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Inheritance of white, black and brown coat colours in alpaca (Vicuna pacos L.)
Small Ruminant Research 99 (2011) 16–19
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Inheritance of white, black and brown coat colours in alpaca (Vicuna pacos L.) A. Valbonesi a,∗ , N. Apaza b , V. La Manna a , M.L. Gonzales b , T. Huanca b , C. Renieri a a b
School of Environmental Sciences, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy Instituto Nacional de Innovacion Agraria (INIA) ILLPA PUNO, Puno, Peru
a r t i c l e
i n f o
Article history: Received 18 January 2011 Received in revised form 8 April 2011 Accepted 11 April 2011 Available online 10 May 2011 Keywords: Alpaca Coat colour Segregation analysis
a b s t r a c t An experimental trial of the segregation of white vs. pigmented and black vs. brown colours in alpacas was conducted at the Peruvian INIA Quimsachata Experimental Station. One hundred and forty five offspring were born from the following matings: 4 white sires × 36 white dams, 4 white sires × 39 pigmented dams, and 9 pigmented sires × 70 pigmented dams. Among these last matings were, 4 black sires × 25 black dams, 2 black sires × 20 brown dams, and 3 brown sires × 25 brown dams. Statistical tests validate that the inheritance of white is due to a single gene which is dominant over pigmentation, without any modifying effect and independent of segregation of black and brown patterns. However, the evidence does not support a simple dominant inheritance of the black vs. brown. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Coat colour is an important selection objective in alpaca (Vicuna pacos). In textile production, full white for dyeing and natural coloured products are both in strong demand. As companion animals, alpacas are bred in a very large variety of colour phenotypes (Lauvergne et al., 1995; Sponenberg, 2006). The list of colour variations includes six pigmentary patterns (wild, red, fawn with dark extremities, badger face, black and tan, and non agouti), two eumelanic types (black and brown), three alterations (full white, grey and dilution) and some possible white spotting patterns (Lauvergne et al., 1995). A scheme for classification was recently defined by ICAR (Renieri et al., 2005; Antonini, 2009). Many genetic systems for interpreting coat colour have been proposed (for a review, see Renieri, 1995). A complete up-to-date system was proposed by Sponenberg (2006). Few designed experiments on phenotype segregation analysis have been carried out and the relationship among
phenotypes becomes often controversial (Toledo and San Martin, 1948; Bustina, 1968; Gandarillas, 1971; Velasco et al., 1978a,b; Valbonesi et al., 2009). White phenotype is dominant for Bustina (1968) and recessive for Gandarillas (1971). Black can be dominant or recessive on brown (Gandarillas, 1971; Velasco et al., 1978a). Finally, spotting seems recessive to solid colour (Velasco et al., 1978a,b). Conservation and diffusion of coloured alpaca is one of the strategic research purposes of the Peruvian National Institute of Agronomic Innovation (Instituto Nacional de Innovación Agraria, INIA, 2011). Several coloured alpaca lines are reared in ILLPA Puno Quimsachata Station, which is the genetic conservation centre. In order to define the inheritance of the main coloured phenotypes, INIA and the School of Environmental Sciences of the University of Camerino, Italy, designed a hierarchical experimental trial involving two segregations: white vs. pigmented animal, and, among the pigmented, black vs. brown animals. 2. Materials and methods 2.1. Experimental station
∗ Corresponding author. Tel.: +39 0737 403452; fax: +39 0737 403446. E-mail address: [email protected] (A. Valbonesi). 0921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2011.04.003
The planned crosses were carried out at the experimental station of the INIA (the Peruvian National Institute for Agronomic Innovation)
A. Valbonesi et al. / Small Ruminant Research 99 (2011) 16–19 located in Quimsachata, Peru. The station, 6282 ha situated at about 4400 m above sea level in the Santa Lucia District of the Puno Department, is committed to alpaca and llama breeding, conservation and genetic improvement. The ecosystem is grassland, typical of the Andean highlands.
