Species identification in salted products of red snappers by semi-nested PCR-RFLP based on the mitochondrial 12S rRNA gene sequence

A molecular approach was developed to distinguish species of red snappers among commercial salted fish products. The specific fragments of the mitochondrial 12S rRNA gene, which were about 450bp, were obtained using the semi-nested polymerase chain reaction (semi-nested PCR). Subsequently, PCR arnplicons were sequenced, aiming to select restriction endonucleases that generated species-specific restriction fragment length polymorphism (RFLP) profiles. Discrimination of red snappers Lutjanus sanguineus, Lutjanus erythopterus from Lutjanus argentimaculatus, Lutjanus malabarius and other morphologically similar fishes such as Lethrinus leutjanus and Pinjalo pinjalo was feasible by one restriction digestion reaction with three endonucleases Hae III, Sca I and SnaB I, however, for discrimination of L. sanguineus and L. erythopterus, another restriction digestion reaction with single restriction endonuclease Mae II was needed. The semi-nested PCR-RFLP was demonstrated to be reliable in species identification of salted fish products in this study. (c) 2005 Elsevier Ltd. All rights reserved.

Characterization and mapping of expressed sequence tags isolated from a subtracted cDNA library of Litopenaeus vannamei injected with white spot syndrome virus

A large number of polymorphic simple sequence repeats (SSRs) or microsatellites are needed to develop a genetic map for shrimp. However, developing an SSR map is very time-consuming, expensive, and most SSRs are not specifically linked to gene loci of immediate interest. We report here on our strategy to develop polymorphic markers using expressed sequence tags (ESTs) by designing primers flanking single or multiple SSRs with three or more repeats. A subtracted cDNA library was prepared using RNA from specific pathogen-free (SPF) Litopenaeus vannamei juveniles (similar to 1 g) collected before (0) and after (48 h) inoculation with the China isolate of white spot syndrome virus (WSSV). A total of 224 clones were sequenced, 194 of which were useful for homology comparisons against annotated genes in NCBI nonredundant (nr) and protein databases, providing 179 sequences encoded by nuclear DNA, 4 mitochondrial DNA, and 11 were similar to portions of WSSV genome. The nuclear sequences clustered in 43 groups, 11 of which were homologous to various ESTs of unknown function, 4 had no homology to any sequence, and 28 showed similarities to known genes of invertebrates and vertebrates, representatives of cellular metabolic processes such as calcium ion balance, cytoskeleton mRNAs, and protein synthesis. A few sequences were homologous to immune system-related (allergens) genes and two were similar to motifs of the sex-lethal gene of Drosophila. A large number of EST sequences were similar to domains of the EF-hand superfamily (Ca2+ binding motif and FRQ protein domain of myosin light chains). Single or multiple SSRs with three or more repeats were found in approximately 61 % of the 179 nuclear sequences. Primer sets were designed from 28 sequences representing 19 known or putative genes and tested for polymorphism (EST-SSR marker) in a small test panel containing 16 individuals. Ten (53%) of the 19 putative or unknown function genes were polymorphic, 4 monomorphic, and 3 either failed to satisfactorily amplify genomic DNA or the allele amplification conditions need to be further optimized. Five polymorphic ESTs were genotyped with the entire reference mapping family, two of them (actin, accession #CX535973 and shrimp allergen arginine kinase, accession #CX535999) did not amplify with all offspring of the IRMF panel suggesting presence of null alleles, and three of them amplified in most of the IRM F offspring and were used for linkage analysis. EF-hand motif of myosin light chain (accession #CX535935) was placed in ShrimpMap's linkage group 7, whereas ribosomal protein S5 (accession #CX535957) and troponin I (accession #CX535976) remained unassigned. Results indicate that (a) a large number of ESTs isolated from this cDNA library are similar to cytoskeleton mRNAs and may reflect a normal pathway of the cellular response after im infection with WSSV, and (b) primers flanking single or multiple SSRs with three or more repeats from shrimp ESTs could be an efficient approach to develop polymorphic markers useful for linkage mapping. Work is underway to map additional SSR-containing ESTs from this and other cDNA libraries as a plausible strategy to increase marker density in ShrimpMap.

