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Journal of Heredity 2004:95(1)
© 2004 The American Genetic Association 95:76-80

Brief Communication

Inheritance of White Head Spotting in Natural Populations of South American Water Rat (Nectomys squamipes Rodentia: Sigmodontinae)

T. I. Axenovich, P. S. D'Andrea, F. Fernandes, C. R. Bonvicino, I. V. Zorkoltseva, and P. M. Borodin

From the Institute of Cytology and Genetics, Russian Academy of Science, Novosibirsk 630090, Russia (Axenovich, Zorkoltseva, and Borodin), Novosibirsk University, Novosibirsk, Russia (Axenovich, Zorkoltseva, and Borodin), Institute of Tropical Medicine of Oswaldo Cruz Institute, Rio de Janeiro, Brazil (D'Andrea), National Museum of the Federal University of Rio de Janeiro, Rio de Janeiro, Brazil (Fernandes), and Division of Genetics, National Institute of Cancer, Rio de Janeiro, Brazil (Bonvicino). We thank Claudia Horta de Almeida for taking care of the captive breeding population of Nectomys squamipes, Juliano Chagas Silva and Stella Franco for help in checking spots in the skins of specimens from the National Museum, and Dr. João Alves for granting access to the mammal collection of the National Museum of Rio de Janeiro. We also thank Rodrigo Mexas for helping with the photographs. This work was supported by research grants from the Brazilian National Research Council (CNPq)/PRONEX Foundation for Promoting Research of the State of Rio de Janeiro (FAPERJ), Oswaldo Cruz Foundation (IOC/PAPES/FIOCRUZ), Russian Foundation for Basic Researches (RFBR), and Federal Program "Russian Universities" (UNIROS).

Address correspondence to P. M. Borodin, Institute of Cytology and Genetics, Novosibirsk 630090, Russia, or e-mail: borodin{at}bionet.nsc.ru.


   Abstract
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 Abstract
Materials and Methods
 Results and Discussion
 References
 
Specimens with white head spots are present at low frequency in the natural populations of South American water rat (Nectomys squamipes) and absent in the sibling species Nectomys rattus. We analyzed the pattern of inheritance of the phenotype using complex segregation analysis of pedigrees of a captive-bred population of N. squamipes. We found that the inheritance of the white head spot in this species can be described within the framework of the major gene recessive model with incomplete penetrance of genotypes.

Studies of genetic variation of morphological traits in natural populations of mammals are essential to understanding their evolution. Application of classical methods of genetic analysis to these studies can meet with serious difficulties. These methods are based on the results of crosses between homozygous purebreds. This simple procedure becomes rather complex in the case of complex traits or those with incomplete penetrance. Special statistical methods have been developed to analyze these kind of traits (Elston 1984; Elston and Stewart 1973). Even these approaches, however, demand crosses of homozygous inbred strains. When these strains are not available, the statistical complexity of the analysis becomes huge (Elston 1981; Ott 1985). This situation is common in genetic analysis of human populations. The general theory of segregation analysis of pedigree data developed by Elston and his colleagues (Elston 1981; Elston and Stewart 1971) has provided a clue for overcoming these difficulties, allowing us to analyze the inheritance of qualitative and quantitative traits in pedigrees. In this case we do not need pure lines. We need to know phenotypes of the specimens and their kinship. This method has been successfully applied to the genetic analysis of various traits in humans. We have shown the efficiency of this approach in analysis of life-history traits of small mammals (Aulchenko et al. 1998, 2002; Axenovich et al. 1998). In this article we demonstrate an application of this method in genetic analysis of a morphological trait: white head spotting in South American water rats (Nectomys squamipes) derived from a natural population.

White spotting is one of the most frequent "mutant" phenotypes detected in many mammal species. Several series of white spotting alleles belonging to different loci have been described in almost all domestic and laboratory mammals, and in several wild species as well (Searle 1968). Recently we detected this phenotype in natural populations of South American water rat. We analyzed its geographic distribution and inheritance.


