GENETIC DIVERSITY IN THE CHINESE PANGOLIN (MANIS-PENTADACTYLA) - INFERRED FROM RESTRICTION ENZYME ANALYSIS OF MITOCHONDRIAL DNAS

Two different forms of Chinese pangolins can be recognized according to the color of their scales, i.e., brown and dusky. We analyzed mitochondrial DNA (mtDNA) purified from the livers of seven dusky and six brown Chinese pangolins from the same locality, using cleavage patterns from 19 restriction enzymes. From the 19 6-bp recognition enzymes used, 51-56 sites were observed. By combining the cleavage patterns for each enzyme, the 13 samples were classified into four restriction types: two in dusky and two in brown Chinese pangolins. The estimated number of nucleotide substitutions per site in dusky and brown types is 0.002, and that between dusky and brown types is 0.012. Divergence between brown and dusky forms began 0.6 Myr ago, provided the mean rate of sequence divergence is 0.02 per Myr in mtDNA. Our results suggest that there is considerable divergence in Chinese pangolins, and brown and dusky Chinese pangolins may be quite different forms or, at least, belong to different maternal groups.

Report on BOBLME Indian mackerel genetic data analysis including final workshop, Phuket, Thailand, 17-19 February 2015

This workshop followed on from two previous workshops held in Colombo, Sri Lanka, 2012 and Kochi, India in 2013. The 14 microsattellite markers had previously been developed for Indian Mackerel (Rastrelliger kanagurta) were used on 31 tissue collections from all eight countries were genotyped in India.

Analysis of genetic diversity in Glyptosternum maculatum (Sisoridae, Siluriformes) populations with AFLP markers

We determined the genetic diversity of geographic populations from three spawning grounds (Nyang River, Lhasa River, Shetongmon Reach of Yarlung Zangbo River) of Glyptosternum maculatum with amplified fragment length polymorphism (AFLP) markers. Five primer combinations detected 332 products, 51 of them (15.4%) were polymorphic in at least one population. The Shetongmon population was found to be the richest in genetic diversity as was indicated by the percentage of polymorphic loci and heterozygosity, followed by the Nyang population and the Lhasa population. The pair-wise genetic distance between populations were all very close, ranging from 0.0015 to 0.0042 with an average of 0.0024. The genetic distance was not proportional to the geographic distance. The analysis of molecular variance demonstrated that all variation occurred within populations. The average estimated fixation index (F (st)) of three populations across all polymorphic loci was -0.0184, indicating the absence of genetic differences among the three sampled populations. The differentiation among populations was not significant, and population structure was weak. Our observations will help identify the genetic relationship among populations as the first approach to understand the genetic diversity of Glyptosternum maculatum.

Amplified fragment length polymorphism analysis to identify the genetic structure of the Gymnocypris przewalskii (Kessler, 1876) population from the Qinghai Basin, China

Amplified fragment length polymorphism (AFLP) was used to analyse the genetic structure of 45 individuals of Gymnocypris przewalskii (Kessler, 1876), an endangered and state-protected rare fish species, from three areas [the Heima (HM), Buha (BH) and Shaliu rivers (SL), all draining into Qinghai Lake]. A total of 563 polymorphic loci were detected. The HM, BH and SL populations have 435, 433 and 391 loci, respectively (Zhu and Wu, 1975), which account for 77.26%, 76.91% and 69.45% of the total number of polymorphic loci of each population, respectively. The Nei indices of genetic diversities (H) of the three populations were calculated to be 0.2869 (HM), 0.2884 (BH) and 0.2663 (SL), respectively. Their Shannon informative indices are 0.4244, 0.4251 and 0.3915, respectively. Research results show that the mean genetic distance between HM and BH is the smallest (0.0511), between BH and SL is the second shortest (0.0608), and between HM and SL is the largest (0.0713), with the mean genetic distance among the three populations being over 0.05. Data mentioned above indicate that the three populations have a certain genetic differentiation. The total genetic diversity (H-t = 0.3045) and the mean value of genetic diversity within the population (H-s = 0.2786) indicate that the variations have mainly come from within the population.

