V. P. E. Phang

Genetic variation inferred from RAPD fingerprinting in three species of tilapia

This study used random amplified polymorphic DNA (RAPD) fingerprinting for estimating genetic variation and species differentiation in three species of tilapia. A 16-mer random primer generated RAPD markers ranging from 250 to 2400 base pairs (bp). Genetic similarity estimates obtained by pairwise comparisons based on the method of Nei and Li (1979) indicated high genetic similarity (mean genetic similarity (± sd), 0.73 (± 0.15) for Nile tilapia; 0.78 (± 0.12) for Mozambique tilapia; and 0.87 (± 0.07) for Aureus tilapia) within each of the tilapia species. The average interspecies genetic similarities obtained among the three species were 0.59 (± 0.07) for Mozambique/Nile tilapia, 0.46 (± 0.09) for Aureus/Nile tilapia and 0.38 (± 0.07) for Aureus/Mozambique tilapia pair. DNA profiles generated in each species of tilapia were unique. A total of 13 RAPD markers differentiating the three species of tilapia were detected. Our study presented RAPD markers as a new class of useful genetic markers for assessment of genetic diversity and species differentiation in tilapia.

Genetic diversity among wild forms and cultivated varieties of Discus (Symphysodon spp.) as revealed by Random Amplified Polymorphic DNA (RAPD) fingerprinting

Abstract Trial-and-error method has been used extensively in the breeding of Discus. There is limited knowledge on the genetic structure of its species complex and also the genetic basis of its stock constitution and management. Random Amplified Polymorphic DNA (RAPD) fingerprinting was used to assess the genetic diversity among four wild forms of Discus: Symphysodon discus (Heckel), S. aequiefasciata aequiefasciata (Wild green), S. a. axelrodi (Wild brown) and S. a. haraldi (Wild blue) and five cultivated varieties of Discus (Turquoise, Pigeon, Ghost, Cobalt and Solid Red). The Mann–Whitney U -test used in the comparisons among the inter-wild form, inter-cultivated variety and between wild form and cultivated variety similarity indices revealed that the gene pool of the cultivated varieties of Discus is smaller than that of the wild Discus forms. Unweighted pair–group method with arithmetic means (UPGMA) phenogram showed that the Heckel Discus ( S. discus ) is genetically the most divergent in relation to the other three wild forms, being 2.89 times further in mean genetic distance from the other three wild forms (Wild green, blue and brown) than Wild green to the other two wild forms (Wild blue and brown). The cultivated varieties is 3.18 times genetically closer to the three S. aequiefasciata wild forms (Wild green, blue and brown) (mean genetic distance=0.033) than to S. discus (Heckel) (mean genetic distance=0.105). This suggests that the S. aequiefasciata wild form is the more likely genetic origin of the cultivated varieties. In addition, there is no distinct clustering of individuals from the same cultivated variety indicating the lack of a genetic basis for the present phenotypic classification of the cultivated varieties. Outcrossing with the wild forms especially, the Heckel Discus is recommended to increase the level of genetic variability in the cultivated varieties.

Genetic Linkage Maps of the Guppy (Poecilia reticulata) : Assignment of RAPD Markers to Multipoint Linkage Groups

Genetic linkage maps of the guppy (Poecilia reticulata) were constructed from independent crosses between the Tuxedo strain and a feral line (Wildtype). Segregation patterns of random amplified polymorphic DNA (RAPD) markers and phenotypic markers were investigated in F2 offspring of Tuxedo ♂♂ × Wildtype ♀♀ and Wildtype ♂♂ × Tuxedo ♀♀ crosses. Among the 300 and 276 RAPD markers scored for the respective crosses, linkages were identified for 230 and 212, respectively. The Tuxedo ♂♂ × Wildtype ♀♀ and Wildtype ♂♂ × Tuxedo ♀♀ maps spanned 2100 Kosambi centiMorgans (cMK) and 1900 cMK, respectively, in 28 linkage groups. Average marker resolution was 10 cMK. Genome length was estimated at 4410 cMK and 4060 cMK for the respective crosses, with an average physical distance of 166 kbp/cMK. Several RAPD markers were closely linked to or mapped onto the loci for the sex-determining region (SdR), and the sex-linked black caudal-peduncle (Bcp) and red tail (Rdt) genes. These primary linkage maps are the initial step toward the construction of a composite high-density map to facilitate map-based cloning and marker-assisted selection of quantitative trait loci that are essential for the development of comprehensive breeding programs for the guppy.