Table 1 Crosses involving full white sires and dams. Rams
Dams (n)
2.3. Segregation analysis Segregation analysis was applied only to segregating families which showed at least one proband, i.e. an individual supposed recessive among the offspring (truncate selection). The tested monofactorial hypotheses were the dominance of the white phenotype on the pigmented and of the black phenotype on the brown. The goodness of fit for the hypotheses was evaluated by means of the maximum-likelihood estimate (MLE) method, i.e. finding the values for the parameters involved in the segregation (i.e. segregation ratio and/or heterozygous frequency), that make the observed values in our set of data seem most probable. The probability Pri (r, s, p) of observing r (r ≥ 1) offspring of a given phenotype, among s total offspring of a cross i, given the probability p resulting from the segregation hypothesis under investigation, was calculated on the logarithmic value of the binomial truncate distribution. The total loglikelihood (L) of a set of mating (C) was then calculated as: L(C) = ˙i ln[Pri (r, s, p)] The probability p has in the present context the meaning of either a segregation ratio or a heterozygous frequency, according to the mating under investigation, for which the symbol R or H, is preferable. MLE of R or H, was obtained by maximizing the total loglikelihood (Lmax ) as a function of R or H, using the solver routine of Excel. The 95% confidence limits (C.L.) of MLE were obtained by finding (again using solver), the two loglikelihood values, L1 and L2 , for which the values −2(L1 − Lmax ) and −2(L2 − Lmax ) were = 5.0238 (i.e. the chi square value with 1 degree of freedom and associated probability = 0.025). The fitting between the value of R or H, postulated according to the segregation hypothesis under investigation, and that resulting from the MLE method, was evaluated by multiplying by a factor of −2 the difference between the L(C) and (Lmax ), so that this quantity will be distributed as the chi square statistic with 1 degree of freedom. Tests were made at the 5% level. In order to confirm the autosomal hypothesis, the segregations were tested for independence of sex chromosome segregation. Segregation data were displayed in the form of a two-way table (contingency table), and the test applied for this table was the G-test of independence with Williams’s correction (Sokal and Rohlf, 2003).
3. Results Table 1 illustrates the results from crosses between four full white sires and 36 full white dams. Because the white
Ln(btd)a
Offspring White
♀
2.2. Animals The available offspring consisted of 145 crias (80 female and 65 males), coming out from mating of 17 sires and 145 dams. The cria born from the following mating: 4 white sires × 36 white dams, 4 white sires × 39 pigmented dams, and 9 pigmented sires × 70 pigmented dams. Among these last matings, 4 black sires were crossed with 25 black dams, 2 black sires with 20 brown dams and 3 brown sires with 25 brown dams. The parents were selected among white, black and brown lines reared at the Quimsachata Station. The brown phenotype includes different patterns with different genetic background (Sponenberg, 2006). The brown considered in this experiment is the most common in the alpaca species. It is a reddish brown pattern, with different Munsel values and chroma notations and with a relatively high amount of pheomelanins (Cecchi et al., 2011). It is completely different from the dark reddish brown pattern, rarely described in alpaca, characterized by a very low amount of pheomelanins and a high amount of brown eumelanins (Cecchi et al., 2011). The reddish brown of the experimental trial is therefore an allelic alternative pattern to uniform black and also differs genetically from the dark reddish brown that expresses the same pattern of black with the substitution of the type of eumelanins from black to brown (Lauvergne et al., 1995; Sponenberg, 2006).
17
Pigmented
♂
♀
♂
443303 058104 1199-M 148102b
8 10 9 9
3 6 4 8
2 3 2 1
0 0 3 0
3 1 0 0
−1.529 −1.495 −1.423
Total
36
21
8
3
4
Lmax c = −4.447
a
Logarithmic values of the binomial truncate distribution, resulting from both the segregation hypothesis (R = 0.25), and the maximumlikelihood estimate of the heterozygous frequency (MLE = 0.94) of the dams involved in the crosses. b Sire not included in the segregation analysis because did not segregate any proband. c Total loglikelihood resulting from the MLE method.
