Zhi-Zhi Liu

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.

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.

Analysis of genetic diversity in wild populations of Trachidermus fasciatus by RAPD and the transformation of two SCAR markers

Firstly, RAPD was conducted to analyze genetic diversity of Trachidermus fasciatus in the Fuchun River population (FR), Yellow River population (YR), Luan River population (LR), and Yalu River population (YL), with 32 polymorphic 10-bp random primers selected from 294 ones. Thirty wild individuals were detected in each population. The results indicated that the genetic diversity of T . fasciatus was relatively rich. The major results were as the following: 1) Altogether, 591 bands were detected and 515 of them were polymorphic, accounted for 87.14%. The range of proportion of polymorphic loci (P) was: FR(89.17%)>YR(87.99%)>YL(86.63%)>LR(83.25%). 2) The Shannons information index(I(T)) and Neis genetic diversity(H(T)) among populations were 0.3393-0.3566 and 0.2157-0.2279, respectively. Compare to other three populations, LR population had relative lower values. If took the populations as a whole, the total Neis genetic diversity(H(T)) and Shannons information index(I(T)) was 0.2336±0.1643 and 0.3710±0.2153, respectively. 3) The value of gene flow (N(m) ) (5.76103-19.84497) were high, indicating certain gene exchange existed among the four populations. But the AMOVA results exhibited significantly differentiation (P<0.05 or P<0.01) among the populations. 4) In the UPGMA tree constructed according to genetic distance, YL and YR populations clustered firstly, then with FR population, and finally they joined to LR population. Obviously, the YL, YR and FR populations had relatively close relationship according to their geographic distance, whereas LR population showed clear divergence to the other three populations. Secondly, out of the five special RAPD bands (S(1225)(525 bp), S(1225)(605 bp), S(1225)(841 bp), S(1345)(695 bp) and S(1345)(825 bp)), SCAR maker SCAR01(560 bp) and SCAR02(443 bp) were successfully transformed from S(1255)(605 bp) and S(1255)(841 bp), respectively. After large samples examination of the two markers, we found the highest frequency (96.67% and 93.33%) in the YL population, higher frequency (83.33% and 90%) in the FR population, high frequency (56.67% and 66.67%) in the YR population, and the lowest frequency (13.33% and 20 %) in the LR population. Therefore, SCAR01(560 bp) and SCAR02(443 bp) can be used as special molecular markers for the population identification between LR and other three populations.

Isolation and characterization of polymorphic microsatellite markers from Japanese grenadier anchovy (Coilia nasus)

We isolated and characterized 12 novel polymorphic microsatellite loci for the Japanese grenadier anchovy (Coilia nasus) using the method of PCR-based isolation of microsatellite arrays. Sixteen individuals of C. nasus from Ariake Bay in Japan were genotyped using 24 PCR primers to characterize the polymorphism at each locus. The average number of the observed alleles per locus was 11.08 (ranging from 8 to 20). The observed heterozygosity values ranged from 0.562 to 1.0, and the expected heterozygosity values ranged from 0.647 to 0.969. Only one of the twelve loci deviated significantly from Hardy–Weinberg equilibrium, and linkage disequilibrium was not found in any pairs of the loci. These markers can be used to investigate the genetic structure of the C. nasus population.

Genetic Structure and Population History of Beleophthalmus petinirostris in Yangtze River Estuary and Its Southern Adjacent Regions

