Characterization of Genome-Wide Microsatellites of Saccharina japonica Based on a Preliminary Assembly of Illumina Sequencing Reads

Microsatellites or simple sequence repeats(SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp(Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N_(50) of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.     

Edible Peanut Worm(Sipunculus nudus) in the Beibu Gulf: Resource,Aquaculture, Ecological Impact and Counterplan

Sipunculus nudus is an important economic species because of its high nutritional and medicinal values. The exploitation and utilization of S. nudus primarily occur in the coastal regions of the Beibu Gulf. However, wild resource of S. nudus is rapidly decreasing because of the overexploitation, which has led to considerable developments of artificial breeding techniques. The cultivation scale of S. nudus has increased in response to successful artificial breeding; however, methods for culturing S. nudus in tidal flats or ponds require further study. Most studies have focused on the breeding, nutrition, medical value and ecological impact of these worms. Studies on the distribution, sediment requirements, nutrition characteristics, breeding techniques and aquaculture ecology of this species are summarized in this paper to promote the development of the aquaculture industry for S. nudus. The high biomass of S. nudus in the Beibu Gulf is positively correlated with the sediment characteristics and water quality of the region. The production of peanut worm has improved to some extent through culturing; however, the nutrient value and ecological environment problems have been observed, which reflect the over exploitation of trace elements and the sediment. These problems will worsen unless they are resolved, and the release of organic materials, nitrogen and phosphorus during harvesting impacts the coastal environment. Moreover, genetic erosion is a potential risk for larvae in artificial breeding programs in tidal flats. Therefore, culturing and collecting methods should be improved and the wild resource conservation should be implemented to promote the sustainable development of the peanut worm.     

Edible peanut worm (<Emphasis Type="Italic">Sipunculus nudus</Emphasis>) in the Beibu Gulf: Resource,aquaculture, ecological impact and counterplan

Sipunculus nudus is an important economic species because of its high nutritional and medicinal values. The exploitation and utilization of S. nudus primarily occur in the coastal regions of the Beibu Gulf. However, wild resource of S. nudus is rapidly decreasing because of the overexploitation, which has led to considerable developments of artificial breeding techniques. The cultivation scale of S. nudus has increased in response to successful artificial breeding; however, methods for culturing S. nudus in tidal flats or ponds require further study. Most studies have focused on the breeding, nutrition, medical value and ecological impact of these worms. Studies on the distribution, sediment requirements, nutrition characteristics, breeding techniques and aquaculture ecology of this species are summarized in this paper to promote the development of the aquaculture industry for S. nudus. The high biomass of S. nudus in the Beibu Gulf is positively correlated with the sediment characteristics and water quality of the region. The production of peanut worm has improved to some extent through culturing; however, the nutrient value and ecological environment problems have been observed, which reflect the over exploitation of trace elements and the sediment. These problems will worsen unless they are resolved, and the release of organic materials, nitrogen and phosphorus during harvesting impacts the coastal environment. Moreover, genetic erosion is a potential risk for larvae in artificial breeding programs in tidal flats. Therefore, culturing and collecting methods should be improved and the wild resource conservation should be implemented to promote the sustainable development of the peanut worm.     

ARTIFICIAL PROPAGATION AND BREEDING OF MARINE FISH IN CHINA

Since the 1990s, artificial propagation and breeding technique of marine fish in China have developed by way of increasing species and fry numbers, with special stress laid on valuable species. Large quantities of artificial fry can meet the needs of both marine cage culture and pond culture for most species. Experimental results obtained by scientists have been put into use in actual production.Fish fry production has entered a period of sustainable development. So far, at least 44 species (21 families) of marine fish have been successfully bred in China. The artificial fry number of large yellow croaker ( Pseudosciaena crocea) exceeded 300 million in 1999. The species whose artificial fry numbers have each surpassed 10 million annually are red drum ( Sciaenops ocellatus ), Japanese seabass ( Lateolabrax japonicus), cuneate drum (Nibea miichthioides ), spring spawning red seabream (Pagrosomus major ) and threebanded sweethp ( Plectorhynchus cinctus ). Millions of artificial fry are bred annually in the species of black porgy ( Sparus macrocephalus ), Russell‘ s snapper ( Lutjanus russeUi ), javelin gnmt ( Pomadasys hasta ), miiuy croaker ( Miichthys miiuy) and skewband gnmt ( Hapalogenys nitens ). The fish in the family Sciaenidae are the main species in artificial propagation and breeding. Some problems and prospects on marine fish culture and stock enhancement are also discussed and some proposals for sustainable development are put forward in this article.     

