Cloning and sequence analysis of Sox genes in a tetraploid cyprinid fish, Tor douronensis

A PCR survey for Sox genes in a young tetraploid fish Tor douronensis (Teleostei: Cyprinidae) was performed to access the evolutionary fates of important functional genes after genome duplication caused by polyploidization event. Totally 13 Sox genes were obtained in Tor douronensis, which represent SoxB, SoxC and SoxE groups. Phylogenetic analysis of Sox genes in Tor douronensis provided evidence for fish-specific genome duplication, and suggested that Sox19 might be a teleost specific Sox gene member. Sequence analysis revealed most of the nucleotide substitutions between duplicated copies of Sox genes caused by tetraploidization event or their orthologues in other species are silent substitutions. It would appear that the sequences are under purifying selective pressure, strongly suggesting that they represent functional genes and supporting selection against all null allele at either of two duplicated loci of Sox4a, Sox9a and Sox9b. Surprising variations of the intron length and similarities of two duplicated copies of Sox9a and Sox9b, suggest that Tor douronensis might be an allotetraploidy.

Induced spawning, larval development and rearing of two indigenous Malaysian mahseer, Tor tambroides and T. douronensis

The Mahseers (Tor spp.) are highly valued freshwater fishes across the Himalayan and South-east Asian regions. Over exploitation of natural stocks because of high demand and the deteriorating environmental conditions have resulted in marked decline of mahseers in the wild. Malaysian mahseers, T. tambroides (Bleeker) and T. douronensis (Valenciennes), locally known as empurau, kelah or belian and semah, respectively, have significant cultural and economic importance but both species are now threatened in the wild because of environmental degradation and over fishing. A captive breeding programme was instigated to attempt to propagate these two species artificially for conservation and aquaculture purposes. Both pond-reared and tank-held T. tambroides and T</i>. douronensis reached sexual maturity in captivity and were successfully induced to spawn using hormone treatments. Ovaprim (0.5 mL kg⁻¹) was the most successful hormone treatment for both species. Pre-treatment of fish with Ovaplant (28–68 μg kg⁻¹, 2–7 weeks before spawning induction) greatly improved the success rate of spawning induction. Repeat spawning (within 4 months of initial spawning) was induced in some captive fish. Use of formalin baths improved hatching by preventing fungal infections. Embryonic development and hatching are described. Juveniles were reared in static greenwater ponds. Tor tambroides reached 142–179 g (max 270 g) in 60 weeks. These results represent the first successful captive spawning and rearing of both species. Options for future research to improve production are discussed.

Mitochondrial DNA diversity of broodstock of two indigenous mahseer species, Tor tambroides and T. douronensis (Cyprinidae) cultured in Sarawak, Malaysia

Tor tambroides and T. douronensis, locally referred to as empurau and semah, respectively, are high valued mahseer species, indigenous to Sarawak, East Malaysia, with an aquaculture potential and of conservational value. Direct sequencing of mitochondrial DNA (mtDNA) 16S rRNA gene region (542 bp) was used to investigate genetic variation of T. tambroides and T. douronensis broodstock collected from different geographic locations in Sarawak and maintained at the Indigenous Fish Research and Production Center (IFRPC), Tarat, Sarawak, Malaysia. A total of 11 unique haplotypes were identified, of which six were detected in T. tambroides, and five in T. douronensis. Overall, nucleotide diversity (π) was low, ranging from 0.000 to 0.006, and haplotype diversity (h) ranged from 0.000 to 0.599. Although the analysis failed to detect genetic variation amongst populations of T. tambroides (significant pairwise FST was found for only one test, but pairwise haplotype frequencies were not statistically significant), substantial inter-population divergence among T. douronensis was recognised, especially those originating from different river systems (pairwise F_ST = 0.754 to 1.000, P &lt; 0.05). Fixed haplotype differences were found in one population of T. douronensis. Average nucleotide divergence between T. tambroides and T. douronensis was 0.018, similar to the amount recognised between T. tambroides and the outgroup T. khudree (0.017). In addition, phylogenetic analysis revealed that the T. douronensis mtDNA consisted of two highly divergent clusters (0.020), one of which is more closely related to T. tambroides rather than with the other group of haplotypes of the conspecifics. The findings from the present study have important implications for aquaculture, management and conservation of these two species. The data also raise some concerns regarding the taxonomic status of T. douronensis, which needs to be addressed.</sub>

Population structure in the highly fragmented range of Tor douronensis (Cyprinidae) in Sarawak, Malaysia revealed by microsatellite DNA markers

1. Understanding the structure of threatened populations, particularly those that exist in degraded or fragmented habitats is crucial for their effective management and conservation. Recently developed methods of individual-based analysis of genetic data provide an unprecedented opportunity to understand the relationships amongst fragmented populations.

2. In the present study, population structure of an important cyprinid species (Tor douronensis), which is indigenous to Sarawak, Malaysia, is investigated as part of an ongoing conservation effort to restore threatened wild populations of the species. The population structure inferred using data from seven autosomal microsatellite loci was generally consistent with geography and habitat fragmentation.

3. The results indicate that there are two well-defined clusters of T. douronensis in Sarawak, namely the 'northeastern' and the 'southwestern' clusters. In addition, a further subdivision was observed in each of the clusters distributed between river systems. Low levels of gene flow were also observed and migrants between habitat fragments were identified, possibly resulting from human-mediated translocations.

4. Implications of the findings for management and conservation of T. douronensis are discussed.

Sox genes evolution in closely related young tetraploid cyprinid fishes and their diploid relative

Previous study and analysis of cytochrome b suggested that polyploidization event in the genus Tor occurred about 10 Mya ago. In order to understand evolutionary fates of Sox gene in the early stage of genome duplication at the nucleotide level, PCR surveys for Sox genes in three closely related cyprinid fishes T douronensis (2n = 100), T qiaojiensis (2n = ?), T sinensis (2n = 100) and their relative T brevifilis (2n = 50) were performed. Totally, 52 distinct Sox genes were obtained in these four species, representing SoxB, SoxC, and SoxE group. As expected, isoforms of some Sox genes correspond with the ploidy of species, such as two copies of Sox9a exist in tetraploid species. Analysis indicated that duplicated Sox gene pairs caused by polyploidization evolved independently of each other within polyploid species. Results of substitution rate showed nearly equal rate of nonsynonymous substitution of duplicated Sox orthologs among different polyploid species and their diploid relative orthologs, suggesting at the early stage of genome duplicated Sox orthologs are under similar selective constraints in different polyploidy species and their diploid relative at the amino acid level. All PCR fragments of Sox genes obtained in this study are not accompanied by obvious increase in mutations and pseudogene formation which means that they are under strong purifying selection, suggesting that they are functional at the DNA level. Cenealogical analysis revealed that T qiaojiensis was tetraploid, and T douronensis, T qiaojiensis as well as T sinensis had an allotetraploid ancestor. (C) 2009 Elsevier B.V. All rights reserved.

Contributions to propagation of two Tor species in Sarawak, Malaysia

The study focuses on the natural spawning ecology, induced spawning of captive broodstock and larval and fry nursing techniques of Tor tambroides and Tor douronensis, two mahseer fish of aquaculture and conservational importance. Both species were induced bred, and the development will help develop commercial aquaculture.