Genetic mapping of laminaria japonica and l. longissima using amplified fragment length polymorphism markers in a "two-way pseudo-testcross" strategy

With a "two-way pseudo-testcross" mapping strategy, we applied the amplified fragment length polymorphism (AFLP) markers to construct two moderate density genetic linkage maps for Laminaria. The linkage maps were generated from the 60 progenies of the F, cross family (Laminaria longissima Aresch. x L. japonica Miyabe) with twenty pairs of primer combinations. Of the 333 polymorphic loci scored in 60 progenies, 173 segregated in a 1:1 ratio, corresponding to DNA polymorphisms heterozygous in a single parent, and the other 58 loci existing in both parents followed a 3:1 Mendelian segregation ratio. Among the loci with 1:1 segregating ratios, 79 loci were ordered in 14 linkage groups (648.6 cM) of the paternal map, and 72 loci were ordered in 14 linkage groups (601.9 cM) of the maternal map. The average density of loci was approximately 1 per 8 cM. To investigate the homologies between two parental maps, we used 58 loci segregated 3:1 for further analysis, and deduced one homologous linkage group. The linkage data developed in these maps will be useful for detecting loci-controlling commercially important traits for Laminaria.

A semi-analytical theory of coastal upwelling and jet

The general forms of the conservation of momentum, temperature and potential vorticity of coastal ocean are obtained in the x-z plane for the nonlinear ocean circulation of Boussinesq fluid, and a elliptic type partial differential equations of second order are derived. Solution of the partial differential equations are obtained under the conditions that the fluid moves along the topography. The numerical results show that there exist both upwelling and downwelling along coastline that mainly depends on the large scale ocean condition. Numerically results of the upwelling (downwelling), coastal jet and temperature front zone are favorable to the observations.

Density-dependent effects on seston dynamics and rates of filtering and biodeposition of the suspension-cultured scallop Chlamys farreri in a eutrophic bay (northern China): An experimental study in semi-in situ flow-through systems

Effects of stocking density on seston dynamics and filtering and biodeposition by the suspension-cultured Zhikong scallop Chlamys farreri Jones et Preston in a eutrophic bay (Sishili Bay, northern China), were determined in a 3-month semi-field experiment with continuous flow-through seawater from the bay. Results showed that the presence of the scallops could strongly decrease seston and chlorophyll a concentrations in the water column. Moreover, in a limited water column, increasing scallop density could cause seston depletion due to scallop's filtering and biodeposition process, and impair scallop growth. Both filtration rate and biodeposition rate of C. farreri showed significant negative correlation with their density and positive relationship with seston concentration. Calculation predicts that the daily removal of suspended matter from water column by the scallops in Sishili Bay ecosystem can be as high as 45% of the total suspended matter; and the daily production of biodeposits by the scallops in early summer in farming zone may amount to 7.78 g m(-2), with daily C, N and P biodeposition rates of 3.06 x 10(-1), 3.86 x 10(-2) and 9.80 x 10(-3) g m(-2), respectively. The filtering and biodeposition by suspension-cultured scallops could substantially enhance the deposition of total suspended particulate material, suppress accumulation of particulate organic matter in water column, and increase the flux of C, N and P to benthos, strongly enhancing pelagic-benthic coupling. It was suggested that the filtering-biodeposition process by intensively suspension-cultured bivalve filter-feeders could exert strong top-down control on phytoplankton biomass and other suspended particulate material in coastal ecosystems. This study also indicated that commercially suspension-cultured bivalves may simultaneously and potentially aid in mitigating eutrophication pressures on coastal ecosystems subject to anthropogenic N and P loadings, serving as a eutrophic-environment bioremediator. The ecological services (e.g. filtering capacity, top-down control, and benthic-pelagic coupling) functioned by extractive bivalve aquaculture should be emphasized in coastal ecosystems. (c) 2005 Elsevier B.V. All rights reserved.