phenotype is supposed to be dominant over the pigmented one, the occurrence of both the phenotypes among the F1 progeny of each male can be explained by the presence of heterozygous male and either heterozygous or homozygous female. As only one offspring can be obtained annually from an alpaca female, the observed segregation ratio for a number of females of unknown genotype is a function of two parameters: (i) the probability that a heterozygous animal generates a pigmented offspring (i.e. the Mendelian transmission probability) and (ii) the probability that an animal is heterozygous. Most value combinations of these two parameters produce the same value of the maximum likelihood. Therefore, a practical approach could be to estimate the value that each parameter assumes, once the other parameter is fixed on a value representing a reasonable choice. By assuming that the segregation ratio is 0.25 (the probability that a heterozygous white female mated to a heterozygous white male produces a pigmented offspring for a simple recessive genetic model), the maximum likelihood estimate of the heterozygous dams involved in the crosses is 0.94, with 95% C.L. = 0.29–1.00. The results from crosses between four full white sires and 39 pigmented dams are shown in Table 2. The segregation hypothesis of 1 white and 1 pigmented individual was tested. The maximum likelihood estimate of the recessive (pigmented) frequency was 0.563, with 95% C.L. = 0.38–0.73. This frequency does not differ significantly from that expected of 0.50 (−2[L(C) − Lmax ] = 0.620; P = 0.431). The single gene hypothesis of black phenotype dominant on the brown one was tested throughout three types of crosses. Table 3 reports the results from crosses between four full black sires and 25 full black dams. The occurrence of both the phenotypes among the F1 progeny of each male can be explained by assuming what has been postulated for the dominance of white over pigmented. It thus results that for observing a segregation ratio of 0.25 (the probability that a heterozygous black female mated to a heterozygous black male produces a brown offspring for a simple recessive genetic model), the maximum likelihood estimate of the heterozygous dams involved in the crosses is 1.00, with 95% C.L. lower limit of 0.46.
18
A. Valbonesi et al. / Small Ruminant Research 99 (2011) 16–19
Table 2 Crosses between full white sires and pigmented dams. Rams
Dams (n)
Observed offspring White
Ln(btd)a
Ln(btd)b
Pigmented
♀
♂
♀
♂
SO-502 EEI-025 322203 370397
9 15 9 6
2 1 6 3
0 4 0 1
5 6 1 1
2 4 2 1
−2.653 −2.390 −1.806 −1.435
−2.090 −1.874 −2.262 −1.747
Total
39
12
5
13
9
L(C)c = −8.284
Lmax d = −7.973
a b c d
Logarithmic values of the binomial truncate distribution resulting from the segregation hypothesis (R = 0.50). Logarithmic values of the binomial truncate distribution resulting from the maximum-likelihood estimate of the segregation ratio (MLE = 0.563). Total loglikelihood resulting from the expected segregation ratio. Total loglikelihood resulting from the MLE method.
Table 3 Crosses involving full black sires and dams. Rams
Dams (n)
Ln(btd)a
Offspring Black
237204 244203 095101 035104 Total
types, the segregations were tested for independence of sex chromosome segregation. The G-test of independence, applied to the segregation data related to crosses between white sires and both white and pigmented dams, was clearly not significant (G = 2.084, P = 0.149). Similar results were obtained for crosses between black sires and both black and brown dams (G = 0.516, P = 0.472).
Brown
♀
♂
♀
♂
6 7 6 6
0 3 3 1
3 2 1 3
1 2 2 0
2 0 0 2
−1.830 −1.023 −1.019 −1.019
25
7
9
5
4
Lmax b = −4.891
4. Discussion and conclusion
a
Logarithmic values of the binomial truncate distribution, resulting from both the segregation hypothesis (R = 0.25), and the maximumlikelihood estimate of the heterozygous frequency (MLE = 1.00) of the dams involved in the crosses. b Total loglikelihood resulting from the MLE method.
The results from the crosses involving two full black sires and 20 brown dams are shown in Table 4. The segregation analysis was not carried out because of the low number of crias (sire 366203) or the lack of segregation (sire EEI – 024). Nevertheless it should be noted that, in the latter cross, the occurrence of an offspring of 15 crias all exhibiting a supposed recessive coat colour (i.e. brown) is an extremely rare event, characterized by a frequency, in a binomial distribution of 3.05 × 10−6 . Lastly, three brown sires were mated with 25 brown dams. All the resulting offspring were brown, in full agreement with what is expected from crosses involving recessive parents, but also consistent with a dominant allele if all animals are homozygous for it. Assuming a single gene two-allele genetic model for both the white/pigmented and the black/brown phenoTable 4 Crosses between full black sires and brown dams. Rams
Dams (n)
Observed offspring Black
Brown
♀
♂
♀
♂
366203 EEI-024a
5 15
1 0
1 0
2 8
1 7
Total
20
1
1
10
8
a
Sire not included in the segregation analysis because did not segregate.
