Source of the fish

Three- to four-week old fry of the Green Variegated (GV) guppy strain were obtained from Swee Hing & Brothers Aquarium Co. in Singapore. Wild-type (WT) guppies were collected from an isolated hill-stream near the Bukit Timah nature reserve in Singapore. Juvenile GV were cultured in 180-liter fibreglass tanks (30 fish/tank) in the aquarium area of the Department of Biological Sciences, National University of Singapore, at temperatures of 25–28°C. WT fry were separated from the collected samples and raised in 30-liter clear plastic tanks (20 fish/tank). Under laboratory conditions, sexual maturation of WT fry usually occurs at 4-6 weeks of age. Juvenile WT were checked daily for developing males which is recognised by the formation of the gonopodium from the anal fin. Males, when spotted, were immediately removed and reared separately from females as virgin females were necessary for the reciprocal crosses.

Description of the fish

Adult males and females of the GV strain have a total length of 3–4 cm and 5–6 cm, respectively. Adult GV males have a green metallic sheen overlying the wild-type male body coloration that comprise small spots or patches of various colors. Only GV males have an orange tail with some yellow streaks, and numerous black spots and patches of different shapes and sizes (Fig. 1A). GV females show wild-type female olive-brown body coloration and faint greyish-brown variegated patterns on a yellowish tail (Fig. 1B). Wild-type guppies are smaller than the domesticated GV strain. Adult WT males are 2–2.5 cm in length while the females are about 3–4 cm. As described earlier, WT males have highly polymorphic color patterns on the body and fins (Fig. 1C), while WT females totally lack any color patterns (Fig. 1D).

Fig. 1

(A) Adult male guppy of the Green Variegated strain. (B) Adult female guppy of the Green Variegated strain. (C) Adult male wild-type guppy. (D) Adult female wild-type guppy. (E) F₁ males showing typical variegated tail patterning (top) and wild-type tail without any patterns (bottom) from mating pair PG9. (F) F₁ female with variegated tail patterning. (G) F₂ males of the VAR (top) and WT (bottom) phenotypes. (H) F₂ females of the VAR (top) and WT (bottom) phenotypes.

Reciprocal crosses

Inheritance of the variegated tail pattern was elucidated by singlepair reciprocal crosses between the GV strain and WT stock, using six-week old mature virgin fish. Each pair was kept in a 3.5-liter breeding tank. Broods were produced 4-6 weeks after mating. Single-pair full-sib F₁ males and F₁ females were mated to obtain the F₂ generation. The following notations were used: GV ♂♂× WT ♀♀ (Table 1A) and WT ♂♂ × GV ♀♀ (Table 2A) for parental crosses, and F₁ ♂♂ × F₁ ♀♀ (Tables 1B, 2B) for full-sib F₁ crosses. Newly born fry were separated and raised to maturity in 3.5-liter clear plastic tanks (five fish/tank). F₁ and F₂ progenies were segregated and scored according to phenotypes and sex. All progenies displaying variegated tail patterns were designated as the VAR phenotype and those without such patterns, WT phenotype.

Table 1

Mating results of crosses between Green Variegated (GV) male and wild-type (WT) female guppies showing observed and expected numbers for each phenotypic class, expected segregation ratios, chi-square goodness-of-fit to the expected ratios and their corresponding adjusted values (χ² adj) after application of Yates' correction for continuity, χ² test for homogeneity, probable genotypes and recombinants for (A) the F₁ generation of single-pair parental crosses, and (B) the F₂ generation of single-pair crosses between full-sib F₁ males and F₁ females. Recombinants (^§) due to crossing-over of the Var gene were not considered in chi-square analyses. (Phenotypes: VAR = tail with variegated patterns, WT = wild-type tail without variegated patterns. Genes: Var = variegated tail pattern gene, Var⁺ = absence of variegated tail pattern gene).

Table 2

Mating results of crosses between wild-type (WT) male and Green Variegated (GV) female guppies showing observed and expected numbers for each phenotypic class, expected segregation ratios, chi-square goodness-of-fit to the expected ratios and their corresponding adjusted values (χ²adj) after application of Yates' correction for continuity, χ² test for homogeneity, probable genotypes and recombinants for the (A) F₁ generation of single-pair parental crosses, and (B) F₂ generation of single-pair crosses between full-sib F₁ males and F₁ females. Recombinants (^#) due to crossing-over of the Var gene were not considered in chi-square analyses. (Phenotypes: VAR = tail with variegated patterns, WT = wild-type tail without variegated patterns. Genes: Var = variegated tail pattern gene, Var⁺ = absence of variegated tail pattern gene).

