Analysis Of The Phenotypes And Genetic Characters Of The Grouper Hybrid

Grouper(Epinephelus)is an important marine economic fish in China due to its delicious meat and rich nutrition,and is mainly cultured in the southeast coast of China,but also in the north of the China.In recent years,on account of the maturity of grouper farming technology and the sharply expansion of market demand for grouper,the scale of grouper farming has expanded dramatically.In the same time,grouper is facing problems such as degradation of germplasm resources,susceptibility to diseases and high deformation rate.The sustainable development of the grouper farming industry requires new grouper species with excellent traits to maintain.As an effective way to breed new varieties,hybrid breeding is an effective means to solve the degradation of biological germplasm genetic resources,and is widely used in grouper breeding.For many hybrid offspring,the study of their phenotype and genetic traits is particularly important.1.Determination of growth and deformities and transcriptome sequencing analysis of Yunlong GrouperThe Yunlong grouper is a novel hybrid grouper obtained by crossing the Epinephelus moara(?)and Epinephelus lanceolatus(?).The Yunlong grouper combines the advantages of both parents,such as fast growth rate and rich nutrition.Compared with the parent,Yunlong grouper shows significant growth advantages and has been confirmed as a novel variety.In this study,male E.lanceolatus and female E.moara were used to establish 28families of Yunlong grouper as well as three families E.moara by selfing of E.moara.The average fertilization rate of the Yunlong grouper families was 55.5%±26.7%and the average deformity rate was 8.3%±0.9%.At 45 to 245 days of age,the growth curves of Yunlong grouper were W=0.0392L^2.8912(R²=0.9869),E.moara's W=0.0255L^3.0216(R²=0.9908),with Yunlong grouper being the allometric growth type and E.moara being the isochronous growth type at this stage.At day 245,the average body weight and body length of Yunlong grouper were(316.7±57.3)g and(22.5±1.7)cm,respectively,and those of E.moara were(123.2±30.2)g and(16.8±1.3)cm.The body weight and body length of Yunlong grouper are 2.6 and 1.3 times that of E.moara,respectively.A 12-month comparative culture of Yunlong grouper with Zhenzhulongdan grouper was conducted,the body weight was 1.3 times of the Zhenzhulongdan grouper,and the length was 1.2 times.The heterosis of Yunlong grouper was obvious.The prevalence of deformed fish in grouper breeding has brought about a large economic loss to the development of grouper farming.In the Yunlong grouper we breed,the average deformity rate was 8.3%±0.9%,and the deformity rate of an individual family was as high as 44.7%.Studies on the occurrence of deformities in grouper are lacking.We therefore sequenced the transcriptome of brain,pituitary and vertebrae of normal and malformed fish from the high malformation rates family using Illumina Hi Seq sequencing technology.A total of 1.05 billion high quality reads were obtained from 18 RNA-seq libraries.87,888 unigenes were assembled and 36,268 unigenes were annotated,the length of unigenes ranging from 201 to 28,922 bp.We performed a comparative transcriptome study of normal and deformed fish and obtained 398,136and 2,341 differentially expressed genes in brain,pituitary and vertebrae,respectively.A total of 706 up-regulated genes and 2,169 down-regulated genes were obtained.GO functional enrichment analysis and KEGG pathway analysis of the differentially expressed genes revealed the involvement of DNA binding,kinase activity,cytokine receptor response,neural ligand receptor action,calcium signaling pathway,extracellular matrix receptor interaction,estrogen signaling pathway and mineral absorption pathway in the development of vertebral malformations.Furthermore,three modules related to malformed traits were identified by weighted gene co-expression network analyses,and some genes with high connectivity were screened,such as cyclin-dependent kinase 4 and E3 ubiquitin-protein ligase RNF19A.In the blue module,most of the genes were found to be associated with OCM,GPIBA and Mmp9,all of which are closely related to vertebral bone development.In addition,validation by q PCR showed that the transcriptome data from this study were of high quality and met the requirements for bioinformatics analysis.2.Mitochondrial structure and evolutionary analysis of hybrid grouperGrouper species are diverse and widely distributed,and mitochondrial DNA has its own unique characteristics and is an important marker for molecular phylogenetic studies.In this study,we present the mitochondrial structure of the hybrid offspring of E.moara(?)