| Penaeus monodon is one of the main shrimp breeds in the world and also the main shrimp species in South China.In recent years,due to the lack of fish meal resources and the rising price of fish meal,the feed cost in P.monodon aquaculture industry has increased year by year,which seriously reduces the profit of P.monodon culture and restricts the development of P.monodon aquaculture in China.Therefore,the breeding of new strains of P.monodon which is suitable for the low fish meal protein in feed is of great significance for the healthy and sustainable development of the P.monodon industry in China.In the program of genetic breeding,accurate estimation of breeding value is the basic guarantee for developing new strains in breeding.It is very important to analyze the transcriptional characteristics and intestinal flora structure of different families under different culture conditions,and estimate the phenotype and intrinsic molecular mechanism correlation of economic traits of the selected families.It is a very important research content in genetic breeding and the basis of developing excellent varieties in breeding.In this paper,the effects of different contents of concentrated dephenolized cottonseed protein instead of fish meal on P.monodon were tested,and the best content of concentrated dephenolized cottonseed protein instead of fish meal protein was determined.On this basis,through the construction of specialized families and the evaluation of growth performance and the estimation of genetic parameters,excellent families were obtained.The F1 generation core breeding group was used to establish F2 families,and the genetic parameters and morphological differences of F2 generation were evaluated,and the breeding values of the new roughage tolerant strain of P.monodon were calculated.The correlation between the quantitative traits of F2 representative type and its contribution to the important economic trait of body weight were analyzed by path analysis.The transcriptome sequencing among different feed groups of the selected families was carried out,and the roughage tolerance was obtained.The results are as follows:(1)To determine the appropriate replacement of fish meal with concentrated dephenolized cottonseed protein in formula feed for P.monodon,the growth experiment of P.monodon(0.85 ± 0.02g)was carried out for 8 weeks and the growth performance,muscle composition,feed utilization,hepatopancreatic digestive enzymes,antioxidant capacity and intestinal traits were detected.According to the nutritional requirements of P.monodon,five kinds of isonitrogenous and isoenergetic feed were designed,which were compared with the control feed(fish meal content was 30%).The dosages of concentrated dephenolized cottonseed protein in feed were5%,10%,15%,20% and 25%,respectively,to replace 16.67%,33.33%,50%,66.67% and 83.33% of fish meal,respectively.The results showed that the growth of fish meal was not significantly affected by protein partial replacement,which indicated that it was feasible to use concentrated dephenolized cottonseed protein partial replacement of fish meal.The experimental group using 20% concentrated dephenolized cottonseed protein instead of fish meal had the best effect.The weight gain rate,specific growth rate,survival rate,apparent digestibility of feed dry matter and feed coefficient were 206.27 ± 12.09%,2.01 ± 0.14% and 60.83 ± 2.05%respectively.There were no significant differences in weight gain rate,specific growth rate,survival rate and apparent digestibility of feed dry matter between the two groups(P>0.05),but the feed coefficient of the two groups was significantly lower than that of the control group(P<0.05).The results showed that the growth performance of P.monodon was not affected by using 66.67% concentrated dephenolized cottonseed protein instead of fish meal,which laid a data foundation for further screening of new strains of P.monodon resistant to roughage.(2)Using the proportion of fish meal in the feed replaced by concentrated dephenolized cottonseed protein obtained in Experiment 1,two kinds of diets were prepared: control group(30% fish meal protein level)and experimental group(10%fish meal protein level and 20% concentrated dephenolized cottonseed protein level).The results showed that: in the same diet,there were significant differences in the body weight of different families of P.monodon.The absolute weight gain rate of the families with the fastest weight gain was 97.16% and 95.46% higher than that of the families with the slowest weight gain in the control group and the experimental group,respectively,but the order of the body weight of the families with different diets was not the same.The average survival rate of P.monodon was 80.59% in the control group and 77.88% in the experimental group.There were significant differences in production performance among families in different feed groups.The highest yield of families in the control group and the experimental group was 100% and 124.44%higher than the lowest.No.10 and No.6 families were in the top 10% in different feed groups,and had good production performance.The results of the experiment laid a data foundation for the breeding of new varieties of P.monodon with roughage tolerance.