and there is a need to identify dedicated high-throughput molecular markers to better exploit this crop. The objective of this study was to develop and evaluate diversity arrays technology (DArT) markers in triticale. DArT marker technology offers a high level of multiplexing. Development of new markers from triticale accessions was combined with mining the large collection of previously developed markers in rye and wheat. Three genotyping arrays were used to analyze a collection of 144 triticale accessions. The polymorphism level ranged from 8.6 to 23.8% for wheat and rye DArT markers, respectively. Among the polymorphic markers, rye markers were the most abundant (3,109) followed by wheat (2,214) and triticale (719). The mean polymorphism information content values were 0.34 for rye DArT markers and 0.37 for those from triticale and wheat. High correlation was observed between similarity matrices derived from rye, triticale, wheat and combined marker sets, as well as for the cophenetic values matrices. Cluster analysis revealed genetic relationships among the accessions consistent with the agronomic and pedigree information available. The newly developed triticale DArT markers as well as those originated from rye and wheat provide high quality markers that can be used for diversity analyses and might be exploited in a range of molecular breeding and genomics applications in triticale.     

Soybean pedigree analysis using map-based molecular markers: recombination during cultivar development

An analysis of the genome structure of soybean cultivars was conducted to determine if cultivars are composed of large regions of chromosomes inherited intact from one parent (indicative of minimal recombination) or if the chromosomes are a mixture of one parent's DNA interspersed with the DNA from the other parent (indicative of maximal recombination). Twenty-one single-cross-derived and 5 single-backcross-derived soybean cultivars and their immediate parents (47 genotypes) were analyzed at 89 RFLP loci to determine the minimal number and distribution of recombination events detected. Cultivars derived from single-cross and single-backcross breeding programs showed an average of 5.2 and 8.0 recombination events per cultivar, respectively. A homogeneity Chi-square test based upon a Poisson distribution of recombination events across 13 linkage groups indicated that the number of recombinations observed among linkage groups was random for the single-cross cultivars, but not for the single-backcross-derived cultivars. A twotailed t-test demonstrated that for some linkage groups, the number of recombinations per map unit exceeded the confidence interval developed from a t-distribution of recombinations standardized for map unit distance. Paired t-tests of the number of recombinations observed between linkage-group ends and the mid-portion of the linkage groups indicated that during the development of the cultivars analyzed in this study more recombinations were associated with the ends of linkage groups than with the middle region. Detailed analysis of each linkage group revealed that large portions of linkage groups D, F, and G were inherited intact from one parent in several cultivars. A portion of linkage group G, in contrast, showed more recombination events than expected, based on genetic distance. These analyses suggest that breeders may have selected against recombination events where agronomically favorable combinations of alleles are present in one parent, and for recombination in areas where agronomically favorable combinations of alleles are not present in either parent.Names are necessary to report factually on the available data; however, the USDA neither guarantees nor warrants the standard of the product, and the use of the name by the USDA implies no approval of the product to the exclusion of others that may also be available. Contribution of the Midwest Area, USDA-ARS, Project No. 3236 of the Iowa Agriculture and Home Economics Experiment Station, Ames, IA 50011. Journal Paper No. J-16533     

Somatic embryogenesis in Abutilon indicum (L.) Sweet and assessment of genetic homogeneity using SCoT markers

This study describes an efficient plant regeneration protocol for Abutilon indicum via somatic embryogenesis from 2,4-dichlorophenoxyacetic acid (2,4-D)-induced leaf-derived callus on MS medium, fortified with 13.32 μM 6-benzyladenine (BA), 2.68 μM α-naphthalene acetic acid (NAA), 200 mgl^?1-activated charcoal, and 11.54 μM ascorbic acid. This combination produced the highest (15.5 ± 0.7) number of somatic embryos after four weeks of culture. Further, the embryogenic calli were transferred to MS medium supplemented with 13.32 μM BA, 1.44 μM gibberellic acid (GA₃), and 3% (w/v) sucrose and showed highest rate of germination (76.3 ± 7.0%). The germinated somatic embryos showed maximum plantlet conversion (62.6 ± 1.90%) on ½ MS medium supplemented with 4.92 μM indole-3-butyric acid and 6.0% sucrose (w/v). The highest frequency of secondary somatic embryogenesis (34.4 ± 0.82) was observed on ½ MS medium, supplemented with 133 μM FeSO₄·7H₂O, 74 μM ethylene diamine tetraacetic acid disodium dihydrate (disodium EDTA), and 15% polyethylene glycol-4000 (PEG-4000) after three weeks of subculture. Scanning electron microscopy observations also substantiated the development of primary and secondary somatic embryos from embryogenic calli. Start codon targeted polymorphism (SCoT) marker analysis of 214 somatic embryo-derived plantlets amplified 167 numbers of bands ranging from 230 to 2125 bp. The homogeneous banding pattern confirmed the genetic uniformity of this sample of somatic embryo-derived plantlets as compared with the donor plant.     

转大麻哈鱼生长激素基因鲤生态安全性检测与分析

评价了转大麻哈鱼（Oncorhynchus Suckley）生长激素基因鲤生态安全性问题及研究转基因鱼对天然野生鲤群体遗传污染程度。通过RAPD和SSLP方法,用265个RAPD标记和35对鲤的微卫星标记对受杂交鲤污染的哈尔滨江段黑龙江鲤群体、未受污染的抚远江段黑龙江鲤群体及模拟转基因鲤占普通鲤群体的1%和10%比例获得繁殖子代等实验群体的DNA样本进行全基因组扫描统计分析得出结论,即转基因鲤占普通鲤群体1%时对普通群体的基因污染程度是微乎其微的,远远低于杂交鲤对野生群体基因污染,转基因鲤占普通鲤群体10%时对普通鲤遗传背景的影响稍有升高,但仍然远远低于杂交鲤对野生群体基因污染程度。总之,在现有的检测技术条件及有效的监控条件下,与杂交鲤相比转基因鲤对鲤野生群体的遗传背景的影响是微弱的,而外来种和杂交种则对生态环境有严重威胁。