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Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

Igawa T, Watanabe A, Suzuki A, Kashiwagi A, Kashiwagi K, Noble A, Guille M, Simpson DE, Horb ME, Fujii T, Sumida M - PLoS ONE (2015)

Bottom Line: It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s.Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains.Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

View Article: PubMed Central - PubMed

Affiliation: Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.

ABSTRACT
The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

No MeSH data available.


Related in: MedlinePlus

Plot of expected heterozygosity (HE) against generation number (t).The curved dotted strain indicates theoretical reduction of heterozygosity via single brother-sister matings for every generation, calculated from the following equation: , where H0 is the heterozygosity of the founder colony, and Ne is effective population size (i.e., number of individuals that contribute to breeding). HE of F0 of N (Gurdon Inst.) (0.509) and 2 are used for H0 and Ne, respectively. Open rectangles indicate mean HE estimated from individual based simulation using VORTEX version 10 [43]. During simulation, allele frequencies of N (Gurdon Inst.) were used as initial allele frequency, and single brother-sister mating was conducted in every generation. The simulation was repeated one hundred times. Bar indicates standard deviation.
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pone.0133963.g005: Plot of expected heterozygosity (HE) against generation number (t).The curved dotted strain indicates theoretical reduction of heterozygosity via single brother-sister matings for every generation, calculated from the following equation: , where H0 is the heterozygosity of the founder colony, and Ne is effective population size (i.e., number of individuals that contribute to breeding). HE of F0 of N (Gurdon Inst.) (0.509) and 2 are used for H0 and Ne, respectively. Open rectangles indicate mean HE estimated from individual based simulation using VORTEX version 10 [43]. During simulation, allele frequencies of N (Gurdon Inst.) were used as initial allele frequency, and single brother-sister mating was conducted in every generation. The simulation was repeated one hundred times. Bar indicates standard deviation.

Mentions: In this study we estimated genetic data on SSLP markers from all chromosomes. Because each chromosome is operated independently during meiosis, it is considered that the lineages of the chromosomes sometimes differ, especially for recent crossing between different strains. Therefore, to check the heterogeneities of these measurements across the chromosome, we conducted exact tests both in each colony and by combining all samples, but no significant differences were detected (data not shown). Comparison of the measurements among colonies highlighted lower polymorphism values for the three inbred Nigerian strains, NA (IAB), NH (IAB), and Golden (IAB). NA and NH in particular showed the lowest and the second lowest HE values, reflecting their longer history of brother-sister mating. Within Ivory Coast strain, TGA blue (NXR) and IC (IAB) showed equally lower values. Inbreeding coefficients or F values are estimated by dividing residual observed heterozygosity (HO) by HE, and thus become minus when HO exceeds HE. All samples used in this study were offspring of single brother-sister pairs, and the number of alleles was limited. In this situation, allelic frequency fluctuates due to sampling, and F for most loci of NA (IAB) and NH (IAB) could not be estimated due to a lack of multiple alleles. Even taking into account these shortcomings, however, observation of heterozygote excess and minus F values in most cases might be explained by heterozygote advantage on marker linked loci. In either case, F was not an appropriate estimator for inferring the inbreeding ratio in this study. Thus we estimated pairwise relatedness (r) within each colony. In theory, r values are estimated from real pedigrees and become 0.5 when the relationship is parent-offspring or brother-sister, and 1.0 when the individuals are identical clones. Our estimated r values within each colony showed a mean of more than 0.5 (Fig 3), which is consistent with the physical sister-brother relationship of the samples. Of these, the three Nigerian strains maintained at IAB showed significantly higher r values, reflecting a smaller value of HE (Table 1). In particular, individuals in the NA and NH strains are nearly identical clones, showing r values of almost 1.0 (Fig 3). These results suggest that these four IAB strains have been successfully maintained to achieve inbred strains. HE values for these strains, however, deviated somewhat from the theoretical and simulation-based predictive reduction curve when brother-sister matings are conducted in every generation (Fig 5). Because loss of alleles and reduction of heterozygosity are stochastic processes, such deviation can be caused by chance. In addition, different allele frequency and heterozygosity in the ancestral colony or at some specific loci might have caused this discrepancy between strains. In order to reduce heterozygosity in NA and NH to establish inbred strains on the same level with the mouse, which retain heterozygosity less than 0.02, at least five or six more generations will be necessary. Alternatively, a more rapid establishment of inbred strains may be accomplished using the gynogenetic technique [34,35].


Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.

