| Japanese flounder （Paralichthys olivaceus） is an important economical fish inChina, Japan, Korea and so on. In recent years, the development of flounderaquaculture has been seriously interfered with bacterial and viral diseases. Toprevent or at least diminish the devastating effects of these diseases, one usefulmethod is to cultivate families or strains with good characters such as fast growth,strong disease resistance, high breeding survival rate（BSR） and so on. To achievethis purpose, four studies have been carried out as follows: firstly, lymphocystisdisease（LD） resistance families and high BSR families were analyzed; secondly,the families of three successive generations with strong resistant to Vibrioanguillarum were screened and analyzed; thirdly, the molecular markers associatedwith resistant to the V. anguillarum and LD were screened; fourthly, two successivegenerations in meiotic gynogenesis families and inbreeding（brother sister mating）strain of the first finial generation（F1） family have been genetically analyzed. Themain results are as followings:1. Comparative analysis of resistant to LD and BSR among flounder familiesIn2010, LD outbreak in some families established in2009. After evaluating thehealth rate of16families and a control group，four families with incidence below40%were selected and one of the four families with0%incidence was F0939.Around580days after hatching （DAHS） in2011, BSR and body mass （BM） weremeasured and seven families were discovered with BSR above55%. At least one parent of the above six families derived from F0750. It indicates that a family with aparent from F0750may effectively improve the BSR. Correlation analysis showedthat it was significantly negative correlation between the incidence of LD and bodymass at the time of infection （r=-0.790）. In the mean time, the survival rate afterinfection with V. anguillarum （VSR） was relatively positive correlation with BSR（r=0.371）.2. Comparative analysis of disease resistance among flounder families of threesuccessive generationsIn order to enhance V. anguillarum disease resistance and screen new familieswith high resistance to V. anguillarum disease, sixty-five families of flounder wereestablished in2012. Forty-three families were selected as candidate materialschallenged with V. anguillarum using intraperitoneal injection. After infection,different families exhibited variable abilities of disease resistance and8families withstrong disease resistance were discovered. In contrast with the control family, thesurvival of one family was very significantly higher （P<0.01） and seven weresignificantly higher（P<0.05）which contained an F3family and a second generationof gynogenesis family. In the mean time, sixteen families with strong resistance to V.anguillarum from the last few years were analyzed. Fourteen of these families wererelated with Chinese resistance stock （selection by spontaneous challenge andintraperitoneal injection of Vibrio anguillarum）. This indicated that the ability ofresistance to Vibrio anguillarum in flounder could inherit to their offsprings.3. Screening of molecular markers associated with resistant to the V. anguillarumand LD（1） A genome scan for quantitative trait loci （QTLs） associated with V. anguillaruminfection resistance in flounder by bulked segregant analysisIn order to speed up the process of disease resistance breeding, it is necessary toscreen some molecular markers associated with V. anguillarum disease resistance. Inorder to map QTLs, a high-density genetic linkage map was employed to detectedQTLs. F1population was established by crossing of F0750and F0743andchallenged with V. anguillarum in2009. A total of178simple sequence repeat （SSRs） markers were selected around every10cM from genetic map. To screeneffectively, bulked segregant analysis （BSA） and QTL mapping were combined todetect association with this disease. One （qVT-0） and three （qVT-1, qVT-2, qVT-3）QTLs which explained more than60%phenotypic variance in the two models werelocated in a new map, respectively. At the same time, qVT-0and qVT-2were at thesame region. The common QTL maybe a major candidate region for disease resistantto V. anguillarum infection.（2） Screening of EST-SSR markers associated with high VSR families in flounderAs most of QTLs were detected from special families, only a few QTLs wereuniversal. In order to filter out some molecular markers associated with V.anguillarum disease resistance witch suited the entire breeding populations, a total offorty-six simple sequence repeats （SSRs） derived from expressed sequence tags（EST）were used to genotype individuals in thirty infected families. In the end, three SSRswere verified to significantly association （P<0.01） with disease resistance.（3） Initial screening of SSR markers associated with LD resistance in flounderTo screen SSR markers associated with resistant to LD, a family of which theincidence of LD was nearly50%was employed as a resource family. In initialscreening, a total of178simple sequence repeat （SSR） markers were selected aroundevery10cM from a high-density genetic linkage map to be used in BSA. Fifty-twoindividuals derived from three families were selected to confirm the associationbetween markers and LD and one SSR（scaffold185597）was verified in the end.4. Genetic analysis of two successive generations in meiogynogenetic family and F1familyFull-sib inbred and gynogenesis are two common methods to make genehomozygous and traits stable. To accurately assess the impact of the two methods,thirty SSRs were employed to analyze the families of F1, the first-generation diploidgynogenesis, the second-generation diploid gynogenesis and the family from whichthe parents of F1came. The results indicated that meiotic gynogenesis of floundercould rapidly reduce the offspring’s genetic polymorphism and increase the genetic similarity between offsprings. As high recombination rate in some loci, it’s difficultto get pure line following simple repeatedly meiotic gynogenesis. But full-sibinbreeding is free from the influence of recombination, it’s necessary to combine thetwo methods to cultivate pure line. In the mean time, parental homozygosity isclosely related with their offspring in meiotic gynogenesis. As a result, selectingindividuals with high homozygosity could rapidly improve breeding program offlounder combining inbreeding with gynogenesis. All these studies laid thefoundation of cultivating a new breed with several good traits. |
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