GFP reporter gene confirms paternity in the androgenote Buenos Aires tetra, Hemigrammus caudovittatus

Abstract

A protocol for successful induction of androgenetic cloning of the Buenos Aires tetra (BT), Hemigrammus caudovittatus, with contrasting gray and golden strains is described. At the intensity of 4.2W/m², UV irradiation for 2.75 min totally inactivated the maternal genome in eggs of gray BT. Following activation by sperm of golden BT, the 25-min-old embryos were shocked at 41°C for 2 min to restore diploidy. Interestingly, the hatching success of the haploid fry was always higher than that of the diploid fry, indicating that the enhanced homozygosity (Y²Y²) is more deleterious than haploidy. Maternal genomic inactivation was confirmed by (i) expression of green fluorescent protein (GFP) gene in the 6-16 hr old live haploid and aneuploid embryos, (ii) golden body color in the diploid fry and adult and (iii) progeny testing. Survival of androgenotes was 10% at hatching and 6% at sexual maturity. Reproductive performance of F₀ and F₁ males (Y²Y²) was superior to that of normal ones (X¹Y²), but that of the F₀ and F₁ females (X²X²) was inferior to the control (X¹X²). Of 21 crosses involving homozygous androgenetic (Y²Y²) males and heterozygous (X¹X²) females, 7 of them (33%) produced 3-9% unexpected female progenies. But only a single cross (14%) generated 3-4% unexpected female progenies, when 7 pairs of homozygous androgenetic (Y²Y²) males and (X²X²) females were crossed. Hence, the paternal autosomes, inherited by the homozygous androgenetic female (X²X²), produced female progenies in significantly less number of crosses, also at lower frequencies than the crosses with heterozygous females (X¹X²), which carried an equal number of paternal and maternal autosomes. However, progenies resulting from the cross between gray female (X¹X²) and golden male (Y²Y²), after undergoing androgenesis, were males, with paternal chromosomes alone, indicating that the presence of Y²Y² appears to override the modifying effect of autosomes, but the paternal or maternal autosomes seemed to override the single Y² present with X¹ or X², and induced the production of unexpected female progenies. Using Double sex Mab3 related transcription factor (DMRT 1)-specific primers, PCR analyses of the genomic DNA of the normal (X¹Y²) and androgenetic males (X¹Y²) produced two amplicons of 237 and 300 bp length. However, they were not detectable in the female (X¹X²) genomic DNA, which amplified only one amplicon of 100 bp. Genomic DNA extracted from the 18 unexpected female progenies expressed the (X¹Y²) genotype-specific banding pattern with two amplicons of 237 and 300 bp length and thereby confirmed that they were genotypic males. A partial sequencing of the male-specific sequence indicated that DMRT 1-specific primer was bound to the fragment of the genomic DNA of the male tetra, although the male-specific sequence of DMRT 1 was not completely detectable.