A unique accumulation of chromosomal variants and coat colors in a small population of the Norway rat at Kanagawa Prefecture, central Japan

Abstract

We researched coat coloration, chromosomal polymorphism, and mitochondrial DNA in a small population of the Norway rat, Rattus norvegicus, in Fujisawa, Kanagawa Prefecture of central Honshu, Japan. In the research area, higher frequency of the non-agouti coat coloration (29.4%), and heteromorphic pairs of subtelocentrics (ST) and telocentrics (T) in the Nos. 3 (57.2%) and 12 (14.3%) homologues were observed. Previously, such coat coloration and chromosomal heterozygosity have been confirmed at lower frequencies in some localities in Japan. In addition, according to the Hardy–Weinberg test for the current zygosities for both homologues, both F_IS values statistically indicating denial of inbreeding (p＞0.05) were apparently different and Fisher’s test revealed the rejection of independent inheritance of both homologues (p＞0.1). Moreover, the non-agouti individuals frequently carried the No. 3 chromosome of ST. These facts would be caused by a certain bias of some combinations of zygosities of both homologues. Furthermore, the mitochondrial DNA results revealed quite lower nucleotide diversity indicating the occurrence of a recent bottleneck. Considering the higher incidence of the non-agouti coat with frequent No. 3 chromosome of ST and such a bias of combinations of both homologue zygosities, it is concluded that inbreeding has been progressed after a bottleneck event in the current research area and a certain No. 3 chromosome of ST carrying a mutant allele causing the non-agouti seems to be accumulated there. Accordingly, the coat and chromosomal tendencies of the research area seem to be unique, that are different from those of other localities.

The Norway rat, Rattus norvegicus, is distributed worldwide as a commensal rodent (Burgin et al. 2020) and is well known as a laboratory animal through domestication (Castle 1947; Suckow et al. 2019; Hulme-Beaman et al. 2021). The native distribution is often considered to be northern China, Mongolia, and southeastern Siberia in evolutionary terms (Suckow et al. 2019; Puckett et al. 2016, Puckett and Munshi-South 2019; Hulme-Beaman et al. 2021). Recent studies suggest that the native distribution probably corresponds to the natural range of the rat, which was established after the Last Glacial Maximum (Yang et al. 2004) by the climatic and environmental changes in northern China during the Late Pleistocene and Quaternary (Hulme-Beaman et al. 2021). In the Japanese Islands, fossils of the Norway rat have been found in the middle to late Pleistocene layers in Honshu, Japan (Kowalski and Hasegawa 1976; Kawamura 1989) and the rats had been introduced to the Japanese Islands before the historical periods. On the other hand, the rats have also been introduced to Japan from overseas since the recent development of transportation methods, such as vessels and planes (Yamada 1998). It is mentioned that the Rattus rats occur in the Japanese Islands as human-dependent and human-independent populations, probably caused by chronologically different introductions such as before and during the historical periods (Dinets and Asada 2021). After the establishment of the rat populations in the Japanese Islands, some genetic characterizations for the R. norvegicus have been performed, mainly from morphological and cytogenetical standpoints.

First, as an interesting topic, polymorphisms in coat coloration under wild and domestic conditions are pointed out. It is well known that the wild coat coloration of the Norway rat is entirely brown or grayish brown as the agouti coloration (Iwasa 2015; Galef and Alberts 2019; Burgin et al. 2020). However, aberrations of the coat coloration such as non-agouti (black), albino, cinnamon, and others have been reported in the laboratory strains of the Norway rat (King 1932; Castle 1947; Suckow et al. 2019; Hulme-Beaman et al. 2021). Such coat color aberrations would be strongly associated to allelic mutations at the agouti locus, as in the house mouse and the locus is located on the No. 3 homologue in the Norway rat (Yamada et al. 1994; Robinson 2013). In the Japanese Islands, the coat coloration of the wild-caught rats has been researched in Hokkaido and Kyoto (Ota and Makino 1950; Nakata et al. 1958). The agouti coat color (wild) was the highest frequency as 99.8% and the non-agouti coat color (black) was lower at 0.02% in Hokkaido, and the wild coat color was 75.5% and the non-agouti coat color was 24.5% in Kyoto. In addition, a yellowish color which had not been observed in the Japanese Islands was recently reported in a wild individual of the Norway rat from Sado Island, Niigata Prefecture of Honshu (Tsunoi et al. 2021).

