Lingbo Ma

The complete mitochondrial genome sequence and gene organization of the mud crab (Scylla paramamosain) with phylogenetic consideration.

The complete mitochondrial genome is of great importance for better understanding the genome-level characteristics and phylogenetic relationships among related species. In the present study, we determined the complete mitochondrial genome DNA sequence of the mud crab (Scylla paramamosain) by 454 deep sequencing and Sanger sequencing approaches. The complete genome DNA was 15,824 bp in length and contained a typical set of 13 protein-coding genes, 22 transfer RNA (tRNA) genes, two ribosomal RNA (rRNA) genes and a putative control region (CR). Of 37 genes, twenty-three were encoded by the heavy strand (H-strand), while the other ones were encoded by light strand (L-strand). The gene order in the mitochondrial genome was largely identical to those obtained in most arthropods, although the relative position of gene tRNA(His) differed from other arthropods. Among 13 protein-coding genes, three (ATPase subunit 6 (ATP6), NADH dehydrogenase subunits 1 (ND1) and ND3) started with a rare start codon ATT, whereas, one gene cytochrome c oxidase subunit I (COI) ended with the incomplete stop codon TA. All 22 tRNAs could fold into a typical clover-leaf secondary structure, with the gene sizes ranging from 63 to 73 bp. The phylogenetic analysis based on 12 concatenated protein-coding genes showed that the molecular genetic relationship of 19 species of 11 genera was identical to the traditional taxonomy.

First mitochondrial genome for the red crab ( Charybdis feriata ) with implication of phylogenomics and population genetics

In this study, we first described the complete mitochondrial genome for the red crab (Charybdis feriata), elucidated its phylogenetic relationship among 20 species within Decapoda, and estimated the population genetic diversity. The mitochondrial genome was 15,660 bp in size and encoded 13 protein-coding genes, 22 transfer RNA (tRNA) genes, and two ribosomal RNA genes. The gene arrangement of the mitochondrial genome was the same as that of its sister species, C. japonica. Phylogenomic analysis suggested that genus Charybdis should be classified into subfamily Portuninae but not into subfamily Thalamitinae. Moreover, a total of 33 haplotypes of complete cytochrome c oxidase subunit I gene were defined in 70 individuals of C. feriata derived from three localities. Haplotype diversity and nucleotide diversity values among three localities indicated a high level of genetic diversity in C. feriata. AMOVA analysis suggested a low level of genetic differentiation among the three localities (FST = 0.0023, P > 0.05). Neutrality tests and mismatch analysis revealed that C. feriata might have undergone a population expansion event that possibly occurred in the last 61,498 to 43,814 years. This study should be helpful to better understand the evolutionary status, and population genetic diversity of C. feriata and related species.

Transcriptome Analysis of the Mud Crab (Scylla paramamosain) by 454 Deep Sequencing: Assembly, Annotation, and Marker Discovery

In this study, we reported the characterization of the first transcriptome of the mud crab (Scylla paramamosain). Pooled cDNAs of four tissue types from twelve wild individuals were sequenced using the Roche 454 FLX platform. Analysis performed included de novo assembly of transcriptome sequences, functional annotation, and molecular marker discovery. A total of 1,314,101 high quality reads with an average length of 411 bp were generated by 454 sequencing on a mixed cDNA library. De novo assembly of these 1,314,101 reads produced 76,778 contigs (consisting of 818,154 reads) with 5.4-fold average sequencing coverage. The remaining 495,947 reads were singletons. A total of 78,268 unigenes were identified based on sequence similarity with known proteins (E≤0.00001) in UniProt and non-redundant protein databases. Meanwhile, 44,433 sequences were identified (E≤0.00001) using a BLASTN search against the NCBI nucleotide database. Gene Ontology (GO) analysis indicated that biosynthetic process, cell part, and ion binding were the most abundant terms in biological process, cellular component, and molecular function categories, respectively. Kyoto Encyclopedia of Genes and Genome (KEGG) pathway analysis revealed that 4,878 unigenes distributed in 281 different pathways. In addition, 19,011 microsatellites and 37,063 potential single nucleotide polymorphisms were detected from the transcriptome of S. paramamosain. Finally, thirty polymorphic microsatellite markers were developed and used to assess genetic diversity of a wild population of S. paramamosain. So far, existing sequence resources for S. paramamosain are extremely limited. The present study provides a characterization of transcriptome from multiple tissues and individuals, as well as an assessment of genetic diversity of a wild population. These sequence resources will facilitate the investigation of population genetic diversity, the development of genetic maps, and the conduct of molecular marker-assisted breeding in S. paramamosain and related crab species.

