Studies On The Application Of DNA Molecular Marker Technique In Genetic Breeding Of Pseudosciaena Crocea

Microsatellites was isolated by twain traditional crossing selection.The gynogenesis were induced by cool-shock and hydrostatic pressure method in this project.Using the AFLP and SSR marker technique,the genetic diversity both in the wild and the domestic populations,and the genetic relationship of artificial gynogenesis were analysed.In addition,the cross testing of Pseudosciaena crocea(♀)and Miichthys mijuy((?))was performed in P.crocea.The results showed as follows:1.An(CA)microsatellites-enriched library was triumphantly constructed by isotope marker for P.crocea.82 clones(57.4%)over 7 core-sequence contained microsatellites.18 of them were sent to GenBank and their accession number were EU022002～EU022019.18 SSR-primers were designed and 13 of these primers were polymorphic.Its average polymorphic information content(PIC)was 0.597 varied from 0.452 to 0.915.These markers were useful for the studies on population genetics and selective breeding of the large yellow croaker.Consequencely,13 polymorphic microsatellit loci were used to analyze the variation and divergence among the wild and two domestic stocks of the large yellow croaker.The expected heterozygosity(He)was higher than the observed heterozygosity(Ho),which indicated that there was relatively higher genetic polymorphism in wild stock.The 2 domestic stocks got together and diverged from the wild stock.Finally,the results indicated that the wild and domestic stocks were different from each other in genetic structure as well as the three stocks had obvious differentiation.2.Two gynogenesis groups(G1,G2)were induced in P.crocea by cool-shock and hydrostatic pressure method.The results showed that the effect of cool-shock method obtained a hatching rate of 35.3%,and a survival rate of 9.9%at 45 days,the effect of cool-shock method was obviously better than hydrostatic pressure method.The AFLP analysis showed the successful ratio was 100%in family G1,87.5%in family G2.The average gynogenetic ratio of the two families was 93.75%.SSR analysis also had simliar results.The homozygosity ratio of the offspring in two families were 87.5%and 76.2% respectively,and the average homozygosity ratio is 81.9%,however,that of control offspring was zero.It was clear that the similarity coefficient between gynogens of two families was the largest,or the genetic distance among gynogens was the shortest between the hybrids of their contrast.The relationship of the gynogens was the closer to the female parent.This study showed that gynogenesis was an effective method to promote gene purification.In addition, microsatellite marker and AFLP marker technique were effective method of gynogenesis verification and genetic analysis.3.The cross testing of Pseudosciaena crocea(♀)and Miichthys mijuy((?))was produced, and obtained the fertilization rat at 56.25%,the hatching rate at 45.24%;the survival rate at 0.65%in fry stage.The genetic analysis on the relationship between the F₁ progenies and their parents were carried out by using AFLP and microsatellite markers.Clear parental specific bands were amplified from all the four microsatellite loci,but none of paternal specific bands were found in all F₁ progenies.In the AFLP results,132 of 143 maternal specific bands presented in filial generation,only 18 of 94 paternal specific bands presented in filial generation.The genetic similarity coefficient was 0.853 between the F₁ progenies and their female parent,0.271 between the F₁ progenies and their male parent.The genetic distance was 0.159 between the F₁ progenies and their female parents,while it was 1.307 between them and their male parents. The results suggested that the F₁ progenies were allogynogens for high genetic identity between the F₁ and the maternal female parent.