   Materials and Methods
Top
 Abstract
 Materials and Methods
Results and Discussion
 References
 
We distinguished three phenotypes: Spot, an easily visible white area on the forehead; Star, a very small white mark visible under close examination; and wild type, with no white hair on the forehead (Figure 1). To determine the geographical distribution of the phenotypes, we analyzed 820 skins of the South American water rat that were collected in various places in Brazil over a century and deposited at the National Museum of Rio de Janeiro. We also examined 155 specimens of N. squamipes trapped from two natural populations, Nova Friburgo and Sumidouro (State of Rio de Janeiro) in 1997–1999.



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  		Figure 1.. White head spotting in N. squamipes (left: Wild; middle, Star; right, Spot)

 
To determine the pattern of inheritance of the phenotype we analyzed pedigrees of a captive-bred population of N. squamipes maintained at the Department of Tropical Medicine, Oswaldo Cruz Institute, Rio de Janeiro. Seven pedigrees containing 190 offspring from 53 crosses (Figure 2) were analyzed. We used the well-known approach for analysis of pedigree data developed by Elston and Stewart (1971). We modified it for the case of qualitative traits with more than two phenotypes. Mendelian inheritance, if present, was presumed to be due to a single autosomal locus with two alleles, S and s. For each genotype g we specified the probabilities P(x|g) of each phenotype x. We considered three phenotypes: Spot, Star, and wild type. Therefore each genotype had two penetrances: vg, probability of Spot; and wg, probability of Star. If an animal was the founder of the pedigree (i.e., its parents were not members of the pedigree), then we specified the prior probability P(g) of each of the genotypes g. Under Hardy-Weinberg equilibrium, the prior probabilities can be described by the frequency of the s allele (q): P(SS) = (1 - q)2, P(Ss) = 2q(1 - q), and P(ss) = q2. Assumption of Hardy-Weinberg equilibrium is common in segregation analysis and was reasonable for the captive-bred colony of N. squamipes. It was rather large and noninbred, the crosses were set up irrespective of the phenotype analyzed, and there was no difference between the phenotypes in fertility (Table 1).



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  		Figure 2.. Pedigrees of the captive-bred population of N. squamipes (filled, Spot; hatched, Star; blank, Wild)

 

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  		Table 1.. Segregation of white head spot phenotypes of N. squamipes.

 
We defined the probability of parents with genotypes gm and gf having an offspring with genotype g as P(g|gmgf). In the case of the major gene diallele model, this probability is described via three transmission probabilities ({tau}g) (i.e., the probabilities of the parent SS, Ss, or ss transmitting allele S to its offspring. Assuming Mendelian inheritance, {tau}g are 1, 0.5, and 0 for SS, Ss, and ss, respectively. Thus the most general model is described by the following 10 parameters:
Frequency of s allele q.
Six parameters of penetrance: wSS, wSs, wss, vSS, vSs, vss.
Transmission probabilities {tau}SS, {tau}Ss, {tau}ss.

The likelihood (LH) of a pedigree containing n members can be written as


where i refers to those individuals whose phenotypes are observed, j to the founders, and k to the offspring.

We tested Mendelian transmission for a putative gene in accordance with Elston (1981). We tested the Mendelian model ({tau}SS = 1, {tau}Ss = 0.5, and {tau}ss = 0) versus the unrestricted model of general transmission. The later model does not fix the rates of transmission of the alleles at Mendelian values, but infers them from experimental data. We also compared the unrestricted model with the environmental hypothesis, assuming that there was no transmission of the major gene (i.e., {tau}SS = {tau}Ss = {tau}ss). Evidence for a major gene could be inferred only if the Mendelian model was not rejected and the environmental model was rejected against the unrestricted model (Elston 1981). We tested hypotheses using the likelihood ratio test (Neyman and Pearson 1928).

The pedigrees under analysis contained several loops (see Figure 2). To calculate the pedigree likelihood we used an approach where the loops were cut and extended by introducing artificial phenocopies of some individuals in a pedigree and then the likelihood was computed conditional on the likelihood of the phenocopies (Stricker et al. 1996; Wang et al. 1996). We used a modified version of our software MAN-1, which we designed for complex segregation analysis of qualitative traits (Axenovich and Ginsburg 1987).


