RAPD And Microsatellite Analysis Of Diploid Gynogens From Allotetraploid Hybrids Of Red Crucian×Common Carp

The formation of allotetraploid hybrid population from red crucian carp (♀ ) (Carassius auratus red var.) × common carp ( ♂ ) (Cyprinus carpio L.) is the first case in the world which both the female and the male of allotetraploids are fertile. They provide important theory evidence and very good experimental materials for studying the mechanism of formation of fish multiploidy in nature. The allotetraploids possesse 4 sets of chromosomes, and produced the diploid gametes. So it is worth making the gynogenetic experiment to elucidate their sex determining system and the diploid eggs' trait. In the present paper, the genetic diversity, genetic purity and incorporation of heterologous genetic material were analyzed in the G₁ and G₂ from F₁₀ allotetraploid hybrids (AT), using random amplified polymorphic DNA (RAPD) assay and microsatellite analysis.1) RAPD and microsatelliteanalysis of the first-generation diploid gynogens (G₁) from F₁₀ allotetraploid hybrids (AT)Thirteen AT individuals and twelve G₁ individuals were randomly selected for the RAPD and microsatellite analysis. From 233 10 mer random primers, a total of twenty-eight random primers were screened for further analysis. A total of 4249 reproducible bands were obtained for the twent-y-eight primers in the two populations. There was average of 6.07 amplified bands per random primer in each individual. From 3 to 10 reproducible amplified fragments ranging in size from 0.55 to 1.9Kb were obtained. In RAPD patterns, some bands were present in the two populations, whereas other bands were species-specific: the RAPD marker (S45-600)was missing from all AT individuals and present in all Gi individuals, and another RAPD marker (SI34-900) was present in all AT individuals but absent in all Gi individuals, which can be used as molecular markers for discriminating between Gj population and AT population. The genetic similarity coefficients of Gi ranged from 0.92 to 0.97 with a mean of 0.96, whereas the genetic similarity coefficients of AT ranged from 0.87 to 0.96 with a mean of 0.91. The average genetic diversity indice of Gi and AT estimated by using Shannon's index of phenotypic diversity and its modified formula were different, ranging 0.25, 0.04 to 0.67, 0.09, respectively., The mean percentage of polymorphic loci was 12.71%, 30.69% in the two populations, respectively.From 31 pairs of microsatellite primers, a total of 11 pairs of microsatellite primers were screened for further analysis^ A total of 28 different alleles were found across the two populations by the tested 11 microsatellites, of which 19, 28 loci were detected in Gi and AT, respectively. The mean number of alleles of Gj (1.73) per locus was lower than that (2.55) of AT. Average expected heterozygosily value (He) of Gi (0.26) was significantly lower than that of AT (0.40). The observed heterozygosity value (Ho) was detected at all microsatellites loci in AT, however, Ho was only detected at six loci in Gj, and was considerately lower than that of AT in all loci. In addition, the mean effective number of alleles of G| (1.60) at 11 microsatellite loci was lower than that (1.88) of AT.Therefore, the results from RAPD assay and microsatellite analysis were in agreement with each other, that is to say, G\ presented drastically lower level of polymorphism than AT.2) RAPD analysis between the first-generation diploid gynogens (d) and their parentsFrom 233 10 mer random primers, a total of 34 random primers were screened for comparatively further analysis. Four Gi individuals, four female AT individuals used to produce Gi and four male scatter scale common carp individuals (SSC) were analyzed using RAPD. A total of 3062 reproducible bands were obtained for the 34 primers in G,, AT and SSC, of which 1054, 1044 and 964 bands were detected in Gj, AT and SSC, respectively. There was average of 7.87, 7.68 and 7.20 bands per random primer in each individual, respectively. From 3 to 11 reproducible amplified fragments ranging in size from 0.5 to 2.1Kb were obtained. The average genetic similarity coefficient between Gi and AT was 0.97, whereas between Gi and SSC was only 0.60. It was no doubted that the genetic material comes mainly from the female AT. The average genetic similarity coefficient between AT and SSC was 0.59. Similar or identical banding patterns were observed between G\ and AT, whereas obvious differences were detected between G| and SSC. However, there were a few identical RAPD special bands that were detected in G| and SSC between two primers (S28, S128), but not detected in AT. The result indicated that heterologous genetic material had incorporated into G\. However, the correct conclusion will be drawn by the further analysis. 3) RAPD analysis between the first-generation diploid gynogens(G]) and the second-generation diploid gyjiogens (G2) from allotetraploidsThe genetic heterogeneity and molecular markers were analyzed by RAPD technique in the G| and G2. Of 134...