Chromosomal mapping of 5S ribosomal RNA genes in the eastern oyster, Crassostrea virginica gmelin by fluorescence in situ hybridization

Chromosomal location of the 5S ribosomal RNA gene was studied in the eastern oyster, Crassostrea virginica Gmelin. using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos, and the FISH probe was made by PCR (polymerase chain reaction) amplification of the 5S rRNA gene and labeled by incorporation of digoxigenin-1 1-dUTP during PCR. Hybridization was detected with fluorescein-labeled antidigoxigenin antibodies. Two pairs of FISH signals were observed on metaphase chromosomes. Karyotypic analysis showed that the 5S rRNA gene cluster is interstitially located on short arms of chromosomes 5 and 6. On chromosome 5, the 5S rRNA genes were located immediately next to the centromere, whereas on chromosome 6, they were located approximately half way between the telomere and the centromere. Chromosomes of C. virginica are difficult to identify because of their similarities in size and arm ratio, and the chromosomal location of 5S rRNA genes provides unambiguous identification of chromosomes 5 and 6. Previous studies have mapped the major rRNA gene cluster (18S-5.8S-28S) to chromosome 2. and this study shows that the 5S rRNA gene cluster is not linked to the major rRNA genes and duplicated during evolution.

Genetic mapping of size-related quantitative trait loci (QTL) in the bay scallop (Argopecten irradians) using AFLP and microsatellite markers

We constructed genetic linkage maps for the bay scallop Argopecten irradians using AFLP and microsatellite markers and conducted composite interval mapping (CIM) of body-size-related traits. Three hundred seventeen AFLP and 10 microsatellite markers were used for map construction. The female parent map contained 120 markers in 15 linkage groups, spanning 479.6 cM with an average interval of 7.0 cM. The male parent map had 190 markers in 17 linkage groups, covering 883.8 cM at 7.2 cM per marker. The observed coverage was 70.4% for the female and 81.1% for the male map. Markers that were distorted toward the same direction were closely linked to each other on the genetic maps, suggesting the presence of genes important for survival. Six size-related traits, shell length, shell height, shell width, total weight, soft tissue weight, and shell weight, were measured for QTL mapping. The size data were significantly correlated with each other. We subjected the data, log transformed firstly, to a principle component analysis and use the first principle component for QTL mapping. CIM analysis revealed one significant QTL (LOD=2.69, 1000 permutation, P<0.05) in linkage group 3 on the female parent map. The mapping of size-related QTL in this study raises the possibility of improving the growth of bay scallops through marker-assisted selection. (c) 2007 Published by Elsevier B.V.

Identification and mapping of amplified fragment length polymorphism markers linked to shell color in bay scallop, Argopecten irradians irradians (Lamarck, 1819)

Amplified fragment length polymorphisms (AFLP) were used to study the inheritance of shell color in Argopecten irradians. Two scallops, one with orange and the other with white shells, were used as parents to produce four F-1 families by selfing and outcrossing. Eighty-eight progeny, 37 orange and 51 white, were randomly selected from one of the families for segregation and mapping analysis with AFLP and microsatellite markers. Twenty-five AFLP primer pairs were screened, yielding 1138 fragments, among which 148 (13.0%) were polymorphic in two parents and segregated in progeny. Six AFLP markers showed significant (P < 0.05) association with shell color. All six loci were mapped to one linkage group. One of the markers, F1f335, is completely linked to the gene for orange shell, which we designated as Orange1, without any recombination in the progeny we sampled. The marker was amplified in the orange parent and all orange progeny, but absent in the white parent and all the white progeny. The close linkage between F1f335 and Orange1 was validated using bulk segregation analysis in two natural populations, and all our data indicate that F1f335 is specific for the shell color gene, Orange1. The genomic mapping of a shell color gene in bay scallop improves our understanding of shell color inheritance and may contribute to the breeding of molluscs with desired shell colors.

