Lu Maixin

Effect of low temperature on genomic DNA methylation in Nile tilapia(Oreochromis niloticus)

In this study,using the methylation sensitive amplified polymorphism(MSAP) method,we globally assessed the effect of low temperature adaptation on the level and variations of cytosine methylation alterations at CCGG sites of genomic DNA in Nile tilapia(Oreochromis niloticus) strains with good coldresistance characters developed by the successive and directional selection breeding of multi-generations. Total of 849 fragments were identified by 18 pairs of se1ective primer combinations of which 411 and 438 were detected in cold-resistance tilapia strain and the control,respectively. Among these fragments,the methylation sites were 72 and 104,and the corresponding total-methylation levels w ere at 17.52% and 23.74%,respectively. The full methylation sites at the internal Cs were 37 and 65,and the corresponding full methylation levels were at 9. 00% and 14. 84%,respectively,and the hemi-methylation sites at the external Cs were 35 and 39,and the corresponding hemi-methylation levels were at 8.52% and 8.90% in coldresistance tilapia strain and the control,respectively. Further analysis of DNA methylation indicated that a statistically decrease in the overall level of total-methylation levels and both methylation types was detected in cold-resistance tilapia strain compared with the control(decrease at 6.22%,5.84% and 0.38%, respectively),and the changes of DNA methylation pattern were recognized mainly in the full methylation sites. The level of decrease in DNA methylation suggested that the levels of genomic DNA methylation were changed during the successive cold stress of multi-generations in tilapia,and it also indicated that DNA methylation alteration in tilapia treated with cold stress was mainly through de-methylation that occurred in some CCGG sites. All these results implied that the change of DNA methylation was closely associated with cold tolerance of tilapia. The present findings are valuable to further explore the application potential of DNA methylation alteration in tilapia genetic improvement and provide a new method and theoretical basis for fish stress resistance breeding.

The genetic structure of four populations of Oreochromis aureus by microsatellite DNA analysis.

Genetic diversity and phylogenetic relationships among four breed varieties of Oreochromis aureus (83), O.aureus (02), O.aureus (05) and red O.aureus were studied using microsatellite analysis technology.19 pairs of microsatellite DNA primers of Oreochromis niloticu registered in GenBank which were selected and proved to amplify successfully on Oreochromis aureus by pre-experiment were used.The PCR reaction condition especially concentration of Mg2+ and annealing temperature were adjusted for each pair of primers.Amplified PCR products were electrophoresed in PAGE and stained according to the silver stain procedures and then imagined by Alphalmager.A total of 82 alleles were obtained from the four populations.The number of alleles for each locus ranged from 2-8, and the average alleles was 4.3 each locus.Average observed heterozygosity and average expected heterozygosity were calculated by POPGENE3.2 software.The results showed that the observed value of average heterozygosity of the four O.aureus populations ranged from 0.154 to 0.391.The expected value of average heterozygosity ranged from 0.181 to 0.428, and the average polymorphism information contents ranged from 0.1513 to 0.3882, respectively.The lowest genetic diversity level was found in the O.aureus (02).The highest genetic similarity index (0.9422) and the lowest genetic distance (0.0596) were found between O.aureus (05) and O.aureus (83), which indicated a closer phylogenetic relationship between these two populations.On the other hand, the lowest genetic similarity index (0.6977) and the highest genetic distance (0.3599) existed between O.aureus (83) and red O.aureus, which suggested a farthest phylogenetic relationship between these two populations.The evaluation results of the genetic deviation index D revealed that there were loci departed from Hardy-Weinberg equilibrium in all the four O.aureus populations.Results of null allele analysis by MicroChecker showed that there were loci showing evidences for null alleles in the populations except red O.aureus population, including one locus for O.aureus (83), two for O.aureus (02) and seven for O.aureus (05).Such results suggested that locus with evidence for null alleles in the populations except red O.aureus might be one of the reasons causing deviation of Hardy-Weinberg equilibrium.Phylogenetic relationship of the four populations was analyzed by using UPGMA methods based on their genetic distances and the results showed that O.aureus (83) and O.aureus (05) populations were clustered in the first group, the O.aureus (02) in the second group, while the red O.aureus was in a separate group.