Culture of the guppy, Poecilia reticulata, in Singapore

Abstract Singapore is known internationally as a breeding centre for the guppy, Poecilia reticulata. About 30 domesticated colour pattern and tail shape varieties are reared, mainly for export. The culture of these varieties at two rural and eight resettled farms is described. All farms are small family concerns. The resettled farms practise monoculture of guppies while the rural ones integrated farming. Guppies are reared in large, shallow, outdoor, cement tanks and nylon cage-nets suspended in ponds. Water in aquaria and ponds has salinities from 0.5 to 9.0‰, at neutral or alkaline pH of 7.0 to 8.3. The sizes and stocking densities of aquaria and nets vary within and between farms. In breeding tanks, a male to female sex ratio of 1:3, 1:4 or 1:10 is maintained. Newborn fry are collected daily and placed in nursery aquaria. After 3 weeks, these fry are sexed: young males are transferred to grow-out cement tanks, and females to grow-out cagenets. Each farm has at least two ponds, one for water storage and the others for cagenet culture. Strains are cultured separately to maintain pure lines. Farmers improve colour patterns, size and fin shapes of the strains by continued mass selection. Four- to 6-month-old guppies are selected as brookstock and the rest which meet export quality are held in stocking aquaria and sold when demand arises. Each farmer prepares his own formulated diet for the fish. Supplemental foods like live tubificiids, commercial dried fish flakes, or floating pellets may be given to adults, and water fleas to fry. The rural farms add pig manure to fertilise the tanks. To remain viable in the future, farmers need to increase productivity by mechanising cleaning, draining, and filling tanks and ponds, feeding, and harvesting of the fish. This will cut down on labour and its increasing cost. Understanding the genetics of economically important characters will also help farmers produce higher quality and more exotic strains.

Sex-Linkage of the Black Caudal-Peduncle and Red Tail Genes in the Tuxedo Strain of the Guppy, Poecilia reticulata

Abstract Two color patterns of the Tuxedo guppy strain commercially cultured in Singapore were subjected to genetic analyses. Gene control of the black caudal-peduncle and red tail color patterns was elucidated by reciprocal crosses between the Tuxedo (TUX) strain and wild-type (WT) stock. F1 progenies were produced by single-pair crossing between TUX and WT, while the F2 generation was obtained from full-sib mating between F1 males and females. F1 and F2 data were segregated according to phenotypes and sex, and tested by chi-square analyses. Both color patterns show single gene inheritance, and are dominantly expressed in both sexes, sex-linked and determined by different loci on the X- and Y-chromosomes. Alleles for the black caudal-peduncle (Bcp) and red tail (Rdt) loci, are dominant over that of the wild-type, Bcp+ and Rdt+, which do not display these color patterns. The typical genotypes for TUX guppies are proposed to be XBcp,RdtYBcp,Rdt for males and XBcp,RdtXBcp,Rdt for females. Heterozygous TUX males have the XBcp,RdtYBcp+,Rdt, XBcp,RdtYBcp+,Rdt+ and XBcp,RdtYBcp,Rdt + genotypes while the females are XBcp,RdtXBcp+,Rdt. The segregation and inheritance of the Bcp and Rdt genes are illustrated by genetic models. Map distances estimated from F1 and F2 recombinants are approximately 3.4, 5.1 and 2.4 map units for the sex-determining region (SdR)–Rdt, SdR–Bcp and Rdt–Bcp, respectively. The gene map order is hypothesized to be SdR–Rdt–Bcp.

Linkage Analysis and Mapping of Three Sex-Linked Color Pattern Genes in the Guppy, Poecilia reticulata

Abstract Three phenotypic color pattern genes of the guppy (Poecilia reticulata), i.e., black caudalpeduncle (Bcp), red tail (Rdt) and variegated tail patterning (Var), were genetically analyzed and mapped. Crosses between the Tuxedo (TUX) and Green Variegated (GV) guppy strains commercially cultured in Singapore were used to determine the gene control of these color patterns. F1 progenies were produced by single-pair reciprocal crossing between TUX and GV, while the F2 generation was obtained from full-sib mating between F1 males and females. F1 and F2 data were segregated according to color phenotypes and sex, and tested by chi-square analyses. The Bcp, Rdt and Var color pattern genes, located at different loci on the X- and Y-chromosomes, showed single gene inheritance and dominant expression in both sexes. Their corresponding recessive alleles, Bcp+, Rdt+ and Var+, do not produce any color patterns. Genotypes of Tuxedo males are proposed to be XBcp,Rdt,Var+YBcp,Rdt,Var+ (type I), XBcp+,Rdt,Var+YBcp,Rdt,Var+ (type II) and XBcp,Rdt,Var+YBcp+,Rdt,Var+ (type III) while females are XBcp,Rdt,Var+XBcp,Rdt,Var+. Green Variegated males and females have the XBcp+,Rdt+,VarYBcp+,Rdt+,Var and XBcp+,Rdt+,VarXBcp+,Rdt+,Var genotypes, respectively. Close linkages of 3.1, 2.3 and 2.2 map units were estimated for the sex-determining region (SdR)–Rdt, Rdt–Bcp, and SdR–Var gene pairs, respectively, while Bcp was approximately 5.1 map units from the SdR. The phenotypic map order of the guppy Y-chromosome is inferred to be Var–SdR–Rdt–Bcp.

