Induction and identification of gynogenesis in Nibea albiflora

Abstract

In order to establish a procedure to induce artificial gynogenesis for genetic improvement and sex control studies in Nibea albiflora,ultraviolet irradiation was used to genetically inactivate the sperm and cold shock was used to induce the chromosome duplication of the eggs.Relative DNA content in newly-hatched larvae of candidate haploid gynogens,candidate diploid gynogens and the control groups from normal fetilization were mensurated,and paternity test was made using SSR markers for confirmation of gynogenesis.The gynogenetic diploid was successfully induced by activating egg development with UV-irradiated sperm combined with cold shock to prevent extrusion of the second polar body.UV irradiation time range was preliminarily determined by an insemination trial with the sperms irradiated by different doses of UV.The fertilized egg hatchability exhibited typical Hertwig effect when sperm irradiation time was 0-100 seconds at a UV intensity of 3 800 μW/(cm2·s).All newly-hatched larvae exhibited typical haploid syndrome when the UV irradiation time was over 60 seconds.In addition,we designed an orthogonal experiment to further optimize UV irradiation time,cold shock initiation time and cold shock duration time.The orthogonal experiment results showed that the hatching rate was highest(16%)in the following conditions:sperm irradiation time was 60 seconds,cold shock temperature was 3-4 ℃,cold shock initiation time was 2 minutes after fertilization and cold shock duration time was 10 minutes.The larvae of the optimal group have the same morphological characteristics and cellular DNA content with normal diploids.A further paternity test with five compatible SSR primers of Larimichthys crocea proved that the larvae in the optimal group were all gynogenetic diploids without paternal specific alleles.