   Results and Discussion
Top
 Abstract
 Materials and Methods
 Results and Discussion
References
 
Geographic Distribution of the Polymorphism
Table 2 shows the geographical distribution of white head spot phenotypes. There are two sibling species of the water rat in South America. N. squamipes is found in the eastern part of Brazil to the south from Rio São Francisco in the Atlantic forest belt in Bahia Espírito Santo Rio de Janeiro and further to the south in Uruguay and the northern provinces of Argentina. Its western border passes through the Cerrado gallery forest of Minas Gerais, São Paulo, and Mato Grosso do Sul. In the west and north, this species borders on the sibling species Nectomys rattus, which occupies Amazonian and north Atlantic forests and also Cerrado and Caatinga in central Brazil. It is quite difficult to distinguish these species using morphological attributes. However, they are true species. They differ from each other karyotypically and their hybrids are sterile (Bonvicino et al. 1996).


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  		Table 2.. Frequency of the white head spot in natural populations of N. squamipes and N. rattus.

 
None of the animals collected from the area of distribution of N. rattus had a white spot. The animals with Spots and Stars were found only in the area occupied by N. squamipes, in the states of Bahia Minas Gerais, Espírito Santo, and Rio de Janeiro. Even in this part of the range the spotted animals are very rare. We did not find spotted animals in the sample taken from São Paulo, which is populated by N. squamipes. The highest frequency of spotted animals was found in the population of Sumidouro (Rio de Janeiro). In this population we did not detect a sex difference in the occurrence of white spots (tST = 0.73). The data on geographic distribution show that the white head spot is present in populations of N. squamipes (although at low frequency) and absent in populations N. rattus.

Segregation Analysis
Table 1 shows the results of crosses between different phenotypic classes. We did not find a significant difference in litter size between different crosses. There was a dependence of segregation in the offspring on the parental phenotypes ({chi}2 = 14.88; df = 6; P <.05). This means that the occurrence of the white spotting is genetically controlled. However, the segregation did not match any simple model of inheritance. Offspring of all phenotypes came from all types of crosses. This may indicate that the same phenotypes are controlled by different genotypes or that the same genotypes are manifested as different phenotypes. This means that the parents involved in each cross present in Table 1 were genetically heterogeneous. These crosses were chosen from complex pedigrees with a loss of information about the descent of their members. To make use of this information we performed a procedure that has been designed for studying genetically heterogeneous crosses—complex segregation analysis.

First, we tested for Mendelian segregation. We compared the Mendelian model to the unrestricted model of general transmission. Table 3 shows that the unrestricted model did not differ significantly from the Mendelian model ({chi}2 = 2.56; df = 3) and was much better than the environmental model ({chi}2 = 10.76; df = 2; P <.005). According to the Elston-Stewart test, we interpret this result as evidence for a major gene controlling head spotting.


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  		Table 3.. Results of segregation analysis of white head spot in N. squamipes.

 
To proceed further we needed to simplify the Mendelian model, because it had too many parameters to be estimated correctly. We found that the estimated probabilities of Star (wg) were almost the same in different genotypes (Table 3, column 2). Therefore we tried to simplify the model by making all these probabilities equal: wSS = wSs = wss. This simplified model (Table 3, column 4) did not differ significantly from the Mendelian model (Table 3, column 2): {chi}2 = 0.48; df = 2. This means that putative locus S controls the polymorphism of Spot, but it is not involved in the control of the Star phenotype. This phenotype occurs with the same frequency in all three genotypes—SS, Ss, ss. We called the simplified model the Spot model.

We then tested recessive and dominant models of inheritance of the Spot phenotype. A comparison of column 4 with columns 5 (dominant Spot model) and 6 (recessive Spot model) of Table 3 shows that the recessive model does not differ significantly from the Spot model ({chi}2 = 1.60; df = 1), while the dominant model is significantly worse than the Spot model ({chi}2 = 3.98; df = 1). Therefore the recessive Spot model is the simplest model.

