Genetic characterization of asymmetric reciprocal hybridization between the flatfishes Paralichthys olivaceus and Paralichthys dentatus

Interspecific reciprocal crosses between the two flatfishes Paralichthys olivaceus and P. dentatus yielded hybrids with different viabilities. Specifically, the hybrids of P. olivaceus female and P. dentatus male (HI) were found to be viable, while the reciprocal hybrids from P. dentatus female and P. olivaceus male (HII) were completely inviable. All the HII individuals showed morphological deformities and died before first feeding. The chromosome analysis showed that HI individuals had the same chromosome number as parents. However, two chromosomes were missing in HII offspring indicating that the latter were aneuploids. Genomic inheritance from the parents to F-1 progeny was also examined by amplified fragment length polymorphism (AFLP) analyses, and the results showed differences between reciprocal hybrids. Almost all AFLP bands (97.71%) observed in parents were passed on to HI individuals. In contrast, only 86.64% of the AFLP bands from parents were scored in HII individuals. Frequency of lost parental bands was thus significantly higher in HII than that in HI and intraspecific crosses, which was probably associated with chromosomal elimination. In addition, higher segregation distortions were found in hybrids than in controls, although these differences were not significant. The present study indicates that chromosomal elimination and loss of AFLP loci occurred in inviable HII individuals, while such genomic changes were not found in viable HI individuals. Possible implications of such difference on genomic changes for asymmetric viability in reciprocal hybrids are discussed.

VEGF gene silencing by cytomegalovirus promoter driven ShRNA expression vector results in vascular development defects in zebrafish

Zebrafish has been generally considered as an excellent model in case of drug screening, disease model establishment, and vertebrate embryonic development study. In this work, the ability of human cytomegalovirus immediate early promoter (CMV promoter)-driven short hairpin RNA (shRNA) expression vector to induce shRNA against VEGF gene in zebrafish was tested, and its effect on vascular development was assed, too. Using RT-qPCR, blood vessel staining, and in situ hybridization, we confirmed certain transcriptional activity and down regulation of gene expression by the vector. In situ hybridization analysis indicated selective inhibition of NRP1 expression in the VEGF gene loss of function model, which might imply in turn that VEGF could not only activate endothelial cells directly but also could contribute to stimulating angiogenesis in vivo by a mechanism that involved up-regulation of its cognate receptor expression in zebrafish. This contributed to a better understanding of molecular mechanisms of cardiovascular development. The system improved the success rate in making inducible knockdown and widened the possibilities for better therapeutic targets in zebrafish.

Expression of muscle-related genes and two MyoD genes during amphioxus notochord development

The notochord is one of the diagnostic features of the phylum Chordata. Despite the similarities in the early morphogenetic patterns of the notochords of various chordates, they are strikingly distinct from one another at the histological level. The amphioxus notochord is one example of an evolutionary novelty because it is made up of muscle cells. Our previous expressed sequence tag analysis, targeting messenger RNAs expressed in the adult amphioxus notochord, demonstrated that many muscle-related genes are expressed there. To characterize amphioxus notochord cells and to gain insights into the myogenic program in the notochord, we determined the spatial and temporal expression patterns of these muscle-related genes during amphioxus development. We found that BbNA1 (notochord actin), Amphi-Trop I (troponin I), Amphi-TPmyosin (tropomyosin), Amphi-MHC2 (myosin heavy chain), Amphi-nMRLC (notochord-specific myosin regulatory light chain), AmphinTitin/MLCK (notochord-specific titin/myosin light chain kinase), Amphi-MLP/CRP3 (muscle LIM protein), and Amphi-nCalponin (notochord-specific calponin) are expressed with characteristic patterns in notochord cells, including the central cells, dorsally located cells, and ventrally located cells, suggesting that each notochord cell has a unique molecular architecture that may reflect its function. In addition, we characterized two MyoD genes (Amphi-MyoD1 and Amphi-MyoD2) to gain insight into the genetic circuitry governing the formation of the notochord muscle. One of the MyoD genes (Amphi-MyoD2) is expressed in the central notochord cells, and the coexistence of Amphi-MyoD2 transcripts along with the Amphi-MLP/CRP3 transcripts implies the participation of Amphi-MyoD2 in the myogenic program in the notochord muscle.

