Genetic Analysis And Genomic Selection In Large Yellow Croaker For Utilization Of Plant Protein

Large yellow croaker(Larimichthys crocea)is an important marine economic fish in China,ranking the top of China’s marine fish farming production.However,the rapid development of the large yellow croaker aquaculture industry has resulted in a large number of bottlenecks problems that restrict the healthy development of the industry,including the problem of over-reliance on trash fish(also known as:low value fish)for aquaculture.The use of trash fish not only has a significant impact on the environment and fishery resources,but also has a negative impact on the health of the farming population and the farming environment due to the lack of quality assurance and the tendency to carry pathogens,which seriously restricts the healthy development of the large yellow croaker aquaculture industry.The popularity of compound feed is still low because it contains more plant protein and affects the digestion and utilization of large yellow croaker.Nowadays,most study focused on improving feed formulations and feed processing techniques to gradually develop full-price compound feeds for large yellow croaker to meet the nutritional requirements of large yellow croaker;promoting the replacement of young fish with compound feeds and increasing the popularization of compound feeds.However,it is also essential to analyze the traits and genetic improvement of large yellow croaker to improve the adaptability and utilization of compound feeds for the farming population.In view of this,the object of this study is to cultivate new strains of large yellow croaker that can efficiently utilize plant protein by genetic analyses of the traits of efficient utilization of plant protein in large yellow croaker and applying genomic selection on breeding techniques.1.Differential responses of the gut and liver transcriptome to high levels of plant proteins in diets for large yellow croakerTo understand how large yellow croaker adapted to high plant protein diets,we analyzed the transcriptome of large yellow croaker liver and hindgut in response to compound feeds in which 30%(PSB),50%(PSC)and 70%(PSD)of fishmeal was replaced by plant protein,respectively.After 120 d of feeding,the growth performance of PSC group was significantly lower than that of PSB and PSD groups(P<0.05),and there was no significant difference between the PSB and PSD groups(P>0.05).We obtained 453,762,877 clean reads,of which 92.05%were successfully mapped to the reference genome of large yellow croaker.In pairwise comparison,521 and 1048 non-redundant differentially expressed genes(DEGs)(|log2FC|≥2,and P<0.05)were obtained in the hindgut and liver,respectively.Gene Ontology(GO)analysis showed that only one GO term,carbohydrate binding(GO:0030246),was co-enriched in both tissues of the two experimental feed groups.KEGG enrichment results showed that metabolism-related pathways such as amino acid metabolism,carbohydrate metabolism,lipid metabolism and energy metabolism were enriched in the hindgut and liver of both feed groups.In the amino acid metabolic pathways,arginine biosynthesis(ko00220)and alanine,aspartate and glutamate metabolism(ko00250)were enriched in both groups,and the enrichment was significant(P<0.05)in the hindgut,but not significant(P>0.05)in the liver.The protein-protein interaction network analysis showed that the interactions between DEGs and their interacting protein partners in the PSC group were strong,and seven hub genes(h2afz,ezh2,hdac7,old,hcfc1,trrap and lmna)with the highest connection level were obtained.These hub genes are involved in important physiological and biochemical processes of amino acids and proteins which play an important role in normal development and cell proliferation of the body.This study increases our understanding of the nutrition metabolism mechanism of high plant protein diets adaptability in large yellow croaker and provides a useful resource for large yellow croaker genetics and breeding.2.Analysis of the regulatory mechanism of intestinal microbiota for utilization of plant proteins in large yellow croakerLarge yellow croaker is a carnivorous fish that requires diets with high level of protein from fishmeal and increasing plant protein sources.Several studies have investigated the replacement of fishmeal with plant protein in large yellow croaker feed,but the effects of plant protein diet on its intestinal microbiota are still unknown.In this study,a total of 8,000 large yellow croakers were cultured in 4 sea cages,and fed with trash fish and compound feeds in which 30%,50%and 70%of fishmeal was replaced by plant protein,respectively.After 120 days of satiety feeding,the experimental fish starved for 24h,and intestinal samples were collected from 12 randomly selected fish in four groups for bacterial community analysis.After metaanalysis,1,602 amplicon sequence variants(ASVs)were obtained from 4 groups.Bacteria composition of the large yellow croaker intestine at the phylum level was similar across the 4 groups,including Proteobacteria,Firmicutes,Actinobacteria,Bacteroidetes,Deinococcus-Thermus,Fusobacteria and Gemmatimonadetes.And