Wen-Qiao Tang

Phylogeographical Analysis on Squalidus argentatus Recapitulates Historical Landscapes and Drainage Evolution on the Island of Taiwan and Mainland China

Phylogeographical analyses on Squalidus argentatus samples from thirteen localities within mainland China and Taiwan were conducted for biogeographic studies, as their dispersal strictly depends on geological evolution of the landmasses. A total of 95 haplotypes were genotyped for mtDNA cyt b gene in 160 specimens from nine river systems. Relatively high levels of haplotype diversity (h = 0.984) and low levels of nucleotide diversity (π = 0.020) were detected in S. argentatus. Two major phylogenetic haplotype groups, A and B, were revealed via phylogenetic analysis. The degree of intergroup divergence (3.96%) indicates that these groups diverged about 4.55 myr (million years) ago. Haplotype network and population analyses indicated significant genetic structure (FST = 0.775), largely concordant with the geographical location of the populations. According to SAMOVA analysis, we divided these populations into four units: Yangtze-Pearl, Qiantang-Minjiang, Jiulong-Beijiang and Taiwan groups. Mismatch distribution analysis, neutrality tests and Bayesian skyline plots indicated a significant population expansion for lineage A and B, approximately dated 0.35 and 0.04 myr ago, respectively. We found strong geographical organization of the haplotype clades across different geographic scales that can be explained by episodes of dispersal and population expansion followed by population fragmentation and restricted gene flow.

Two Origins of Blastemal Progenitors Define Blastemal Regeneration of Zebrafish Lower Jaw

Zebrafish possess a remarkable ability to regenerate complicated structures by formation of a mass of undifferentiated mesenchymal cells called blastema. To understand how the blastema retains the original structural form, we investigate cellular transitions and transcriptional characteristics of cell identity genes during all stages of regeneration of an amputated lower jaw. We find that mesenchymal blastema originates from multiple sources including nucleated blood cells, fibroblasts, damaged muscle cells and pigment cells. These cells are transformed into two populations of blastemal progenitors: foxi1-expression and isl1-expression, before giving rise to cartilage, bone, and muscle. Time point- based transcriptomal analysis of 45 annotated Hox genes reveal that five 3′-end Hox genes and an equal number of 5′-end Hox genes are activated largely at the stage of blastema reformation. RNA in situ hybridization shows that foxi1 and pax3a are respectively expressed in the presumptive mandible skeletal region and regenerating muscle at 5 dpa. In contrast, hoxa2b and hoxa11b are widely expressed with different domain in chondrogenic blastema and blastema mesenchyme. Knockdown foxi1 changes the expression patterns of sox9a and hoxa2b in chondrogenic blastema. From these results we propose that two origins of blastemal progenitors define blastema skeleton and muscle respecifications through distinct signaling pathways. Meanwhile, the positional identity of blastema reformation is implicated in mesenchymal segmentation and characteristic expression pattern of Hox genes.

De novo transcriptomes of olfactory epithelium reveal the genes and pathways for spawning migration in japanese grenadier anchovy (Coilia nasus).

Background Coilia nasus (Japanese grenadier anchovy) undergoes spawning migration from the ocean to fresh water inland. Previous studies have suggested that anadromous fish use olfactory cues to perform successful migration to spawn. However, limited genomic information is available for C. nasus. To understand the molecular mechanisms of spawning migration, it is essential to identify the genes and pathways involved in the migratory behavior of C. nasus. Results Using de novo transcriptome sequencing and assembly, we constructed two transcriptomes of the olfactory epithelium from wild anadromous and non-anadromous C. nasus. Over 178 million high-quality clean reads were generated using Illumina sequencing technology and assembled into 176,510 unigenes (mean length: 843 bp). About 51% (89,456) of the unigenes were functionally annotated using protein databases. Gene ontology analysis of the transcriptomes indicated gene enrichment not only in signal detection and transduction, but also in regulation and enzymatic activity. The potential genes and pathways involved in the migratory behavior were identified. In addition, simple sequence repeats and single nucleotide polymorphisms were analyzed to identify potential molecular markers. Conclusion We, for the first time, obtained high-quality de novo transcriptomes of C. nasus using a high-throughput sequencing approach. Our study lays the foundation for further investigation of C. nasus spawning migration and genome evolution.

Comparative Evolution of S7 Intron 1 and Ribosomal Internal Transcribed Spacer in Coilia nasus (Clupeiformes: Engraulidae)

Coilia nasus is widely distributed in the Yangtze River, the coastal waters of China, Korea and the Ariake Sound of Japan. Several ecotypes exist and this provides a useful model for the study of comparative diversity between molecular markers. Here we analyze and compare the nucleotide sequences between single-copy ribosomal protein S7 gene intron 1 (rpS7) and multiple-copy ribosomal internal transcribed spacer 1 (ITS1) in this species to compare the phylogenetic signal of the two nuclear genes. Nucleotide substitutions among the two gene sequences and partial sequence of mitochondrial cytochrome c oxidase subunit I (COI) gene were also analyzed. A total of 115 clones for rpS7 and 122 clones for ITS1 were obtained from 37 specimens. The nucleotide sequence length is 741 to 743 bp for rpS7 and 334 to 348 bp for ITS1. Intra- and inter-specimen variation in rpS7 results from nucleotide substitution, while such variation in ITS1 is mainly due to different numbers of short base repeats. The content of G + C is lower in rpS7 (43.5%) than in ITS1 (68.2%). Our results indicate that the proportion of the sequence variable sites is higher in rpS7 (61) than in ITS1 (23); the informative parsimony of rpS7 is evidently higher than that of ITS1 (26 vs. 2); the overall ratio between transitions and transversions in ITS1 is slightly lower than in rpS7, but remarkably lower than in COI. These results suggest that rpS7 is more suitable than ITS1 as a marker for genetic divergence of this group. Furthermore, gene flow is observed between the different geographic populations of C. nasus from the phylogeny of this species based on rpS7, showing that rpS7 has more evolutionary characteristics for understanding the processes of genomic evolution at the intraspecific level.

