Identification And Application Of The Molecular Markers Related To Growth Performance In Huanghe Carp New Strain F3

Huanghe carp is one of the oldest and most popular cultured freshwater fish species in world.In order to improve its economic contribution,we have created a new,fast-growing strain common carp with higher growth rate and better poly unsaturated fatty acid in muscle for many years.However,marker assistant breeding can shorten the generation interval of germplasm improvement and speed up the selection response.Owing to higher cost for sequence,together with BLUP,pre-selection of candidate individuals,low density array,they can become an optional choice under the consideration of selection cost and efficiency.Using such method,the Huanghe carp new strain F3 from F2 was produced in this study.Further more,molecular markers and differential expression genes,pathway which were related with the Huanghe carp new strain advantaged performance were explored from DNA and RNA level.In addition,the functionof candidate gene IGF2 a and IGF2 b was proved by gene knocking out in zebrafish.The results could enrichthe molecular marker pools,and provide the retical theory and technology for the breeding of Huangcarp new strain and discovering its better performance mechanism.The results also could provide a reference for other similar aquatic fish breeding.(1)In this study,450 fish consisted of parents in F2 and their offspring were collected,which were from Nanquan farm in Fresh water Fisheries Research Center in Chinese Fishery Academy.Finally,5 microstallites primers filtered and 4 functional gene regional amplification primers were used to observe the corresponded genotypes.Correlationship between molecular markers and body weight of Huanghe carp new strain and genetic effect analysis of such markers was explored.The results showed that,Koi42 and 4 SNPs from the functional genes have significant effect on the carp’s body weight.13 candidate parents which have over two genotypes with higher body weight were found.These individuals have better growth performance compared to those without markers.Further analysis by multiple step-wise regressions was conducted in order to make sure which markers can be implicated in practical breeding together effectively.COI626,D-Loop 253 and Koi42 were left by regression analysis and AIC criteria,where Koi42 has the higher contribution to the body weight variation among 3 markers.It is interesting that Koi42 is related with sex by Fisher exact test.Genetic analysis supply the information Koi42 plays its role by additive effect(P< 0.1).Among many epitasis components,significant effect between D-Loop 253 additive effect and Koi42 additive effect was found.These results illustrated that Koi42 with its interaction prater D-Loop 253 could be used to marker assistant breeding or multiple markers based co-selection.(2)Although with the help of marker assistant breeding the growth performance at eight months has been improved by 20.84% in F3 and polyunsaturated FAs were more abundant in the selected group,more candidate loci were still demanded in the breeding progress based on the molecular assisted breeding technology in(1).In order to know whether the molecular markers existed in better performance of Huanghe carp new stran,a 2b-RAD sequence assay as a means of investigating the quantitative trait loci in common carp were explored.A total of 4,953,017,786 clean reads were generated for 250 specimens from 25 families(each family has two parents and eight progenies,average reads/specimen = 19,812,071)with Bsa XI Restriction Enzyme.From these,56,663 SNPs were identified,covering 50 chromosomes and 3,377 scaffolds.Principal component analysis indicated that selection and control groups are relatively clearly distinct.Top 1% of Fst values were selected as the threshold signature of artificial selection.Among the 244 identified loci,genes associated with sex-related factors and nutritional metabolism(especially fat metabolism)was annotated.Eighteen QTL were associated with growth parameters.Body length at three months of age and body weight(both at 3 and 8 months)were controlled by polygenic effects,but body size(length,depth,width)at eight months of age was controlled mainly by several loci with major effects.Importantly,a single shared QTL(IGF2 gene: IGF2 aand IGF2b in annotation)partially controlled the body length,depth,and width.(3)Based on the above results in(2),it illustrated that IGF2 gene play key role in Huanghe carp new strain growth performance.In order to validated this,CRISPR–Cas9 method was used to produce the IGF2 a and IGF2 b knock-out zebrafish.For the male IGF2 a knock-out zebrafish has lower body length/body width.Beside this,harvest performance was also measured to determine the IGF2 a and IGF2 b promoting growth function.Both IGF2 a and IGF2 b,in Huanghe carp new strain female have the higher body length/body width compared to male.(4)Besides the gene markers,expression of differential genes should be also focused on for exploring the mechanism for growing fast.To better understand the impacts of gene regulation in intestinal tissue on growth,we conducted a comparative RNA-Seq transcriptome analysis between intestine samples of selected and control groups(and corroborated selected results by PCR).In total,we found 106 differentially expressed genes(DEGs).Only the endocytosis pathway was significantly enriched in DEGs,with two upregulated genes: il2 rb and ehd1.The latter is involved in the growth hormone/insulin-like growth factor(Gh/IGF)axis,which plays a key role in the regulation of growth in animals.Tll2,which is known to be associated with intestinal regeneration,was extremely highly upregulated in both transcriptomic(infinite)and q PCR(610.70)analyses.Two of the upregulated genes are associated with the fatty acid metabolism,several genes are likely to be indicators of heightened transcription levels,several are associated with metabolic and developmental roles,several with neuronal functions(including two with vision),several with the immune system,and two downregulated genes with the development of vasculature.The higher growth rate of the selected group is likely to be at least partially attributed to increased endocytosis efficiency and genetically-driven behavioural differences(higher aggression levels).