(3)In this study,the genetic parameters and G × E interaction effects of body weight and survival traits were analyzed after 56 days of polyculture with two diets:control group(Diet A,30% fish meal protein level)and experimental group(Diet B,10% fish meal protein level and 20% concentrated dephenolized cottonseed protein level).The results showed that the growth performance of P.monodon in Diet A group was better than that in Diet B group.The estimated heritability of body weight was0.53 ± 0.12 and 0.39 ± 0.09 in the two groups respectively,which was high heritability;the estimated heritability of living traits was 0.38 and 0.22 respectively,which was also medium high heritability.The interaction effect between genotype and environment(G × E)was estimated.The results showed that there was a high genetic correlation(0.84-0.92)in body weight and viability between the two groups.The ratio of G × E variance component to additive genetic variance component was less than0.5,and the G × E effect was not significant.The results showed that although the growth and survival traits of P.monodon were different under different diets,the interaction between genotype and diet condition was not significant.Therefore,it was not necessary to establish different breeding lines for different diet conditions in the range of 10%-30% dietary fish meal protein level.(4)Fifteen whole sib families of the second generation roughage tolerant P.monodon were constructed by artificial mating in 2020,and the growth characteristics and genetic parameters were evaluated.The results showed that the coefficient of variation of growth traits was 11.52-47.53%,which indicated that there was high genetic variation in this trait.The heritability of growth traits of P.monodon F2 population ranged from(0.25±0.03)to(0.41±0.13),belonging to medium and high heritability(P<0.01).The heritability of body length and weight were(0.38±0.11)and(0.41±0.13),respectively.The genetic correlation between body weight and body length was the highest(0.99),while the genetic correlation between head chest width and first abdominal segment height was the lowest(0.71).There was significant correlation between phenotypic data of growth traits,belonging to medium and high correlation.The results showed that the F2 population of P.monodon has high potential for genetic improvement,and a better breeding effect can be obtained by family selection combined with individual selection.There is a high genetic positive correlation between growth traits,so body length and body weight can be selected as the key traits of growth selection and included in the comprehensive selection index,and the other growth traits can be indirectly selected through positive genetic correlation and the production performance is improved.(5)The effects of different fish meal protein levels on the transcription group of P.monodon were studied.The m RNA was enriched by total RNA extraction,the c DNA library was amplified and constructed,and the high-throughput sequencing of illustra was carried out,and the transcriptional books were assembled.The results showed that there were 586 genes differentially expressed in different genetic backgrounds(comparison among families),including 520 up regulated genes and 66 down regulated genes.Further analysis shows that the top 10 GO annotation items with the most significant difference were obtained.The results showed that the most significant gene groups of differential enrichment genes mainly involved in biological processes such as protein transport,protein digestion and absorption,lipid absorption,and other related molecular functions of signal transduction,such as calcium ion binding activity and G-protein coupled receptor,which were located in the extracellular region and cell connection and other cell sites.The KEGG pathway,which was significantly enriched,was mainly related to protein transport,including calcium pathway and glutamate synaptic conduction.Differential expression genes mainly involve the related receptors,regulatory proteins and subunits in signal transduction pathway,absorption of various channel proteins and transporters in osmotic regulation,and stress response design of various regulatory factors and enzymes.(6)Hi-Seq sequencing technology and bioinformatics analysis were used to construct gene sequencing libraries of intestinal microflora of two P.monodon families with different fish meal protein levels.At the genus level,the relative distribution of intestinal microflora of P.monodon in different feed groups was different.The higher abundance of intestinal microflora in high fish meal protein diet group was Cetobacterium,Paenibacillus and Romboutsia,while the higher abundance of intestinal microflora in low fish meal protein diet group was Cebacteria,Enterovibrio and Plesiomonas.According to the results of comparative analysis among different families,the dominant phyla of intestinal tract of P.monodon in X family were Proteus(58.23%)and Firmicutes(31.75%),and the abundance of other phyla was low(below 5.00%).Bacteroides(57.12%),Fusobacteria(22.54%)and Bacteroides(11.47%)were the main phyla in Y family.It is expected to improve the digestion and absorption function of P.monodon by increasing the abundance of Firmicutes and Proteus in intestinal tract,and improve the utilization rate of low fish meal level feed. |
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