Background
A critical feature of Medaka laboratory husbandry has been the routine inbreeding of wild individuals from the Southern medaka population to isogenic strains pioneered by Hyodo-Taguchi in the 1980s. Some of these strains are now in their 80th brother-sister mating, and importantly, there are routine protocols for creating an inbred strain from the wild. At least 8 isogenic strains derived from single wild catches are available from the medaka NBRP stock center. Together with other features discussed elsewhere this makes medaka an ideal candidate for generation of a vertebrate inbred genetic reference panel. Characteristion of heterozygosity and phenotypes in existing inbred lines suggests that we are likley to achieve good homozygosity. This work is described in full detail in the G3 publication.
Sampling and Characterisation of Founders
We sampled fish from two Southern Japanese locations : irrigation canals in the Takashi Hongo and the Kiyosu areas near Toyohashi (Aichi Prefecture) in July 2010. By analysis of the mitotype of 50 and 109 individuals from the two sites respectively, we detected an unusual Northern Japanese mitotype in the Takashi Hongo population likely to be the result of human-mediated dispersal (e.g. fish discarded from aquariums). Therefore we settled on fish from the Kiyosu population for further analysis.
We used genotyping and high throughput sequencing to characterise population structure and sequence diversity. Phylogentic analysis of 8 trios showed that the parental genotypes were largely equidistant from each other without visible structure.
Estimating the LD for the medaka Kiyosu population from the 16 parental sample genotypes, showed that the median r2 between pairs of loci gradually drops with increasing distance, reaching a minimum at around 12.5 kb. ~85% of SNP pairs with r2 > 0.8 mapped to the same gene, with ~37% mapping to the same exon, so that fine mapping in medaka should be largely possible to the resolution of single genes and frequently even to a single exon. FThe mean haplotype block size was 712 bp (median 259 bp) with a maximum of ~78 kb. Overall these data indicate that a mapping panel generated from this population was likely to have favourable LD properties for mapping traits.
Inbreeding Progress
We have currently started the 4th generation of inbreeding. We expect at least 9 generations will be required and will be testing the panel extensively at that stage. Please come back later for further updates, and access arrangements once the panel is completed. We will also provide updates via Twitter.
Characterising Existing Inbred Lines
Inbreeding and Heterozygosity
To illustrate the likely properties of the inbred lines that will form the Medaka genetic reference panel, we characterised the genomes of four inbred lines relative to the reference line HdrR by high throughput sequencing. The lines are HNI and Kaga from Northern Japan, HSOK from South Korea and Nilan from Taiwan. The inbred lines were predominantly highly homozygous (see right) with few regions of high heterozygosity and mean heterozygosity levels around 100 fold lower (average around 3x10-5) than a wild catch (1.5x10-3). There are few blocks of high heterozygosity consistent with a wild origin. The Nilan strain is not as inbred as the others (heterozygosity at 2x10-4 with more blocks of wild-like heterozygosity consistent with far fewer cycles of inbreeding. Chromosome 1 harbors the sex determination locus, and as the individuals sequenced for Nilan, Kaga and HNI, were male, there is a stretch of residual heterozygosity is centered around the known sex determination locus. We conclude that inbreeding in medaka can create highly homozygous individuals, with the expected exception of the sex chromosome.
| Strain | Mean heterozygosity | Generation |
|---|---|---|
| Wild catch females | 0.00163 | 1 |
| Wild catch males | 0.00163 | 1 |
| Nilan | 0.0002032 | 16 |
| HSOK | 0.0000227 | 30 |
| Kaga | 0.0000527 | 35 |
| HNI | 0.0000374 | 70 |
Heterozygosity estimates for inbred lines.
Phenotypic Diversity
An important feature of a population derived inbred panel is that it should exhibit appreciable phenotypic variation across the individual lines. To characterize one set of phenotypes we took advantage of existing Southern inbred lines since these are likely to be representative of lines generated from the Kiyosu population. Using light microscope-based imaging combined with an automatic annotation algorithm we derived a number of morphometric features across four Southern (HdrR, Icab, HNCMH2, HO5), two Northern strains (Kaga, HNI) and the Kiyosu population (panel A, below). Of the 7 phenotypes analyzed, after normalising for body length, 4 show greater than 30% broad sense heritability i.e. the differences between strains explained 30% or more of the variance (panel B). An example is shown in Panel C where a fish from the HdrR strain has substantially smaller eyes relative to body length than the Icab strain (compare box plot in panel D). The broad sense heritability estimate is similar to analogous morphometric measurements between inbred mouse strains, suggesting that medaka populations have similar levels of phenotypic variation. Many other established phenotypes that differ between medaka strains have been observed and further work will be needed to examine the suitability of this population for each phenotype. Given the high diversity of alleles in the Southern population we expect many phenotypes to have least some genetic variance in this population.
Analysis of Morphometric pehnotypes in inbred lines