Chromosomal mapping of major ribosomal rRNA genes in the hard clam (Mercenaria mercenaria) using fluorescence in situ hybridization

Karyotype and chromosomal location of the major ribosomal RNA genes were studied in the hard clam (Mercenaria mercenaria Linnaeus) using fluorescence in situ hybridization (FISH). Metaphase chromosomes were obtained from early embryos. Internal transcribed spacers (ITS) between major RNA genes were amplified and used as FISH probes. The probes were labeled with digoxigenin-11-dUTP by polymerase chain reaction and detected with fluorescein-labeled anti-digoxigenin antibodies. FISH with the ITS probes produced two to four signals per nucleus or metaphase. M. mercenaria had a haploid number of 19 chromosomes with a karyotype of seven metacentric, four metacentric or submetacentric, seven submetacentric, and one submetacentric or subtelocentric chromosomes (7M + 4M/SM + 7SM + 1SM/ST). Two ITS loci were observed: one located near the centromere on the long arm of Chromosome 10 and the other at the telomere of the short arm of Chromosome 12. FISH signals on Chromosome 10 are strong and consistent, while signals on Chromosome 12 are variable. This study provides the first karyotype and chromosomal assignment of the major RNA genes in M. mercenaria. Similar studies in a wide range of species are needed to understand the role of chromosomal changes in bivalve evolution.

Chromosomal mapping of the major ribosomal RNA genes in the dwarf surfclam (Mulinia lateralis Say)

Chromosomal location of the major ribosomal RNA genes (rRNA) were studied in the dwarf surfclam (Mulinia lateralis, Say) using fluorescence in situ hybridization (FISH). FISH probes for the rRNA genes were made by polymerase chain reaction (PCR), labeled with digoxigenin-11-dUTP and detected with fluorescein-labeled antidigoxigenin antibodies. Mulinia lateralis had a diploid number of 38 chromosomes and all chromosomes were telocentric. FISH with the rRNA probe produced positive and consistent signals on two pairs of chromosomes: Chromosome 15 with a relative length of 4.6% and Chromosome 19, the shortest chromosome. Both loci were telomeric. The rRNA location provides the first physical landmark of the M. lateralis genome.

Genetic diversity of the subterranean Gansu zokor in a semi-natural landscape

We studied the relationship between genetic diversity of the subterranean Gansu zokor Myospalax cansus and habitat variability in the Loess Plateau, Qinghai Province, China. We used a combination of geographic information systems and molecular techniques to assess the impact of habitat composition and human activities on the genetic diversity of zokor populations in this semi-natural landscape. Although they occurred relatively infrequently in the landscape, woodland and high-coverage grassland habitats were the main positive contributors to the genetic diversity of zokor populations. Rural residential land, plain agricultural land and low-coverage grassland had a negative effect on genetic diversity. Hilly agricultural land and middle-coverage grassland had little impact on zokor genetic diversity. There were also interactions between some habitat types, that is, habitat types with relatively better quality together promoted conservation of genetic diversity, while the interaction between (among) bad habitat types made situations worse. Finally, habitat diversity, measured as patch richness and Shannon's diversity index, was positively correlated with the genetic diversity. These results demonstrated that: (1) different habitat types had different effects on the genetic diversity of zokor populations and (2) habitat quality and habitat heterogeneity were important in maintaining genetic diversity. Habitat composition was closely related to land use thus emphasizing the importance of human activities on the genetic diversity of subterranean rodent populations in this semi-natural landscape. Although the Gansu zokor was considered to be a pest species in the Loess Plateau, our study provides insights for the management and conservation of other subterranean rodent species.