Second, another interesting topic of the genetic characterization shows chromosomal features. Rattus norvegicus carries the standard karyotype of 2n=42 as a diploid chromosome number with slight polymorphisms (Yong 1969; Committee for a Standardized Karyotype of Rattus norvegicus 1973; Yosida 1973; Levan 1974; Gamperl 1980; Diaz de la Guardia et al. 1981; Iwasa 2015). According to previous studies with their chromosome numbering (Yosida and Amano 1965; Yosida and Harada 1985), the No. 3 autosomal pair (here the No. 3 homologue) of the Norway rat shows polymorphisms, either of a homomorphic pair of telocentrics (T/T), that of subtelocentrics (ST/ST), and a heteromorphic pair carrying both telocentrics and subtelocentrics (ST/T). To date, the combinations of the No. 3 homologue have been researched in Osaka, Shizuoka, and Hokkaido prefectures and Miyake Island of Tokyo Metropolis and the T/T pair is obviously higher than the ST/ST pair (Yosida and Harada 1985; Yosida and Amano 1965; Tsuchiya 1980; Yosida 1986). However, information on the No. 3 homologue is still scarce because previous studies have been conducted in only four areas in the Japanese Islands. In addition, studies of the No. 3 homologue and the coat coloration of the Norway rat in the Japanese Islands have been conducted in only a few localities and a relationship between these traits are still unclear. Moreover, the same polymorphisms consisting of ST and/or T are also known in the No. 12 homologue in the Norway rat but the polymorphic state in detail is uncertain because of its smaller size (Tsuchiya 1979; Diaz de la Guardia et al. 1981). Therefore, it is necessary to study the chromosomes in conjunction with coat coloration in order to evaluate the background for occurrence of these features.

In this study, in order to obtain new additional information on the coat coloration and the chromosomal polymorphisms of the Norway rat, we collected rat samples in Fujisawa, Kanagawa Prefecture, which has not been studied before, and analyzed the frequencies of the polymorphic state and the coat coloration in the Fujisawa area. Considering the agouti locus located on the No. 3 homologue, we discussed a background of higher frequencies of aberrant coat coloration and a relationship between coat coloration and the zygosity of the No. 3 homologue.

Materials and methods

In total, 17 individuals of the Norway rat were caught alive using mesh-type live traps baited with oatmeal and a mixture of peanut butter and flour at the interior farm structures of the Nihon University Farm and Suda Farm in Fujisawa, Kanagawa Prefecture, Japan during 2019–2022 (Table 1, Fig. 1). All of the rats examined in this study were listed in Table 1 and were stored as specimens (skin and skeleton; MAI series) in the authors’ laboratory.

Table 1. The Norway rat samples examined in this study.

Sampling locality	Specimen no.	Coat color	No. 3*	No. 12*	MtDNA
Nihon Univ., Kameino, Fujisawa, Kanagawa Pref. (35°22″33′N, 139°27″58′E)	MAI-2602	Non-agouti	ST/ST	ST/ST	nd
	MAI-2637	Non-agouti	T/T	ST/ST	N1
	MAI-2638	Non-agouti	nd	nd	nd
	MAI-2685	Agouti	T/T	T/T	N1
	MAI-2686	Agouti	ST/T	ST/ST	N1
	MAI-2687	Non-agouti	ST/ST	ST/ST	N1
	MAI-2694	Agouti	T/T	ST/ST	N1
	MAI-2699	Agouti	ST/ST	ST/ST	N1
	MAI-2700	Agouti	ST/T	ST/ST	N1
	MAI-2701	Agouti	ST/T	T/T	N1
	MAI-2702	Agouti	ST/T	ST/ST	nd
	MAI-2703	Agouti	ST/T	ST/T	nd
	MAI-2740	Agouti	nd	nd	N2
	MAI-2741	Agouti	nd	nd	N2
	MAI-2747	Agouti	ST/T	ST/ST	N1
Suda Farm, Ishikawa, Fujisawa, Kanagawa Pref. (35°22″31′N, 139°27″30′E)	MAI-2727	Non-agouti	ST/T	ST/ST	N1
	MAI-2734	Agouti	ST/T	ST/T	N1
Heterozygosity			60.0%	14.3%

*Combination of the Nos. 3 and 12 homologues: T, telocentrics; ST, subtelocentrics.

