Use of microsatellite markers in molecular analysis of segregating populations of papaya (Carica papaya L.) derived from backcrossing.

Brazil is the world leader in papaya production. However, only a small number of cultivars are registered for commercial planting, mainly owing to delays in obtaining cultivars and the high costs of the field phase of breeding programs. These costs can be reduced when molecular tools are combined with conventional breeding methods. In the present study, we conducted a molecular analysis of a self-fertilized population of a first backcrossing generation of BC1S1 papaya plants via microsatellite markers both to monitor the level of homozygosity and the gene/allele transfer that confers the Golden trait (fruit color) and to assess the parental genomic proportion in the genotypes studied. Based on the analysis of 20 polymorphic microsatellite loci, 19 genotypes with the Golden trait belonging to BC1S1 were evaluated in addition to the parental genotypes. Genetic distance was estimated through weighted index. The genotypes were then grouped using the hierarchical nearest neighbor method, and the analysis of principal coordinates was used to measure the proportion of parental genomes in the segregating genotypes. The mean value of the inbreeding coefficient was 0.36. The analysis of the principal coordinates revealed that on average, 64% of the recurrent parent genome was present in the population. Together, the analyses allowed the selection of 3 individuals for the next backcross cycle (33BC1S1-18, 34BC1S1-16, and 37BC1S1-10). These individuals had a higher proportion of the recurrent parent and were grouped close to the recurrent parent in the cluster analysis.

EGL-36 Shaw channels regulate C. elegans egg-laying muscle activity.

The C. elegans egl-36 gene encodes a Shaw-type potassium channel that regulates egg-laying behavior. Gain of function [egl-36(gf)] and dominant negative [egl-36(dn)] mutations in egl-36 cause reciprocal defects in egg laying. An egl-36::gfp reporter is expressed in the egg-laying muscles and in a few other tissues. Expression of an egl-36(gf) cDNA in the egg-laying muscles causes behavioral defects similar to those observed in egl-36(gf) mutants. Gain of function EGL-36 subunits form channels that are active at more negative potentials than wild-type channels. The egl-36(gf) alleles correspond to missense mutations in an amino terminal subunit assembly domain (E138K) and in the S6 transmembrane domain (P435S), neither of which were previously implicated in the voltage dependence of channel activation. Altogether, these results suggest that EGL-36 channels regulate the excitability of the egg-laying muscles.

Midbrain dopaminergic neurons from postnatal rat in long-term primary culture.

Midbrain dopamine neurons project extensively throughout the vertebrate forebrain and influence a wide variety of brain functions. These neurons, which are believed to form a major brain reward system, are involved in initiation and control of motor programs, addictive behaviors, and determination of mood. Given their critical role in behavioral function, relatively little is known about their fundamental cellular physiological and pharmacological properties. A long-term dissociated culture system for postnatal rat dopamine neurons was developed to permit both acute and chronic studies of these cells. Dopamine neurons were dissociated from slices of ventral midbrain from neonatal rat pups and maintained in cell culture for several months. The dopaminergic phenotype was confirmed by catecholamine fluorescence and by tyrosine hydroxylase immunocytochemistry. After four weeks in culture, dopamine neurons had cell bodies 10-40 microns in diameter, displayed either fusiform or multipolar morphology, and had processes with varicosities of 0.5-2 microns in diameter. Electrophysiological recordings were made from 71 dopamine neurons identified by 5,7-dihydroxytryptamine fluorescence after six to 67 days in culture. The neurons had resting potentials of -51 +/- 5 mV, broad action potentials with durations of 2.9 +/- 1.3 ms, and the majority of the neurons (65%) displayed anomalous rectification. Most dopamine neurons in culture fired spontaneously in a pacemaker-like manner with a frequency of 2.3 +/- 1.3 Hz, or in a bursting pattern, typically having two to seven action potentials per burst. All neurons tested had glutamate and gamma-aminobutyric acid receptors, and 90% of neurons responded to dopamine or quinpirole with inhibition of firing, suggesting the presence of dopamine autoreceptors. Some neurons were inhibited by concentrations of quinpirole as low as 10 nM. The results show that midbrain dopamine neurons can be maintained in dissociated cell culture for periods of several months. These neurons can be identified prior to electrophysiological recording, and they express many of the physiological characteristics that have been reported for midbrain dopamine neurons in vivo.

Voltage-dependent calcium channels in rat midbrain dopamine neurons: modulation by dopamine and GABAB receptors.

1. Voltage-dependent calcium channels were studied with whole cell voltage-clamp recordings from neurons enzymatically dispersed from the ventral mesencephalon of rat brains (postnatal days 3-10) and identified as dopamine neurons by 5,7-dihydroxytryptamine autofluorescence. 2. Dopamine neurons had large high-threshold calcium currents activated by depolarizations positive to -50 mV. Different components of calcium channel current were not readily distinguishable by voltage dependence or kinetics, but pharmacological experiments showed the existence of different channel types. The overall current had significant components blocked by nimodipine (28%), by omega-conotoxin GVIA (22%), and by omega-agatoxin-IVA (omega-Aga-IVA) (37%), and there was a significant amount of current (16%) remaining in saturating concentrations of all three blockers. 3. High-threshold calcium current was reversibly reduced by the gamma-aminobutyric acid-B (GABAB) receptor agonist baclofen and by dopamine and the D2 receptor agonist quinpirole. Inhibition by GABAB or dopamine agonists developed and reversed within seconds. 4. Quinpirole reduced both omega-conotoxin-sensitive and omega-Aga-IVA-sensitive components of calcium current. 5. With physiological ionic conditions, inward calcium currents were outweighed by outward currents, in part through calcium-activated potassium channels activated by omega-conotoxin-sensitive and omega-Aga-IVA-sensitive calcium entry.