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.
The guppy, Poecilia reticulata Peters, is a fresh- and brackish-water ovoviviparous poecilid fish native to Trinidad, Barbados, Venezuela, Guyana and North-Eastern Brazil (Haskins and Haskins, 1951; Yamamoto, 1975). This fish is known for its striking sexual dimorphism. Wild-type males are smaller than females and their anal fin is modified into a copulatory organ called the gonopodium. The complex polymorphic spots and patches of color on the body and fins of wild guppies are expressed only by sexually mature males. The female guppy is devoid of color patterns, being a uniform olive-brown with hyaline fins (Haskins and Haskins, 1951). The wild-type guppy was introduced into Singapore and other parts of South-East Asia in the late 1930s for mosquito control (Herre, 1940).
The guppy became popular among aquarists and hobbyists who developed many exotic strains by intensive selection of spontaneous mutant genes that affect the coloration as well as the shape and size of the body and fins (Dzwillo, 1959; Kirpichnikov, 1981; Fernando and Phang, 1985). In Singapore, commercial culture of fancy guppy strains began in the early 1950s. About 30–40 different strains are reared in monoculture farms (Fernando and Phang, 1985). The guppy plays an important role in the ornamental fish industry of Singapore.
The guppy is unique among other teleosts in that almost all the genes encoding for color patterns are sex-linked and sex-limited. There are 23 pairs of chromosomes in the guppy, 22 of which are autosomal and one the sex chromosomes. Male guppies are heterogametic (XY) while the females are homogametic (XX) (Winge, 1922a, b; Winge and Ditlevsen, 1947). It is the first species known to have Y-linked inheritance of color genes (Schmidt, 1920). Kirpichnikov (1981) documented 17 Y-linked genes that are only passed from father to son (one-sided masculine inheritance), 15 that are X- and Y-linked (found in both males and females but expressed only in males as they are sex-limited and hormone-mediated), and one that is autosomal dominant. Several of these genes, e.g., Maculatus (Ma), Armatus (Ar) and Pauper (Pa) influence sex determination in the guppy (Schmidt, 1920; Winge, 1922a,b, 1927, 1934; Winge and Ditlevsen, 1947). These are usually located close to or within a sex-determining region (designated as SdR) on the Y-chromosome, and are presumed to be tightly linked with a gene for maleness (Winge, 1927, 1934; Winge and Ditlevsen, 1947; Kirpichnikov, 1981). The SdR may also represent, by itself, a dominant factor for male-determination and possibly has a recessive female-determining region at a similar position on the X-chromosome. Genes for background body coloration such as blond (bb), gold (gg), albino (aa) and blue (blbl) are, however, autosomally inherited and recessive to their wild-type alleles (Haskins and Druzba, 1938; Goodrich et al., 1944, 1947; Kirpichnikov, 1981).
Color patterns on the body and fins of domesticated guppies take the form of single colors, snakeskin-like reticulations and variegated mosaic patterns of two or more colors (Nayudu, 1975, 1979; Kirpichnikov, 1981; Fernando and Phang, 1989, 1990; Phang et al., 1989a,b, 1990; Phang and Fernando, 1991; Khoo et al., 1999). The ease with which new strains can be developed from spontaneous mutation makes the guppy a suitable species for investigating the genetic control of color polymorphism (Dzwillo, 1959; Yamamoto, 1975; Nayudu, 1979; Kirpichnikov, 1981; Fernando and Phang, 1985). Expression of phenotypic color patterns in cultured strains has been found to be determined by dominant X- and Y-linked genes (Dzwillo, 1959; Nayudu, 1975, 1979; Kirpichnikov, 1981; Fernando and Phang, 1989, 1990; Phang et al., 1989a, b, 1990; Phang and Fernando, 1991). These genes may consequently be used as genetic (phenotypic) markers to map the sex chromosomes of the guppy (Winge, 1927, 1934; Winge and Ditlevsen, 1947; Nayudu, 1975, 1979; Kirpichnikov, 1981; Purdom, 1993).