Fig. 1. The current research area.

(A) Farm structures of Nihon University and (B) Suda Farm in Fujisawa, Kanagawa Prefecture, Japan.

Furs of the current rats were prepared as flat skin specimens. The dorsal and ventral coat colorations of the fur were determined as agouti (wild) or non-agouti (black), according to previous findings (Ota and Makino 1950; Nakata et al. 1958; Ohno et al. 1994). On the basis of previous and the current data (Table 2), the chi-square test was performed to compare the frequencies of the agouti and the non-agouti coats between Fujisawa and Sapporo, and Kyoto.

Table 2. Comparisons of the frequencies of the coat colorations of the Norway rats.

Sampling locality	No. of individuals examined	Agouti (%)	Non-agouti (black) (%)	Dilution (%)	Silver (%)	Albino (%)	Piebald (%)
Kyoto1	445	336 (75.5)	47 (10.6)	0 (—)	0 (—)	0 (—)	62 (13.9)
Fujisawa*	17	12 (70.6)	5 (29.4)	0 (—)	0 (—)	0 (—)	0 (—)
Hokkaido2	4171	4163 (99.8)	1 (0.0)	1 (0.0)	1 (0.0)	4 (0.1)	1 (0.0)

¹Nakata et al. (1958); ²Ota and Makino (1950); *, current study.

The current chromosomal preparation were performed using bone marrow cells. Bone marrow cells were cultured in minimum essential medium (MEM, Nissui) including 15% calf serum containing colcemid (final concentration: 0.025 µg mL⁻¹) at 37°C for 37 min. After centrifugation, these cells were subsequently treated in the 0.075 M KCl hypotonic solution at 37°C for 20 min. Finally, the cells were fixed with modified Carnoy’s fixative (methanol : acetic acid=3 : 1) three times. The present numbering for karyotyping was done according to previous system (Yosida and Harada 1985; Yosida 1986). Based on the polymorphic states of Nos. 3 and 12 homologues, we performed the Hardy–Weinberg test for each homologue and the Fisher’s test for an independent inheritance state of both homologues using Genepop ver. 4.7 (Raymond and Rousset 1995; Rousset 2008), to evaluate a random mating state in the present research area. In addition, the frequencies between the zygosities of No. 3 homologue and the coat coloration were statistically evaluated.

A total of thirteen rats were used for the present DNA analysis (Table 1). DNA was extracted from the liver tissue using a Wizard^® Genomic DNA Purification Kit (Promega) after proteinase K digestion (10 µL of pK 300 mAU mL⁻¹+490 µL pK buffer, in total, at 37°C overnight). A fragment of the mitochondrial cytochrome b gene (Cytb, 1,140 bp) was amplified by PCR using primers L14724 and H15915 (Irwin et al., 1991). The PCR mixture contained 2.5 mM MgCl₂, 0.2 mM each dNTP, 0.05 mM primers, and 0.5 units of Ex Taq^® polymerase (TaKaRa) and amplification was done for 40 cycles of 30 s at 96°C for denaturation, 30 s at 50°C for annealing and 30 s at 72°C for extension, according to Myoshu and Iwasa (2018). The PCR products were directly sequenced using the BigDye Terminator Cycle Sequencing Kit ver. 3.1 (ABI) and an automated sequencer (ABI). The nucleotide diversity (π) was calculated based on the Tamura-Nei model (Tamura and Nei 1993) using MEGA11 (Tamura et al. 2021), to evaluate the bottleneck event in the research area.