Polymorphic Microsatellite Loci Isolated from the Squalidus argentatus Using PCR-Based Isolation of Microsatellite Arrays (PIMA)

Squalidus argentatus (Sauvage and Dabry de Thiersant 1874) is a small-sized freshwater fish which is distributed in Mainland China, Hainan Island and Taiwan. The populations of S. argentatus have dropped sharply probably due to overharvesting and water pollution recently. Eleven polymorphic microsatellite markers were developed for the cyprinid fish S. argentatus. These new markers were tested on 43 individuals collected from Yangtze River and Qiantang River. The number of alleles, observed and expected heterozygosity per locus, in two populations ranged from 3 to 14, from 0.333 to 0.954 and from 0.480 to 0.928, respectively. Only two loci are significantly deviated from Hardy–Weinberg expectations due to the heterozygote deficiency. No significant linkage disequilibrium was detected between the pairwise comparisons of these loci. These polymorphic microsatellite loci will enable us to study the genetic variation, population structure, and conservation genetics of this species in the future.

A simple method for isolation of microsatellites from the mudskipper (Boleophthalmus pectinirostris), without constructing a genomic library

The mudskipper Boleophthalmus pectinirostris is an important aquaculture species in China. It is endangered due to the loss and fragmentation of natural habitat, seawater pollution and overfishing. In this study, ten polymorphic microsatellite loci were isolated by the 5′-anchored polymerase chain reaction technique. Population genetic parameters were estimated on 60 wild individuals collected from Jiuduansha wetland in Yangtze River estuary. The number of alleles ranged from 3 to 14. The observed(HO) and expected(HE) heterozygosities ranged from 0.2500 to 0.8500 and from 0.3679 to 0.8692, respectively. This suite of microsatellite loci will facilitate future studies of the genetic status of wild and hatchery bred populations of B. pectinirostris.

PCR and magnetic bead-mediated target capture for the isolation of short interspersed nucleotide elements in fishes.

Short interspersed nucleotide elements (SINEs), a type of retrotransposon, are widely distributed in various genomes with multiple copies arranged in different orientations, and cause changes to genes and genomes during evolutionary history. This can provide the basis for determining genome diversity, genetic variation and molecular phylogeny, etc. SINE DNA is transcribed into RNA by polymerase III from an internal promoter, which is composed of two conserved boxes, box A and box B. Here we present an approach to isolate novel SINEs based on these promoter elements. Box A of a SINE is obtained via PCR with only one primer identical to box B (B-PCR). Box B and its downstream sequence are acquired by PCR with one primer corresponding to box A (A-PCR). The SINE clone produced by A-PCR is selected as a template to label a probe with biotin. The full-length SINEs are isolated from the genomic pool through complex capture using the biotinylated probe bound to magnetic particles. Using this approach, a novel SINE family, Cn-SINE, from the genomes of Coilia nasus, was isolated. The members are 180–360 bp long. Sequence homology suggests that Cn-SINEs evolved from a leucine tRNA gene. This is the first report of a tRNALeu-related SINE obtained without the use of a genomic library or inverse PCR. These results provide new insights into the origin of SINEs.

The complete mitogenome of the oriental sucking barb, Garra orientalis (Cypriniformes, Cyprinidae): repetitive sequences in the control region

Abstract The oriental sucking barb, Garra orientalis, is a small to moderate-sized freshwater fish. In this study, the complete mitochondrial genome of G. oriental was successfully sequenced for the first time with total length of 17,288 bp. The genome structure consists of 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes and a control region. Moreover, tandem repeat unit ranged from 259 to 260 bp (repeated 3–4 times) was identified in the control region among G. orientalis individuals.

Complete mitochondrial genome of Osteochilus salsburyi (Cypriniformes, Cyprinidae).

Osteochilus salsburyi (Cypriniformes, Cyprinidae) is a small-sized fish of significant economic value. In this paper, the complete mitogenome sequence of O. salsburyi was first determined. It is 16,599 bp in length and consists of a typical vertebrate genome structure including 13 protein-coding genes, 2 ribosomal RNA genes, 22 transfer RNA (tRNA) genes, and a control region. Except for eight tRNA and ND6 genes, all other mitochondrial genes are encoded on the heavy strand. The overall base composition of the heavy strand in descending order is A (33.0%), T (26.5%), C (25.4%), and G (15.0%), with a slight AT bias of 59.5%. This information of O. salsburyi mitogenome could contribute not only to the development of efficient conservation strategy on vulnerable genetic diversity but also to the identification of useful genetic markers for distinction across species boundary.

Isolation and characterization of polymorphic microsatellite markers in the fish Garra orientalis (oriental sucking barb)

Abstract14 Polymorphic microsatellite markers were developed in the Garra orientalis. These new markers were tested in 23 individuals collected from the Wanquan River in the Hainan Island. The number of alleles (NA), polymorphism information content, observed and expected heterozygosity per locus ranged from 8 to 25, 0.6788 to 0.9342, 0.5652 to 1.0000 and 0.7179 to 0.9585, respectively. Four loci are deviated from Hardy–Weinberg equilibrium due to the heterozygote deficiency. These markers can be used to study genetic diversity, population structure, conservation and rational utilization of this species.