Genetic analysis of selected Sargassum fusiforme (Harvey) Setchell (Sargassaceae,Phaeophyta) strains with RAPD and ISSR markers

Since the 1980s, Sargassum fusiforme has been cultivated in Zhejiang, South China, and nowadays it becomes one of the important commercial seaweeds in China. With traditions of eating habits in the East Asian countries, this brown alga is used as food, because it contained functional oligo/polysaccharides and chemical components, and was regarded playing roles in antioxidant activities and regulating immunology. Through over 15 years' selection, breeding and cultivation, we obtained three strains with good traits and testified their characters during the production, which included the cultivars with high yield and other two good characters, either all the selected strains were applied in the Sargassum production. To avoid confusion during the selection and nursery, it was preferred to establish one fingerprint for distinguishing the Sargassum cultivars from different strains. Random amplified polymorphic DNA(RAPD) and inter-simple sequence repeat(ISSR) methods were adopted to analyze the genetic diversities of the selected S. fusiforme strains. With that, one fingerprint with RAPD markers was constructed, and one sequence characterized amplified region(SCAR) marker to S.fusiforme was obtained. It is indicated that the applied fingerprint could be valid in S. fusiforme genetic and germplasm justification, and will be positive to molecular marker assistance in its selection and cultivation.     

A Preliminary Genetic Linkage Map of Sinonovacula constricta(Lamarck, 1818) Based on Microsatellites Derived from RAD Sequencing

Sinonovacula constricta is one of the important economic aquaculture species in China. In this study, we constructed genetic linkage maps of S. constricta based on 300 microsatellite markers derived from RAD-seq using an F1 full-sib family. The female map contained 204 microsatellites assigned to 22 linkage groups, which covered 1529.5 cM with an average interval of 10.3 cM. The male consisted of 187 microsatellites in 19 linkage groups corresponding to the haploid chromosome number(n(28)19), which spanned 1429.3 cM with an average interval of 8.7 cM. The genome coverage was approximately 83.5% and 81.4%, respectively. An integrated map was constructed according to the common markers in parental linkage groups, which had a total length of 1683.8 cM with an average interval of 7.3 cM. The genome coverage of the integrated map was approximately 86.3%. The genetic linkage map would form the foundation for further studies on the quantitative trait loci(QTL), as well as accelerating the breeding process of this species.     

16S rRNA Gene Phylogenesis of Culturable Predominant Bacteria from Diseased Apostichopus japonicus(Holothuroidea,Echinodermata)

Cultured Apostichopusjaponicus in China suffers from a kind of skin ulceration disease that has caused severe economic loss in recent years. The disease, pathogens of which are supposed to be bacteria by most researchers, is highly infectious and can often cause all individuals in the same culture pool to die in a very short time. The 16S rRNA gene phylogenesis of the culturable bacteria from the lesions of diseased individuals was conducted to study the biodiversity of the bacterial communities in the lesions and to identify probable pathogen(s) associated with this kind of disease. S. japonica samples were selected from a hatchery located in the eastern part of Qingdao, China. Bacterial universal primers GM5F and DS907R were used to amplify the 16S rRNA gene of bacteria colonies, and touchdown PCR was performed to amplify the target sequences. The results suggest that γ- proteobacteria (Alteromonadales and Vibrionales) of CFB group, many strains of which have been also determined as pathogens in other marine species, are the predominant bacterial genera of the diseased Apostichopusjaponicus individuals.     