Results

In the current rat samples (n=17), 70.6% (n=12) individuals were agouti-colored (wild) and 29.4% (n=5) ones non-agouti colored (black) (Table 1, Fig. 2). All of the dorsum and venter of the current agouti coats showed brawn color and light grayish beige color, respectively, as typical agouti coat coloration of the Norway rat (Fig. 2). On the other hand, all of the non-agouti coats showed a complete blackish color in both dorsum and venter (Fig. 2). The current chi-square test revealed that the observed frequencies of the agouti and non-agouti coat colorations were significantly differed from the expected ones, when comparing between Fujisawa and Sapporo (χ²=34.351, p＜0.001) and between Fujisawa and Kyoto (χ²=8.056, p＜0.05). Therefore, the frequency of the non-agouti coat as 29.4% in Fujisawa was obviously higher than that in Sapporo (10.6%) and Kyoto (0.0%).

Fig. 2. Typical appearances of dorsum and venter.

(A) an agouti coat (specimen no.: MAI-2747) and (B) a non-agouti (black) coat (specimen no.: MAI-2637). Bar indicates 20 mm.

The diploid chromosome numbers of all fourteen individuals analyzed were 2n=42 (Fig. 3) as in previous work (Tsuchiya 1981). In all of the present rat samples, there are three types of zygosity in the No. 3 homologue, ST/ST (21.4%, n=3), T/T (21.4%, n=3), and ST/T (57.2%, n=8) (Table 1, Fig. 3), as reported in previous studies (Yosida and Amano 1965; Tsuchiya 1980, 1981; Yosida and Harada 1985; Yosida 1986). In fact, the heteromorphic pair with ST/T was the most frequent No. 3 homologue in the current rat individuals (Table 1). In addition, there are three types in the No. 12 homologue, ST/ST (71.4%, n=10), T/T (14.3%, n=2), and ST/T (14.3%, n=2) (Table 1, Fig. 3) and the homomorphic pair with ST/ST was the most frequent in the No. 12 homologue (Table 1). On the basis of the heterozygosities of these Nos. 3 and 12 homologues, we evaluated the random mating condition in the present research area by Hardy–Weinberg test. F_IS (Weir and Cockerham 1984) was 0.1789 for the No. 3 homologue (p=0.6150) and 0.6000 for the No. 12 homologue (p=0.0632) and both values were significantly not larger than zero (α=0.05 by probability test) (Table 3). In addition, Fisher’s test showed χ²=6.4962 and p=0.1650.

Fig. 3. Typical karyotypes.

(A) ST/ST and ST/ST (MAI-2602), (B) T/T and T/T (MAI-2685), and (C) ST/T and ST/T (MAI-2703) of the Nos. 3 and 12 homologues, respectively, in the current rat samples.

Table 3. Results by Hardy–Weinberg exact test.

	No. individuals			FIS		Fisher
	ST/ST	T/T	ST/T	W & C*	p	χ2	p
No. 3 homologue	3	3	8	0.1789	0.6150	6.4962	0.1650
No. 12 homologue	10	2	2	0.6000	0.0632

*Weir & Cockerham’s (1984) estimate.

The No. 3 homologue zygosities of individuals with the agouti coat (n=10) were ST/ST (10.0%), T/T (20.0%), and ST/T (70.0%), and those with the non-agouti coat (n=4) ST/ST (50.0%), T/T (25.0%), and ST/T (25.0%). The chi-square test indicated that the zygosity frequencies observed in the No. 3 homologue were significantly different from those expected (χ²=48.538, p＜0.001), and that ST/ST and ST/T were more and less frequent, respectively, in the non-agouti coat individuals than in the agouti ones. On the other hand, the ST and T frequencies of the No. 3 homologue were calculated in each coat coloration and were 45.0% and 55.0% in the agouti coat and 62.5% and 37.5% in the non-agouti coat, respectively. The chi-square test showed that the ST and T frequencies observed in the No. 3 homologue were also significantly different from those expected (χ²=6.159, p＜0.05) and the ST was more frequent in the non-agouti than in the agouti. In fact, the zygotic patterns and the frequencies of ST and T in the No. 3 homologue were different between the agouti and non-agouti coats.

There were two haplotypes with N1 and N2 in the current Cytb sequences (Table 1). The N1 haplotype was completely identical to that of an individual from South Africa (accession no.: MH794457) and there was only one substitution between N1 and N2 haplotypes at site 474 (N1=T, N2=C). Based on the Cytb sequences, the nucleotide diversity was calculated to be π=0.0002478 in the research area.