In our experimental model the inheritance of white is due to a single gene segregation, without any modifying effect. It is independent and completely dominant over pigmented animals, without any difference in segregation on black and brown colours. This hypothesis is fully supported by the segregations observed in crosses involving white sires and pigmented dams, as well as in crosses of white parents, assuming that the frequency of heterozygous females ranges from 29% up to 100%. The result is in agreement with the conclusion of Bustina (1968). The Gandarillas hypothesis of white recessivity can be justified assuming that the white defined by him is probably due to the spotting segregation or to another recessive mechanism. White in mammals arises from improper melanoblast development or survival, reflecting absence of mature melanocytes. White can be caused by defects at various stages of melanocytes development, including proliferation, survival, migration, invasion of the integument, hair follicle entry and melanocytes stem cell renewal (Bennett and Lynn Lamoreux, 2003). Many white traits have been identified in mouse and man, and 10 of the genes have been cloned (Baxter et al., 2004). The hypothesis is that the gene for white in alpaca is among these loci, with special emphasis on Kit gene (Zhang et al., 2009). The inheritance of black and brown phenotypes is uncertain. The hypothesis of dominance of black over brown does not seem fully supported by the segregations observed in crosses involving black sires and brown dams, because of the occurrence of only brown crias in one of the two crosses. On the other hand, the occurrence of both black and brown crias in crosses of black parents, can be explained only by assuming that the black coat is dominant over the brown. This hypothesis is substantiated also by the absence of black offspring in the crosses involving
A. Valbonesi et al. / Small Ruminant Research 99 (2011) 16–19
brown parents. Specific research for defining the genetic variation on MC1R and ASIP is in progress (Bathrachalam et al., 2010). Lastly, the hypothesis of a complete independence of the segregations related to coat colour (white or pigmented) and the sex of the animals is fully confirmed by the results of the G-test of independence. Acknowledgements The authors thank Prof. S. Presciuttini (University of Pisa) for his valuable help in reviewing the statistical analysis, as well as Prof. P. Sponenberg and another anonymous reviewer for their advice and suggestions. References Antonini, M., 2009. ICAR Guidelines for Alpaca Shearing Management, Fibre Harvesting and Grading. ICAR Technical Series No. 13, pp. 91–98. Bathrachalam, C., La Manna, V., Renieri, C., La Terza, A., 2010. Asip and MC1R genes in Alpaca. In: 9th WCGALP , Leipzig, Germany, PP2-154, p. 238. Baxter, L.L., Hou, L., Loftus, S.K., Pavan, W.J., 2004. Spotlight on spotted mice: a review of white spotting mouse mutants and associated human pigmentation disorders. Pigment Cell Res. 17, 215–224. Bennett, D.C., Lynn Lamoreux, M., 2003. The color loci of mice—a genetic century. Pigment Cell Res. 16, 333–344. Bustina, C.V., 1968. Herencia del palaje en alpacas. Tesis Facultad de Medicina Veterinaria y Zootecnia UTA, Puno, Peru. Cecchi, T., Valbonesi, A., Passamonti, P., Renieri, C., 2011. Quantitative variation of melanins in alpaca. In: EAAP Annual Meeting 2011 , Stavanger, Norway, abstract 59.
19
Gandarillas, H., 1971. Identificacion preliminar de los genes involucrados en la herencia del colour de las llamas y alpacas. Div. Invest. Agric. Estac. Exp. Patacamaya, Boliva, Bol. Exp. N◦ 19, La Paz, Bolivia, p. 29. Instituto Nacional de Innovación Agraria (INIA), 2011. http://www. inia.gob.pe/investigagion/Recursos Zoogeneticos. Lauvergne, J.J., Renieri, C., Frank, E.N., 1995. Identification of some allelic series for coat colour in domestic camelids of Argentina. In: Gerken, M., Renieri, C. (Eds.), Proceedings of the 2nd European Symposium on South American Camelids. Camerino, pp. 39–50. Renieri, C., 1995. Pigmentation in domestic mammals, with reference to fine fibre producing species. In: Laker, J.P., Bishop, S.C. (Eds.), Genetic Improvement of Fine Fibre Producing Animals. EFFN Occ. Publ., Peebles, UK, pp. 113–136 (1). Renieri, C., Antonini, M., Frank, E.N., 2005. Fiber Recording Systems in Camelids. ICAR Technical Series No. 11, pp. 131–144. Sponenberg, P., 2006. Genetics of fiber type and coat color. In: Hoffman, E. (Ed.), The Complete Alpaca Book. Bonny Doon Press, 121 McGivern Way, Santa Cruz, California 95060, pp. 539–560. Toledo, L., San Martin, M., 1948. Alpacas y Vicunas y su plan de mejoramiento. Lanas y Lanares 3 (10–11), 47–50. Sokal, R.R., Rohlf, F.J., 2003. Biometry, third ed. Freeman WH and Company, New York. Valbonesi, A., Apaza Castillo, N., La Manna, V., Gonzales Castilli, M.L., Huanca Mamani, T., Renieri, C., 2009. Inheritance of white, black and brown coat color in alpaca by segregation analysis. In: EAAP Book of Abstracts, p. 317. Velasco, J.M., Condorena, N.A., Novoa, C.M., Sumar, J.K., Franco, E., 1978a. Herencia de colores en alpacas. Centro de Investigacion IVITA, pp. 1–3. Velasco, J.M., Condorena, N.A., Novoa, C.M., Sumar, J.K., Franco, E., 1978b. Herencia del color y tipo de fibra en alpacas. Resumenes de Proyectos de Investigacion (1975–1979). UNMSM, tommo II, pp. 1–127. Zhang, Q., Jiang, J., Fan, R., Dong, C., 2009. Cloning expression and bioinformatics analysis of Kit gene in alpacas (Lama pacos). Acta Veterinaria Zootechnica Sinica 40 (4), 493–499.