Chromosomal mapping of 5S and 18S-5.8S-25SrRNA genes in Saccharina japonica(Phaeophyceae) as visualized by dual-color fluorescence in situ hybridization

It has been reported that there was a linkage of 5 S rRNA gene to 18 S-5.8 S-25 S rRNA gene in a few of species in Ochrophyta.In regard to the usual two positions of linked 5 S rDNA to the 3' end of 25 S rDNA,two pairs of primers were designed for amplification to verify this linkage of two genes in a kelp cultivar of Saccharina japonica,one of species in Ochrophyta.This result supplemented the previous report that 5 S rDNA was unlinked to 25 S rDNA in this kelp.In order to simultaneously visualize this unlinkage of two genes,dual-color fluorescence in situ hybridization(FISH) technique was applied to the cytogenetics of S.japonica.Dual-color FISH images showed that two and four hybridization signals were present in the kelp gametophyte and sporophyte,respectively,metaphase nuclei hybridized simultaneously with the labeled probes of 18 S rDNA and 5 S rDNA.Both haploid and diploid karyotypes in decreasing length of chromosomes showed that 18 S-5.8 S-25 S rDNA was localized at the interstitial region of Chromosome 23,whereas 5 S rDNA resided at the sub-telomeric region of Chromosome 27.These karyotypes suggested that the kelp nuclear genome had only one locus of each rRNA gene,and their loci on different chromosomes indicated the physical unlinkage of 5 S rDNA to 18 S-5.8 S-25 S rDNA in this kelp.Therefore,dual-color FISH seems to be a powerful technique for the discrimination and pairing of chromosomes featured in both small size and nearly identical shape in S.japonica.     

Wetland resource protection and rational utilization in China

Wetlands of China cover an area of 63 million ha, among which, natural wetlands 25 million ha, including mires, shallow lakes, etc., artificial wetlands 38 million ha. Besides these, there are a lot of rivers and islands, stretching 18,000 km of coastal line and coastal zone below the tidal within the water depth of 6 m. Based on the climatic regional differences, biota similarity and biodiversity abundance, China’s wetlands can be divided into 9 main regions. Wetlands not only have huge functions to regulate mankind’s living environment, but also are the important and precious natural resources. For a long period, since the contradiction between population and resources, energy, grain is getting more and more obvious, China’s wetlands are facing to serious disturbances, such as large-scale reclamation, overhunting, industrial waste water pollution and species introducing. They have brought about the wetland quantity reducing and quality falling, furthermore have effects on sustainable development. For the special national conditions of China, higher population pressure, China should carry out wetland resource protection and rational utilization, not only protection.     

Partial function prediction of sulfate-reducing bacterial community from the rhizospheres of two typical coastal wetland plants in China

Sulfate-reducing bacteria(SRB) are ubiquitous anaerobic microorganisms that play significant roles in the global biogeochemical cycle.Coastal wetlands,one of the major habitats of SRB,exhibit high sulfate-reducing activity and thus play significant roles in organic carbon remineralization,benthic geochemical action,and plant-microbe interactions.Recent studies have provided credible evidence that the functional rather than the taxonomic composition of microbes responds more closely to environmental factors.Therefore.in this study,functional gene prediction based on PacBio single molecular real-time sequencing of 16 S rDNA was applied to deterrmine the sulfate-reducing and organic substrate-decomposing activities of SRB in the rhizospheres of two typical coastal wetland plants in North and South China:Zostera japonica and Scirpus mariqueter.To this end,some physicochemical characteristics of the sediments as well as the phylogenetic structure,community composition,diversity,and proportions of several functional genes of the SRB in the two plant rhizosphere s were analyzed.The Z.japonica meadow had a higher dis similatory sulfate reduction capability than the S.mariqueter-comprising saltmarsh,owing to its larger proportion of SRB in the microbial community,larger proportions of functional genes involved in dissimilatory sulfate reduction,and the stronger ability of the SRB to degrade organic substrates completely.This study confirmed the feasibility of applying microbial community function prediction in research on the metabolic features of SRB,which will be helpful for gaining new knowledge of the biogeochemical and ecological roles of these bacteria in coastal wetlands.     

