Polymorphisms Analyzing Of The Main Farming Regions Of Takifugu Rubripes And Isolation Of Gender‐Specific Microsatellite Markers

Takifugu rubripes is a high-nutritional and economic valued specie, which is one ofthe most important marine fish of China. However, in recent years, development of theaquaculture has been slowed down due to serious germplasm degradation, so geneticimprovement is inmininent. Firstly, this study summarized the breeding、culturing andgenetic researching of major economic species of Takifugu, the molecular markers andgenetic research status、research of tetrodotoxin of Takifugu rubripes. Secondly, thisstudy carried out the screening of gender-related molecular markers of rubrippes, so itlaid the foundation for the gender controlling technology and male nurturing of Takifugurubripes. Besides,this study carried out genetic diversity analysis of the main farmingregions of rubripes, so we can generally understand the germplasm resources of theseplaces, this study will be foundations of selective breeding、high yielding and highquality of Takifugu rubripes.The following results were received from this study:In order to investigate resources of the main breeding areas in China and know theresource conditions of Takifugu rubripes, twenty pairs of microsatellite primers weretested on five culture populations from different area in China (wendeng group:WD;laizhou group:LZ; Japanese group of2009:J1; Japanese group of2011:J2; broodstockof tanghai:THQ)by way of microsatellite marker analysis. It showed that12pairs(60%) display polymorphism. The number of alleles of these five groups range from2to8, the average observed heterozygosity were0.27、0.26、0.18、0.25、0.20; the averageexpected heterozygosity were0.55、0.57、0.69、0.71、0.62respectively; the order ofpolymorphism information content (PIC) were J2(0.65)＞J1(0.63)＞THQ(0.55)＞LZ(0.51)＞WD(0.48). The results showed that (1)genetic distance between WD andLZ is0.2336, it is the lowest of all; the genetic distance between LZ and J1is0.6242,and it is the highest of all.(2)the genetic diversity of WD was moderate(0.25＜PIC＜ 0.5), LZ、J1、J2and THQ were relative high(PIC≥0.5). The analysis showed thatgenetic diversity of Takifufu rubripes of main breeding region is very high, andtherefore, the groups have good potential for genetic improvement.In addition, the polymorphism of70pairs of microsatellite markers were screenedand62pairs of markers which had stable amplification products were gained, the ratiowas88.6%. At the same time,44pairs of polymorphic primers were gained, the ratiowas62.8%.Gender-related microsatellite markers were screened from62pairs ofmicrosatellite markers which had stable amplification products. Microsatellitescombined with bulked segregation analysis (BSA) were used to screen the genderdifferences of44individuals (22for male and female each) of Takifugu rubripes. Twogene pools were constructed using22individuals for male and female separately, genepools were used to scan totally70pairs of microsatellite primers, specific DNAfragments were amplified by4pairs of primers(Fms42、70、89、134). Then, the specificDNA fragments amplified by the4pairs of primers were verified for the44individualsconstructing the gene pools, the results showed that the4primers amplified in male andfemale individuals did not differ significantly.70pairs of primers were used to detectmicrosatellite polymorphism in the22male and female individuals,44pairs ofpolymorphic primers were gained, the ratio was62.8%. The results showed that thepolymorphism information content (PIC) of male and female group were0.61and0.56respectively, the two groups all showed a high degree of polymorphism(PIC≥0.5),indicating high genetic diversity and genetic potential in the male and female groups. Studies on Feeding behavior and Morphological Development ofEpinephelus moara in early development stage Besides, some researches were done to analiyze the feeding behavior andmorphological developments of Epinephelus moara in early development stages. Weused ecological method of simple random sampling to observe morphological features,growth changes and feeding conditions during larval development of Epinephelusmoara. The results showed that, under hatching conditions at dissolved oxygen (DO)6.2mg/L, temperature20℃, salinity30, pH7.9, light100lx, it took45hours to hatchthe larvae. The yolk of four day-old larvae almost disappeared and these lava startedconsuming oyster larvae. The8—13day-old larvae ate both the oyster larvae andS-rotifer; the14—22day-old larvae consumed L-rotifer; the23—66day-old larvae andjuvenile can be fed with brine shrimp. At about67days, the larvae metamorphosiscompleted and began to eat compound feed. The results of morphological data showthat, total length, body weight and the age larval, juvenile and young E. moara showedsignificant correlations. The relationship between total length and age (in days) can beexpressed as L=2.8058e0.0287D; the relationship between body weight and age can beexpressed as W=0.0004e0.0825D; the relationship between total length and weight canbe expressed as W=0.001e0.103L, the cross-correlation coefficient is0.895,0.905,0.798, respectively. The feeding results of E. moara showed that, under conditions ofartificial cultivation, the intake amount of5day-old larvae reached90%, the satiationrate of18day-old larvae was100%. As the age increased, the plump coefficient ofstomach increased too, the time for feeding from empty to full and digesting food fromfull to empty decreased. The results also showed the larvae reached the peak of feedingat2:00pm under continuous light; under natural diurnal cycles, they reached the peak at4:00—6:00pm; under continuous dark conditions, there was no obvious peak of feeding.The results indicate that the larvae had obvious circadian rhythm of feeding, they atemainly during the day at any circumstance.