January 1, 2015;
Inbreeding Ratio and Genetic Relationships among Strains of the Western Clawed Frog, Xenopus tropicalis.
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.
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Fig 1. General pedigree chart of X. tropicalis strains.
Frogs facing left represent the Ivory Coast strain and those facing right are Nigerian strains. Frogs colored black indicate strains used in this study. The arrows and figures next to frogs indicate transportation of animals and the years when those were held (if there is a record).
Fig 2. Alignment of physical and genetic linkage maps of X. tropicalis.
Left = FISH cytogenetic map . Middle = physical map from full nucleotide sequence data (JGI X. tropicalis genome assembly 7.1). Right = genetic map based on linkage analysis . The sixty loci used in our analyses are indicated on the right. All maps are size proportional. Physical nucleotide units are mega base (Mbp), and genetic map units are centimorgan (cM).
Fig 3. Observed distribution of pairwise r estimates among X. tropicalis strains.
Box plots represent the distribution of relatedness values (r) of each strain. Mean and SD values are described in the parentheses below the labels.
Fig 4. Neighbor joining trees and comparison of the control region structures of mitochondrial genomes.
(A) Neighbor joining trees of the strains based on genetic distances (DA) of SSLP data (left) and p-distance of mitochondrial haplotypes (2,328 nucleotide sites) (right). Numbers on branches indicate percent bootstrap probability (>50%). The values for SSLP tree are given in order for DA/DPS. (B) Comparison of the control region structures of mitochondrial genomes. Structures of the control region and alignments of identified putative origin of H-strand replication (OH), conserved sequence blocks (CSB-1-3), and repeat motifs are shown. (C) Neighbor joining tree of the strains and wild individuals based on p-distance of mitochondrial haplotypes (1,109 nucleotide sites).
Fig 5. 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 . 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.