Biopotentials of marine sponges from China oceans: past and future

An extensive literature survey of over 17 Journals was carried out on Chinese sponges and their natural products in the period from 1980 to 2001. This review is thus intended to provide the first thorough overview of research on marine sponges from China Ocean territories. Information is provided about the rather-limited taxonomic study of Chinese marine sponges, with an analysis on their distribution and diversity. Research findings on the natural products and their bioactivity screening from Chinese sponges are summarized. The weaknesses, gaps and problems in the past R&D program of Chinese sponges are identified, which point to the future opportunities in exploiting these huge untapped sponge resources. The report is expected to serve as an entry point for understanding Chinese sponges and for furthering R&D on their bioactive compounds for new drug development. (C) 2003 Elsevier Science B.V. All rights reserved.

Effects of cell density, light intensity and mixing on Undaria pinnatifida gametophyte activity in a photobioreactor

An on-line controlled 7 1 sterilizable photobioreactor was used for the optimisation of a culture of gametophytes of Undaria pinnatifida. The gametophytes, which had been stored for three years in a culture cabinet at 16 degreesC, could rapidly grow in the photobioreactor under controlled conditions. The rate of increase of dissolved oxygen and pH were used to monitor the photosynthetic activity. Optimal gametophytes density changed varying the light intensity. The optimal cell densities were 3.24 and 3.45 g FW l(-1) when the cultures were exposed to 61.7 and 82.3 muE m(-2) s(-1), respectively. The optimal cell density was higher under a high photon flux density (PFD) than under low PFD. On the other hand, the optimal light intensities were different for different cell density cultures. The light saturation point was higher at high cell density cultures than at low cell density cultures. The optimal rotational speed was 150 rpm for high cell density culture in the photobioreactor. (C) 2003 Elsevier B.V. All rights reserved.

An estimate of divergence time of parazoa and eumetazoa and that of cephalochordata and vertebrata by aldolase and triose phosphate isomerase clocks

Previously we suggested that four proteins including aldolase and triose phosphate isomerase (TPI) evolved with approximately constant rates over long periods covering the whole animal phyla. The constant rates of aldolase and TPI evolution were reexamined based on three different models for estimating evolutionary distances, It was shown that the evolutionary rates remain essentially unchanged in comparisons not only between different classes of vertebrates but also between vertebrates and arthropods and even between animals and plants, irrespective of the models used, Thus these enzymes might be useful molecular clocks for inferring divergence times of animal phyla, To know the divergence time of Parazoa and Eumetazoa and that of Cephalochordata and Vertebrata, the aldolase cDNAs from Ephydatia fluviatilis, a freshwater sponge, and the TPI cDNAs from Ephydatia fluviatilis and Branchiostoma belcheri an amphioxus, have been cloned and sequenced, Comparisons of the deduced amino acid sequences of aldolase and TPI from the freshwater sponge with known sequences revealed that the Parazoa-Eumetazoa split occurred about 940 million years ago (Ma) as determined by the average of two proteins and three models, Similarly, the aldolase and TPI clocks suggest that vertebrates and amphioxus last shared a common ancestor around 700 Ma and they possibly diverged shortly after the divergence of deuterostomes and protostomes.

Lack of genetic differentiation in the shrimp Penaeus chinensis in the northwestern pacific

Genetic differentiation of the shrimp Penaeus chinensis in the Yellow Sea and Bohai Sea was investigated using the mitochondrial control region (CR). RFLP of a partial CR segment (613 bp) shows that 106 out of 122 (86.9%) individuals from six sampling localities along the coast of northern China and the west coast of the Korean Peninsula share the same haplotype, and the haplotype frequencies among localities are not significantly different. The findings are further confirmed by sequencing the complete CR. Divergence of the complete CR (992 bp) is less than 1.6% in 14 individuals from the six localities. F-statistics based on RFLP data and the TCS network of sequencing data suggest little genetic differentiation of P. chinensis in the Yellow Sea and Bohai Sea. Mismatch analysis suggests a rapid expansion of P. chinensis population to the Yellow Sea and the Bohai Sea, which probably occurred with the rapid rise in sea level after the last glacial maximum. Despite the lack of genetic heterogeneity, we propose that P. chinensis populations in this region should be treated as separate management units, as fishery management programs have to be applied on a local basis by different governments.

