| The marble goby,(Oxyeleotris marmoratus, Bleeker), is also well known as the sand goby, which belongs to the family Eleotridae (Sleepers). It is an important commercial fish in some Southeast Asia countries such as Thailand, Malaysia, Indonesia. In this study, microsatellite molecular markers were isolated by Magnetic-bead enrichment method in Oxyeleotris marmoratus. And these microsatellite markers were used to analysis the genetic variation in hybrid progenies and backcross progenies from Oxyeleotris lineolatus(♀) and Oxyeleotris marmoratus(♂). Then, the population genetic diversity of marble goby were inferred from mitochondrial DNA and microsatellite analysis.We also used the microsatellite markers developed from Oxyeleotris marmoratus do the applicability in applicability of microsatellite markers developed from lineolatus and Odontobutis potamophila. The major results and conclusions were as follows:(1) Isolation and detection of microsatellite markers in Oxyeleotris marmoratusMicrosatellite molecular markers were isolated by Magnetic-bead enrichment method in Oxyeleotris marmoratus.146clones were sequenced from all the1032positive clones. Eighty one sequences (55.5%) contained microsatellite in these clones and forty three sequences contained more than10times microsatellite repeat units. There are52perfect type microsatellites,27imperfect type microsatellites and2compound type microsatellites. These microsatellites contained not only CA and GA repeat unit but a few other type unit (TAC) be detected. Thirty eight microsatellite primer pairs were designed and synthetized. Twenty nine loci showed clear and good repeatability bands after testing. A population of48individuals was tested with the29loci:The average observed heterozygosity (Ho) and expected heterozygosity (He) was0.63and0.43respectively. There are seven highly polymorphic loci, forteen moderate polymorphic loci revealed, seven low-grade polymorphic loci and one monomorphic loci in the population. Overall, most of the microsatellite loci from O. marmoratus are abundant polymorphic. (2) Microsatellite marker analysis of genetic variation in hybrid progenies and backcross progenies from Oxyeleotris lineolatus(♀) and Oxyeleotris mannoratus(♂)20pairs of microsatellite primers were used to study genetic variation of Oxyeleotris lineolatus(♀), Oxyeleotris marmoratus(♂),their hybrid progenies and backcross progenies. The results indicated as follows:Ninety-five alleles in total were detected from four stocks,the lowest average numbers of alleles was in O. lineolatus stock(1.6087), and the highest is in hybrid progeniey stock(2.9130). The average effective number of alleles was O. marmoratus(2.2570)> hybrid progeniey F1(2.1516)> backcross progeniey F2(2.1061)> O. lineolatus(1.5164). The mean levels of observed heterozygosity(Ho), expected heterozygosity(He), polymorphism information content(PIC) and Shannon (I*) in four stocks were0.4891～0.7767,0.2714～0.5193,0.3473～0.4458and0.3662～0.7993, respectively. Among all parameters, the O. lineolatus stock was always the lowest, the highest average observed heterozygosity and Shannon appeared in the hybrid progeniey F1stock, the highest levels of average expected heterozygosity and average polymorphism-information content were in backcross progeniey F2stocks and O.marmoratus stocks, in addition, all stocks’ average observed heterozygosities were, higher than their average expected heterozygosities. Chi-square tests showed that most loci were deviation from Hardy-Weinberg equilibrium(P<0.05). An UPGMA tree which on a basis of Nei’s genetic distance demonstrated that the hybrid progeniey F1and backcross progeniey F2grouped together, and then grouped together with O. lineolatus, O. marmoratus comprised the other clade. Not only hybrid progenieies but also backcross progenies all showed definite maternal effect.(3) Population genetic diversity of marble goby(Oxyeleotris marmoratus) inferred from mitochondrial DNA and microsatellite analysisThree populations of marble goby(Oxyeleotris marmoratus) were collected from Vietnam, and Guangdong and Hainan Provinces in China. Genetic variation and population structure were assessed by mitochondrial DNA (mtDNA) and nuclear microsatellite variation. A total of855bp of mitochondrial control region was sequenced and fourteen microsatellite loci were genotyped for91individuals. Twenty haplotypes were detected among all samples based on mtDNA. The h.20was the most widespread haplotype among three populations, comprising61.5%of all samples. A moderate level of haplotypic diversity (h=61.6%), low level of nucleotide diversity (π=0.311%), average expected heterozygosity (He=0.472-0.504) and polymorphism information content (PIC=0.454-0.497) were observed in our studies. Phylogenetic trees of haplotypes were constructed by NJ method based on mitochondrial control regions. Neither significant genealogical branches nor geographic cluster were observed. Nei’s genetic distance between HS and GS populations was much smaller than that between either of them and the YS population from the microsatellite, which was consistent with their geographical distance. The non-significant Tajima’s D values suggested that all populations were at equilibrium except the YS population. Analysis of molecular variance (AMOVA) revealed a significant difference within populations and no significant difference among populations for both mtDNA and microsatellites. The significant values of FsT from two kinds of analysis methods suggested that the marble goby in Vietnam might be genetically differentiated populations from Guangdong and Hainan populations. However, a wide range of gene flow exists among all, populations, especially between Guangdong and Hainan populations.(4) Applicability of microsatellite markers developed from Oxyeleotris marmoratus in Oxyeleotris lineolatus and Odontobutis potamophilaUsing Microsatellite DNA-polymerase chain reaction (STR-PCR), fourteen microsatellite primers had been developed by our laboratory were tested for cross-amplification in two species Oxyeleotris lineolatus and Odontobutis potamophila.14loci were amplified successfully in Oxyeleotris lineolatus and10loci were amplified successfully in Odontobutis potamophila. The polymorphisms of these scorable loci were assessed using corresponding population individuals, and the results showed that9loci were polymorphic in Oxyeleotris lineolatus and6loci were polymorphic in Odontobutis potamophila. The average of three populations observed heterozygosity was0.3416, the average of expected homozygosty was0.5788, and the average of PIC was0.5369. What was more,3loci(H27,H142and H97) revealed highly polymorphic(PIG>0.5) in the three populations, respectively. The number of alleles per polymorphic locus ranged from6to22in these three loci. In addition, we found a genotype-specific locus(H97) which was differences average allelic length between genera Odontobutis and Oxyeleotris. Therefore, developed microsatellite markers of Oxyeleotris marmoratus applied to cross-amplification in closely related species was feasible and valid. |
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