Igawa T, Watanabe A, Suzuki A, Kashiwagi A, Kashiwagi K, Noble A, Guille M, Simpson DE, Horb ME, Fujii T, Sumida M - PLoS ONE (2015)

Plot of expected heterozygosity (HE) against generation number (t).The curved dotted strain indicates theoretical reduction of heterozygosity via single brother-sister matings for every generation, calculated from the following equation: , where H0 is the heterozygosity of the founder colony, and Ne is effective population size (i.e., number of individuals that contribute to breeding). HE of F0 of N (Gurdon Inst.) (0.509) and 2 are used for H0 and Ne, respectively. Open rectangles indicate mean HE estimated from individual based simulation using VORTEX version 10 [43]. During simulation, allele frequencies of N (Gurdon Inst.) were used as initial allele frequency, and single brother-sister mating was conducted in every generation. The simulation was repeated one hundred times. Bar indicates standard deviation.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4519292&req=5

pone.0133963.g005: Plot of expected heterozygosity (HE) against generation number (t).The curved dotted strain indicates theoretical reduction of heterozygosity via single brother-sister matings for every generation, calculated from the following equation: , where H0 is the heterozygosity of the founder colony, and Ne is effective population size (i.e., number of individuals that contribute to breeding). HE of F0 of N (Gurdon Inst.) (0.509) and 2 are used for H0 and Ne, respectively. Open rectangles indicate mean HE estimated from individual based simulation using VORTEX version 10 [43]. During simulation, allele frequencies of N (Gurdon Inst.) were used as initial allele frequency, and single brother-sister mating was conducted in every generation. The simulation was repeated one hundred times. Bar indicates standard deviation.
Mentions: In this study we estimated genetic data on SSLP markers from all chromosomes. Because each chromosome is operated independently during meiosis, it is considered that the lineages of the chromosomes sometimes differ, especially for recent crossing between different strains. Therefore, to check the heterogeneities of these measurements across the chromosome, we conducted exact tests both in each colony and by combining all samples, but no significant differences were detected (data not shown). Comparison of the measurements among colonies highlighted lower polymorphism values for the three inbred Nigerian strains, NA (IAB), NH (IAB), and Golden (IAB). NA and NH in particular showed the lowest and the second lowest HE values, reflecting their longer history of brother-sister mating. Within Ivory Coast strain, TGA blue (NXR) and IC (IAB) showed equally lower values. Inbreeding coefficients or F values are estimated by dividing residual observed heterozygosity (HO) by HE, and thus become minus when HO exceeds HE. All samples used in this study were offspring of single brother-sister pairs, and the number of alleles was limited. In this situation, allelic frequency fluctuates due to sampling, and F for most loci of NA (IAB) and NH (IAB) could not be estimated due to a lack of multiple alleles. Even taking into account these shortcomings, however, observation of heterozygote excess and minus F values in most cases might be explained by heterozygote advantage on marker linked loci. In either case, F was not an appropriate estimator for inferring the inbreeding ratio in this study. Thus we estimated pairwise relatedness (r) within each colony. In theory, r values are estimated from real pedigrees and become 0.5 when the relationship is parent-offspring or brother-sister, and 1.0 when the individuals are identical clones. Our estimated r values within each colony showed a mean of more than 0.5 (Fig 3), which is consistent with the physical sister-brother relationship of the samples. Of these, the three Nigerian strains maintained at IAB showed significantly higher r values, reflecting a smaller value of HE (Table 1). In particular, individuals in the NA and NH strains are nearly identical clones, showing r values of almost 1.0 (Fig 3). These results suggest that these four IAB strains have been successfully maintained to achieve inbred strains. HE values for these strains, however, deviated somewhat from the theoretical and simulation-based predictive reduction curve when brother-sister matings are conducted in every generation (Fig 5). Because loss of alleles and reduction of heterozygosity are stochastic processes, such deviation can be caused by chance. In addition, different allele frequency and heterozygosity in the ancestral colony or at some specific loci might have caused this discrepancy between strains. In order to reduce heterozygosity in NA and NH to establish inbred strains on the same level with the mouse, which retain heterozygosity less than 0.02, at least five or six more generations will be necessary. Alternatively, a more rapid establishment of inbred strains may be accomplished using the gynogenetic technique [34,35].

Bottom Line: It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s.Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains.Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

View Article: PubMed Central - PubMed

Affiliation: Institute for Amphibian Biology, Graduate School of Science, Hiroshima University, Higashi-Hiroshima, Hiroshima, Japan.

ABSTRACT
The Western clawed frog, Xenopus tropicalis, is a highly promising model amphibian, especially in developmental and physiological research, and as a tool for understanding disease. It was originally found in the West African rainforest belt, and was introduced to the research community in the 1990s. The major strains thus far known include the Nigerian and Ivory Coast strains. However, due to its short history as an experimental animal, the genetic relationship among the various strains has not yet been clarified, and establishment of inbred strains has not yet been achieved. Since 2003 the Institute for Amphibian Biology (IAB), Hiroshima University has maintained stocks of multiple X. tropicalis strains and conducted consecutive breeding as part of the National BioResource Project. In the present study we investigated the inbreeding ratio and genetic relationship of four inbred strains at IAB, as well as stocks from other institutions, using highly polymorphic microsatellite markers and mitochondrial haplotypes. Our results show successive reduction of heterozygosity in the genome of the IAB inbred strains. The Ivory Coast strains clearly differed from the Nigerian strains genetically, and three subgroups were identified within both the Nigerian and Ivory Coast strains. It is noteworthy that the Ivory Coast strains have an evolutionary divergent genetic background. Our results serve as a guide for the most effective use of X. tropicalis strains, and the long-term maintenance of multiple strains will contribute to further research efforts.

No MeSH data available.


Related in: MedlinePlus