Two color patterns of the Tuxedo guppy strain, namely, black caudal-peduncle and red tail, were genetically investigated in this study. The loci for the black caudal-peduncle and red tail genes on the sex chromosomes are proposed to be Bcp and Rdt, respectively. Genetic map distances of these genes from the sex-determining region were determined from recombination rates. Our study forms part of an on-going effort to link color pattern genes to the X- and Y-chromosomes and autosomes of the guppy using phenotypic markers (Winge, 1927, 1934; Winge and Ditlevsen, 1947; Dzwillo, 1959; Nayudu, 1975, 1979; Kirpichnikov, 1981; Fernando and Phang, 1989, 1990; Phang et al., 1989a,b, 1990; Phang and Fernando, 1991; Purdom, 1993; Khoo et al., 1999) and recently, molecular markers by Random Amplified Polymorphic DNA (RAPD) fingerprinting (Foo et al., 1995).
MATERIALS AND METHODS
Source of the fish
Three- to four-week old fry of the Tuxedo (TUX) guppy strain were obtained from Chin Lam Brothers Tropical Fish Farm in Singapore. The common name, Tuxedo, for this strain was given by guppy breeders. Wild-type (WT) feral guppies were collected from an isolated hill-stream near the Bukit Timah nature reserve in Singapore. Juvenile TUX 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 are detected by gonopodial formation of the anal fin. Males, when spotted, were immediately removed and reared separately from females as virgin females were essential for the reciprocal crosses.
Description of the fish
Adult males and females of the TUX strain have a total length of 3–4 cm and 5–6 cm, respectively. Adult TUX males have black (melanic) or dark grey pigmentation on the caudal-peduncle region which masks normal wild-type male body coloration, and a caudal fin that ranges from blood-red to orange-red in color (Fig. 1A). Some TUX males may have a metallic blue or green sheen overlying the black caudal-peduncle. TUX females show drab wild-type olive-brown body coloration and grey caudal-peduncle with red tinges of varying intensity on an opaque greyish-white tail (Fig. 1B). Wild-type guppies are smaller than the domesticated TUX strain. Adult WT males are 2–2.5 cm in length and 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 are devoid of color patterns (Fig. 1D).
Inheritance of the black caudal-peduncle and red tail color patterns was elucidated by single-pair reciprocal crosses between the TUX 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: TUX♂♂×WT♀♀(Table 1A) and WT♂♂×TUX♀♀(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). All F1 and F2 progenies were segregated and scored according to phenotypes and sex. Progenies displaying color patterns such as Tuxedo, red tail and black caudalpeduncle were designated as the TUX, RT and BCP phenotypes, respectively, and those without such color patterns, WT phenotype. Tuxedo males of parental crosses were typed using Roman numerals (I, II, III, IV and V) according to their putative alleles following segregation of their F1 and F2 progenies. This was to facilitate description of the crosses.
Mating results of crosses between Tuxedo (TUX) males and wild-type (WT) females 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 (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 Rdt and Bcp genes were not considered in chi-square analyses. (Phenotypes: TUX♂♂=Tuxedo males with black caudal-peduncle and red tail; TUX♀♀=Tuxedo females with grey caudal-peduncle and faint red tinges on an opaque greyish-white tail; RT = red tail without black caudal-peduncle; BCP=black caudal-peduncle without red tail; WT = wild-type coloration without red tail and black caudal-peduncle. Genes: Bcp=black caudal-peduncle gene; Bcp+=absence of black caudal-peduncle gene; Rdt=red tail gene; Rdt+=absence of red tail gene). A. TUX♂♂×WT♀♀ (Parental Cross)
Mating results of crosses between wild-type (WT) males and Tuxedo (TUX) females 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 (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 Rdt and Bcp genes were not considered in chi-square analyses. (Phenotypes: TUX♂♂= Tuxedo males with black caudal-peduncle and red tail; TUX♀♀= Tuxedo females with grey caudal-peduncle and faint red tinges on an opaque greyish-white tail; RT = red tail without black caudal-peduncle; BCP = black caudal-peduncle without red tail; WT = wild-type coloration without red tail and black caudal-peduncle. Genes: Bcp = black caudal-peduncle gene; Bcp+ = absence of black caudal-peduncle gene; Rdt = red tail gene; Rdt+ = absence of red tail gene).
