Genetic Diversity Of Isochrysis Galbana And Directional Breeding For High DHA And EPA Strains

Isochrysis galbana is an important food for aquaculture,which has been widely used in breeding of shellfish and sea cucumber.Its' significant nutritional value lies not only in high DHA content,but also in the ability of synthesizing a small amount of EPA.The results showed that,as the essential fatty acids,suitable amount of DHA and EPA could significantly increase the hatching rate of aquatic seedlings and enhance the immunity of adults.However,after years of using,the germplasmoften appears to degrade.The growth rate is decreasing and the DHA and EPA contents are reduc ing.Therefore,it is necessary to process the breeding work to find a good strain which has fast growth rate and high DHA and EPA levels.In order to better carry out the work for purification and culture of I.galbana,it is necessary to optimize the agar solid medium.The effects of different concentration of agar,different kinds of culture medium and different source of nitrogen on the growth of I.galbana were studied by using the orthogonal design of 3 factors and 3 levels.The effects of vitamins and antibiotics on the formation of algae were compared by single factor optimization.The results showed that the concentration of agar could affect the formation of algae,and the suitable concentration of agar was 0.6%-1.0% in solid plate.While different kinds of medium and nitrogen source only had small effects on the formation of algae.It showed that it is not necessary to add Vitamin B1 or B12 to solid culture and adding antibiotics to solid culture would have a significant inhibition,but it can not completely remove the bacteria.It is recommended to use F / 2 medium with 1% agar and vitamin-free during the purification process of I.galbana(OA-3011).I.galbana is excellent unicellular bait for the larvae of many aquaculture animals.Thus screening for the I.galbana strains with rapid growth rate and high content of DHA and EPA is very important for the breeding of aquaculture animals.However,most of the I.galbana strains in China derive from a common ancestor called I.galbana OA-3011 and different strains o f I.galbana have developed after so many years of geographical isolation.Whether the traits of these strains have changed is unknown.In order to evaluate the status of the phenotypic traits of different geographic isolations for future selective breeding,comparative analysis among five strains of I.galbana(FACHB-861,OA3011-QD,OA3011-LK,OA3011-AY and OA3011-LZ)on their taxonomy,growth and fatty acid composition was conducted.Different strains were cultured under identical conditions for six days until arriving at stationary phase.Genomic DNA was extracted for the PCR sequencing of 18 S rDN A gene.OD660 was recorded daily for growth curve plotting.When entering stationary phase,cultures were harvested by centrifuge for biomass determination and fatty acid analysis conducted by Gas Chromatograph.Phylogenetical analysis of 18 S rDN A genes suggested that FACHB-861 was distantly separated with OA-3011 strains.OA3011-LK,OA3011-AY and OA3011-LZ were the same in sequence,but OA3011-QD differed from other OA-3011 strains at one position of sequence where single base substitution happened.The growth curve showed the five strains reached stationary phase at different time.FACHB-861 showed a distinct pattern of growth compared with the other four OA-3011 strains.The final biomass concentrations were significantly different between I.galbana FACHB-861 and OA-3011 strains as well as OA3011-LK,OA3011-AY and OA3011-LZ(P<0.05),while the growth rates showed no significant differences among strains(P?0.05).The total fatty acid(TFA)content of FACHB-861 and OA3011-LZ were significantly lower than that of O A3011-LK,OA3011-QD and OA3011-AY(P<0.05).The fatty acid profile of FACHB-861 was obviously different from OA-3011 strains.However,a conspicuous variation in fatty acid compositions was also observed among OA-3011 strains,indicating a wide and frequent quantitative phenotypic variation among the strains of I.galbana.Together,these variations resulted from geographic isolations provided the feasibility for selective breeding of I.galbana strains.I.galbana strains from Yantai city(OA3011-YT)and Hainan province(OA3011-HN)were chose to produce self-fertilizing and hybridization by using their glue group cells.The optimized medium was used to acquire single clone.Then the F1 cells were obtained in the F / 2 liquid medium.The superior I.galbana strains were screened by measuring the growth rate,relatively fat content and fatty acid content.The F2 cells were obtained by repeating the above steps.Finally,the fatty acids of F2 cells were analyzed.The results showed that the trait of high DHA and EPA contents of F1 was kept to F2 and the lowest levels of DHA,EPA and PUFA in F2 generation were improved compared to F1.The simulated hybrids F1 performed well in the growth,the content of fatty acids and the feasibility of improving content of DHA and PUFA.Finally,four strains with high DHA content and two stra ins with high EPA content were obtained,all of which have fast growth rate.Further development,research and utilization of these strains will be done.In view of the above,the necessity and feasibility of breeding for high DHA and EPA content of I.galbana strains are obvious.Although there are reports about mutation breeding of this alga,it is necessary to choose selective breeding as a basic scientific research.Because it is not only possible to obtain the related data about the quantitative traits of the I.galbana,but also to increase the understanding of its genetic diversity and to provide excellent strains for further mutation breeding and transgenic breeding.All these improvments will enhance the germplasm resources of I.galbana.