Statistical analyses

Observed phenotypic distributions were tested for goodness-of-fit with predicted proportions using the chi-square (χ²) test (Sokal and Rohlf, 1981; Strickberger, 1990). Since observed and expected numbers in the phenotypic classes and sample sizes were small (n< 200), Yates' (1934) correction for continuity was included in the calculation of χ² to improve the approximation to the χ² distribution, as shown by the χ²_adj values. The χ² test for homogeneity was used to determine whether there were significant differences among the phenotypic frequencies, and if the observations were sufficiently uniform and the population homogeneous after the data was pooled. The correction for continuity was not incorporated into the test for homogeneity because calculated χ² values had to be summed and χ²_adj values were not additive (Sokal and Rohlf, 1981; Strickberger, 1990). Following Winge (1922b, 1923, 1927, 1934), Nayudu (1979), Phang et al. (1989a, b, 1990), and Phang and Fernando (1991), individuals with exceptional coloration due to crossing-over of the Var gene between the X- and Y-chromosomes were not considered in chi-square analyses. Recombination frequency and map distance between the Var locus and sex-determining region (designated as SdR), proposed by Schmidt (1920) and Winge (1922a, b, 1927, 1934) to be found on the Y-chromosome, were estimated according to Strickberger (1990), Phang et al. (1990), Phang and Fernando (1991), and Purdom (1993).

Cross between GV males and WT females

Four mating pairs of GV♂♂ × WT♀♀ (PG1, PG4, PG5 and PG10) gave a total of 47 male and 63 female F₁ offspring in 13 broods, with an expected ratio of 1:1 (Table 1A). F₁ male progenies showed variegated tail patterns (VAR phenotype) and wild-type male body coloration (WT) (Fig. 1E), while F₁ females had faint greyish-brown variegated patterns on the tail and wild-type olive-brown background body coloration (Fig. 1F). In this typical cross, F₁ males and females could have inherited the variegated pattern only from the GV male parent. Table 1A also shows one mating pair, PG3, which gave almost exclusively all male F₁ offspring (18 males and one female). This is due to the instability of the genetic mechanism of sex determination in the guppy since a large number of weak additive male and female sex genes present in the autosomes and X-chromosome may alter the balance of sex (Nayudu, 1979; Kirpichnikov, 1981). The collective effect of these genes may be more pronounced than that of the sex factors on the sex chromosomes, thus producing more male offspring and vice versa. Another mating, PG9, produced only WT males (Fig. 1E) and VAR females (Table 1A). This is attributed to the possibility that the GV male parent was heterozygous for the Var gene and possessed the recessive allele, Var⁺, on the Y-chromosome.

The F₂ generation comprised a total of 252 VAR males, 114 VAR females and 103 WT females (Fig. 1G, H) with the observed numbers agreeing with the expected 2:1:1 ratio (Table 1B). A slight exception occurred in mating PG4 where a higher number of VAR males caused significant difference at the 5% level but not at 1% (Table 1B). This was rectified when Yates' (1934) correction for continuity was applied. Three mating pairs (PG1, PG4 and PG5) gave eight exceptional males with WT body and tail coloration among the F₂ individuals (Table 1B). We speculate that the lack of variegated tail patterning in these males was due to crossing-over of the Var gene from the Y- to the X-chromosome in the F₁ VAR male parents. Since only the Var locus and sex-determining region (SdR) are considered in this study, the recombination frequency between Var and SdR is estimated to be 3.077%, i.e., eight WT male recombinants of a total 260 F₂ males (Table 1B). For mating pair PG9, the F₂ generation consisted of VAR males, WT males, VAR females and WT females (Fig. 1G, H) in a 1:1:1:1 expected ratio (Table 1B). In addition, homogeneity χ² values showed that the pooled F₁ and F₂ generations of PG1, PG4, PG5 and PG10 were uniform and did not form heterogeneous populations (Table 1A, 1B). χ² tests were not carried out for PG3 as the solitary F₁ female did not produce any F₂ progenies (Table 1B). The F₁ and F₂ results gave evidence that the putative genotype typical of GV males is X_VarY_Var while that of the heterozygous male of mating pair PG9 is X_VarY_Var⁺. A genetic model illustrating the segregation of the dominant Var gene is presented in Fig. 2.

Fig. 2

Schematic diagram of the proposed genetic models illustrating the segregation of the Var tail color pattern gene, phenotypes and genotypes of the parents (P), F₁ and F₂ progenies, and recombinants (R) that occur in reciprocal crosses between the Green Variegated (GV) and wild-type (WT) guppies. Mating pair PG9 in which the GV ♂♂ parent was heterozygous for the Var gene and its F₁ and F₂ progenies (indicated as⁹) are shown within thin borders to distinguish them from the typical GV ♂♂× WT♀♀cross.