The complete mitochondrial genomes of two common shrimps (Litopenaeus vannamei and Fenneropenaeus chinensis) and their phylogenomic considerations

Complete mitochondrial genomes have proven extremely valuable in helping to understand the evolutionary relationships among metazoans. However, uneven taxon sampling may lead to unclear or even erroneous phylogenetic topologies. The decapod crustaceans are relatively well-sampled, but sampling is still uneven within this group. We have sequenced the mitochondrial genomes of two shrimps Litopenaeus vannamei and Fenneropenaeus chinensis. As seen in other metazoans, the genomes contain a standard set of 13 protein-coding genes, 22 transfer RNA genes, two ribosomal RNA genes and an AT-rich non-coding region. The gene arrangements are consistent with the pancrustacean ground pattern. Both the pattern of gene rearrangements and phylogenomic analyses using concatenated nucleic acid and amino acid sequences of the 13 mitochondrial protein-coding genes strengthened the support that Caridea and Palinura are primitive members of Pleocyemata. These sequences, in combination with two previously published penaeid mitochondrial genomes, suggest that genera within the family Penaeidae have the following relationship: (((Penaeits + Fenneropenaett.) + Litopeiiaelts) + Marsupenaeus). The analyses of nucleic acid and amino acid sequences of the mitochondrial genomes also strongly support the monophyly of Penaeidae, Brachyura and Pleocyemata. In addition, the analyses of the average Ka/Ks in the 13 mitochondrial protein-coding genes of penaeid shrimps indicated a strong purifying selection within this group.

Microsatellite-centromere distances and microsatellite diversity in different ploidy classes of Chinese shrimp (Fenneropenaeus Chinensis)

This is the first report of microsatellite-centromere mapping in this commercial species Fenneropenaeus Chinensis, and will be important for providing fixed points in the linkage groups of genetic maps. Triploid Chinese shrimp was induced by heat shock. The fertilized eggs were treated either by retention of the first polar body or the second polar body to produce Meiosis I (MI) or Meiosis II (MII) triploid. The triploidy status in each Chinese shrimp could be confirmed by nine polymorphic microsatellite loci, in which the parents with different alleles and the female parents were each heterozygous. The nine loci were mapped in relation to their centromeres in three MII triploid families, which were induced by retention of the second polar bodies after fertilization with sperm. Microsatellite-centromere (M-C) distances ranged from 9.6 cM to 37 cM under the assumption of complete interference. Information on the positions of centromeres in relation to the microsatellite loci will represent a contribution towards assembly of genetic maps in F. chinensis. Twelve polymorphic microsatellites were used to assess the heterozygosity and allelic diversity in different ploidy classes. As expected, triploids were significantly more polymorphic than diploids. The diploids had an average heterozygosity and allelic diversity value of 0.86, whereas the triploids heterozygosity averaged 0.93 and had allelic diversity value of 1.29. However, MI triploids were not significantly more polymorphic than MII in the microsatellite loci.

Lineage-specific domain fusion in the evolution of purine nucleotide cyclases in cyanobacteria

Cyclic nucleotides (both cAMP and cGMP) play extremely important roles in cyanobacteria, such as regulating heterocyst formation, respiration, or gliding. Catalyzing the formation of cAMP and cGMP from ATP and GTP is a group of functionally important enzymes named adenylate cyclases and guanylate cyclases, respectively. To understand their evolutionary patterns, in this study, we presented a systematic analysis of all the cyclases in cyanobacterial genomes. We found that different cyanobacteria had various numbers of cyclases in view of their remarkable diversities in genome size and physiology. Most of these cyclases exhibited distinct domain architectures, which implies the versatile functions of cyanobacterial cyclases. Mapping the whole set of cyclase domain architectures from diverse prokaryotic organisms to their phylogenetic tree and detailed phylogenetic analysis of cyclase catalytic domains revealed that lineage-specific domain recruitment appeared to be the most prevailing pattern contributing to the great variability of cyanobacterial cyclase domain architectures. However, other scenarios, such as gene duplication, also occurred during the evolution of cyanobacterial cyclases. Sequence divergence seemed to contribute to the origin of putative guanylate cyclases which were found only in cyanobacteria. In conclusion, the comprehensive survey of cyclases in cyanobacteria provides novel insight into their potential evolutionary mechanisms and further functional implications.

A genetic linkage map of Pacific white shrimp (Litopenaeus vannamei): sex-linked microsatellite markers and high recombination rates

Pacific white shrimp (Litopenaeus vannamei) is the leading species farmed in the Western Hemisphere and an economically important aquaculture species in China. In this project, a genetic linkage map was constructed using amplified fragment length polymorphism (AFLP) and microsatellite markers. One hundred and eight select AFLP primer combinations and 30 polymorphic microsatellite markers produced 2071 markers that were polymorphic in either of the parents and segregated in the progeny. Of these segregating markers, 319 were mapped to 45 linkage groups of the female framework map, covering a total of 4134.4 cM; and 267 markers were assigned to 45 linkage groups of the male map, covering a total of 3220.9 cM. High recombination rates were found in both parental maps. A sex-linked microsatellite marker was mapped on the female map with 6.6 cM to sex and a LOD of 17.8, two other microsatellite markers were also linked with both 8.6 cM to sex and LOD score of 14.3 and 16.4. The genetic maps presented here will serve as a basis for the construction of a high-resolution genetic map, quantitative trait loci (QTLs) detection, marker-assisted selection (MAS) and comparative genome mapping.