Genetic Basis of the Variegated Tail Pattern in the Guppy, Poecilia reticulata

Abstract Variegated patterns on the caudal fin are a common and popular trait in guppy strains commercially cultured in Singapore. Gene control of this highly variable mosaic pattern of black spots and patches of different shapes and sizes on a brightly colored tail fin was elucidated by reciprocal crosses between the Green Variegated (GV) strain and wild-type (WT) stock. F1 progenies were produced by single-pair crossing between GV and WT, while the F2 generation was obtained from full-sib mating between F1 males and F1 females. Data for the F1 and F2 generations were segregated according to phenotypes and sex, and tested by chi-square analyses. Inheritance of variegated tail patterns appears to be determined by a single locus on the X- and Y-chromosomes. Genotypes of males and females of the GV strain are proposed to be X VarYVar and XVarXVar, respectively. The allele for variegated tail patterning, Var, is dominant over that of the wild-type, Var+, which does not exhibit these patterns. Recombination frequency between the Var locus and sex-determining region (SdR) in male guppies was estimated to be about 1.9% (map distance ≈1.9 map units). The segregation and mode of inheritance of the Var gene are illustrated by genetic models.

Genetic Diversity Within and Among Feral Populations and Domesticated Strains of the Guppy (Poecilia reticulata) in Singapore

Genetic variability within and among feral populations and cultured strains of the guppy (Poecilia reticulata) was investigated by random amplification of polymorphic DNA (RAPD) fingerprinting. Feral guppies were collected from 6 isolated populations (BT, Bukit Timah; NS, Nee Soon; TS, Tuas; MF, Mount Faber; KR, Kranji; LI, laboratory-inbred feral line), while the Tuxedo and Green Variegated strains were sampled from 2 guppy farms in Singapore. Pairwise genetic distances analyzed by unweighted pair-group method with arithmetic means revealed distinct clustering of guppy individuals into their respective populations and strains. Percentage polymorphic loci ranged from 54.96% (TS) to 68.70% (KR), while average heterozygosity ranged from 0.220 (GV) to 0.271 (KR). In contrast, TS guppies had the highest (0.850) intrapopulation genetic similarity (S), whereas KR had the lowest (0.781). Among populations and strains, S ranged from 0.703 (between GV and LI) to 0.809 (between NS and MB). The GV strain S was closer to TX (0.784) than to the feral guppies. Bootstrapped genetic distance trees depicted 3 major nodes comprising BT-TS, NS-MF, and TX-GV. Principal coordinate analysis also differentiated the 6 feral populations from the 2 cultured strains.

Cloning and Tissue Expression of 6-Pyruvoyl Tetrahydropterin Synthase and Xanthine Dehydrogenase from Poecilia reticulata

Guppy is a popular ornamental fish owing to its diverse body and fin coloration. More than 40 established color varieties have been selectively bred. The complementary DNAs for 2 enzymes that are involved in the de novo synthesis of pteridines and purines, which are important for the production of color pigments, were cloned from the caudal fin. Two cDNA isoforms for 6-pyruvoyl tetrahydropterin synthase (PTPS), with an open reading frame of 130 and 147 amino acids, respectively, were cloned from the Red Tail variety. The deduced amino acid sequence of the longer isoform shows an overall identity of about 65% to the mammalian PTPS sequences. The cDNA for xanthine dehydrogenase (XDH) was cloned from the Yellow Tail variety, and consists of an open reading frame of 1331 amino acids. Although it shows a higher overall identity to bovine aldehyde oxidase (AO; 54%) than to chicken XDH (51%), it has a NAD-binding domain that is specific to XDHs. Northern blot analysis indicated that both PTPS and XDH messenger RNAs were highly expressed in the liver, but absent in the muscle. In the caudal fins, guppy varieties with a higher proportion of xanthophores and erythrophores showed higher expression of PTPS, while XDH mRNA levels were too low to indicate obvious differential expression among the color guppy varieties. The results implied that high expression of PTPS is correlated with the biosynthesis of pteridines in the erythrophores and xanthophores, while the association between the putative guppy XDH with specific chromatophores is less clear.

Interaction between the Autosomal Recessive bar Gene and the Y-Linked Snakeskin Body (Ssb) Pattern Gene in the Guppy, Poecilia reticulata

Abstract Many color varieties of the guppy, Poecilia reticulata, are commercially cultured in Singapore for the aquarium industry. In the group of guppy varieties called Snakeskin, males characteristically have snakeskin-like reticulations on the body and caudal fin. The snakeskin pattern on the body of male Snake-skin guppies is due to a Y-linked gene (Ssb). Female guppies, being homogametic (XX), do not carry the Ssb gene. About 90% of Yellow Snakeskin males have the typical snakeskin pattern on their bodies and tails. The remaining males are different in that the snakeskin body pattern has been modified into four or five vertical bars on the caudal-peduncle region. F1 and F2 results of single-pair reciprocal matings of the Yellow Snakeskin variety show that a single gene is responsible for the vertical bar pattern. This gene, bar, is autosomal recessive. In the homozygous condition (barbar), it interacts with the Y-linked Ssb gene to give vertical barring patterns on the caudal-peduncle of Yellow Snakeskin males. This pattern is not expressed when the dominant allele, bar+, is present.