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Inheritance of white, black and brown coat colours in alpaca (Vicuna pacos L.) A. Valbonesi a,∗ , N. Apaza b , V. La Manna a , M.L. Gonzales b , T. Huanca b , C. Renieri a a b
School of Environmental Sciences, University of Camerino, Via Gentile III da Varano, 62032 Camerino, Italy Instituto Nacional de Innovacion Agraria (INIA) ILLPA PUNO, Puno, Peru
a r t i c l e
i n f o
Article history: Received 18 January 2011 Received in revised form 8 April 2011 Accepted 11 April 2011 Available online 10 May 2011 Keywords: Alpaca Coat colour Segregation analysis
a b s t r a c t An experimental trial of the segregation of white vs. pigmented and black vs. brown colours in alpacas was conducted at the Peruvian INIA Quimsachata Experimental Station. One hundred and forty five offspring were born from the following matings: 4 white sires × 36 white dams, 4 white sires × 39 pigmented dams, and 9 pigmented sires × 70 pigmented dams. Among these last matings were, 4 black sires × 25 black dams, 2 black sires × 20 brown dams, and 3 brown sires × 25 brown dams. Statistical tests validate that the inheritance of white is due to a single gene which is dominant over pigmentation, without any modifying effect and independent of segregation of black and brown patterns. However, the evidence does not support a simple dominant inheritance of the black vs. brown. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Coat colour is an important selection objective in alpaca (Vicuna pacos). In textile production, full white for dyeing and natural coloured products are both in strong demand. As companion animals, alpacas are bred in a very large variety of colour phenotypes (Lauvergne et al., 1995; Sponenberg, 2006). The list of colour variations includes six pigmentary patterns (wild, red, fawn with dark extremities, badger face, black and tan, and non agouti), two eumelanic types (black and brown), three alterations (full white, grey and dilution) and some possible white spotting patterns (Lauvergne et al., 1995). A scheme for classification was recently defined by ICAR (Renieri et al., 2005; Antonini, 2009). Many genetic systems for interpreting coat colour have been proposed (for a review, see Renieri, 1995). A complete up-to-date system was proposed by Sponenberg (2006). Few designed experiments on phenotype segregation analysis have been carried out and the relationship among
phenotypes becomes often controversial (Toledo and San Martin, 1948; Bustina, 1968; Gandarillas, 1971; Velasco et al., 1978a,b; Valbonesi et al., 2009). White phenotype is dominant for Bustina (1968) and recessive for Gandarillas (1971). Black can be dominant or recessive on brown (Gandarillas, 1971; Velasco et al., 1978a). Finally, spotting seems recessive to solid colour (Velasco et al., 1978a,b). Conservation and diffusion of coloured alpaca is one of the strategic research purposes of the Peruvian National Institute of Agronomic Innovation (Instituto Nacional de Innovación Agraria, INIA, 2011). Several coloured alpaca lines are reared in ILLPA Puno Quimsachata Station, which is the genetic conservation centre. In order to define the inheritance of the main coloured phenotypes, INIA and the School of Environmental Sciences of the University of Camerino, Italy, designed a hierarchical experimental trial involving two segregations: white vs. pigmented animal, and, among the pigmented, black vs. brown animals. 2. Materials and methods 2.1. Experimental station
∗ Corresponding author. Tel.: +39 0737 403452; fax: +39 0737 403446. E-mail address: [email protected] (A. Valbonesi). 0921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2011.04.003
The planned crosses were carried out at the experimental station of the INIA (the Peruvian National Institute for Agronomic Innovation)
A. Valbonesi et al. / Small Ruminant Research 99 (2011) 16–19 located in Quimsachata, Peru. The station, 6282 ha situated at about 4400 m above sea level in the Santa Lucia District of the Puno Department, is committed to alpaca and llama breeding, conservation and genetic improvement. The ecosystem is grassland, typical of the Andean highlands.