Multiplex immune-related genes expression analysis response to bacterial challenge in mud crab, Scylla paramamosain

Crabs lack an acquired adaptive immune system and host defense is believed to depend entirely on innate, non-adaptive mechanisms to resist invasion by pathogens. Discovery of immune-related factors are helpful for understanding the molecular response of crabs to pathogens. The mud crab Scylla paramamosain is an important marine species for aquaculture in China because of its high nutritional value for humans. In recent years, the crab is prone to being infected by microbes with the enlargement of breeding scale. In this study, eight immune-related genes were analyzed by multiplex genes expression analysis using the GenomeLab GeXP analysis system (Beckman Coulter). The expression levels of all the detected genes rose after challenged by the live bacteria, but the levels of only four genes (C-type lectin, alpha 2-macroglobulin, HSP70 and thioredoxin 1) increased after challenge in heat-killed bacteria group. So the live bacteria were more effective in motivating expressions of immune factors than heat-killed bacteria. However, the transcript of C-type lectin firstly increased at 1 h after challenge in both heat-killed and live bacteria group. This indicated that C-type lectin was a quite susceptive immune factor responding to external pathogen. In group challenged by live bacteria, the genes of alpha 2-macroglobulin, HSP40, thioredoxin 1 and prophenoloxidase activating factor (PPAF) showed response earlier than the other genes. The rise of PPAF expression preceded prophenoloxidase (proPO), which suggested that PPAF might trigger production of proPO transcripts in the early stage of phenoloxidase reaction system. C-type lectin, proPO, thioredoxin 1, HSP40, and alpha 2-macroglobulin are very important immunity factors in response to bacterial infection. According to the result of heat-killed group, HSP70 is a sensitively inductive factor to foreign stimulus compared with the other genes. The multi-gene analysis presented an alternative approach for screening of immune-related genes, and provided a more global overview of genes transcript alteration in response to bacterial challenge.

Novel Polymorphic Microsatellite Markers in Scylla paramamosain and Cross-Species Amplification in Related Crab Species

Abstract Scylla paramamosain is a commercially important crab species in China. Here we reported 12 novel polymorphic microsatellite markers isolated in S. paramamosain using PCR-based isolation of microsatellite arrays (PIMA). The analysis of genetic variability was performed in a sample of 29 individuals. A total of 82 alleles were detected with an average of 6.8 alleles per locus. The number of alleles, polymorphism information content (PIC), observed and expected heterozygosity per locus ranged from three to 12, from 0.42 to 0.88, from 0.38 to 1.00 and from 0.48 to 0.91, respectively. Three loci significantly deviated from Hardy-Weinberg equilibrium after Bonferroni correction (P < 0.0042) and no significant linkage disequilibrum between pairs of loci was found. Cross-species amplification of these markers was evaluated in three closely related crab species S. tranquebarica, S. olivacea, and S. serrata. This study will potentially be useful for stock management, constructing of a genetic linkage map, mapping economically important quantitative trait loci (QTL), and evaluating the population genetic diversity of S. paramamosain.

cDNA Cloning and Expression Analysis of Gustavus Gene in the Oriental River Prawn Macrobrachium nipponense

The gustavus gene is required for localizing pole plasm and specifying germ cells. Research on gustavus gene expression will advance our understanding of the biological function of gustavus in animals. A cDNA encoding gustavus protein was identified and termed MnGus in the oriental river prawn Macrobrachium nipponense. Bioinformatic analyses showed that this gene encoded a protein of 262 amino acids and the protein belongs to the Spsb1 family. Real-time quantitative PCR analyses revealed that the expression level of MnGus in prawn embryos was slightly higher at the cleavage stage than at the blastula stage, and reached the maximum level during the zoea stage of embryos. The minimum level of MnGus expression occurred during the perinucleolus stage in the ovary, while the maximum was at the oil globule stage, and then the level of MnGus expression gradually decreased with the advancement of ovarian development. The expression level of MnGus in muscle was much higher than that in other tissues in mature prawn. The gustavus cDNA sequence was firstly cloned from the oriental river prawn and the pattern of gene expression was described during oocyte maturation, embryonic development, and in other tissues. The differential expression patterns of MnGus in the embryo, ovary and other somatic tissues suggest that the gustavus gene performs multiple physiological functions in the oriental river prawn.

Identification of Transcriptome-Derived Microsatellite Markers and Their Association with the Growth Performance of the Mud Crab (Scylla paramamosain)

Microsatellite markers from a transcriptome sequence library were initially isolated, and their genetic variation was characterized in a wild population of the mud crab (Scylla paramamosain). We then tested the association between these microsatellite markers and the growth performance of S. paramamosain. A total of 129 polymorphic microsatellite markers were identified, with an observed heterozygosity ranging from 0.19 to 1.00 per locus, an expected heterozygosity ranging from 0.23 to 0.96 per locus, and a polymorphism information content (PIC) ranging from 0.21 to 0.95 per locus. Of these microsatellite markers, 30 showed polymorphism in 96 full-sib individuals of a first generation family. Statistical analysis indicated that three microsatellite markers were significantly associated with 12 growth traits of S. paramamosain. Of these three markers, locus Scpa36 was significantly associated with eight growth traits, namely, carapace length, abdomen width (AW), body height (BH), fixed finger length of the claw, fixed finger width of the claw, fixed finger height of the claw, meropodite length of pereiopod 2, and meropodite length of pereiopod 3 (MLP3) (P<0.05). Locus Scpa75 was significantly associated with five growth traits, namely, internal carapace width, AW, carapace width at spine 8, distance between lateral spine 2 (DLS2), and MLP3 (P<0.05). Locus Spm30 was significantly associated with BH, DLS2, and body weight (P<0.05). Further analysis suggested a set of genotypes (BC at Scpa36, BC and BD at Scpa75, and AC at Spm30) that have great potential in the selection of S. paramamosain for growth traits. These findings will facilitate the development of population conservation genetics and molecular marker-assisted selective breeding of S. paramamosain and other closely related species.