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 χ2adj 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 χ2adj values were not additive (Sokal and Rohlf, 1981; Strickberger, 1990). Following Winge (1922b, 1923, 1927, 1934), Nayudu (1979), Phang et al. (1989a, b, 1990), Phang and Fernando (1991), and Khoo et al. (1999), individuals with exceptional coloration due to crossing-over of the black caudal-peduncle (Bcp) and red tail (Rdt) genes between the X- and Y-chromosomes were not considered in chi-square analyses.
Recombination frequencies and map distances between Bcp and Rdt, and the sex-determining region (SdR) were estimated according to Strickberger (1990), Phang et al. (1990), Phang and Fernando (1991), Purdom (1993) and Khoo et al. (1999). Winge's (1922b, 1927, 1934) “zig-zag line diagram” method was applied to test all possible linkage combinations between Bcp, Rdt and SdR, and map these loci in sequential order. Due to the infrequency of single crossing-over in the guppy compared to the fruitfly, Drosophila (Winge, 1927, 1934; Purdom, 1993), double crossing-over was excluded from calculations of map distances. All crossovers in this study were thus regarded as single crossovers.
Segregation and recombination in TUX × WT F1 and F2 offspring
Three mating pairs TUX♂♂×WT♀♀(PT5, PT9 and PT10) produced 40 male and 34 female F1 offspring in 8 broods (Table 1A). F1 males exhibited the black caudal-peduncle and red tail color patterns of their TUX male parents (Fig. 1E), while F1 females had a grey caudal-peduncle and an opaque greyish-white tail (Fig. 1F). F1 males and females could have inherited the black caudal-peduncle and red tail color genes only from their TUX male parents (designated as type I). Table 1A also shows three other crosses in which the TUX male parents were heterozygous for black caudal-peduncle and red tail. To facilitate description of these crosses and their offspring, these TUX males were labelled as types III, IV and V. Type II TUX males were not observed in this study although they were found among crosses between the Tuxedo and Green Variegated guppy strains that we carried out in a later study (Khoo et al., submitted). For type III, five mating pairs gave 18 broods of 116 red tail (RT) males and 130 TUX females (Fig. 1E, F, Table 1A). Four F1 broods of 33 WT males and 35 TUX females were produced by the cross between a type IV TUX male and a WT female (mating pair PT1), while 25 BCP males and 29 TUX females were obtained from mating pair PT6 (type V TUX male) (Fig. 1E, F, Table 1A). For all four types (I, III, IV and V) of TUX male parents, the F1 male to female ratio was consistent with the expected ratio of 1:1 (Table 1A).
The F2 generation of type I comprised 205 TUX males, 113 TUX females and 105 WT females with the observed numbers conforming to the expected phenotypic ratio of 2:1:1 (Fig. 1E, F, Table 1B). Four F2 phenotypes that consisted of 108 TUX and 121 RT males, and 120 TUX and 113 WT females were obtained from 11 single-pair full-sib F1 crosses of type III (Fig. 1E, F, Table 1B). These phenotypes fitted the hypothetical ratio of 1:1:1:1. Mating pair PT1 (type IV) also gave four F2 phenotypes that were made up of 13 broods consisting of 61 TUX and 54 WT males, and 58 TUX and 48 WT females that agreed with the 1:1:1:1 ratio (Fig. 1E, F, Table 1B). For mating pair PT6 (type V), two full-sib F1 crosses produced nine broods of 64 TUX males, 65 BCP males, 54 TUX females and 60 WT females that corresponded to the expected 1:1:1:1 ratio (Fig. 1E, F, Table 1B). Homogeneity χ2 tests carried out for TUX males of types I and III showed that the F1 and F2 observations were uniform and did not form heterogeneous populations after being pooled (Table 1A, B).
F1 and F2 data for TUX♂♂×WT♀♀showed that the dominantly expressed black caudal-peduncle and red tail color patterns are due to single genes that are found at two different loci, Bcp and Rdt, respectively, on the sex chromosomes. Homozygous TUX male parents (type I) were elucidated to have the XBcp,RdtYBcp,Rdt genotype (Table 1, Fig. 2). When TUX males were heterozygous at the Bcp and Rdt loci, these individuals possessed the XBcp,RdtYBcp+,Rdt (type III), XBcp,RdtYBcp+,Rdt+ (type IV) and XBcp,RdtYBcp,Rdt+ (type V) genotypes (Table 1, Fig. 2). The segregation of the Bcp and Rdt alleles in the TUX♂♂ ×WT♀♀cross is presented schematically by a genetic model in Fig. 2.