Table 1 Crosses involving full white sires and dams. Rams
Dams (n)
2.3. Segregation analysis Segregation analysis was applied only to segregating families which showed at least one proband, i.e. an individual supposed recessive among the offspring (truncate selection). The tested monofactorial hypotheses were the dominance of the white phenotype on the pigmented and of the black phenotype on the brown. The goodness of fit for the hypotheses was evaluated by means of the maximum-likelihood estimate (MLE) method, i.e. finding the values for the parameters involved in the segregation (i.e. segregation ratio and/or heterozygous frequency), that make the observed values in our set of data seem most probable. The probability Pri (r, s, p) of observing r (r ≥ 1) offspring of a given phenotype, among s total offspring of a cross i, given the probability p resulting from the segregation hypothesis under investigation, was calculated on the logarithmic value of the binomial truncate distribution. The total loglikelihood (L) of a set of mating (C) was then calculated as: L(C) = ˙i ln[Pri (r, s, p)] The probability p has in the present context the meaning of either a segregation ratio or a heterozygous frequency, according to the mating under investigation, for which the symbol R or H, is preferable. MLE of R or H, was obtained by maximizing the total loglikelihood (Lmax ) as a function of R or H, using the solver routine of Excel. The 95% confidence limits (C.L.) of MLE were obtained by finding (again using solver), the two loglikelihood values, L1 and L2 , for which the values −2(L1 − Lmax ) and −2(L2 − Lmax ) were = 5.0238 (i.e. the chi square value with 1 degree of freedom and associated probability = 0.025). The fitting between the value of R or H, postulated according to the segregation hypothesis under investigation, and that resulting from the MLE method, was evaluated by multiplying by a factor of −2 the difference between the L(C) and (Lmax ), so that this quantity will be distributed as the chi square statistic with 1 degree of freedom. Tests were made at the 5% level. In order to confirm the autosomal hypothesis, the segregations were tested for independence of sex chromosome segregation. Segregation data were displayed in the form of a two-way table (contingency table), and the test applied for this table was the G-test of independence with Williams’s correction (Sokal and Rohlf, 2003).
3. Results Table 1 illustrates the results from crosses between four full white sires and 36 full white dams. Because the white
Ln(btd)a
Offspring White
♀
2.2. Animals The available offspring consisted of 145 crias (80 female and 65 males), coming out from mating of 17 sires and 145 dams. The cria born from the following mating: 4 white sires × 36 white dams, 4 white sires × 39 pigmented dams, and 9 pigmented sires × 70 pigmented dams. Among these last matings, 4 black sires were crossed with 25 black dams, 2 black sires with 20 brown dams and 3 brown sires with 25 brown dams. The parents were selected among white, black and brown lines reared at the Quimsachata Station. The brown phenotype includes different patterns with different genetic background (Sponenberg, 2006). The brown considered in this experiment is the most common in the alpaca species. It is a reddish brown pattern, with different Munsel values and chroma notations and with a relatively high amount of pheomelanins (Cecchi et al., 2011). It is completely different from the dark reddish brown pattern, rarely described in alpaca, characterized by a very low amount of pheomelanins and a high amount of brown eumelanins (Cecchi et al., 2011). The reddish brown of the experimental trial is therefore an allelic alternative pattern to uniform black and also differs genetically from the dark reddish brown that expresses the same pattern of black with the substitution of the type of eumelanins from black to brown (Lauvergne et al., 1995; Sponenberg, 2006).
