Comparison of genetic diversity between wild-caught broodstock and hatchery-produced offspring populations of the vulnerable Korean kelp grouper (Epinephelus bruneus) by microsatellites.

The kelp grouper Epinephelus bruneus (Perciformes: Haemulidae), is one of the most economically important fishery resources in Korea. This fish is regarded as a target for prospective aquaculture diversification; therefore, maintenance of stock quality is important. To investigate the effects of current artificial reproduction in a hatchery facility, genetic variation in wild-caught broodstock and hatchery-produced offspring of kelp grouper was analyzed using eight polymorphic nuclear microsatellite DNA loci; 77 alleles were identified. Allelic variability ranged from 2 to 22 in the broodstock and from 1 to 10 in the offspring. The average observed and expected heterozygosities were 0.620 and 0.623 in the broodstock and 0.600 and 0.513 in the offspring, respectively. The possibility of a recent genetic bottleneck was suggested in both populations of E. bruneus. The minor, but significant, genetic differentiation (FST = 0.047, P < 0.05) observed was mainly due to statistically significant reductions in the number of alleles in the offspring compared with the broodstock, suggesting that these genetic changes could be related to genetic drift. Our results demonstrate the usefulness of microsatellite markers to monitor genetic variation and raise concerns about potential harmful genetic effects of inappropriate hatchery procedures. Therefore, genetic variation between broodstock and offspring in a hatchery should be monitored in both breeding and release programs as a routine hatchery operation, and inbreeding should ideally be controlled to improve kelp grouper hatchery management. Our data provide a useful genetic basis for future planning of sustainable culture and management of E. bruneus in fisheries.

Development of microsatellite markers for the kelp grouper Epinephelus bruneus by 454 pyrosequencing and transfer to related species.

The kelp or longtooth grouper (Epinephelus bruneus), which inhabits Eastern Asia, is the most economically important of 11 grouper species that inhabit the Southern Sea near Jeju Island in Korea. This species is listed as vulnerable by the International Union for the Conservation of Nature and Natural Resources because of a rapid decrease in its resources. We developed microsatellite markers for E. bruneus using the pyrosequencing technique for applications in resource management and aquaculture. In addition, we tested the cross-species transferability of the microsatellite markers in four species belonging to the Epinephelus genus. Among 66,452 simple sequence repeats, 64 loci containing more than eight CA or TG repeats were randomly selected for primer synthesis; 45 primer sets (75.0%) produced polymerase chain reaction (PCR) products of 100-300 bp and were selected as candidates. After primary testing with four E. bruneus fish, 28 polymorphic loci were selected as the final microsatellite markers, and 23 sets showing clear amplification of polymorphic loci were used to analyze 71 fish. These loci have allele numbers ranging from 2 to 23. Null alleles were detected at three loci, and three loci showed an excess of homozygotes in the Hardy-Weinberg equilibrium test. Of the three species used for cross-species transfer of these markers, Epinephelus moara showed the highest transferability (92.9%) and polymorphism (67.9%), followed by Epinephelus fuscoguttatus (75.0 and 67.9%, respectively) and Epinephelus septemfasciatus (57.1 and 46.4%, respectively). These results suggested that these microsatellite loci should be valuable tools for population genetic studies of the species Epinephelus.

Identification of process operating state with operational map in municipal wastewater treatment plant.

This work was performed to develop an operational map for the objective diagnosis of the process operating states of a municipal wastewater treatment plant, for which multivariate statistical analysis techniques were applied. PCA (principal component analysis) was used to reduce the dimension of the data sets obtained from the field municipal wastewater treatment plant. A K-means clustering analysis was used to classify the group according to the property of the process operating state. A Fisher's linear discriminant analysis was used to derive the discriminant function of each classified group. An operational map was developed by scatter-plotting the derived principal components (PCs) on a two-dimensional coordinate according to the classified groups. Using the new data sets not used for developing the operational map, the practical usefulness of the operational map and discriminant function in diagnosing the process operating state were evaluated. Hence, the process operating state could be easily and quickly diagnosed and the dynamic trend of the process operating state was also able to be estimated using the operational map.

Determination of the three-dimensional structure of hordothionin-alpha by nuclear magnetic resonance.

The high-resolution three-dimensional solution structure of the plant toxin hordothionin-alpha obtained from korean barley was determined by using two-dimensional NMR techniques combined with distance geometry and restrained molecular dynamics. Experimentally derived restraints including 292 interproton distances from nuclear Overhauser effect measurements, 16 hydrogen bond restraints together with four disulphide bridge restraints were used as input to calculations of distance geometry and restrained molecular dynamics. Also included in the calculations were 36 phi and 17 chi 1 torsion angles obtained from 33JHN alpha and 3J alpha beta coupling constants in double quantum filtered COSY and primitive exclusive COSY experiments, respectively. The overall protein fold is similar to crambin and purothionin-alpha 1. Two alpha-helices running in opposite directions are found on the basis of 3JHN alpha and 3J alpha beta and deuterium exchange rates for backbone NH protons, and encompass residues 7-18 and 22-28. These two helices are connected by a turn and form a 'helix-turn-helix' motif. A short stretch of an anti-parallel beta-sheet exists between residues 1-4 and 31-34. the two protein termini of hordothionin-alpha are 'well-anchored'; the N-terminus of the protein is immobilized by this short beta-sheet whereas the C-terminus is 'pasted' to the carbonyl group of Cys-4 by a very stable hydrogen bond. The average root-mean-square differences for the backbone and heavy atoms after the restrained molecular dynamics calculations are 0.62 and 1.16 A respectively. These numbers represent a significant improvement over the corresponding values for the previous NMR structures of other thionins. The distance violation from the experimental interproton distances for the final structures is 0.14 for all atoms.