Crossing-over between the sex-determining region (SdR), and the black caudal-peduncle (Bcp) and red tail (Rdt) loci at the parental and F1 levels for TUX♂♂×WT♀♀produced exceptionally colored F1 and F2 recombinants (Table 1). These individuals which expressed TUX, RT, BCP and WT phenotypic colorations in males, and RT and BCP in females, deviated from the predicted phenotypes for types I, III, IV and V. Type I produced three RT and three WT recombinant F2 males (Fig. 1E, Table 1B). The crossover frequency calculated from the percentage of crossover males out of the total number of F2 males (3/211×100%) was 1.422% for SdR–Rdt and Rdt–Bcp, respectively. Occurrence of RT recombinant F2 males indicated that the Y-chromosome of the guppy may possibly have a map order of SdR–Rdt–Bcp as these recombinants could not be produced using Winge's (1922b, 1927, 1934) “zig-zag line diagram” method if the gene order had been either SdR–Bcp–Rdt or Bcp–SdR–Rdt (Figs. 2, 3). A map distance of 1.802 map units was estimated for Rdt–Bcp from two RT and two BCP recombinant F2 females of 222 F2 females for type I (Fig. 1G, Table 1B). Recombinant F1 TUX males and F2 RT females of type III were not used to estimate map distances because crossovers could have taken place at SdR–Rdt and Rdt–Bcp, making it impossible to determine the actual region of crossover. Of 240 F2 females of this cross, three BCP female recombinants gave a crossover frequency of 1.250% between Rdt and Bcp (Fig. 1G, Table 1B).
For mating pair PT1 (type IV), 5.405 map units were estimated to separate the Rdt locus from SdR and Rdt from Bcp, respectively (Table 1A). Longer SdR–Rdt and Rdt–Bcp map distances for type IV compared to types I and III was due to frequent crossing-over which yielded two TUX and two BCP recombinant F1 males out of only 37 F1 males (Fig. 1E, Table 1A). The presence of F1 BCP male recombinants in this cross also indicated a gene sequence of SdR–Rdt–Bcp (Figs. 2, 3). An F1 BCP recombinant female of 30 F1 females for mating pair PT6 (type V) gave a crossover rate of 3.333% between SdR and Rdt (Fig. 1G, Table 1A). Seven F2 recombinant males that had WT coloration of 136 F2 males of PT6 further supported an order of SdR–Rdt–Bcp, giving an approximate distance of 5.147 map units between SdR and Bcp (Fig. 1E, Table 1B). The mean genetic map distance between the SdR and Rdt, calculated from F1 and F2 recombinant data (Table 1), was 3.387±1.992 map units while the Bcp locus appeared to be about 5.147 map units from the SdR (Fig. 3).
Segregation and recombination in WT×TUX F1 and F2 offspring
Three mating pairs (PB2, PB5 and PB6) of the reciprocal cross, WT♂♂×TUX♀♀, gave nine F1 broods of 69 males and 82 females, all of which had black caudal-peduncle and red tail color patterns typical of the Tuxedo (TUX) phenotype (Fig. 1E, F). The observed numbers of F1 male to female offspring agreed with the expected ratio of 1:1 (Table 2A). With the exception of four males with BCP phenotype, the F2 progeny of this cross segregated into 93 TUX males, 93 WT males and 183 TUX females according to the expected phenotypic ratio of 1:1:2 (Fig. 1E, F, Table 2B). Results for mating pair PB4 were not included with those of the above three mating pairs because it produced a very high number of F2 recombinants (11 BCP males and 16 BCP females) (Table 2B). PB4, however, satisfied the expected F1 male to female ratio of 1:1 with 26 TUX males and 29 TUX females (Table 2A). In addition, three full-sib single-pair F1 crosses of PB4 generated 63 TUX males, 51 WT males and 105 TUX females that were consistent with the F2 phenotypic ratio of 1:1:2 (Table 2B). Tuxedo females of mating pairs PB1 and PB3 were inferred to be heterozygous at the Bcp locus since they gave a total of 24 TUX males, 19 RT males, 18 TUX females and 19 RT females that conformed to the expected F1 ratio of 1:1:1:1 (Table 2A). After pooling of the F1 and F2 data for WT♂♂× TUX♀♀, homogeneity χ2 tests showed that each pooled population was homogeneous and uniform (Table 2A, B).