In order to analysis the genetic structure and demographic history of Beleophthalmus petinirostris , the random amplified polymorphic DNA (RAPD) technology and mitochondrial cytochrome b (Cyt b) gene sequences were used in the study. The results showed that there was high level genetic diversity in the populations of B. pecinirostris in Yangtze River estuary and its southern wet-land adjacent regions. (1) RAPD was conducted to analysis the genetic variation of B. pecinirostris in Jiuduansha (JDS), Dinghai（DH）and Xiapu（XP）for 20 individuals per population, with 30 polymorphic 10-based random primers selected from 100 ones. In total, 236, 270 and 274 bands were obtained for JDS, DH and XP, respectively and there was 301useful loci. The range of proportion of polymorphic loci (P), Nei’s gene diversity (H) and Shannon diversity index (I ) was: 93.02%–96.3%, 0.3890–0.4219, 0.5618–0.6044, respectively. The results of AMOVA showed significantly genetic differentiation (Fst=0.03004-0.03254, P<0.05) but there was only 3.2% genetic variation among populations. (2) Results of Cyt b gene: the total Cyt b gene length was 1 141 bp and 33 haplotypes were obtained from 42 individuals collecting in 4 different regions, which included JDS, DH, XP and Cixi (CX). The average haplotype diversity (h) and nucleotide diversity (π) was 0.9184, 0.0048, respectively. The high level of Nm values (14.50–30.79) and low Fst values (0.0043–0.07814), the average K2-P distances (0.0040–0.0056), as well as the AMOVA analysis (total Fst=0.0193, P>0.05) all revealed that the B. pecinirostris had no obviously geographical differentiation. However, the phylogenetic tree estimated by neighbor-joining (NJ) method indicated that the individuals of B. pecinirostris formed two monophyletic lineages, and the two lineages were not correlated with geographical populations. Neutrality tests, mismatch distribution and network analysis suggested a late Pleistocene population expansion for B. pecinirostris (0.057–0.023 million years ago).

Development and characterization of thirteen polymorphic microsatellite markers in the fish Osteochilus salsburyi

Osteochilus salsburyi (Cypriniformes, Cyprinidae) is a small-sized fish of significant economic value. In this study, thirteen polymorphic microsatellite markers were developed in O. salsburyi and were examined on twenty 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 10 to 21, 0.8020 to 0.9354, 0.7500 to 1.0000 and 0.8436 to 0.9628, respectively. No locus was found to significantly deviate from Hardy–Weinberg equilibrium. These markers would be suitable for studies on conservation genetics and population structure of O. salsburyi.

Developmental stages and migratory routes of larvae Japanese eel from southeast coastal estuaries of China

A total of 534 larvae of Anguilla japonica were collected from 9 estuaries in Guangdong,Fujian,Zhejiang,Shanghai and Jiangsu,China,during Jan.-Mar.of 2008.Their pigmentation stages were determined according to the distribution of pigments on the body surface.The age of leptocephali at metamorphosis(Tm),time between metamorphosis to arrival at estuaries(Tt.m),and age of glass eels(Tt) arriving at the 9 sites were counted according to the number of daily growth increments in the sagitta.The mean Tm was 102 d(90 d to 112 d) and progressively increased from south to north.The homogeneity tests for Tt,Tm and Tt-m among nine estuaries were conducted by Scheffes multiple comparison method.According to sampling location and Tm value,leptocephali were divided into three homgeneous groups.Group I was located in two southern sampling sites(Xinhui and Shantou) and had the lowest Tm.In contrast,group III in the 3 northern sampling sites(Jiuduansha,Cixi,Dafeng) had the highest Tm.Group II were in the four mid-sites(Fuqing,Ningde,Wenzhou,Taizhou).Similar result was acquired by R-cluster dendrogram of nine sampling sites and Q-cluster dendrogram of every sample based on Tm.These results indicated that the leptocephalus might migrate in three populations along the North Equatorial Current from their spawning ground to the northeastern continental shelf waters of Philippines,to Kuroshi Current and then to the estuaries of China for metamorphosis.The fast-growing leptocephali metamorphosed earlier,and migrated to the southern estuaries and then entered into Guangdong coastal rivers,while the slow-growing ones metamorphosed later,migrated to the Changjiang estuary and the northern estuaries of the range.The remaining leptocephali migrated to the middle coastal waters of East China Sea.The difference of leptocephali metamorphosis age between Xinhui [(91.87±1.36) d] and Dafeng [(106.00±3.22) d] was 14.13 d.If the leptocephali were drifted by the Kuroshio Current at a velocity of 96 km/d,the dispersal distance of the leptocephali for 14.03 d would be 1 350 km,approximately equal to the distance between Xinhui and Dafeng.These results suggest that glass eel of Chinese estuaries was passively transported by the Kuroshi Current from the south to north at their leptocephali stage.