Depression of net ecosystem CO2 exchange in semi-arid Leymus chinensis steppe and alpine shrub

Uptake and release of carbon in grassland ecosystems is very critical to the global carbon balance and carbon storage. In this study, the dynamics of net ecosystem CO2 exchange (FNEE) of two grassland ecosystems were observed continuously using the eddy covariance technique during the growing season of 2003. One is the alpine shrub on the Tibet Plateau, and the other is the sem-arid Leymus chinensis steppe in Inner Mongolia of China. It was found that the FNEE of both ecosystems was significantly depressed under high solar radiation. Comprehensive analysis indicates that the depression of FNEE in the L. chinensis steppe was the results of decreased plant photosynthesis and increased ecosystem respiration (R-eco) under high temperature. Soil water stress in addition to the high atmospheric demand under the strong radiation was the primary factor limiting the stomatal conductance. In contrast, the depression of FNEE in the alpine shrub was closely related to the effects of temperature on both photosynthesis and ecosystem respiration, coupled with the reduction of plant photosynthesis due to partial stomatal closure under high temperature at mid-day. The R,c of the alpine shrub was sensitive to soil temperature during high turbulence (u* > 0.2 m s(-1)) but its FNEE decreased markedly when the temperature was higher than the optimal value of about 12 degrees C. Such low optimal temperature contrasted the optimal value (about 20 degrees C) for the steppe, and was likely due to the acclimation of most alpine plants to the long-term low temperature on the Tibet Plateau. We inferred that water stress was the primary factor causing depression of the FNEE in the semi-arid steppe ecosystem, while relative high temperature under strong solar radiation was the main reason for the decrease of FNEE in the alpine shrub. This study implies that different grassland ecosystems may respond differently to climate change in the future. (c) 2006 Elsevier B.V All rights reserved.

Meat characteristics of Qinghai yak and semi-wild yak

Six domestic yaks aged 6 years, comprising three culling male and three culling female yaks, from the Qinghai Lake area and three male semi-wild yaks aged 6 months from Datong Yak Farm were used to detect the levels of amino acids, mineral elements, residual heavy metals and pesticides in yak meat. The results showed that there was little difference in amino acid levels for different types of yak by age and sex. The meat of the adult male yak was 7.35 mg/kg higher in calcium (P < 0.05) and 124 mg/kg higher in phosphorus (P < 0.01) than that of the adult female yak. There was an obvious difference in zinc content, and there was also a significant difference for copper and sulfur between adult male and female yaks (P < 0.01). Iron was 8.85 mg/kg higher in semi-wild yaks than in domestic yaks, but there were no differences for other minerals. The levels of residual heavy metals and residual pesticides were well within those allowed by the National Standard of China. Furthermore, the data showed that introducing wild yak's blood did not result in a quality change in the domestic yak meat. Yak meat from Qinghai Plateau has the characteristics of being non-polluted and rich in amino acids in comparison with local yellow cattle meat.

Assessing the potential of VEGETATION sensor data for mapping snow and ice cover: a Normalized Difference Snow and Ice Index

The VEGETATION (VGT) sensor in SPOT 4 has four spectral bands that are equivalent to Landsat Thematic Mapper (TM) bands (blue, red, near-infrared and mid-infrared spectral bands) and provides daily images of the global land surface at a 1-km spatial resolution. We propose a new index for identifying and mapping of snow ice cover, namely the Normalized Difference Snow/Ice Index (NDSII), which uses reflectance values of red and mid-infrared spectral bands of Landsat TM and VGT. For Landsat TM data, NDSII is calculated as NDSIITM =(TM3 -TM5)/(TM3 +TM5); for VGT data, NDSII is calculated as NDSIIVGT =(B2- MIR)/(B2 + MIR). As a case study we used a Landsat TM image that covers the eastern part of the Qilian mountain range in the Qinghai-Xizang (Tibetan) plateau of China. NDSIITM gave similar estimates of the area and spatial distribution of snow/ice cover to the Normalized Difference Snow Index (NDSI=(TM2-TM5)/(TM2+TM5)) which has been proposed by Hall et al. The results indicated that the VGT sensor might have the potential for operational monitoring and mapping of snow/ice cover from regional to global scales, when using NDSIIVGT.