Discussion

While the sample size of this study was too small for general population genetics and its related field to study, we try to evaluate why the non-agouti coat coloration frequently appear and its relationship with the state of the No. 3 homologue. The current 12 and five individuals carried agouti and non-agouti coats, respectively (Table 2, Fig. 2). The current frequency of the non-agouti coat coloration was significantly higher than previous findings (Table 2) (Ota and Makino 1950; Nakata et al. 1958). In general, it is thought that the non-agouti coat coloration is thought to be due to recessive homozygosity, a/a, at the agouti locus in laboratory mice and rats (Vrieling et al. 1994; Kuramoto et al. 2001; Grandin and Deesing 2014). Although we did not analyze allele types at the agouti locus for the current rat samples, it is assumed that the non-agouti coloration of the current rat samples would be also resulted from such a genetic trait. Considering the higher frequency of the non-agouti coat color, it is suggested that the recessive homozygosity, such as a/a, has been accumulated in the current research locality. On the basis of the fact that the non-agouti coloration was detected only in rats with the N1 haplotype, rat individuals of the maternal group with N1 might frequently carry the recessive a allele in the locality.

The diploid chromosome number of the present rats were 2n=42 (Fig. 3) as in previous studies (Yosida and Amano 1965; Yosida and Harada 1985; Tsuchiya 1980; Yosida 1986). The heteromorphic pair with ST/T and the homomorphic pair with ST/ST were the most frequent Nos. 3 and 12 homologues, respectively, in the current analysis (Table 1). However, the frequency of the combination of the No. 3 homologue in Fujisawa was apparently different from that in other localities (Table 4). It has been suggested that the Norway rats inhabiting the Japanese Islands had originated from the Chinese and Korean populations carrying T/T on the No. 3 homologue (Yosida and Amano 1965). Therefore, the Hokkaido and Osaka populations frequently carry the T/T combination, as 74.7% in Hokkaido and 64.6% in Osaka and it is considered that the T/T combination has been maintained in both areas as a founder effect (Yosida and Amano 1965). However, in Shizuoka, which is adjacent to Kanagawa Pref. and Fujisawa, the frequency of T/T tends to be lower than in other localities, such as 39.5% in Shizuoka and 21.4% in Fujisawa (Table 4). On the other hand, the frequency of ST/ST was 7.0% in Shizuoka and 3.5% in Hokkaido, and 8.3% in Osaka (Yosida and Amano 1965; Tsuchiya 1980; Yosida and Harada 1985), but it was 21.4% in the Fujisawa population, which seems to be higher than in other localities (Table 4). Therefore, the highest frequency of ST/T, the higher frequency of ST/ST, and the lower frequency of T/T tend to be unique characteristics of the Fujisawa population. For example, T/T of the No. 3 homologue has been frequently obtained from Spain (Diaz de la Guardia et al. 1981) but ST/ST of the No. 3 homologue was frequently recognized in the laboratory rat which had been established in the United States (Yosida and Amano 1965). Therefore, it is suggested that such ST/ST has been newly introduced from overseas, probably the United States, to several localities in the Japanese islands.

Table 4. Comparisons of the frequencies of the combination of the No. 3 homologue of the Norway rats.

Sampling locality	No. of individuals examined	Combination of No. 3 homologue (%)
		ST/ST	ST/T	T/T
Hokkaido1	87	3 (3.5)	19 (21.8)	65 (74.7)
Miyake Is.2	5	0 (0.0)	2 (40.0)	3 (60.0)
Fujisawa*	14	3 (21.4)	8 (57.2)	3 (21.4)
Shizuoka3	43	3 (7.0)	23 (53.5)	17 (39.5)
Osaka4	40	4 (8.3)	10 (27.1)	26 (64.6)

¹Tsuchiya (1980); ²Yosida (1986); ³Yosida and Amano (1965); ⁴Yosida and Harada (1985); *, current study.

