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/m<SUP>2</SUP>, 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&#176;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<SUP>2</SUP>Y<SUP>2</SUP>) 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<SUB>0</SUB> and F<SUB>1</SUB> males (Y<SUP>2</SUP>Y<SUP>2</SUP>) was superior to that of normal ones (X<SUP>1</SUP>Y<SUP>2</SUP>), but that of the F<SUB>0</SUB> and F<SUB>1</SUB> females (X<SUP>2</SUP>X<SUP>2</SUP>) was inferior to the control (X<SUP>1</SUP>X<SUP>2</SUP>). Of 21 crosses involving homozygous androgenetic (Y<SUP>2</SUP>Y<SUP>2</SUP>) males and heterozygous (X<SUP>1</SUP>X<SUP>2</SUP>) 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<SUP>2</SUP>Y<SUP>2</SUP>) males and (X<SUP>2</SUP>X<SUP>2</SUP>) females were crossed. Hence, the paternal autosomes, inherited by the homozygous androgenetic female (X<SUP>2</SUP>X<SUP>2</SUP>), produced female progenies in significantly less number of crosses, also at lower frequencies than the crosses with heterozygous females (X<SUP>1</SUP>X<SUP>2</SUP>), which carried an equal number of paternal and maternal autosomes. However, progenies resulting from the cross between gray female (X<SUP>1</SUP>X<SUP>2</SUP>) and golden male (Y<SUP>2</SUP>Y<SUP>2</SUP>), after undergoing androgenesis, were males, with paternal chromosomes alone, indicating that the presence of Y<SUP>2</SUP>Y<SUP>2</SUP> appears to override the modifying effect of autosomes, but the paternal or maternal autosomes seemed to override the single Y<SUP>2</SUP> present with X<SUP>1</SUP> or X<SUP>2</SUP>, 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<SUP>1</SUP>Y<SUP>2</SUP>) and androgenetic males (X<SUP>1</SUP>Y<SUP>2</SUP>) produced two amplicons of 237 and 300 bp length. However, they were not detectable in the female (X<SUP>1</SUP>X<SUP>2</SUP>) genomic DNA, which amplified only one amplicon of 100 bp. Genomic DNA extracted from the 18 unexpected female progenies expressed the (X<SUP>1</SUP>Y<SUP>2</SUP>) 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