F1 and F2 results for WT♂♂×TUX♀♀confirmed the observations of the reciprocal cross (TUX♂♂×WT♀♀) that two single sex-linked genes, black caudal-peduncle (Bcp) and red tail (Rdt), are responsible for the dominant expression of the Tuxedo color pattern. TUX female parents (including PB4) used in this study were found to be homozygous for Bcp and Rdt, and had the XBcp,RdtXBcp,Rdt genotype (Table 2, Fig. 2). Conversely, the putative genotype for heterozygous TUX females of mating pairs PB1 and PB3 was XBcp,RdtXBcp+, Rdt (Table 2, Fig. 2). The segregation of Bcp and Rdt is illustrated by a genetic model in Fig. 2.
Four F2 males of mating pair PB2 had a black caudal-peduncle and black tail instead of a red tail (Fig. 1E, Table 2B). In this instance, Bcp could have crossed over from the X- to the Y-chromosome in the F1 male parent, and also from Y to X in the F1 female parent to produce male BCP recombinants (Fig. 2). Since there were four BCP males of a total 190 F2 male individuals, the crossover frequency between Bcp and Rdt was estimated to be 2.105% (2.105 map units) (Table 2B). As mentioned earlier, mating pair PB4 produced a very high number of F2 BCP male (11) and female (16) recombinants out of 125 F2 males and 121 F2 females (Table 2B, Fig. 2). From these crossover data, the map distance between Bcp and Rdt was averaged to be 11.012±3.128 map units. Simultaneous occurrence of BCP males and females suggested a gene order of SdR–Bcp–Rdt. These results contradicted those of the reciprocal cross (TUX♂♂×WT♀♀) which pointed to a map order of SdR–Rdt–Bcp. Moreover, Bcp and Rdt seemed to be too far apart (11.012± 3.128 map units) for PB4 compared to the 2.397±1.714 map units estimated from mating pairs PB2, PB5 and PB6, and TUX♂♂×WT♀♀ (Tables 1, 2, Fig. 3).
Inheritance of the black caudal-peduncle and red tail color patterns
Observations for all parental (TUX♂♂×WT♀♀and WT ♂♂×TUX♀♀, Tables 1A, 2A) and full-sib (F1♂♂×F1♀♀, Tables 1B, 2B) crosses indicate that the black caudal-peduncle (Bcp) and red tail (Rdt) color pattern genes are responsible for the Tuxedo phenotype of the guppy. This study demonstrates that these two color patterns are simple sex-linked traits controlled by single genes: the Bcp allele is dominant for black caudal-peduncle over Bcp+, and Rdt is dominant for red tail over Rdt+. Wild-type or feral guppies do not display these color patterns as they have the recessive Bcp+ and Rdt+ genes (Fig. 1C, D). Using Tuxedo males that were heterozygous for Bcp and Rdt, we have also shown that the expression of Bcp and Rdt is dominant in both males and females (Tables 1, 2, Figs. 1, 2). This study confirms our preliminary findings that the black caudal-peduncle and red tail colour genes have a dominant mode of inheritance (Fernando and Phang, 1989, 1990, Phang et al., 1990). The typical genotypes for the Tuxedo guppy are thus inferred to be XBcp,RdtYBcp,Rdt for males (type I) and XBcp,RdtXBcp,Rdt for females. Tuxedo males that are heterozygous have the XBcp,RdtYBcp+,Rdt (type III), XBcp,RdtYBcp+,Rdt+ (type IV) and XBcp,RdtYBcp,Rdt + (type V) genotypes while the females are XBcp,RdtXBcp+,Rdt. The presence of these heterozygous individuals suggests that the black caudal-peduncle and red tail color patterns have not yet become “fixed” traits among cultured stocks of the Tuxedo strain.