The Clam Cyclina sinensis (Gmelin) Phylogeography Study with 28S rRNA Gene and Potential of Nuclear rRNA Genes in Genetic Assessments of Molluscs

Intraspecific diversity of molluscan species is usually studied based on maternally inherited mitochondrial DNA,from which only part of the evolutionary history can be reflected.Some nuclear ribosomal RNA genes such as 28S rRNA represent poten-tial candidates that can be easily applied in phylogeography because of lacking intraindividual variation.However,considering their low polymorphism,genetic appraisals on whether and how they can be used in population studies are necessary.Here,we applied a short 28S rRNA to assess genetic patterns of the clam Cyclina sinensis along the coast of China and compared the results with a for-mer study based on COI and ITS-1 analyses.The results revealed the 28S rRNA data set was characterized by an extremely low level of variation,with only seven haplotypes defined for 93 individuals.Haplotype and nucleotide diversity for each population was al-most the lowest when compared with the other two markers.However,the distribution of two dominant haplotypes showed clear geo-graphic patterns,and significant population differentiation was revealed between the East China Sea and the South China Sea.These patterns were highly concordant with findings of the former study that populations of C.sinensis were historically separated by land bridges among sea basins.Our study suggested that although the nuclear rRNAs have shortcomings such as low variation,they have advantages including lack of intraindividual variation and high amplification rates.Applying rRNA genes can enrich the toolbox of nuclear markers in molluscan phylogeographic studies.     

Preliminary Analysis of Population Genetic Diversity of Cultivated Laminaria japonica Sporophyte via AFLP Technique

The amplified fragment length polymorphic DNA (AFLP) technique was adopted to estimate the population genetic polymorphism among 30 sporophytes of Laminaria japonica collected from a cultivating farm in Rongcheng,China.Three methods were used for genomic DNA extraction from Laminaria japonica sporophyte and only the products obtained using the improved genomic DNA extraction kit method proved qualified for AFLP analysis.The parameters of the method were optimized.Samples of forty milligrams and the cell lysis time of 120 min were suggested to replace the parameters recommended by the manufacturer.Thirty individuals of Laminaria japonica from the same cultivating site were investigated using one pair of selective primers.A total of 21 loci were obtained and 17 of them were polymorphic.The mean percent age of polymorphic loci of this population was 80.95%.The Nei's gene diversity (H) within this population was 0.3028 and the average Shannon's Information index (I) was 0.4498.A genetic distance matrix among different individuals was constructed as well.Through this study,an applicable AFLP genetic analysis working system for Laminaria japonica sporophyte was established.The results of this research also revealed a high level of genetic diversity within the studied population.     

Characterization of genome-wide microsatellites of <Emphasis Type="Italic">Saccharina japonica</Emphasis> based on a preliminary assembly of Illumina sequencing reads

Microsatellites or simple sequence repeats (SSR) function widely and locate dependently in genome. However, their characteristics are often ignored due to the lack of genomic sequences of most species. Kelp (Saccharina japonica), a brown macroalga, is extensively cultured in China. In this study, the genome of S. japonica was surveyed using an Illumina sequencing platform, and its microsatellites were characterized. The preliminarily assembled genome was 469.4 Mb in size, with a scaffold N₅₀ of 20529 bp. Among the 128370 identified microsatellites, 90671, 25726 and 11973 were found in intergenic regions, introns and exons, averaging 339.3, 178.8 and 205.4 microsatellites per Mb, respectively. These microsatellites distributed unevenly in S. japonica genome. Mononucleotide motifs were the most abundant in the genome, while trinucleotide ones were the most prevalent in exons. The microsatellite abundance decreased significantly with the increase of motif repeat numbers, and the microsatellites with a small number of repeats accounted for a higher proportion of the exons than those of the intergenic regions and introns. C/G-rich motifs were more common in exons than in intergenic regions and introns. These characteristics of microsatellites in S. japonica genome may associate with their functions, and ultimately their adaptation and evolution. Among the 120140 pairs of designed microsatellite primers, approximately 75% were predicted to be able to amplify S. japonica DNA. These microsatellite markers will be extremely useful for the genetic breeding and population evolution studies of kelp.     

Taxonomic and genetic status of lancelet in Weihai coastal waters based on mitochondrial DNA sequence

Lancelets (subphylum Cephalochordata) are a transitional species between invertebrates and vertebrates. They are currently listed in the Second Order of Protected Animals in China. Lancelets were first documented in the waters around the city of Weihai (Shandong, China) in 2002. However, little is known about the phylogeny of this population. We analyzed the sequences of cytochrome b (Cyt b) and cytochrome oxidase c subunit I (CO I) genes from samples collected from coastal waters in the cities of Weihai and Qingdao (~150 km to the south). We analyzed 176 sequences, of which 150 were novel sequences and 26 were obtained from GenBank. Our results suggest that (1) lancelets in the two cities belong to the species Branchiostoma japonicus and have a high level of genetic diversity; (2) there is a high level of gene flow and low level of genetic differentiation between lancelets from the two cities; (3) demographic expansion occurred an estimated 1.1 million years (Ma) ago (mid Pleistocene) for lancelets in Weihai-Qingdao; and (4) the divergence between B. belcheri and B. japonicus was estimated at between 37.75 Ma (early Oligocene)-46.5 Ma (late Eocene).     