Genetic variation analysis within and among six varieties of common carp (Cyprinus carpio L.) in china using microsatellite markers

Five microsatellites were used to study the genetic diversity and genetic structure of one wild and five domestic varieties of common carp in China (the Yangtze River wild common carp, Xingguo red carp, purse red carp, Qingtian carp, Russian scattered scaled mirror carp and Japanese decorative carp). All loci in this study showed marked polymorphism with the number of alleles ranging from 4 to 13. Domestic varieties (except Xingguo red carp) showed less genetic diversity than the Yangtze River wild common carp in terms of allelic diversity. Population differentiation was assessed and each combination of populations displayed significant differentiation (P < 0.05) with the exception of that between the Yangtze River wild common carp and Xingguo red carp. Genetic distance analysis (Nei's standard genetic distance and pairwise F-st distance) showed that the largest distance was between Russian scattered scaled mirror carp and the Yangtze River wild common carp and the smallest distance was between the Yangtze River wild common carp and Xingguo red carp. However, among six populations Japanese decorative carp displayed the highest level of variability in terms of heterozygosity.

Genetic heterogeneity analysis and RAPD marker detection among four forms of Atrina pectinata Linnaeus

Pen shell (Atrina pectinata Linnaeus) can be distinguished into four forms based on the morphololgic characteristics. Genetic similarity, and heterogeneity were analyzed among the four forms by random amplified polymorphic DNA (RAPD) technique using 24 10-nucleotide-long primers. Of these primers, 22 pruners produced well-identifiable RAPD band patterns. Significant differences in RAPD band patterns were revealed among the four forms. A total of 198 polymorphic fragments were scored from 22 pruners. and they are specific for one form, shared by two or three forms. Several pruners, such as S451, S453 S463 S464, S470. S473 and S474, produced abundant band patterns and provided sufficient information for reliable discrimination of the four forms. The average genetic distances and phylogenetic relationships were calculated and analyzed according to the distinguishable fragments. The data indicate that pen shells of form G and form Y are similar not only among individuals within the same form, but also between individuals from the two forms, and that shells of form T and form S are highly divergent. The constructed phylogenetic free matches the average genetic distances. Three clusters were clearly distinguishable, in which two were corresponding to form S and form T respectively and one included forms G and Y. This Study will be benefit to further studies oil the taxonomy and selective breeding of Pinnid species. It is suggested that the four forms of pen shell should be categorized to at least two species taxonomically.

Genetic heterogeneity and ploidy level analysis among different gynogenetic clones of the polyploid gibel carp

Background: Some triploid and tetraploid clones have been identified in the gynogenetic gibel carp, Carassius auratus gibelio Bloch, by karyotypic and cytologic analyses over many years. Further, 5-20% males and karyotypic diversity have been found among their natural and artificial populations. However, the DNA contents and the relation to their ploidy level and chromosome numbers have not been ascertained, and whether normal meiosis occurs in spermatogenesis needs to be determined in the different clones. Methods: The sampled blood cells or sperms were mixed with blood cells from chicken or individual gibel carp and fixed in 70% pre-cooled ethanol overnight at 4degreesC. The mixed cell pellets were washed 2-3 times in 1x phosphate buffered saline and then resuspended in the solution containing 0.5% pepsin and 0.1 M HCl. DNA was stained with propidium iodide solution (40 mug/mL) containing 4 kU/ml RNase. The measurements of DNA contents were performed with Phoenix Flow Systems. Results: Triploid clones A, E, F, and P had almost equal DNA content, but triploid clone D had greater DNA content than did the other four triploid clones. DNA content of clone M (7.01 +/- 0.15 pg/nucleus) was almost equal to the DNA content of clone D (5-38 +/- 0.06 pg/nucleus) plus the DNA content of common carp sperm (1.64 +/- 0.02 pg/nucleus). The DNA contents of sperms from clones A, P, and D were half of their blood cells, suggesting that normal meiosis occurs in spermatogenesis. Conclusions: Flow cytometry is a powerful method to analyze genetic heterogeneity and ploidy level among different gynogenetic clones of polyploid gibel carp. Through this study, four questions have been answered. (a) The DNA content correlation among the five triploid clones and one multiple tetraploid clone was revealed in the gibel carp, and the contents increased with not only the ploidy level but also the chromosome number. (b) Mean DNA content was 0.052 pg in six extra chromosomes of clone D, which was higher than that of each chromosome in clones A, E, F, and P (about 0.032 pg/ chromosome). This means that the six extra chromosomes are larger chromosomes. (c) Normal meiosis occurred during spermatogenesis of the gibel carp, because DNA contents of the sperms from clones A, P, and D were almost half of that in their blood cells. (d) Multiple tetraploid clone M (7.01 +/- 0.15 pg/nucleus) contained the complete genome of clone D (5.38 +/- 0.06 pg/nucleus) and the genome of common carp sperm (1.64 +/- 0.02 pg/nucleus). Cytometry Part A 56A:46-52, 2003. (C) 2003 Wiley-Liss, Inc.

