| Large yellow croaker Pseudosciaena crocea is the largest artificial breeding and culture species ofmarine fish in China. In recent years, reared P. crocea have begun to degrade in germplasm and loss ofgenetic diversity. It is needed to improve the germplasm and breed superior variety. Gynogenesis is aspecial type of sexual reproduction whereby the chromosomes of offspring are exclusively inherited fromthe mother. It is a valuable tool both in genetic research and aquaculture, e.g., producing monosexpopulation and pure lines, revealing sex-determing mechanisms, constructing genetic maps. Althoughgynogenetic diploids of P. crocea have been induced by UV-irradiated homologous sperm, some researcheshave reported that paternal genetic material was revealed into some offspring, which may due to theinadequacy of UV-irradiation. To ensure that any surviving offspring are truly gynogenetic, using suitableheterologous sperm is an alternative. In this study, gynogenesis was induced in P. crocea using untreatedMiichthys miiuy sperm or UV-irradiated Nibea albiflora sperm, and cold-shock was performed to diploidizethe female chromosome. By altering the initial time, temperature and duration, cold shock at3-4℃for10-13min which started in2-3min after fertilization was found as the optimum protocol for retaining thesecond polar body and producing meiotic diploids P. crocea with heterologous sperm. Under the abovecondition, the morphologic of gynogenetic P. crocea induced by unirradiated M. miiuy and UV-irradiated N.albiflora sperm were all similar compared to normal P. crocea, and cytogenetic analysis showed all thegynogenetic larveas were diploid with~48chromosomes. The results of microsatellite DNA analysisshowed that gynogenetic progeny were exclusive maternal inheritance.The embryonic development and SSR patterns were compared between the two groups of gynogenetic P.crocea which were induced by UV-irradiated homologous sperm and untreated heterologous (M. miiuy)sperm, respectively. The fertilization and hatching rate of heter-gynogenesis was41.33±0.58%and36.67±0.58%, respectively, and both higher than the rate of homo-gynogenesis, which was20.67±1.52%and24.67±0.58%, respectively. But the survival rate was opposite, which was3.28±0.09%forheter-gynogenesis and6.93±0.15%for homo-gynogenesis. In the two gynogenesis groups, the embryoswhich were failed to inhibit the extrusion of the polar body show obvious haploid syndrome; the embryoswhich were successful diploidized had the same process with the normal diploid embryos. However, thedeveloping speed of gynogenetic diploid was slower than control group: the homo-and heter-gynogenesisembryos delayed3min and2min at blastodisc stage,35min and45min at low blastula stage,55min and1h at late gastrula stage,50min and1h45min at heartbeat stage, and1h and1h30min at hatching stage,respectively. The delay may be due to the disturbance of cell division cycle caused by cold shock. Inhomo-gynogenesis, the analysis of SSR primes KPC9and KPC45showed in24tested offsprings,83.3%(20/24) had maternal bands only;12.5%(3/24) individuals had paternal bands in the two loci, suggestingthey derived from normal fertilization; and1individual showed paternal band in locus KPC9but absent inlocus KPC45, suggesting there were genetic leakage of the paternal fish. In heter-gynogenesis, the analysisof SSR prime KPC43showed18tested offsping all have maternal specific band only. The results showedheterologous sperm can avoid paternal gene inflowing to offspring. In gynogenesis induction, the selectionof homo-or heterologous sperm should accord to their relative merits.In order to analyze gynogenetic P. crocea induced by sperm of M. miiuy, as well as manage and protect M.miiuy and study the phylogeny in Sciaenidae, SSR markers were isolated for M. miiuy using genomiclibrary enriched by magnetic beads, and subsequently these markers were cross-amplified in P. crocea andused to analyze the gynogenetic P. crocea induced by sperm of M. miiuy. In total,247positive clones weresequenced and got138unique sequences that contained microsatellite motifs. Thirty-seven primer pairswere designed for35sequences and amplified in30M. miiuy individuals. Thirty primer pairs wereeffective, and within the tested population, twenty-four were polymorphic with an average of4.33allelesper locus (range from2to8). The observed (Ho) and expected (He) heterozygosity ranged from0.0667to 1.0000(mean0.6361) and0.3828to0.8139(mean0.6443), respectively. The mean PIC value was0.576,ranging from0.348to0.823. Seventeen loci showed high polymorphism (PIC>0.5). Eight loci weresignificant deviations from HWE, which may be due to the small sample size. Twelve out of the30primerpairs showed amplification in the different genus fish P. crocea and ten showed polymorphic with anaverage of5.10alleles per locus (range from2to8). The observed (Ho) and expected (He) heterozygosityranged from0.0000to0.8667(mean0.5200) and0.0655to0.8492(mean0.6053), respectively. The meanPIC value was0.555, ranging from0.062to0.815. Six loci showed high polymorphism (PIC>0.5). Fourloci were significant deviations from HWE. Except MM007(the homologous was51.08%between the twospecies), the sequence alignment of microsatellite flanking regions of other11loci showed highlyhomologous (range from89.63%to99.36%) between the two species. The gynogenetic P. crocea inducedby sperm of M. miiuy were analyzed through four cross-amplified primer pairs, and no specific paternalbands were found in the offspring. The results of four SSR markers indicate the genetic material of M.miiuy sperm did not revealed to the gynogenetic P. crocea as neither chromosome nor DNA fragment.The karyotype showed there was no identifiable sex chromosome in P. crocea. The inability to determinethe genotypic sex of P. crocea is a problem in aquaculture. In this study, AFLP technique was used toanalyse the bands between female and male P. crocea. Sixty-three out of64primer combinations couldamplify clear bands, but only the primer combination E-ACC/M-CTG got a band wihch was present in allmales and absent in all females. The result indicates there were only a few genomic differences betweenfemale and male P. crocea. Alignment showed this male-specific candidate band was two differentnucleotide sequences which were both188bp, and BLAST showed there was no homologous sequence inGenBank. Two PCR primers were designed, but amplification showed bands were appeared both in femaleand male P. crocea. We speculate that this sex-difference AFLP band came from the interaction ofrestriction enzyme recognition sites, DNA sequence modification (e.g, methylation) and other factors.In conclusion, this study established a technique that inducing gyonogenetic P. crocea by heterologoussperm (M. miiuy and N. albiflora) and using cold-shock to diploidize the female chromosome, verifiedgynogenesis through morphology, cytogenetic and SSR markers analysis, compared the embryonicdevelopment and SSR patterns between two group of gynogenetic P. crocea which were induced byhomologous sperm and heterologous (M. miiuy) sperm, isolated and characterized SSR markers for M.miiuy, verified the gynogenetic P. crocea using M. miiuy SSR markers which were cross-amplifed in P.crocea besides P. crocea SSR markers, screened a male-specific ALFP band. The result of this study wasuseful both for theoretical research and application research. Nevertheless, the growth, development(especially gonadal development) of gynogenetic offspring and identification of genotypic sex in P. croceaare needed to be further investigated. |
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