Thus the results of complex segregation analysis indicate that the inheritance of the white head spot in N. squamipes can be described within the framework of a major gene recessive model with incomplete penetrance of genotypes. The estimates of the penetrance obtained from segregation analysis were as follows: the Spot phenotype occurs in 8% of SS, 8% of Ss, and 75% of ss animals.

We try to interpret the incomplete penetrances of both normal and mutant genotypes from the point of view of the threshold model, which can be inferred from data on developmental genetics of white spotting in mammals. Skin pigmentation is organized through a series of complementary processes. After migration of melanoblasts out of the neural crest to the epidermis and hair follicle, these cells mature into melanocytes. It has been shown that white spotting appears to be due to the absence of melanocytes at locations in the body where they are normally present. A failure of melanoblasts to reach particular locations can be determined by different mechanisms. They may be melanoblast specific or target tissue specific, or both (Jackson 1994; Quevedo and Holstein 1992; Silvers 1979; Wilkie et al. 2002). It has also been shown that the extent of the white spotted areas and the locations of these areas are influenced by modifying polygenes (Lamoreux 1999, 2000). To differentiate into melanocytes, melanoblasts have to arrive at the destinations at the proper stage of development before differentiation of the hair follicles. This moment is the threshold, and therefore the continuous variation in the time of melanoblast arrival is manifested as two alternative phenotypes: the white spot is either present or absent (Hirobe 1992). The threshold model may explain why some SS and Ss water rats produce the Spot, why some ss homozygotes do not have the Spot and why the Star occurs sporadically in animals of any genotype.

Let us assume that there is individual variation in the time of melanoblast arrival at the front of the head due to genetic and environmental effects. In ss homozygotes, the distribution of the time of melanoblast arrival is shifted substantially to later dates. The majority of the homozygotes have a white spot, but those from the left tail of the distribution are wild type. In the majority of SS and Ss animals the melanoblasts arrive in time and no white spotting occurs. However, in some of them the melanoblasts arrive after the deadline. These animals have Spot. Apparently not all melanoblasts in each animal arrive at its head absolutely synchronously. Some latecomers may cause the Star phenotype.

Of course, this scheme is an oversimplification of a very complex process of coat color development. There are many variables besides the migration rate of melanoblasts that may influence the size of white head spotting and the chance of its occurrence. Within each species there is individual variation in the number and location of melanoblast progenitor cells, their proliferative potential, routes of migration, and the pattern of their progeny distribution and mixing. This individual variation is complicated further by a clonal variation (Jackson 1994; Wilkie et al. 2002). However, in the context of our discussion, it is important that each species has its own limit of individual variation. The individuals falling into these limits have wild-type coat color. Mutations may shift the limit substantially and lead to occurrence of visible polymorphism for white spotting. However, slight quantitative changes (genetic, environmental, or stochastic) of the variables discussed above may shift them beyond the species-specific limit and lead to occurrence of the spots. These spots may be inherited with incomplete penetrance (like Spot in the case considered) or may not be heritable at all (like Star).

We carried out the segregation analysis of head spotting using phenotype distribution in the complex pedigree of a captive-bred colony of N. squamipes. It may also be applied to genetic analysis of various morphological traits in natural populations, because modern methods of parent testing make it possible to reconstruct kinship between the members of a population and their pedigrees.


   Footnotes
 
Corresponding Editor: Stephen O'Brien Back

Received August 13, 2002
Accepted July 31, 2003


   References
Top
 Abstract
 Materials and Methods
 Results and Discussion
 References
 

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    Axenovich TI, Rogatcheva MB, Oda SI, Borodin PM, 1998. Inheritance of male hybrid sterility in the house musk shrew (Suncus murinus, Insectivora, Soricidae). Genome. 41:825-831.[Medline]

    Bonvicino CR, D'Andrea PS, Cerqueira R, Seuánez HN, 1996. The chromosomes of Nectomys (Rodentia Cricetidae) with 2n = 52, 2n = 56, and interspecific hybrids (2n = 54). Cytogenet Cell Genet. 73:190-193.[Web of Science][Medline]

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