×Epinephelus tukula(?)and analyse the phylogeny of the mitochondrial DNA of the grouper.The mitochondrial DNA of the hybrid offspring was 16,695 bp in length,including 13 PCGs,2 r RNA genes,22 t RNA genes,1 replication start site and 1control region.The composition and order of the genes is consistent with other vertebrates.Of these 13 PCGs,12 PCGs are distributed in the heavy strand and ND6encodes in the light strand.The mitochondrial whole genome of the hybrid offspring has a high AT in base usage,with positive values for AT and negative values for GC.There are three types of stop codons,including T(ND2,COXII,ND3,ND4 and Cytb),TA(COXIII)and the remains is TAA.All t RNAs except t RNA^Ser(AGN)could not form a classical cloverleaf pattern due to the lack of a point classical loop.Phylogenetic tree analysis revealed that Yunlong grouper,the offspring of E.moara(?)×E.tukula(?)were most closely related to E.moara,showing that the mitochondria of the hybrid offspring followed the maternal inheritance,which is a guideline for genetic research,species identification,phylogeny and hybridization breeding.3.Analysis of the phenotypes and genetic characters of the hybrid Epinephelus akaara?×Epinephelus tukula?In the hybridization breeding process of grouper,the viability of the hybrid combination and the genetic characteristics of the hybrid offspring need to be evaluated.A new hybrid combination of E.akaara?and E.tukula?was investigated in terms of growth and development,chromosome analysis,mitochondrial analysis and microsatellite analysis.At water temperature of 20.3-24.7°C and a salinity of 30,the hybrid offspring E.akaara?×E.tukula?took 40 h 55 min to hatch out of the membrane and complete the embryonic development.E.akaara is 41 h 18 min.The post-embryonic development was completed in four periods:the early larvae(1-3 d),the late larvae(4-31 d),the larval stage(32-57 d)and the juvenile stage(58 d-).The weight of one-year-old hybrid offspring reaches(176.6±28.7)g and the total length reached(22.4±1.1)cm,while E.akaara weighed(81.4±15.0)g and reached(17.5±1.0)cm in total length.The weight of the hybrid offspring is 2.17 times that of E.akaara and 1.28 times of the full length.Compared to E.akaara,hybrid offspring had black patches attached to the body surface,with inconspicuous stripes and a similar body shape to red-spotted grouper.Chromosome preparations were prepared by the head kidney-colchicine method for four-month-old E.akaara and the hybrid E.akaara?×E.tukula?.Use oil mirror to observe and split phase statistics.The hybrid offspring had a chromosome number of2n=48,account for 72%and a karyotype formula of 2n=3sm+3st+42t.The E.akaara had a chromosome number of 2n=48,account for 70%and a karyotype formula of2n=4sm+6st+38t.The karyotype of E.tukula was found to be 2n=2sm+46t,indicating that the genetic material of the hybrid offspring was derived from one set of chromosomes from each parent.The mitochondrial DNA of the hybrid offspring was16,928 bp in length and contained 13 PCGs,2 r RNA genes,22 t RNA genes,1replication initiation site and 1 control region.Similar to other vertebrates.For protein-coding genes,the most frequently used gene codon was the codon encoding leucine.The phylogenetic analysis of hybrid offspring shows that mitochondrial DNA follows maternal inheritance in inheritance.Genetic traits were analyzed using 24 pairs of microsatellite markers for E.akaara,E.tukula and the hybrid offspring.For these 24 pairs of microsatellite loci,the average frequency of major alleles was 0.4297 and the average shannon polymorphism information content was 0.6531,of which 20 loci were highly polymorphic with an average of 1.4793.The average intra-group inbreeding coefficient,total population inbreeding coefficient and inter-group divergence coefficient and gene flow for E.akaara,E.tukula and the hybrid offspring were 0.0713,0.3097,0.2566,0.7242.The highest mean effective allele counts for the three populations were for E.akaara(3.5883),followed by hybrid offspring(3.3288)and E.tukula(1.9306).Mean observed heterozygosity sizes were hybrid offspring(0.6447),E.akaara(0.5089)and E.tukula(0.3097).The mean expected heterozygosity size was hybrid offspring(0.6569),E.akaara(0.5603),and E.tukula(0.3479),and the polymorphic information content of the three populations ranged from 0.3042-0.5898,with the highest being the hybrid offspring.Cluster analysis indicated high genetic similarity and close genetic distance between the hybrid offspring and the female E.akaara.