Development of microsatellite markers in potato and their use in phylogenetic and fingerprinting analyses.

Three genomic libraries were constructed using a mixture of DNA from Solanum phureja Juz. & Buk., and S. chacoense Bitt. Two of the libraries were enriched for ATT and GT repeats (a 27-fold enrichment was achieved). In total, 3500 clones of the conventional library, 1,000 of the library enriched for ATT, and 12,000 of the one enriched for GT were screened with five different repeat motifs, and a total of 18 primer pairs was obtained. Another group of 12 primer pairs was obtained from the SSR-containing sequences in the public databases (18 SSR-containing sequences were utilized). From among 30 newly developed primer pairs, 12 previously published ones, and 12 pairs developed for tomato, 7 were used to identify 12 different potato cultivars and introductions, and 12 were used to study phylogenetic distance among seven wild and cultivated potato species. Two SSR markers were sufficient to discriminate the 12 cultivars. The mean number of alleles per polymorphic locus was 5 for the 12 cultivars and 4.5 for the seven species. The results obtained in this study confirm those achieved in similar studies in other plant species regarding the abundance and use of SSR markers in identifying species and cultivars.     

Temperature Gradient Measurements by Using Thermoelectric Effect in CNTs-Silicone Adhesive Composite

This work presents the fabrication and investigation of thermoelectric cells based on composite of carbon nanotubes (CNT) and silicone adhesive. The composite contains CNT and silicon adhesive 1∶1 by weight. The current-voltage characteristics and dependences of voltage, current and Seebeck coefficient on the temperature gradient of cell were studied. It was observed that with increase in temperature gradient the open circuit voltage, short circuit current and the Seebeck coefficient of the cells increase. Approximately 7 times increase in temperature gradient increases the open circuit voltage and short circuit current up to 40 and 5 times, respectively. The simulation of experimental results is also carried out; the simulated results are well matched with experimental results.     

Phylogeny of the hyper‐diverse rove beetle subtribe Philonthina with implications for classification of the tribe Staphylinini (Coleoptera: Staphylinidae)

With 71 genera and over 2700 described species, Philonthina is the most speciose subtribe of rove beetle tribe Staphylinini and forms a major component of the largest remaining higher systematics challenge in Staphylinini, the ‘Staphylinini propria’ clade. A related systematics issue concerns the position of the genus Holisus (Hyptiomina), which was recovered within the Neotropical philonthine lineage in several recent analyses of morphology. With the aims of resolving the phylogeny of Philonthina and the position and, thus, validity of Hyptiomina, we performed phylogenetic analyses of the tribe Staphylinini based on molecular (six genes, 4471 bp) and morphological (113 characters) data including 138 taxa from all relevant lineages of Staphylinini. We found that ‘Staphylinini propria’ is a monophylum consisting of six lineages: current subtribes Anisolinina, Philonthina, Staphylinina and Xanthopygina; and two new subtribes, Algonina Schillhammer and Brunke and Philothalpina Chatzimanolis and Brunke. While the previously hypothesized Neotropical lineage of Philonthina was corroborated, Holisus was recovered as a separate subtribe, outside of Philonthina, within an informal ‘Southern Hemisphere clade’. Based on our analyses, we propose tentative new concepts of the polyphyletic genera Belonuchus and Philonthus. We propose the following taxonomic changes: synonymy of the subtribes Staphylinina Latreille (valid name) and Eucibdelina Sharp; resurrection of genera Barypalpus Cameron and Trapeziderus Motschulsky from synonymy with Rientis Sharp and Belonuchus Nordmann, respectively; transfer of 38 Belonuchus species, 16 Hesperus Fauvel species and one Philonthus Stephens species to Trapeziderus as new combinations; transfer of two Hesperus species to Eccoptolonthus Bernhauer as new combinations; transfer of one Belonuchus species to Paederomimus Sharp as a new combination; and transfer of Pridonius Blackwelder new status from its position as a subgenus of Quedius (subtribe Quediina) to Philonthina as a genus, and new combinations for its two described species.     

Identification of molecular markers associated with leptine in reciprocal backcross families of diploid potato

