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.     

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.     

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.     

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.     

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.     

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.     

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.     

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.     

An assessment of European pig diversity using molecular markers: Partitioning of diversity among breeds

Genetic diversity within and between breeds (and lines) of pigs was investigated. The sample comprised 68 European domestic breeds (and lines), including 29 local breeds, 18 varieties of major international breeds, namely Duroc, Hampshire, Landrace, Large White and Piétrain, and 21 commercial lines either purebred or synthetic, to which the Chinese Meishan and a sample of European wild pig were added. On average 46 animals per breed were sampled (range 12–68). The genetic markers were microsatellites (50 loci) and AFLP (amplified fragment length polymorphism, 148 loci). The analysis of diversity showed that the local breeds accounted for 56% of the total European between-breed microsatellite diversity, and slightly less for AFLP, followed by commercial lines and international breeds. Conversely, the group of international breeds contributed most to within-breed diversity, followed by commercial lines and local breeds. Individual breed contributions to the overall European between- and within-breed diversity were estimated. The range in between-breed diversity contributions among the 68 breeds was 0.04–3.94% for microsatellites and 0.24–2.94% for AFLP. The within-breed diversity contributions varied very little for both types of markers, but microsatellite contributions were negatively correlated with the between-breed contributions, so care is needed in balancing the two types of contribution when making conservation decisions. By taking into account the risks of extinction of the 29 local breeds, a cryopreservation potential (priority) was estimated for each of them.     

Identification of unique alleles and assessment of genetic diversity of rabi sorghum accessions using simple sequence repeat markers

Genetic diversity among 42 sorghum accessions representing landraces (19), advanced breeding lines (16), local cultivars (2) and release varieties (5) with 30 simple sequence repeat (SSR) markers revealed 7.6 mean number of alleles per locus showing 93.3% polymorphism and an average polymorphism information content of 0.78 which range from 0.22 (Xtxp12) and 0.91(Xtxp321). The average heterozygosity and effective number of alleles per locus were 0.8 and 6.65 respectively. Cluster analysis based on microsatellite allelic diversity clearly demarcated the accessions into ten clusters. A total of 24 unique alleles were obtained from seven SSR loci in 23 accessions in a size range of 110–380 bp; these unique alleles may serve as diagnostic tools for particular region of the genome of respective genotypes. Selected SSR markers from different linkage groups provided an accurate way of determining genetic diversity at the molecular level.     

Human muscle hardness assessment during incremental isometric contraction using transient elastography

The aim of this study was to investigate the relationship between biceps brachii hardness using the transient elastography technique, and its activity level by quantifying the surface electromyographic signal (sEMG). Ten healthy subjects volunteered for this protocol. To assess the maximal biceps brachii myoelectric activity (sEMG-RMSm), subjects had to achieve their maximal voluntary contraction trial during an elbow flexion effort. They were then asked to perform an isometric biceps sEMG-RMS ramp trial in elbow flexion from 0% to 50% of their sEMG-RMSm in 120 s. A low-frequency pulse was sent every 5 s during all trials by an innovative shear elasticity probe previously placed over the belly of the biceps brachii allowing the calculation of a transverse shear modulus. The main results of this study were (i) the finding of a systematic linear relationship between the biceps brachii transverse shear moduli and the corresponding sEMG-RMS values. This was not the case when plotting transverse shear modulus versus the elbow flexion torque production. Therefore, the computation of a hardness index from the slope of individual transverse shear modulus-sEMG-RMS linear relationship was enabled; (ii) It was also found that the higher is the rest shear modulus, the lower is the hardness index, indicating that the transverse shear modulus change during contraction depends on its level at rest. Therefore, this non-invasive technique could be useful in the medical field to explore deep muscles which are unreachable by classical testing methods. It could also be applied for the follow-up of neuromuscular diseases inducing stiffness changes such as in Duchenne muscular dystrophy.     

Genetic variation assessment of acid lime accessions collected from south of Iran using SSR and ISSR molecular markers

Iran has a long history of acid lime cultivation and propagation. In this study, genetic variation in 28 acid lime accessions from five regions of south of Iran, and their relatedness with other 19 citrus cultivars were analyzed using Simple Sequence Repeat (SSR) and Inter-Simple Sequence Repeat (ISSR) molecular markers. Nine primers for SSR and nine ISSR primers were used for allele scoring. In total, 49 SSR and 131 ISSR polymorphic alleles were detected. Cluster analysis of SSR and ISSR data showed that most of the acid lime accessions (19 genotypes) have hybrid origin and genetically distance with nucellar of Mexican lime (9 genotypes). As nucellar of Mexican lime are susceptible to phytoplasma, these acid lime genotypes can be used to evaluate their tolerance against biotic constricts like lime “witches’ broom disease”.