Analysis Of Growth,Low Temperature Tolerance Phenotype And Genetic Traits Of Two Hybrid Groupers

Grouper is an important marine economic fish in China.In recent years,it has been widely cultured in China.In 2019,the culture output of grouper reached 183,127 tons,ranking second in marine fish culture.The rapid development of the aquaculture industry has brought about rapid economic development.However,some traits are in severe decline in many species,and germplasm resources are seriously degraded after many years of artificial breeding.Nowadays,various new breeding methods have been applied to aquatic animals,including gene editing,gynogenesis,polyploid induction.However,hybridization is still a widely used and effective breeding method.This study explored whether the two new hybrid groupers have heterosis in growth rate and low temperature tolerance.1.Karyotype analysis of hybrid Epinephelus tukula,Epinephelus awoara and Qinglan grouperhybrid grouper showed heterosis in terms of faster growth and stronger stress resistance,and chromosome research of hybrid offspring is one of the key methods to explore heterosis.To explore the difference in karyotypes between hybrid progeny and parents,we prepared chromosome specimens of Qinglan grouper and maternal progeny by colchicine injection in vivo.The results showed that the chromosome number of E.tukula was 2n=48,the karyotype formula was 2n=2sm+46t,NF=50,and the arm ratio was 1.76±0.11;the chromosome number of E.awoara was 2n=48,the karyotype formula was 2n=48t,NF=48;the chromosome number of Qinglan grouper was 2n=48,the karyotype formula was 2n=1sm+47t,NF=49,and the arm ratio was 1.75±0.29.Furthermore,we prepared gonadal sections of 16-month-old Qinglan grouper to investigate whether the heterochromosome of Qinglan grouper was related to sex.The results showed that there were a large number of oocytes in the gonads,which indicated that the heterochromosomes of Qinglan grouper had no relationship with sex.Based on the above results,it is speculated that the cause of heterochromosomes in hybrid progenies is that the male parent provided 23 telocentric chromosomes and one submetacentric chromosome,whereas the female parent provided 24 telocentric chromosomes.These results not only provide a method for the preparation of chromosome specimens of grouper,but also provide abundant data for chromosome polymorphism,genetic variation,hybrid breeding,and the germplasm improvement of hybrid progenies.2.Metamorphosis and skeletal development of hybrid Epinephelus awoara（♀）and E.tukula（♂）progeniesHere,we explored the metamorphosis and skeletal development of hybrids between E.awoara（♀）and E.tukula（♂）.Currently,many varieties with good characters have been produced by cross breeding,which plays an important role in the development of aquaculture.However,few basic studies have focused on the developmental biology of hybrid progeny.We studied the morphological characteristics and body structure of larvae and juveniles of the hybrid and measured their body length.The results showed that the metamorphosis process of the hybrid grouper was comparable to that of other groupers.Moreover,using wholemount bone-staining technology,the ontogeny and ossification of the cranium,appendage skeleton and vertebral column were observed and described in the larvae and juveniles.Newly hatched larvae did not have a skeleton.Head cartilage and the pectoral fin developed at3 days after hatching（DAH）,and the total length（TL）was 2.74±0.06 mm.Cartilage of the dorsal fin and pelvic fin developed at 15 DAH（3.90±0.97 mm TL）.The vertebral column and caudal fin cartilage appeared at 18 DAH（4.69±0.58 mm TL）.Cartilage was observed in the anal fin at 22 DAH（6.16±1.76 mm TL）.The second dorsal fin spine and pelvic fin spine were fully ossified between 24 and 26 DAH（8.01±1.41–9.29±1.64 mm TL）.The first skeletal part that completed ossification was the pelvic fin,which occurred at the 44 DAH（24.64±3.63 mm TL）.The vertebral column was fullyossified at 47 DAH（29.70±1.97 mm TL）.The dorsal fin and anal fin were ossified at 51 DAH（34.64±1.92 mm TL）and the cephalic skeleton,caudal fin and pectoral fin were fully ossified at 68 DAH（41.89±2.16 mm TL）.These results provide abundant biological data on the metamorphosis and skeletal development of larvae and juvenile groupers,as well as analysis of the growth and survival rate advantage of hybrid offspring3.Heterosis analysis of growth in Jinhu grouperHybridization is a widely used and effective breeding method in grouper breeding.In this study,we constructed full sib families of brown-marbled grouper（E.fuscoguttatus）,Jinhu grouper and Hulong grouper（E.fuscoguttatus♀×E.lanceolatus♂）,and compared the growth characteristics of the three groupers under the condition of industrialized culture.The results showed that the body weight of Jinhu grouper was1.49 times that of Hulong grouper,and the body weight of Jinhu grouper was 2.03 times that of E.fuscoguttatus.What’s more,the survival rate of Jinhu grouper was significantly higher than that of Hulong grouper and E.fuscoguttatus.The relationship between body length and body weight of 15-month-old Jinhu grouper and E.fuscoguttatus is W=0.0487L^2.8466（R²=0.9671）and W=0.0514L^2.8835（R²=0.9678）,respectively.The relationship