Phenotype,Gonad Development And Related Genetic Analysis Of Hybrid Progeny Of Grouper

Grouper is a rare Marine fish in the world,and also an important fish for Marine fishing and aquaculture in China.Its aquaculture output ranks the forefront of Marine fish and plays a pillar role in the industrial development.In recent years,the scale of grouper breeding is gradually expanding,which directly leads to a sharp increase in the demand for larvae.However,the artificial breeding technology of grouper is difficult,the survival rate of seedlings is low,and large-scale production may lead to the degradation of germplasm resources,which is difficult to meet the social needs.Therefore,under the premise of sustainable development of grouper,cross breeding is an effective method.This scientific problem can be solved by cross-breeding and breeding some varieties with high survival rate,fast growth rate and strong disease resistance.Yun Long grouper is a new varieties obtained through hybridization in recent years,which combines the characteristics of rapid growth and strong disease resistance of its parents.At present,the new variety has been in many coastal provinces for large-scale breeding,some of the seedlings have been exported abroad,has been recognized by the majority of farmers.The relevant studies on hybrid of Epinephelus lanceolatus♀×Epinephelus moara♂F1 Grouper of Yun long in this subject are summarized as follows:（1）Early development of hybrids of Epinephelus lanceolatus♀×Epinephelus moara♂and growth characteristics of reciprocalHybridization is the most widely used and effective way of artificial breeding in aquaculture.This study use Epinephelus lanceolatus as female parent,E.moara as male parent by artificial insemination technology has carried on the interspecific cross experiment,and developmental sequence of fertilized egg,growth of larvae,juvenile and young fish was observed and studied,and then with the parent and the hybrid F1generation Yunlong grouper（E.moara♀×E.lanceolatus）contrasted the phenotypic traits.Results showed the reverse crossing F1 generation can develop normally to juvenile stage,and its has four phenotypes.The growth comparison results showed that the growth rate of the positive and negative cross F₁ generation was between that of the parents,which was faster than that of the E.moara,which was slower than that of the E.lanceolatus,and the growth rate of the reverse cross F₁ generation was slower than that of the orthogonal F₁ generation.The results provide a basis for the formation of hybrid dominant traits and genetic analysis of grouper.（2）Analysis on ploidy and gonadal development of Epinephelus moara♀and Epinephelus moara♀×Epinephelus lanceolatus♂Grouper is a hermaphrodite,female first mature bony fish,4 to 5 years of age can reach sexual maturity and ovarian development is complete,in the reproductive season can be divided into batches of eggs.But hybrid offspring Epinephelus moara♀×Epinephelus lanceolatus♂has been more than 5 years old,there is still no sign of sexual maturity.In order to find out the scientific problem that hybrid F1 E.moara♀×E.lanceolatus♂develops to the fifth instar but does not reach sexual maturity and does not spawn,on the one hand,this study put forward the hypothesis of sterility caused by triploid produced by natural hybridization,and used the method of DNA content determination by flow cytometry to verify whether hybrid F1 E.moara♀×E.lanceolatus♂is triploid.On the other hand,the gonad development degree of Grouper of different ages was observed through pathological tissue sections.The results showed that the fluorescence value of 175 Groupers in the experimental group was 101.3±8.01by flow cytometry.The fluorescence value of 6 E.moara groupers in the control group was 104.2±9.77,and the ratio between them was 1.02.The results showed that no polyploid was found among the 175 hybrid F1 E.moara♀×E.lanceolatus♂,all of them were conventional diploid.At the same time observe the hybrid F1 E.moara♀×E.lanceolatus♂of gonad slice of different ages,from the original gonads form initial stage to the age of five,find that the growth rate of primitive gonads was similar to that of E.moara♀,3-5 year-old hybrid F1 E.moara♀×E.lanceolatus♂ovaries stay in theⅢfinal toⅣearly period for a long time,compared to the age of 5 E.moara grouper has to lay eggs,experiencedⅤperiod after ovarian appear shrivel,oocyte degradation also to 4 phase,hybrid F1 E.moara♀×E.lanceolatus♂gonad development significantly slower than the homozygous maternal E.moara♀.The results laid a foundation for the polyploid detection and polyploid breeding of hybrid grouper in the future,and systematically observed and recorded the gonad development of hybrid F1 E.moara♀×E.lanceolatus♂,which provided a reliable experimental material for future experiments.（3）Contrastive analysis of pituitary transcriptome of Yunlong grouper and E.moaraIn this study,Illumina high-throughput sequencing technology was used to perform transcriptome sequencing and bioinformatics analysis on pituitary tissues of Yunlong grouper and E.moara in order to analyze the molecular mechanisms related to their growth and reproductive development.The results showed that a total of 7736differentially expressed genes were obtained,among which 2412 genes were up-regulated and 5324 genes were down-regulated in pituitary tissue samples（EM-VS-EML）.Then GO functional annotations and KEGG analysis,screening the related to the growth and reproductive development path,the result shows that the neural activity associated with growth of ligand and receptor interaction pathways（Neuroactive ligand-receptor interaction）the difference of gene enrichment degree is higher,its growth hormone,growth hormone,GH)and prolactin（prolactin,PRL）common control.Among them,GH protein and PRL protein in pituitary gland of Yunlong grouper were up-regulated by 28 and 14 respectively compared with those in pituitary gland of E.moara.It is preliminarily speculated that this process of neuroactive ligand-receptor interaction is related to the cross dominance of reproductive development.Among the screening of reproductive and developmental pathways,the enrichment degree of oxytocin signaling pathway is the highest.This pathway is mainly affected by oxytocin receptor（OTR）and released into circulation under the action of a series of enzymes such as phosphatidylinositol phospholipase C（PLC）and protein kinase C（PKC）.The average number of oxytocin receptors（OTR）in morel grouper was6.7,while that in morel grouper was 0.Phosphatidyl inositol phospholipase C（PLC）E.moara have an average of 2982 and yunlong grouper is only 20.3,protein kinase C（PKC）and peptide chain elongation factor 2（EEF2）were also significantly lower,presumed to oxytocin-oxytocin receptor interaction may be caused by the process of the main reasons for the sluggish development in yunlong grouper,but also need to be further verified experiment.In this study,the growth advantages and reproductive disadvantages of grouper were studied at the molecular level,which provided valuable references for future molecular breeding and reproductive regulation.