High genetic diversity and low differentiation in mud crab (Scylla paramamosain) along the southeastern coast of China revealed by microsatellite markers

SUMMARY The mud crab (Scylla paramamosain) is a carnivorous portunid crab, mainly distributed along the southeastern coast of China. Mitochondrial DNA analysis in a previous study indicated a high level of genetic diversity and a low level of genetic differentiation. In this study, population genetic diversity and differentiation of S. paramamosain were investigated using nine microsatellite markers. In total, 397 wild specimens from 11 locations on the southeastern coast of China were sampled and genotyped. A high level of genetic diversity was observed, with the number of alleles, and the observed and expected heterozygosity per location in the range 7.8–9.6, 0.62–0.77 and 0.66–0.76, respectively. AMOVA analysis indicated a low level of genetic differentiation among the 11 locations, despite the fact that a statistically significant fixation index (FST) value was found (FST=0.0183, P<0.05). Out of 55 pairwise location comparisons, 39 showed significant FST values (P<0.05), but all of them were lower than 0.05, except for one between Sanmen and Shantou locations. No significant deficiency of heterozygotes (inbreeding coefficient FIS=0.0007, P>0.05) was detected for all locations except Sanmen and Zhanjiang. Cluster analysis using UPGMA showed that all locations fell into one group except Sanmen. Significant association was found between genetic differentiation in terms of FST/(1–FST) and the natural logarithm of geographical distance (r2=0.1139, P=0.02), indicating that the genetic variation pattern closely resembled an isolation by distance model. This study supports the proposal of high genetic diversity and low genetic differentiation in S. paramamosain along the southeastern coast of China.

The Gut Microbial Community of Antarctic Fish Detected by 16S rRNA Gene Sequence Analysis

Intestinal bacterial communities are highly relevant to the digestion, nutrition, growth, reproduction, and a range of fitness in fish, but little is known about the gut microbial community in Antarctic fish. In this study, the composition of intestinal microbial community in four species of Antarctic fish was detected based on 16S rRNA gene sequencing. As a result, 1 004 639 sequences were obtained from 13 samples identified into 36 phyla and 804 genera, in which Proteobacteria, Actinobacteria, Firmicutes, Thermi, and Bacteroidetes were the dominant phyla, and Rhodococcus, Thermus, Acinetobacter, Propionibacterium, Streptococcus, and Mycoplasma were the dominant genera. The number of common OTUs (operational taxonomic units) varied from 346 to 768, while unique OTUs varied from 84 to 694 in the four species of Antarctic fish. Moreover, intestinal bacterial communities in individuals of each species were not really similar, and those in the four species were not absolutely different, suggesting that bacterial communities might influence the physiological characteristics of Antarctic fish, and the common bacterial communities might contribute to the fish survival ability in extreme Antarctic environment, while the different ones were related to the living habits. All of these results could offer certain information for the future study of Antarctic fish physiological characteristics.

Transcriptome Sequencing, De Novo Assembly and Differential Gene Expression Analysis of the Early Development of Acipenser baeri.

The molecular mechanisms that drive the development of the endangered fossil fish species Acipenser baeri are difficult to study due to the lack of genomic data. Recent advances in sequencing technologies and the reducing cost of sequencing offer exclusive opportunities for exploring important molecular mechanisms underlying specific biological processes. This manuscript describes the large scale sequencing and analyses of mRNA from Acipenser baeri collected at five development time points using the Illumina Hiseq2000 platform. The sequencing reads were de novo assembled and clustered into 278167 unigenes, of which 57346 (20.62%) had 45837 known homologues proteins in Uniprot protein databases while 11509 proteins matched with at least one sequence of assembled unigenes. The remaining 79.38% of unigenes could stand for non-coding unigenes or unigenes specific to A. baeri. A number of 43062 unigenes were annotated into functional categories via Gene Ontology (GO) annotation whereas 29526 unigenes were associated with 329 pathways by mapping to KEGG database. Subsequently, 3479 differentially expressed genes were scanned within developmental stages and clustered into 50 gene expression profiles. Genes preferentially expressed at each stage were also identified. Through GO and KEGG pathway enrichment analysis, relevant physiological variations during the early development of A. baeri could be better cognized. Accordingly, the present study gives insights into the transcriptome profile of the early development of A. baeri, and the information contained in this large scale transcriptome will provide substantial references for A. baeri developmental biology and promote its aquaculture research.