Phenotypic map of Bcp, Rdt and the SdR
Our results prove that the Bcp and Rdt alleles are able to cross over from the X- to the Y-chromosome and vice versa since male and female recombinants of the TUX, RT, BCP and WT phenotypes were obtained from both F1 and F2 offspring of TUX♂♂×WT♀♀(types I, III, IV and V) and WT♂ ♂×TUX♀♀(Tables 1, 2, Figs. 1, 2). 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 (1922a, b, 1923). Subsequent analyses by Winge (1927, 1934), Winge and Ditlevsen (1938), Dzwillo (1959), Nayudu (1975, 1979) and Kirpichnikov (1981) showed that the X- and Y-chromosomes of the guppy are cytologically indistinguishable and possibly share undifferentiated homologous regions along the lengths of their chromatids. Therefore, crossing-over occurs between the sex chromosomes, and recombination rates of up to 10% have been recorded between the Vitellinus (Vi) and Elongatus (El), and Doppelschwert (Ds) and Pigmentierte caudalis (Cp) genes (Winge, 1927, 1934; Winge and Ditlevsen, 1947; Dzwillo, 1959; Kirpichnikov, 1981; Purdom 1993). In our study, the occurrence of recombination shows that Bcp and Rdt are located within homologous regions on the X- and Y-chromosomes, and are approximately 5.147 and 3.387±1.992 map units, respectively, away from the sex-determining region (SdR) (Tables 1, 2, Fig. 3).
From the F1 and F2 recombinant data (Tables 1, 2), genetic map distances of 3.387±1.992, 5.147 and 2.397±1.714 map units were obtained for SdR–Rdt, SdR–Bcp and Rdt–Bcp, respectively (Fig. 3). As expected, the map distance between SdR and Bcp (5.147 map units) is close to the sum of distances (3.387+2.397=5.784 map units) for SdR–Rdt and Rdt–Bcp (Fig. 3). Estimates for crossing-over between two loci that are far apart are, however, never exactly the sum of the estimates for smaller regions amidst them (Purdom, 1993). This is because a crossover between two loci usually inhibits a second crossover from occurring in an adjacent region (Strickberger, 1990). Crossing-over at SdR–Bcp, SdR–Rdt and Rdt–Bcp will thus influence each other, thereby affecting their frequency of occurrence. Despite this, our results show that Rdt is closer to SdR than Bcp because lower crossover frequencies (shorter map distances) were obtained between Rdt and SdR than from Bcp to SdR for all crosses (Tables 1, 2). Recombinant data for Tuxedo male parents of types I, IV and V also provides evidence that Rdt lies between the SdR and Bcp, hence indicating a gene map order of SdR–Rdt–Bcp (Fig. 3). The exception is mating pair PB4 which suggests a sequence of SdR–Bcp–Rdt. The latter may possibly result from translocation of a segment of the sex chromosomes that contain at least one of these color genes onto another, or pericentric inversion of a region that has two of these genes (Purdom, 1993).
In conclusion, the black caudal-peduncle (Bcp) and red tail (Rdt) genes of the domesticated Tuxedo guppy strain (1) occur as single genes at two different loci, and are (2) dominantly expressed, (3) X- and Y-linked, and (4) fully capable of crossing-over from the Y- to the X-chromosome and vice versa since they are situated in an undifferentiated homologous region on these chromosomes. Genetic map distances for the sex-determining region (SdR)–Rdt, SdR–Bcp and Rdt–Bcp are estimated to be 3.4, 5.1 and 2.4 map units, respectively. We therefore propose a phenotypic map of SdR–Rdt–Bcp (Fig. 3) for the Y-chromosome of the guppy. The Bcp and Rdt loci are also inferred to be at similar positions on the X-chromosome that was postulated by Winge (1927, 1934) to have a corresponding feminine segment of the SdR (Fig. 3). Our findings for the black caudal-peduncle and red tail genes, and the sex-determining region should prove valuable for the mapping of color pattern genes onto the sex chromosomes of the guppy as initiated by Winge (1927, 1934), and carried on by Winge and Ditlevsen (1947), Dzwillo (1959), Nayudu (1975, 1979), Kirpichnikov (1981), Fernando and Phang (1989, 1990), Phang et al. (1989a, b, 1990), Phang and Fernando (1991), Purdom (1993) and Khoo et al. (1999).
This project was funded by research grants from the European Commission (CI1*-CT94-0021) and National University of Singapore (RP3972366 and RP3981303) to V. P. E. Phang (P. I.), W. -K. Chan and T. M. Lim.