Hybrid origin of gynogenetic clones and the introgression of their mitochondrial genome into sexual diploids through meiotic hybridogenesis in the loach,Misgurnus anguillicuadatus

In a few Japanese populations of the loach Misgurnus anguillicaudatus (Teleostei: Cobitidae), clonal diploid lineages produce unreduced diploid eggs that normally undergo gynogenetic reproduction; however the origin of these clones remains elusive. Here, we show the presence of two diverse clades, A and B, within this loach species from sequence analyses of two nuclear genes RAG1 (recombination activating gene 1) and IRBP2 (interphotoreceptor retinoid-binding protein, 2) and then demonstrate heterozygous genotypes fixed at the two loci as the evidence of the hybrid nature of clonal lineages. All the clonal individuals were identified by clone-specific mitochondrial DNA haplotypes, microsatellite genotypes, and random amplified polymorphic DNA fingerprints; they commonly showed two alleles, one from clade A and another from clade B, whereas other wild-type diploids possessed alleles from either clade A or B. However, we also found wild-type diploids with clone-specific mitochondrial DNA and nuclear genes from clade B. One possible explanation is an introgression of a clone-specific mitochondrial genome from clonal to these wild-type loaches. These individuals likely arose by a cross between haploid sperm from bisexual B clade males and haploid eggs with clone-specific mtDNA and clade B nuclear genome, produced by meiotic hybridogenesis (elimination of unmatched A genome followed by meiosis after preferential pairing between two matched B genomes) in clone-origin triploid individual (ABB)

Development of nuclear DNA markers to characterize genetically diverse groups of Misgurnus anguillicaudatus and its closely related species

Repetitive DNA sequences, ManDra and ManBgl, were isolated from the DraI and BglII digests of the genomic DNA of Misgurnus anguillicaudatus, respectively. A primer set of ManDra distinguished two genetically different groups (A and B) of M. anguillicaudatus by specific electrophoretograms. A primer set of ManBgl amplified the DNA of M. anguillicaudatus and M. mizolepis. The individuals of M. anguillicaudatus were divided into two groups depending on the fragment sizes, in which the groups A and B (B-1 and B-2) showed 400 and 460 bp, respectively. M. mizolepis was distinguished by a different pattern (400-, 460-, and 510-bp fragments). PCR-RFLP analyses of recombination activating gene 1 gave a clear difference between A or B-2 (443-bp fragment) and B-1 groups (296- and 147-bp fragments). Clonal lineages and hybrids between B-1 and B-2 groups could be identified by appearance of three fragments (443, 296, and 147 bp). The combined analyses using the above three nuclear markers discriminated among nuclear genomes of genetic groups (A, B-1 and B-2) of M. anguillicaudatus and M. mizolepis. In several localities, natural hybridizations between the group B-1 and B-2 loaches and introgressions of clonal mitochondrial genomes into the group B-1 loaches were detected