Overview on seagrasses and related research in China

Seagrass research in China is still in its infancy. Even though there has been progress recently, there is still a great deal of research needed to gain a better understanding of seagrass. In this article we review and discuss the advances in seagrass research in China from two aspects: (1) seagrass species and their distribution; (2) seagrass research in China, including studies on their taxonomy, ecology, photosynthesis, applications in aquaculture, salt-tolerance mechanisms and other research topics. A total of 18 seagrass species belonging to 8 genera are distributed in nine provinces and regions in China (including Hong Kong and Taiwan), as well as the Xisha and Nansha Archipelagos. They can be divided into two groups: a North China Group and a South China Group. Based on the seagrass distribution, the Chinese mainland coast can be divided into three sections: North China Seagrass Coast, Middle China Seagrass Coast, and South China Seagrass Coast. Ecological studies include research on seagrass communities, nutrient cycling in seagrass ecosystems, genetic diversity, pollution ecology and research in the key regions of Shandong, Guangdong, Guangxi, and Hainan. Seagrass species and their locations, community structure, ecological evaluation, epiphytes, ecological functions and threats in the key regions are also summarized. Other studies have focused on remote sensing of seagrass, threatened seagrass species of China, and pollen morphology of Halophila ovalis.     

3种柔鱼线粒体基因与核核糖体基因片段序列比较分析

为准确检测柔鱼（Ommαstrephesbartram川、茎柔鱼（ Dosidicus gigas）与阿根廷滑柔鱼（ IIIex argentinus ） 的种间遗传差异,对线粒体16SrRNA、细胞色素b（Cytb）与编码核糖体大亚基的基因（28SrDNA）片段序列进 行测定。经比对获得同源片段序列的长度分别为444、430、464坤,其中16SrRNA与28SrDNA基因片段上分别存在3处和47处碱基插入/缺失。核昔酸组成分析表明;3种柔鱼在3个基因片段上的核音酸组成差异不显著, 在线粒体2个基因片段上的A＋T含量（16SrRNA;69.90%、72.01%、74.66%; Cytb; 63.61%、68.91%、71.65% ） 均明显高于C＋C含量（16SrRNA;30.10%、27.99%、25.34%; Cytb; 36.39%、31.09%、28.35% ）,而在28SrDNA 基因片段上的A＋T含量（37.16%、36.74%、38.29% ）明显低于C＋C含量（62.84%、63.26%、61.71 %）0 3种柔鱼 在28SrDNA基因片段上检测到的核昔酸替代率最低,为6.68%,而蛋白质编码基因Cytb核昔酸替代率最高,为 20.93%,核营酸替代均发生在密码子第3位点上,而且未引起氨基酸替代。基于邻接法、最大简约法与最大似然 法重建的系统树显示,柔鱼与茎柔鱼的亲缘关系较近。根据C严b基因片段序列分析,柔鱼与茎柔鱼和阿根廷滑柔鱼的分歧时间分别为653-790万a和765 - 925万a,种间分化事件发生在中新世至上新世间。     

THE SPREAD OF AGRICULTURE IN CHINA

At pressent, China has about 100 million ha of farmland and 1.1 billion inhabitants, of which, about 80% are still engaged in agriculture. In geographical distribution, primitive shifting cultivation was at first mainly scattered in small patches in Eastern Monsoon China, especially concentrated in the middle and lower reaches of the Huanghe (Yellow) River and the Changjiang (Yangtze) River. The ratio of agriculture to industry in production value changed from 7:3 in the early 1950s to 3:7 in the middle 1980s. Agricultural Regionalization is based upon agricultural development as well as physical regionalization in China. Seven first-level agricultural regions might be identified.