The Relationship Between The Inviability Of Cyprinid Hybrid(Blunt-Snout Bream♀×Goldfish♂)and Cytonuclear Conflict

Hybrids between species are usually unviable or sterile.One possible mechanism causing reproductive isolation is incompatibility between genes from different species.Intragenomic conflicts and cytonuclear incompatibilities are as engines of speciation.In this thesis,basing on the hybridization between goldfish(carassius auratus red var.)and blunt-snout bream(megalobrama amblycephala),we found that the offspring produced by hybridization goldfish(♀)and blunt-snout bream(♂)are survival.However,100% of the reciprocal offspring are unviable.With this phenomenon and theory,we boldly hypothesized that cytonuclear may be one of the most important factor for the lethality of reciprocal hybrid.To test our hypothesis,in this study we conducted investigation of mitochondrial DNA in four representative stages(blastula,gastrula,heartbeat and fry)of embryo of hybridization of blunt-snout bream(♀)and goldfish(♂)(BG)with the control of hybridization goldfish(♀)and blunt-snout bream(♂)(GB),goldfish and blunt-snout bream.Firstly,we detected the change of parental mtDNA during embryonic development and tried to exploring the relationship between mtDNA change and embryonic lethality.Additionally,we also used the technology of microinjection to search the effect of foreign mtDNA and its change during embryogenesis.Furthermore,we also took advantage of high throughput sequencing,and we sequenced four representative embryonic stage of these four fish.We hope to obtain some genes related to the lethality of BG.By analyzing the hybrids of goldfish and blunt-snout bream,we observed the behavior of paternal mitochondria in embryo and also elucidated the relationship between cytonuclear conflict and hybrid lethality.In this thesis,we have obtained some results and conclusions as follows: 1.To explore the change between experimental group and control group,we designed species specific primers for goldfish and snout-blunt bream based on cytb gene.We systematically investigated the change of parental mtDNA during embryogenesis.We found that paternal mtDNA shows late elimination and transcriptional quiescence in cyprinid fishes.Paternal mtDNA was present in zygotes but absent in larvae and adult organs of goldfish and blunt-snout bream,demonstrating paternal mtDNA delivery and elimination for maternal mtDNA inheritance.Surprisingly,paternal mtDNA remained detectable up to the heartbeat stage,suggesting its late elimination leading to embryonic heteroplasmy up to advanced embryogenesis.Most importantly,we never detected the cytb RNA of paternal mtDNA at all stages when paternal mtDNA was easily detectable,which reveals that paternal mtDNA is transcriptionally quiescent and thus excludes its effect on the development of heteroplasmic embryos.Therefore,paternal mtDNA in cyprinids shows late elimination and transcriptional quiescence.Clearly,transcriptional quiescence of paternal mtDNA represents a new mechanism for maternal mtDNA inheritance and provides implications for treating mitochondrion-associated diseases by mitochondrial transfer or replacement.2.Mitochondrion(MT)and its DNA(mtDNA)show maternal inheritance in most eukaryotic organisms,in which sperm mtDNA is usually delivered into the egg during fertilization but eliminated shortly after fertilization.Mouse mtDNA in microinjected sperm or liver MT can persist until or post birth.Fertilization-delivered fish sperm mtDNA exhibits late elimination and transcriptional quiescence in cyprinid fish embryos.Here we report the fate of MT injection-delivered foreign mtDNA in cyprinid embryos.Goldfish and zebrafish MT from heart,liver and sperm were microinjected reciprocally into the zebrafish and goldfish zygotes and examined for mtDNAs and their RNAs during embryogenesis until birth.Rather than disappearance and transcriptional quiescence at the heartbeat stage for fertilization-delivered sperm mtDNA,these MT injection-delivered foreign mtDNAs were capable of persistence and transcription until birth.Amazingly,MT-injected zebrafish embryos developed normally,demonstrating that heteroplasmy does not affect cyprinid embryogenesis.Therefore,persistence and transcription of foreign mtDNA are dependent on the delivery strategy rather than MT source,suggesting the presence of sperm factor(s)responsible for elimination and transcriptional quiescence of fertilization-delivered sperm mtDNA but not MT injection-delivered foreign mtDNA prior to birth.These findings provide insights into mechanisms underlying mtDNA fate and heteroplasmy in normal and disease processes.Hybridization between two species often causes cytonuclear conflict in hybrids,and then leads to hybrid unviable,sterile and abnormal in phenotype.Recently,many researchers have done a lot of studies on cytonuclear incompatibility.However,there are still few direct evidences for cytonuclear incompatibility.Both blunt-snout bream(2n=48)and goldfish(2n=100)belong to cyprinidea,but belong to two different subfamily of cultrinea and cyprinea.In this study,we artificially crossed blunt-snout bream and goldfish.Intriguingly,we found that the hybrid of BG was abnormal and will be die in ten days after hatching.Taking into the consideration of maternal genomic greatly larger than paternal genomic in hybrid GB,and mitochondria are strictly maternally genetic,we think that the conflict of cytonuclear in GB is weaker than in BG.so we hypothesized that one of the most important reason for hybrid lethality may be due to the incompatibility between the maternal mitochondrial DNA and the bigger paternal genomic.To test our hypothesis,we microinjected mitochondria isolated from goldfish sperm.The result showed that the destiny of BG was not changed after microinjecting paternal mitochondria.However,fortunately the degree of abnormality was greatly released and even some fries could swim normally.Therefore,we safely concluded that cytonuclear conflict have strongly influence on energy supply in BG,which may lead to BG abnormal.The cause of hybrid lethality is co-effect of cytonuclear and nuclear-nuclear interaction.Taken these experiments together,we concluded that though paternal mitochondrial DNA is late eliminated,its transcription is quiescent during embryogenesis,which prevents paternal mitochondrial gene contributing to the development of embryo.Meanwhile,there is no relationship between BG lethality and paternal mtDNA late elimination.Additionally,we attempted to microinject paternal mitochondria to one cell embryo of BG to change the destiny of hybrid lethality,but it failed.These results show that we did not find the reason of embryo lethality by conducting experiment on mitochondria.Therefore,to further our study on the relationship between hybrid lethality and cytonuclear conflict,taking advantage of high throughput sequencing,we conducted RNA-seq on four representative embryos of the four species.By investigating gene expression globally and comparing BG embryonic gene expression to the other species,we obtained many differentially expressed genes.Meanwhile,we constructed a mimetic reference transcriptome of hybrid fish.Then we analyzed differential expression of parental genes in hybrid fish and we obtained parental gene expression in fish hybrid embryo.These transcriptomic analysis on early embryonic stages greatly facilitate us to explore incompatibility between parental nuclear genes and maternal mitochondrial genes.It is also provided a crucial foundation for the further study on the relationship between cytonuclear conflict and cyprinid hybrids lethality.