| "Dongyoul" Haliotis diversicolor is a hybrid of H.diversicolor Taiwan population(as maternal group)and H.diversicolor Japan population(as paternal group),the hybrid has a high level of resistance heterosis,showing higher overwinter survival rate than its parental genotypes.At present,the "Dongyoul" hybrid has been widely used in abalone production,it partially solved the winter disease outbreak problem in H.diversicolor.However,physiological basis and molecular mechanisms of this high survival rate heterosis are not well understood.To utilize heterosis in abalone production more scientifically and effectively,also for a clearer understanding of the molecular mechanism of shellfish heterosis,physiological and molecular analysis and comparison of the cold resistance and disease resistance features in "Dongyoul" genotype(DY),and its parental genotypes:H.diversicolor Taiwan population(TW)and H.diversicolor Japan population(JP)were conducted.The main works are showed as follows:(1)Evaluation of cold resistance ability of 3 H.diversicolor genotypes1.The survival rates of three groups:TW,JP and DY were studied under factory overwinter breeding and acute cold treatment.Results showed that after factory overwinter breeding,survival rates of DY,JP and TW were separately 95.56,93.56 and 37.56%;12h-LTso of DY,RB and TW were separately 3.10,3.34 and 4.15℃,and 24h-LT50 of DY,RB and TW were separately 5.70,5.69 and 6.58℃.DY showed heterosis in survival rate under cold stress.2.Change of abalone heart rate under low temperature stress was studied.The slope of the Arrhenius curve of abalone heart beat and temperature(ADR),and the theoretical temperature when abalone heart rate is approximately(AZT),were used as the evaluation index of abalone’s cold resistance.Results showed that the ADR of DY,JP and TW were 9.00,9.78 and 10.07,respectively,and AZT of DY,JP and TW were-13.75-10.67 and-9.53℃,respectively.DY showed heterosis in ADR and AZT,indicating that DY is more stable in heart beating under cold stress,is more capable of tolerance to extremely low temperature.3,The effect of short term(7 days)cold exposure on the variation of oxygen consumption rate,ammonia excretion rate and fatty acids contents and composition in 3 H.diversicolor genotypes were determined.Results showed that under low temperature stress,DY got lower changes in oxygen consumption and ammonia excretion rates,compared to the parental genotypes,suggesting that DY’s respiratory metabolism was less affected by cold stress;Also,the unsaturation index of DY(270.72%)was also lower than TW genotype(232.63%),showing that DY’s membrane function was also less affected by cold stress.4,Using entropy method and the membership function method,based on the above indexes,a comprehensive evaluation system of abalone cold resistance is established.The average membership value is used to represent the comprehensive cold resistance ability of abalone.Results showed that the average membership values of DY,JP and TW were 0.834,0.627 and 0.252,respectively,which agreed with their cold resistance phenotypes.It indicated that DY was superior to the parental population in comprehensive cold tolerance.(2)Evaluation of disease resistance ability of 3 H.diversicolor genotypes1,After exposure to the pathogenic bacteria immersion for 7 days,the survival rates of DY,JP and TW were 93.33,84.44 and 36.67%,DY showed heterosis.2,The effect of bacteria immersion on the non-specific immune indexes(total hemocytes count(THC),phagocytosis,respiratory burst and phenoloxidase(PO)activity)in 3 H.diversicolor genotypes were determined.Results showed that the reduction rate of THC was lower in DY(-8.26%)than JP(-15.12%)and TW(-24.48%),the increase rate of phagocytic activity and respiratory burst were higher in DY(70.69%and 163.50%)than that of the parent group:JP(28%and 146.32%)and TW(-2.86%and 106.26%),DY showed over parent heterosis.The increase rate of PO in DY was lower than that in parents,indicating that the disease resistance heterosis of DY was mainly caused by its higher efficiency in cellular immune functions.3,The disease resistance comprehensive membership values of DY,JP and TW were separately 0.777,0.707 and 0.087,indicating that DY shows heterosis on comprehensive immune ability.(3)Transcriptional analysis of heterosis in "Dongyoul" H.diversicolorUsing high-throughput sequencing and bioinformatics techniques,the transcriptome changes of 3 H.diversicolor genotypes under cold exposure were analyzed and compared,the results are as follows:1,Under low temperature stress,the overall gene expression pattern of DY was more similar to JP,but quite different from that of TW;At the 3 time points during cold exposure(Oh,6h and 7D),2186,688 and 574 non-additively expressed genes in DY were identified,in which positive and negative overdominance expressed genes accounted for two largest groups.Functional analysis further indicated that the positive dominance genes include cold resistance related genes,such as lipid metabolism,transmembrane protein,antioxidant,low temperature protection,epigenetic modification,etc.These genes were expressed positive-overdominance in DY,and may contributed to DY’s organ and tissue’s responses to cold stress.2,Gene expression pattern of DY was also biased towards JP under bacterial stress,and different from that of TW.At the Oh and 24h post infection,264 and 105 NAG were identified.In the 24h NAG,number of positive-overdominance genes composed main part of the NAG,indicated that many genes showed positive-overdominance expression in hybrid challenged with bacteria.Functional analysis of these positive-overdominance genes showed that they were responsible for cell phagocytosis,pathogen clearance,antioxidation and nutrition immunology.This indicated that DY could perform better in cellular and humoral immune functions under the stress of bacteria,and could better eliminate the excess free radicals,resulting in disease resistance heterosis.(4)Exploration in epigenetic mechanism of heterosis in DYTo explore the epigenetic mechanism of heterosis in DY,genome-wide methylation sequencing and small RNA sequencing in 3 H.diversicolor genotypes were conducted.Results showed that there were significant differences between DY and parental genotypes in the whole genome methylation rate(TW>JP>DY),and the expression level of small RNA(DY>JP>TW),this indicated that epigenetic regulation of gene transcription in DY was more active;the methylation status of the positive-overdominance genes identified in transcriptional analysis were further investigated.And found that 19.50%～29.58%of these genes exist methylation modification difference.These results suggest that the differences in the whole genome methylation status,and sRNA expression between the 3 genotypes of H.diversicolor,may lead to their difference in mRNA expression,and through a series of physiological and biochemical processes,finally lead to the phenotypic differences between 3 H.diversicolor genotypes,resulting in heterosis in "Dongyoul" H.diversicolor. |
PDF Full Text Request