Genetic divergence between Cyprinus carpio carpio and Cyprinus carpio haematopterus as assessed by mitochondrial DNA analysis, with emphasis on origin of European domestic carp

Although common carp is the major fish species in Asian and European aquaculture and many domestic varieties have occurred, there is a controversy about the origination of European domestic common carp. Some scientists affirmed that the ancestor of European domestic common carp was Danube River wild common carp, but others considered it might be Asian common carp. For elucidating origination of European domestic common carp, we chose two representative European domestic common carp strains (German mirror carp and Russian scattered scaled mirror carp) and one wild common carp strain of Cyprinus carpio carpio subspecies (Volga River wild common carp) and two Asian common carp strains, the Yangtze River wild common carp (Cyprinus carpio haematopterus) and traditionally domestic Xingguo red common carp, as experimental materials. ND5-ND6 and D-loop segments of mitochondrial DNA were amplified by polymerase chain reaction and analyzed through restriction fragment length polymorphism (RFLP) and sequencing respectively. The results revealed that HaeIII and DdeI digestion patterns of ND5-ND6 segment and sequences of control region were different between European subspecies C. carpio carpio and Asian subspecies C. carpio haematopterus. Phylogenetic analysis showed that German mirror carp and Russian scattered scaled mirror carp belonged to two subspecies, C. carpio carpio and C. carpio haematopterus, respectively. Therefore, there were different ancestors for domestic carp in Europe: German mirror carp was domesticated from European subspecies C. carpio carpio and Russian scattered scaled mirror carp originated from Asian subspecies C. carpio haematopterus.

PCR-RFLP analysis of mitochondrial DNA ND5/6 region among 3 subspecies of common carp (Cyprinus carpio L.) and its application to genetic discrimination of subspecies

Mitochondrial DNA ND5/6 region was studied by PCR-RFLP analysis among ten representative strains belonging to three subspecies (Cyprinus carpio carpio, Cyprinus carpio haematopterus and Cyprinus carpio rubrofuscus) of common carp (Cyprinus carpio L.). A total of 2.4 kb fragment was amplified and subjected to restriction endonuclease analysis with nine restriction endonucleases subsequently. The results indicated that each subspecies owned one hyplotype and four restriction enzymes (Dde I, HaeIII, Taq I and Mbo I) produced diagnostic restriction sites which could be used for discriminating the three subspecies and as molecular genetic markers for assistant selective breeding of common carp.

RAPD and AFLP techniques for the analysis of genetic relationships in two genera of Decapoda

RAPD was used fur analysing three (sub-)species of mitten crabs (Eriocheir sinensis, E. japonicus, and E. japonicus hepuensis) and three populations of E. sinensis. The results show that their relationships on DNA level are similar to the classical taxonomic hypotheses (Dai, 1991). No diagnostic RAPD marker could be found, but there were statistically significant genetic differences among these taxa (P < 0.001) or populations (P < 0.001). That is, the intraspecific similarities were larger than the interspecific similarities; the intrasubspecific similarities were larger than the intraspecific similarities; and the intrapopulational similarities were larger than the interpopulational similarities. In AFLP analysis, no significant genetic difference has been found between E. sinensis and E. japonicus, but AFLP markers among four species of Macrobrachium (M. rosenbergii. M. nipponense, M. hainanense, and M. asperulum) were found. The DNA similarities among these four species of Macrobrachium are in accordance with morphological similarities.