Comparative molecular population genetics of phycoerythrin locus in Prochlorococcus

As the only remainder type of phycobiliproteins in Prochlorococcus, the actual role of phycoerythrin still remains unknown. Previous studies revealed that two different forms of phycoerythrin gene were found in two ecotypes of Prochlorococcus that are specifically adapted to either high light (HL) or low light (LL) conditions. Here we analyze patterns of phycoerythrin nucleotide variation in the HL- and LL-Prochlorococcus populations. Our analyses reveal a significantly greater number of non-synonymous fixed substitutions in peB and peA than expected based on interspecific comparisons. This pattern of excess non-synonymous fixed substitutions is not seen in other five phycoerythrin-related genes (peZ/V/Y/T/S). Several neutrality statistical tests indicate an excess of rare frequency polymorphisms in the LL-Prochlorococcus data, but an excess of intermediate frequency polymorphisms in the HL-Prochlorococcus data. Distributions of the positively selected sites identified using the likelihood ratio test, when mapped onto the phycoerythrin tertiary structure, reveal that HL- and LL-phycoerythrin should be under different selective patterns. These findings may provide insights into the likely role of selection at the phycoerythrin locus and motivate further research to unveil the function of phycoerythrin in Prochlorococcus.

The complete mitochondrial genome of the mantid shrimp Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda): Novel non-coding regions features and phylogenetic implications of the Stomatopoda

The complete mitochondrial (mt) genome sequence of Oratosquilla oratoria (Crustacea: Malacostraca: Stomatopoda) was determined; a circular molecule of 15,783 bp in length. The gene content and arrangement are consistent with the pancrustacean ground pattern. The mt control region of O. oratoria is characterized by no GA-block near the 3' end and different position of [TA(A)]n-blocks compared with other reported Stomatopoda species. The sequence of the second hairpin structure is relative conserved which suggests this region may be a synapomorphic character for the Stomatopoda. In addition, a relative large intergenic spacer (101 bp) with higher A + T content than that in control region was identified between the tRNA(Glu) and tRNA(Phe) genes. Phylogenetic analyses based on the current dataset of complete mt genomes strongly support the Stomatopoda is closely related to Euphausiacea. They in turn cluster with Penaeoidea and Caridea clades while other decapods form a separate group, which rejects the monophyly of Decapoda. This challenges the suitability of Stomatopoda as an outgroup of Decapoda in phylogenetic analyses. The basal position of Stomatopoda within Eumalacostraca according to the morphological characters is also questioned. (C) 2010 Elsevier Inc. All rights reserved.

Screening of genes related to ovary development in Chinese shrimp Fenneropenaeus chinensis by suppression subtractive hybridization

The ovary of triploid shrimp Fenneropenaeus chinensis was apparently impaired compared to that of the diploid shrimp at the same age. Therefore triploid shrimp ovary is possible to be taken as a model to understand the mechanism of ovary development of shrimp compared to that of the ovary of diploid shrimp at the same age. In the present study, a suppression subtractive hybridization (SSH) technique was applied to identify differentially expressed genes in the ovary between diploid and triploid shrimp. For the forward library (RNA from the ovary of triploid shrimp as the tester), 54 genes were identified. For the reverse library (RNA from the ovary of diploid shrimp as the tester), 16 genes were identified. The identified genes encoded proteins with multiple functions, including extracellular matrix components, cytoskeleton, cell growth and death, metabolism, genetic information processing, signal transduction/transport or immunity related proteins. Eleven differentially expressed genes were selected to be confirmed in the ovaries of triploid and diploid shrimp by semi-quantitative RT-PCR. Genes encoding spermatogonial stem-cell renewal factor, cytochrome c oxidase subunits I and II, clottable protein, antimicrobial peptide and transposase showed up-regulated expressions in the ovary of triploid shrimp. Genes encoding tubulin, cellular apoptosis susceptibility protein, farnesoic acid O-methyltransferase, thrombospondin and heat shock protein 90 genes showed higher expressions in the ovary of diploid shrimp. The differential expressions of the above genes are suggested to be related to the ovary development of shrimp. It will provide a new clue to uncover the molecular mechanisms underlying the ovarian development in penaeid shrimp. (C) 2010 Elsevier Inc. All rights reserved.