17
Pigmented
♂
♀
♂
443303 058104 1199-M 148102b
8 10 9 9
3 6 4 8
2 3 2 1
0 0 3 0
3 1 0 0
−1.529 −1.495 −1.423
Total
36
21
8
3
4
Lmax c = −4.447
a
Logarithmic values of the binomial truncate distribution, resulting from both the segregation hypothesis (R = 0.25), and the maximumlikelihood estimate of the heterozygous frequency (MLE = 0.94) of the dams involved in the crosses. b Sire not included in the segregation analysis because did not segregate any proband. c Total loglikelihood resulting from the MLE method.
phenotype is supposed to be dominant over the pigmented one, the occurrence of both the phenotypes among the F1 progeny of each male can be explained by the presence of heterozygous male and either heterozygous or homozygous female. As only one offspring can be obtained annually from an alpaca female, the observed segregation ratio for a number of females of unknown genotype is a function of two parameters: (i) the probability that a heterozygous animal generates a pigmented offspring (i.e. the Mendelian transmission probability) and (ii) the probability that an animal is heterozygous. Most value combinations of these two parameters produce the same value of the maximum likelihood. Therefore, a practical approach could be to estimate the value that each parameter assumes, once the other parameter is fixed on a value representing a reasonable choice. By assuming that the segregation ratio is 0.25 (the probability that a heterozygous white female mated to a heterozygous white male produces a pigmented offspring for a simple recessive genetic model), the maximum likelihood estimate of the heterozygous dams involved in the crosses is 0.94, with 95% C.L. = 0.29–1.00. The results from crosses between four full white sires and 39 pigmented dams are shown in Table 2. The segregation hypothesis of 1 white and 1 pigmented individual was tested. The maximum likelihood estimate of the recessive (pigmented) frequency was 0.563, with 95% C.L. = 0.38–0.73. This frequency does not differ significantly from that expected of 0.50 (−2[L(C) − Lmax ] = 0.620; P = 0.431). The single gene hypothesis of black phenotype dominant on the brown one was tested throughout three types of crosses. Table 3 reports the results from crosses between four full black sires and 25 full black dams. The occurrence of both the phenotypes among the F1 progeny of each male can be explained by assuming what has been postulated for the dominance of white over pigmented. It thus results that for observing a segregation ratio of 0.25 (the probability that a heterozygous black female mated to a heterozygous black male produces a brown offspring for a simple recessive genetic model), the maximum likelihood estimate of the heterozygous dams involved in the crosses is 1.00, with 95% C.L. lower limit of 0.46.
18
A. Valbonesi et al. / Small Ruminant Research 99 (2011) 16–19
Table 2 Crosses between full white sires and pigmented dams. Rams
Dams (n)
Observed offspring White
Ln(btd)a
Ln(btd)b
Pigmented
♀
♂
♀
♂
SO-502 EEI-025 322203 370397
9 15 9 6
2 1 6 3
0 4 0 1
5 6 1 1
2 4 2 1
−2.653 −2.390 −1.806 −1.435
−2.090 −1.874 −2.262 −1.747
Total
39
12
5
13
9
L(C)c = −8.284
Lmax d = −7.973
a b c d
Logarithmic values of the binomial truncate distribution resulting from the segregation hypothesis (R = 0.50). Logarithmic values of the binomial truncate distribution resulting from the maximum-likelihood estimate of the segregation ratio (MLE = 0.563). Total loglikelihood resulting from the expected segregation ratio. Total loglikelihood resulting from the MLE method.
Table 3 Crosses involving full black sires and dams. Rams
Dams (n)
Ln(btd)a
Offspring Black
237204 244203 095101 035104 Total
types, the segregations were tested for independence of sex chromosome segregation. The G-test of independence, applied to the segregation data related to crosses between white sires and both white and pigmented dams, was clearly not significant (G = 2.084, P = 0.149). Similar results were obtained for crosses between black sires and both black and brown dams (G = 0.516, P = 0.472).
Brown
♀
♂
♀
♂
6 7 6 6
0 3 3 1
3 2 1 3
1 2 2 0
2 0 0 2
−1.830 −1.023 −1.019 −1.019
25
7
9
5
4
Lmax b = −4.891
4. Discussion and conclusion
a
Logarithmic values of the binomial truncate distribution, resulting from both the segregation hypothesis (R = 0.25), and the maximumlikelihood estimate of the heterozygous frequency (MLE = 1.00) of the dams involved in the crosses. b Total loglikelihood resulting from the MLE method.