between body length and body weight of 12-month-old Jinhu grouper and Hulong grouper is W=0.1129L^2.6022（R²=0.9611）and W=0.1178L^2.6543（R²=0.9603）,respectively.We sequenced the transcriptome of brain,liver and muscle of Jinhu grouper and E.fuscoguttatus using Illumina Hi Seq sequencing technology.Moreover,we performed a comparative transcriptome study of them.The results showed that 1448（441 up-regulated genes and 1007 down-regulated genes）,443（248up-regulated genes and 195 down-regulated genes）and 203（87 up-regulated genes and116 down-regulated genes）differentially expressed genes（DEGs）were obtained in brain,liver and muscle.GO functional enrichment analysis and KEGG pathway analysis of the DEGs revealed the involvement of single-organism metabolic process,oxidation-reduction process,protein binding,c AMP signaling pathway,MAPK signaling pathway,Fox O signaling pathway and Growth hormone synthesis,secretion and action in the cell growth,body growth and organic metabolism.Through the screening of growth related genes,it was found that igf-1 and igfbp1 genes were closely related to growth and development.4.Construction of a high-density genetic linkage map and QTL mapping for growth traits in Jinhu grouperQTL mapping is an effective method for marker assisted selection（MAS）in animal breeding,and high-density linkage genetic maps are necessary for fine mapping of QTLs.In this study,high-density genetic maps of Jinhu grouper were constructed using RAD technology.Sex-average,female-specific,and male-specific linkage maps were composed of 24 linkage groups（LGs）.The genetic map developed in this study had the highest number of SNPs among reported genetic maps for groupers.The sex-average linkage map contained 2759 bin makers（150,468 SNPs）and spanned1627.28?c M,with an average marker interval of 0.59?c M.The female-specific（E.fuscoguttatus）linkage map contained 648 bin makers（38,269 SNPs）and spanned1414.68?c M,with an average marker interval of 2.22?c M.The male-specific（E.tukula）linkage map contained 2126 bin makers（112,015 SNPs）and spanned 1839.88?c M,with an average marker interval of 0.86?c M.In total,45 QTLs comprising 318 SNPs for growth-related traits were identified,including 10 QTLs for body weight,6 QTLs for full length,7 QTLs for body length,14 QTLs for head length,5 QTLs for body height,and 3 QTLs for body thickness.In addition,56 candidate genes related to growth,metabolism,bone formation,muscle development,and neural regulation were identified,including eight genes associated with growth in other fish.Among these,gpr126,fam20a,fam20c,sox9b,foxm1,foxl1,myf6,zbtb38,and sl should be a focus of further research.These results provide a foundation for fine QTL mapping in grouper,and theoretical guidance for exploring the causes of heterosis,which is of great significance for MAS in the breeding of hybrids and male and female parents.5.Physiological and molecular mechanism responses to low temperature stress in two hybrid grouper and their female parentsE.fuscoguttatus,Jinhu grouper,Qinglan grouper and E.awoara stopped feeding at 19℃,16℃,12℃ and 12℃,respectively,and reached semi-lethal temperature at11℃,9℃,7.5℃ and 6.5℃,respectively.Histopathological analysis showed that the damage degree of gill,muscle and liver tissue increased with the decrease of temperature.The activities of total superoxide dismutase（T-SOD）,total antioxidant capacity（T-AOC）and glutathione peroxidase（GSH-Px）in liver and serum decreased first and then increased with the temperature decreasing;content of malondialdehyde（MDA）in liver and serum increased first and then decreased;The activities of lysozyme（LZM）in serum increased first and then decreased,and immunoglobulin M（Ig M）had no significant change.We sequenced the transcriptome of brain,liver and muscle of Jinhu grouper and E.fuscoguttatus in different temperature using Illumina Hi Seq sequencing technology.Moreover,we performed a comparative transcriptome study of them.The results showed that most of the DEGs were enriched in the liver.Through the trend analysis of the DEGs between Jinhu grouper and E.fuscoguttatus from 26℃-16℃-half lethal temperature,we found that two patterns of continuously down-regulated genes and continuously up-regulated genes were significantly enriched in the brain,liver and muscle of the two groupers,and the number of genes is large in the two trends.KEGG pathway analysis of the continuously down-regulated genes revealed the involvement of significant enrichment of metabolic pathways.KEGG pathway analysis of the continuously up-regulated genes revealed the involvement of significant enrichment of AMPK signaling pathway,m TOR signaling pathway,Fox O signaling pathway.atm,bcl6,gadd45 and other genes in Fox O signaling pathway were significantly up-regulated,which may be related to low temperature stress.In addition,the high expression of hsp70 and aqp1 genes at 16℃ may contribute to the high cold tolerance of Jinhu grouper.In conclusion,Jinhu grouper and Qinglan grouper have heterosis of fast growth and high survival rate.In addition,the Jinhu grouper has the heterosis of low temperature tolerance,and the Qinglan grouper has inherited the low temperature tolerance of the E.awoara.These two groupers have broad prospects for popularization.