Evaluation Of Genetic Parameters And Screening Related Genes Of Cold Tolerance Traits In Shrimp

Shrimp is an ectotherm and cannot regulate temperature by itself.Temperature,as an important environmental factor,can directly affect the growth,reproduction,metabolism and survival of shrimp.Fenneropenaeus chinensis is a cold-water shrimp,and its optimum growth temperature range is 18³0°C.When the water temperature is lower than 4°C,it will cause a large number of death.Litopenaeus vannamei is poorly tolerant to low temperatures,and the suitable temperature for growth is 25³5°C.When the water temperature is lower than 18°C,it will stop ingestion.The low temperature limits the breeding season and area of shrimp and affects the production safety of the breeding industry.Moreover,the use of boiler heating to maintain water temperature not only increases costs but also pollutes the environment.In order to solve the problems caused by low temperatures and accelerate the development of new varieties of low-temperature resistant shrimp.This study used F.chinensis and L.vannamei as experimental materials,and low-temperature stress was achieved by cooling the refrigerator.Then using ASReml-R to evaluate the genetic parameters of its low temperature and growth traits and calculations predict genetic progress.Then Illumina HiSeq 2500 was used to screen differentially expressed genes and low temperature-related metabolic pathways under low temperature stress.The main results are summarized as follows:1.Evaluation of heritability for growth and cold tolerance traits in 40-day-old F.chinensis juvenilesIn three generations,the heritability of body weight?BW?of F.chinensis juveniles ranged from 0.078±0.124 to 0.680±0.233,and the heritability of body length?BL?ranged from 0.131±0.133 to 0.825±0.246.Across generations,the heritability of BW and BL respectively was 0.253±0.0888 and 0.206±0.0844,which were moderate heritability and had significant difference from 0?P<0.01?.In G₁₀,G₁₁,and G₁₂2 generations,the heritability of cold tolerance traits?CDH,TAD?was between 0.054±0.074 and 0.265±0.091,both of them are medium and low heritability.Across generations,the heritability of cooling degree hours?CDH?and temperature at death?TAD?were 0.077±0.038 and 0.076±0.029,both of them were low heritability and were all significantly different from 0?P<0.01?.2.Correlation analysis of growth traits and cold tolerance traits in 40-day-old F.chinensis juvenilesThe genetic correlation and phenotypic correlation of BW/BL were highly positively correlated.In generations,the genetic correlation of BW/BL was form0.956±0.055 to 0.987±0.003,and the phenotypic correlation was form 0.867±0.009 to 0.9408±0.0040,which were highly positively related and were significantly different from 0?P<0.01?.Across generations,the genetic correlation of BW/BL was0.910±0.008,and the phenotypic correlation was 0.902±0.004,which was highly positively correlated and significantly different from 0?P<0.01?.The genetic correlation and phenotypic correlation of CDH/TAD were highly negatively correlated.In generations,the genetic correlation of CDH/TAD was between-0.671±0.364 and-0.912±0.216,and the phenotypic correlation was between-0.547±0.017 and-0.874±0.005,which were negatively correlated and significantly different from 0?P<0.05?.Across generations,the genetic correlation of CDH/TAD was-0.874±0.100,and the phenotypic correlation was-0.864±0.00337,which was highly positively correlated and significantly different from 0?P<0.05?.In generations,the genetic correlation between growth traits?BW,BL?and CDH was between-0.986±0.477 and 0.9914±0.7010,and the phenotypic correlation coefficient was between 0.0381±0.0299 and 0.109±0.0291,all positive phenotypic correlations.The genetic correlation coefficients between growth traits and TAD was between-0.374±0.310 and 0.576±0.422,and the phenotypic correlation coefficients was between-0.366±0.324 and-0.076±0.032.There was a negative phenotypic correlation between growth traits and TAD.Across generations,the genetic correlations between the growth traits?BW,BL?and CDH were-0.095±0.371 and-0.123±0.411,both of them were negatively related.And the phenotypic correlation coefficients were 0.113±0.017 and 0.125±0.017,both of them were significant positive phenotypic correlation?P<0.01?.The genetic correlation coefficients between growth traits and TAD were-0.241±0.283 and 0.703±0.604,and the phenotypic correlation coefficients were-0.118±0.015 and-0.136±0.015.There was significant negative phenotype correlation?P<0.01?.3.Screening differentially expressed genes related to cold tolerance traits of40-day-old F.chinensis juvenile based on high-throughput transcriptome sequencingAnalysis of transcriptome information of F.chinensis juveniles in cold stress group?4°C?and normal temperature group?18°C?.The results showed,a total of127,939 Unigenes were obtained by splicing and the average length was 1,190 bp.And 35%of Unigene has no annotations in seven databases including Nr and Swiss-prot.A total of 1,698 differentially expressed genes?DEGs?were screened,of which 920 were up-regulated and 778 were down-regulated.GO and KEGG enrichment analysis revealed that DEGs were enriched in the synthesis of spliceosomes,ribosomes,bile secretion,ABC transport pathway,and cellular nitrogen compound biosynthetic process.This study screened eight genes that may be associated with cold tolerance traits.Five of them were up-regulated and expressed as ABCC3,ASAH,GST,C-type lectin,and HSP70,Three down-regulated expressions were?-BBH,?-HEXA,and ACSBG2.Eight differential genes were randomly selected and verified by real time RT-PCR.The results showed that the trend of their expression was consistent with the sequencing results,which proved the accuracy of the sequencing results.4.Screening differentially expressed genes related to low-temperature tolerance of L.vannamei based on high-throughput transcriptome sequencingThis study analyzed the transcriptome data of hepatopancreas tissues in the cold stress?18°C?and normal temperature?24°C?groups of L.vannamei.A total of 50,921Unigenes were obtained by transcriptome sequencing.The average length was 828 bp and N50 was 1,589 bp.Of these,28.13%of Unigene were known genes.A total of243 cold stress-related genes were screened out,of which 89 were up-regulated and154 were down-regulated.Functional enrichment analysis revealed that the differentially expressed genes were more abundant in the binding,catalytic activity,peroxisome,lysosomes,arginine and proline metabolism,biosynthesis of amino acid.According to the Q-value,top 10 genes including ABCB6,C-type lectin and GS,et al.real time RT-PCR was used to verify transcriptome sequencing results.The results of RNA-seq and real time RT-PCR showed that the expression of ten different genes showed the same tendency.This proved that the result of the gene differential expression based on the sequence of transcriptome sequences is credible.5.This study found that the ABC gene and the C-type lectin gene were selected candidate genes related to low temperature resistance both in F.chinensis and L.vannamei.Therefore,it is speculated that ABC gene and C-type lectin gene may be involved in regulating the molecular mechanism of shrimp response to low temperature stress.