Mitochondrial and nuclear inheritance in an androgenetic line of rainbow trout, Oncorhynchus mykiss

Mitochondrial and nuclear inheritance of clonal rainbow trout produced by androgenesis were evaluated using DNA sequencing and amplified fragment length polymorphism (AFLP) analysis. All individuals were propagated using sperm from a single OSU clonal line sex inversed female crossed with multiple egg sources whose nuclear material was destroyed using Cobalt(60) gamma radiation. A heat shock was administered to developing haploid embryos to prevent the first cleavage and restore diploidy. Sequencing of 4713 continuous base pairs was performed on four rainbow trout and one cutthroat trout mitochondrial haplotypes spanning the ND 5 protein subunit of Complex I through the control region. Total sequence variation between haplotypes ranged from 0.34% to 6.14% with rainbow trout haplotype variation between 0.34% and 0.85%. No evidence was found to indicate point mutations associated with irradiation. DNA sequence for coded proteins was converted to amino acid sequence using the standard mammalian mitochondrial code to determine conservative and non-conservative amino acid substitutions. Total amino acid variation ranged between 0.60% and 3.10% with rainbow trout haplotype variation from 0.60% to 1.12% with non-conservative amino acid substitutions present between all mitochondrial haplotypes. AFLP analysis was performed on 119 clonal individuals to determine levels of nuclear genetic variation within the clonal line. Twelve primer sets revealed nine polymorphic bands among the 493 readable bands. Seventy individuals showed between one and five polymorphic loci with nuclear AFLP band variation ranging from 0.0% to 1.01% among all possible pairs. DNA from 12 individuals containing variable loci was re-extracted and AFLP amplifications repeated. Repeated primer sets showed little variation between extractions with polymorphic loci present in all repeated primer sets. AFLP primer sets having polymorphic loci were run using OSU by Arlee doubled haploids to determine map locations on the published linkage map of rainbow trout. Three of the nine polymorphic loci had differential polymorphism between the parents to allow mapping. Two loci mapped to linkage group II 46.2 and 32.6 centimorgans away from the putative centromere while the third locus was unassignable. The large distances between the two mapped loci and the putative centromere, along with the inability to assign the third locus, support the possibility that identified variable loci result from tetrasomic inheritance. The type of tetrasomic inheritance present is the result of distal crossover between homologous chromosomes during spermatogenesis, secondary tetrasomy. The low level of genetic variation found within the OSU clonal line is comparable to reported genetic variation within inbred lines of mice, zebrafish, and other species. (C) 2002 Elsevier Science B.V. All rights reserved.