The results from the crosses involving two full black sires and 20 brown dams are shown in Table 4. The segregation analysis was not carried out because of the low number of crias (sire 366203) or the lack of segregation (sire EEI – 024). Nevertheless it should be noted that, in the latter cross, the occurrence of an offspring of 15 crias all exhibiting a supposed recessive coat colour (i.e. brown) is an extremely rare event, characterized by a frequency, in a binomial distribution of 3.05 × 10−6 . Lastly, three brown sires were mated with 25 brown dams. All the resulting offspring were brown, in full agreement with what is expected from crosses involving recessive parents, but also consistent with a dominant allele if all animals are homozygous for it. Assuming a single gene two-allele genetic model for both the white/pigmented and the black/brown phenoTable 4 Crosses between full black sires and brown dams. Rams
Dams (n)
Observed offspring Black
Brown
♀
♂
♀
♂
366203 EEI-024a
5 15
1 0
1 0
2 8
1 7
Total
20
1
1
10
8
a
Sire not included in the segregation analysis because did not segregate.
In our experimental model the inheritance of white is due to a single gene segregation, without any modifying effect. It is independent and completely dominant over pigmented animals, without any difference in segregation on black and brown colours. This hypothesis is fully supported by the segregations observed in crosses involving white sires and pigmented dams, as well as in crosses of white parents, assuming that the frequency of heterozygous females ranges from 29% up to 100%. The result is in agreement with the conclusion of Bustina (1968). The Gandarillas hypothesis of white recessivity can be justified assuming that the white defined by him is probably due to the spotting segregation or to another recessive mechanism. White in mammals arises from improper melanoblast development or survival, reflecting absence of mature melanocytes. White can be caused by defects at various stages of melanocytes development, including proliferation, survival, migration, invasion of the integument, hair follicle entry and melanocytes stem cell renewal (Bennett and Lynn Lamoreux, 2003). Many white traits have been identified in mouse and man, and 10 of the genes have been cloned (Baxter et al., 2004). The hypothesis is that the gene for white in alpaca is among these loci, with special emphasis on Kit gene (Zhang et al., 2009). The inheritance of black and brown phenotypes is uncertain. The hypothesis of dominance of black over brown does not seem fully supported by the segregations observed in crosses involving black sires and brown dams, because of the occurrence of only brown crias in one of the two crosses. On the other hand, the occurrence of both black and brown crias in crosses of black parents, can be explained only by assuming that the black coat is dominant over the brown. This hypothesis is substantiated also by the absence of black offspring in the crosses involving
A. Valbonesi et al. / Small Ruminant Research 99 (2011) 16–19
brown parents. Specific research for defining the genetic variation on MC1R and ASIP is in progress (Bathrachalam et al., 2010). Lastly, the hypothesis of a complete independence of the segregations related to coat colour (white or pigmented) and the sex of the animals is fully confirmed by the results of the G-test of independence. Acknowledgements The authors thank Prof. S. Presciuttini (University of Pisa) for his valuable help in reviewing the statistical analysis, as well as Prof. P. Sponenberg and another anonymous reviewer for their advice and suggestions. References Antonini, M., 2009. ICAR Guidelines for Alpaca Shearing Management, Fibre Harvesting and Grading. ICAR Technical Series No. 13, pp. 91–98. Bathrachalam, C., La Manna, V., Renieri, C., La Terza, A., 2010. Asip and MC1R genes in Alpaca. In: 9th WCGALP , Leipzig, Germany, PP2-154, p. 238. Baxter, L.L., Hou, L., Loftus, S.K., Pavan, W.J., 2004. Spotlight on spotted mice: a review of white spotting mouse mutants and associated human pigmentation disorders. Pigment Cell Res. 17, 215–224. Bennett, D.C., Lynn Lamoreux, M., 2003. The color loci of mice—a genetic century. Pigment Cell Res. 16, 333–344. Bustina, C.V., 1968. Herencia del palaje en alpacas. Tesis Facultad de Medicina Veterinaria y Zootecnia UTA, Puno, Peru. Cecchi, T., Valbonesi, A., Passamonti, P., Renieri, C., 2011. Quantitative variation of melanins in alpaca. In: EAAP Annual Meeting 2011 , Stavanger, Norway, abstract 59.
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