On the other hand, ST/ST was the highest frequency in the No. 12 homologue examined in this study (Table 1), although T/T and ST/T have been frequently recognized in Spain (Diaz de la Guardia et al. 1981). No findings of the No. 12 homologue have been reported in detail in other localities of the Japanese Islands and the frequencies of the three combinations tended not to be equilibrium and the frequencies were different between Nos. 3 and 12 homologues (Table 4). The result of Fisher’s test (p=0.1650) indicates that independent inheritance of both Nos. 3 and 12 homologues is statistically denied, and this fact would be caused by a certain bias of some combinations between both homologue zygosities, such as quite higher frequency of ST/ST in the No. 12 homologue. On the other hand, the Hardy–Weinberg test was carried out for the Nos. 3 and 12 homologues, and the F_IS values of both homologues did not indicate positive inbreeding at least statistically (Table 3). In general, a deviation of the F_IS value from zero means progress of inbreeding (Weir and Cockerham 1984; Nishimura et al. 2007). In the current results, the F_IS values between Nos. 3 and 12 homologues were apparently not similar and this difference would be caused by such a bias for combinations of both homologue zygosities as suggested by the above Fisher’s test results. In fact, certain combinations of recessive lethal alleles, caused by some kinds of combinations of both homologue zygosities can lead to inviability, and the postulated presence of such a bias can explain that inbreeding has progressed among individuals since the founder individuals, considering that the current research locality is narrower, as a small population, due to the location of the present sampling sites surrounded by residential areas.

The zygosity state of the No. 3 homologue carrying the agouti locus (Yamada et al. 1994; Robinson 2013) was statistically evaluated between the agouti and non-agouti coat individuals. The agouti coat individuals frequently carried the ST/T heterozygosity (70.0%), whereas the non-agouti individuals frequently carried the ST/ST homozygosity (50.0%). In addition, the ST frequency was significantly higher in the non-agouti coat individuals (62.5%) than in the agouti coat individuals (45.0%). Considering these facts, the No. 3 chromosome of ST may be related to the expression of non-agouti coat. For example, in the current research area, it is estimated that a particular No. 3 chromosome of ST may frequently carry the recessive a allele causing the non-agouti expression.

Based on the Cytb results, π=0.0002478 is quite low in the current research area. In general, such a low value of π (＜0.005) indicates that a population has recently experienced a bottleneck (Grant and Bowen 1998). In addition, the F_IS results indicate a tendency toward inbreeding as a result of random mating in the research area. In general genetics, if ST and T are neutral and equal for viability, ST/T would be expected to be the most common combination by random mating (Frankham 2019). However, in the No. 3 homologue, ST/T and T/T were actually frequent in the areas studied, and ST/ST was hardly confirmed (Yosida and Amano 1965; Tsuchiya 1980; Yosida and Harada 1985; Yosida 1986) (Table 4). Such bias of the frequent ST/T and T/T can imply a situation lays out of random mating and it is estimated that the ST/ST homozygosity may be a certain negative combination for viability, such as frequent non-agouti coat colorations, in the rat populations. Actually, the ST/ST frequency was higher in the non-agouti coat individuals than in the agouti individuals. On the other hand, ST/ST was frequent in the No. 12 homologue (Table 1) and ST/ST may be a certain positive combination for viability in the rat populations, contrary to the No. 3 homologue. In addition, as mentioned above, a non-agouti colored coat expressed by the a/a homozygosity at the agouti locus seems to be usually rare in natural populations in mammals and would result from inbreeding causing a frequent recessive homozygosity at the agouti locus (Green 1966; Frankham et al. 2017). Nevertheless, considering the current higher frequency of the non-agouti coat, it does not seem to be a negative phenotype for the viability in the rat because the current habitat consists mainly of the interior farm structures with rich resources that inhibit predator attacks (such as feral cats and crows). Therefore, it is assumed that the rats with the non-agouti coat can inherit generations at the current research locality, even though inbreeding has progressed after a bottleneck event.

Acknowledgments

We are grateful to staffs of Nihon University Farm and Suda Farm for cooperation to the current rat samplings.

Author contributions

Hayakawa and Iwasa designed this study. Hayakawa, Myoshu, and Iwasa contributed to collection of the current specimens. Hayakawa mainly performed experimental works, data collection and data analyses. Hayakawa and Iwasa wrote the manuscript and Myoshu checked it.

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