Solanum phureja clone 1-3 and S. chacoense clone 80-1 have a zero and high leptine content in their foliage, respectively. An F(1) hybrid (CP2) was intermediate for the trait, but self-incompatible. Two reciprocal backcross families, PBCp ( phu 1-3 x CP2) and PBCc (CP2 x phu 1-3), and a family of monoploids derived by anther culture of CP2, were characterized for leptine as the aglycon, acetylleptinidine (ALD), content in leaves by gas chromatography. ALD was present in 43 of 87 genotypes in the PBCp backcross, implying simple genetic control by a dominant gene. However, the ALD levels were low compared to CP2. In the PBCc backcross, only 7 of 42 genotypes expressed ALD at a level generally higher than in PBCp. This ratio was significantly different from the 1:1 segregation observed in the reciprocal backcross and suggests a cytoplasmic influence. ALD levels in the CP2 monoploids ranged from 0 to 8,968 &mgr;g.g(-1) of dry weight (dw) with 18 individuals expressing ALD and five with 0 ALD content. Ten high (mean ALD = 546 &mgr;g.g(-1) of dw) and ten low (mean ALD = 0) individual plants within PBCp and seven high (mean ALD = 3,037 &mgr;g.g(-1) of dw) and eight low (mean ALD = 0) individual plants within PBCc were used for bulk segregant analysis (BSA) using 214 RAPD (randomly amplified polymorphic DNA) primers. Three RAPD primers (OPQ-2, OPT-16 and OPT-20) amplified bands exclusively in bulks containing DNA mixes of high ALD producers in both PBCp and PBCc populations. These results suggest that these markers were associated in coupling to ALD content. ANOVAs for ALD content verified association between the markers and the trait. A CAPS (cleaved amplified polymorphic sequence) marker, GP82A, was also significantly associated with ALD production in both the monoploid and the PBCp populations. None of the RAPD markers was associated to ALD in the monoploids but one was associated in repulsion. The monoploid data indicate the likelihood of a recessive gene(s) that controls leptine production, but the backcross data indicate the action of modifying loci.     

Variable optimization for the formation of three-dimensional self-organized heart muscle

Cardiac tissue-engineering research is focused on the development of functional three-dimensional (3D) heart muscle in vitro. These models allow the detailed study of critical events in organogenesis, such as the establishment of cell–cell communication and construction and modification of the extracellular matrix. We have previously described a model for 3D heart muscle, termed cardioids, formed by the spontaneous delamination of a cohesive monolayer of primary cells in the absence of any synthetic scaffolding material. In an earlier publication, we have shown that, upon electrical stimulation, cardioids generate a twitch force in the range of 200–300 μN, generate a specific force (twitch force normalized to total cross-sectional area) of 2–4 kN/m², and can be electrically paced at frequencies of up to 10 Hz without any notable fatigue. We have two objectives for the current study: model development and model optimization. Our model development efforts are focused on providing additional characterization of the cardioid model. In this study, we show for the first time that cardioids show a pattern of gene expression comparable to that of cells cultured in two dimensions on tissue culture plastic and normal mammalian heart muscle. Compared with primary cardiac cells cultured on tissue culture plastic, the expression of α-myosin heavy chain (MHC), β-MHC, SERCA2, and phospholamban was significantly higher in cardioids. Our second objective, model optimization, is focused on evaluating the effect of several cell culture variables on cardioid formation and function. Specifically, we looked at the effect of plating density (1.0–4.0 × 10⁶ cells per cardioid), concentration of two adhesion proteins (laminin at 0.2–2.0 μg/cm² and fibronectin at 1–10 μg/cm²), myocyte purity (using preplating times of 15 and 60 min), and ascorbic acid stimulation (1–100 μl/ml). For our optimization studies, we utilized twitch force in response to electrical stimulation as our endpoint metric. Based on these studies, we found that cardioids formed with a plating density in the range 3–4 × 10⁶ cells per cardioid generated the maximum twitch force, whereas increasing the surface adhesion protein (using either laminin or fibronectin) and increasing the myocyte purity both resulted in a decrease in twitch force. In addition, increasing the ascorbic acid concentration resulted in an increase in the baseline force of cardioids, which was recorded in the absence of electrical stimulation. Based on the model development studies, we have shown that cardioids do indeed exhibit a gene expression pattern similar to normal mammalian heart muscle. This provides further validity for the cardioid model. Based on the model optimization studies, we have identified specific cell culture regimes which support cardioid formation and function. These results are specific to the cardioid model; however, they may be translated and applied to other tissue-engineering models. Collectively, the work described in this study provides insight into the formation of functional 3D heart muscle and the effect of several cell culture variables on tissue formation and function.     

Improved androgenesis of interspecific potato and efficiency of SSR markers to identify homozygous regenerants

Three interspecific diploid potato hybrids between selections of Solanum phureja Juz. & Buk. and S. chacoense Bitt. were used in anther culture experiments to construct a monoploid family. Different aspects of the anther culture process were affected by the treatments, such as: growing conditions of donor plants, ways of preparing the anther culture medium, and conditions of anthers in culture. Genotype and date of culture initiation were among the most significant sources of variation. Significant improvements in anther culture response were achieved by growing plants at 30°C and by a heat shock of 35°C for 12 h given to anthers in culture, which gave an increase of up to 40% in embryo yield. However, the heat shock reduced the plant regeneration rate. The majority of regenerated plants was diploid, suggesting that there were several recessive lethal alleles in heterozygous status in the anther-donor. Among the regenerants, the homozygotes could be successfully identified by simple sequence repeat analysis, using eight polymorphic primer pairs. This revised version was published online in June 2006 with corrections to the Cover Date.