Lachnospiraceae and Alcaligenaceae were the most abundant Proteobacteria.Differential abundance analysis with edgeR showed that the abundance of pathogenic bacteria Lawsonia was significantly higher and the abundance of lactic acid bacteria Lactobacillus significantly was significant lower in all 3 compound feed groups comparing with the group only fed by trash fish.The characteristic gut microbiota Rhodospirillales was identified in the 30%fishmeal replacement group by LEfSe analysis.PICRUSt standardized the ASV abundance table,and then a total of 16 KEGG pathways were significantly identified.Among them,5 pathways were related to the immune system,such as "antigen processing and presentation" and "NOD-like receptor signaling pathway".Histological examination showed that goblet cells significantly increased in the 50%and 70%replacement groups.Our study aims to evaluate the impact of different proportions of plant protein in diets on gut microbiota during the fattening period of large yellow croaker.3.Effects of replacing fishmeal with plant protein on growth traits of L.croceaTwo kinds of experimental feed,C and P,were prepared according to the principle of equal nitrogen and equal energy.The content of fishmeal was 28.00%and 10.00%,and the content of plant protein was 47.81%and 63.11%,respectively.2048 large yellow croakers that had been injected with PIT tags were divided into 2 breeding cages,and were cultured with C and P feeds for 206 days with the same feeding management,respectively.Body weight and body length of group C and P were measured respectively at 0,84 and 206 days.To eliminate the differences in initial body weight and influence of initial body weight on weight gain,weight gain rate(WGR)was used as the primary analytical trait.Fin clips were collected and were sequenced using Double-digest restriction-associated(ddRAD)approach.The experiment results showed that the overall survival rates of groups C and P were 47.10%and 46.69%,respectively,with no significant difference in survival rate at any stage between groups C and P(P>0.05).On day 84 of the experiment(August to November,high temperature period),there was no significant difference in weight gain rate(WGR1)and specific growth rate(SGR1)between groups C and P(P>0.05).From day 85 to 206(November to March of the following year,low temperature period),the weight gain rate(WGR2)and specific growth rate(SGR2)of group C were significantly higher than those of group P(P<0.05).The correlation coefficients between weight gain rate,specific growth rate and initial body weight were all extremely low(|r|<0.15),indicating that the effect of initial body weight on both weight gain rate and specific growth rate has been eliminated.The correlation coefficients between specific growth rate(SGR)and weight gain rate(WGR)were all greater than 0.998,showing a high correlation between the two traits.4.Genome-wide association analysis of traits for utilization of plant proteins in large yellow croakerIn this section,a total of 891 individuals and 34,994 SNPs passed the quality control procedure for genome wide association study(GWAS).A total of 36 SNPs associated with WGR were identified,and a significant peak was found at 11.9-18.9 Mb on chromosome 18.The trait of plant protein utilization efficiency in large yellow croaker is a complex trait controlled by micro-effects and multiple genes.This study provides theoretical reference data for the genome selection of plant protein utilization in large yellow croaker.5.Genomic prediction revealed the potential for genetic improvement of large yellow croaker adapting to high plant protein dietA total of 891 individuals and 34,994 SNPs passed the quality control procedure and used for genomic prediction analyses.The heritability of each trait was also calculated.The heritability of WGR1(weight gain rate from 0 to 84 days)in the P group was 0.16.The predictive ability of GBLUP and BayesB for WGR1 in P group was 0.10 and 0.11,respectively.In this paper,the genome selection technology was used to select the parents with the top 6%of the genome estimated breeding value to construct a new strain of large yellow croaker for utilization of plant protein.This study provided a reference for genetic improvement of efficient plant protein utilization in large yellow croaker by using genomic selection.In this paper,the transcriptional regulation mechanism,intestinal microflora regulation mechanism and genome-wide association analysis of utilization of plant proteins in large yellow croaker were analyzed,which provided an important theoretical support for the promotion and popularization of compound feed of large yellow croaker.The development of genome selection breeding for efficient utilization of plant protein in large yellow croaker is expected to provide a solution for the popularization of large yellow croaker compound feed from the perspective of genetics and genetic breeding.It is of great significance to reduce feed cost of large yellow croaker cultivation,fully replace trash fish with compound feed,protect ecological fishery resources and realize green and sustainable development of large yellow croaker cultivation industry.