Cross between WT males and GV females

Eight matings (PW1–PW8) of the reciprocal cross, WT♂♂ × GV♀♀, gave 23 F₁ broods consisting of 162 males and 168 females, all with variegated tail patterns (VAR phenotype) albeit fainter and less distinct in the females (Fig. 1E, F). The observed number of male to female offspring conformed to the expected 1:1 ratio (Table 2A). With the exception of three recombinant females with WT phenotype, the F₂ progeny of this cross segregated into 181 VAR males, 174 WT males and 387 VAR females (Fig. 1G, H) according to the 1:1:2 hypothetical ratio (Table 2B). The F₁ and F₂ progenies also formed homogeneous populations after their respective observations were pooled (Table 2A, 2B). The occurrence of three F₂ females for mating pairs PW1, PW5 and PW6 which had hyaline tails instead of the expected faint greyish-brown variegated patterns on a yellowish tail could be caused by crossing-over of the Var gene from the X- to the Y-chromosome in the F₁ VAR male parents (Table 2B). Since there were three WT female recombinants of a total of 390 F₂ females, the crossover frequency between the Var locus and SdR in this case is 0.769% (Table 2B). The F₁ and F₂ results therefore confirmed that the GV female parents used in this study were homozygous at the Var locus and possessed the genotype X_VarX_Var. The genetic model for the segregation of Var in this reciprocal cross is shown in Fig. 2.

The Var gene

Results of all parental (GV♂♂ × WT♀♀ and WT♂♂ × GV♀♀, Tables 1A, 2A) and full-sib (F₁♂♂ × F₁♀♀, Tables 1B, 2B) crosses indicate that the highly variable variegated tail pattern of the Green Variegated guppy strain is a simple sex-linked trait controlled by a single gene with two alleles, Var dominant for variegated tail pattern over Var⁺ recessive for wild-type which do not exhibit these patterns (Fig. 2). This is the first study that reports the expression of the variegated tail pattern gene, Var, was dominant in both sexes. Our results also show that the Var allele is able to cross over from the Y- to the X-chromosome and vice versa as male and female recombinants of the WT phenotype were obtained from the F₂ progenies of GV♂♂ × WT♀♀ and WT♂♂ × GV♀♀, respectively (Tables 1B, 2B). The phenomenon of alleles migrating between the X- and Y-chromosomes as a result of crossing-over was first documented in the guppy by Winge (1923, 1927) and Dzwillo (1959). This confirms that the sex chromosomes of the guppy share undifferentiated regions of homology along their chromatids in which pairing and crossing-over can occur (Winge, 1923, 1934; Winge and Ditlevsen, 1938; Dzwillo, 1959; Nayudu, 1975, 1979; Kirpichnikov, 1981).

A mean crossover frequency of 1.923 (±1.632)% was obtained (Tables 1B, 2B) between the Var locus and sex-determining region (SdR) in the Y-chromosome of male guppies. The phenotypic map distance was thus approximately 1.923 (±1.632) map units. Winge (1927), Winge and Ditlevsen (1947), and Kirpichnikov (1981) proposed that genes found within or close to the SdR are either alleles of one of the same locus (allelomorphic) or belong to the so-called supergene family, i.e., a group of closely located and tightly linked loci with completely suppressed crossing-over. Our results indicate that Var is most likely found in a homologous region about 1.923 map units from the SdR (Tables 1B, 2B). This suggests that Var is situated close to the SdR since crossing-over between other genes, e.g., Vitellinus (Vi) and Elongatus (El), and Doppelschwert (Ds) and Pigmentierte Caudalis (Cp) occur with a frequency of up to 10% (Winge, 1927, 1934; Winge and Ditlevsen, 1947; Dzwillo, 1959; Kirpichnikov, 1981; Purdom, 1993). As initiated by Winge (1927, 1934), and later Winge and Ditlevsen (1947), Dzwillo (1959), Nayudu (1975, 1979), Kirpichnikov (1981), Phang et al. (1989a, b, 1990), Phang and Fernando (1991), and Purdom (1993), the recombinant data for Var and SdR reported here should contribute to the mapping of additional color pattern genes onto the sex chromosomes of the guppy.

In conclusion, the variegated tail pattern gene, Var, of domesticated selected guppy strains (1) shows dominant expression, and is (2) both X- and Y-linked, (3) fully capable of crossing-over from the Y- to the X-chromosome and vice versa, and (4) located about 1.9 map units from the sex-determining region. The variegated tail pattern trait may thus prove valuable as a genetic marker in linkage analyses and investigations of color patterns in the guppy.