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 4.2 W/m(2), 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 degrees 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 (Y(2)Y(2)) 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 F(0) and F(1) males (Y(2)Y(2)) was superior to that of normal ones (X(1)Y(2)), but that of the F(0) and F(1) females (X(2)X(2)) was inferior to the control (X(1)X(2)). Of 21 crosses involving homozygous androgenetic (Y(2)Y(2)) males and heterozygous (X(1)X(2)) 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 (Y(2)Y(2)) males and (X(2)X(2)) females were crossed. Hence, the paternal autosomes, inherited by the homozygous androgenetic female (X(2)X(2)), produced female progenies in significantly less number of crosses, also at lower frequencies than the crosses with heterozygous females (X(1)X(2)), which carried an equal number of paternal and maternal autosomes. However, progenies resulting from the cross between gray female (X(1)X(2)) and golden male (Y(2)Y(2)), after undergoing androgenesis, were males, with paternal chromosomes alone, indicating that the presence of Y(2)Y(2) appears to override the modifying effect of autosomes, but the paternal or maternal autosomes seemed to override the single Y(2) present with X(1) or X(2), 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 (X(1)Y(2)) and androgenetic males (X(1)Y(2)) produced two amplicons of 237 and 300 bp length. However, they were not detectable in the female (X(1)X(2)) genomic DNA, which amplified only one amplicon of 100 bp. Genomic DNA extracted from the 18 unexpected female progenies expressed the (X(1)Y(2)) 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.