Chromosomal Localization And Molecular Organization Analysis Of RDNA Clusters In The Hybrid Lineage Derived From Carassius Auratus Red Var.(♀)× Megalobrama Amblycephala(♂)

Polyploidy species have played a major role in the evolution and the adaptation of eukaryotes.Hybridization is one of the primary mechanisms for the origin of species leading to the formation of polyploids.In previous study,the hybrid lineage derived from Carassius auratus red var.（RCC）（♀）×Megalobrama amblycephala（BSB）（♂）were obtained.The rRNA gene is a classic locus for the study of genetic evolution of species.Thus,the chromosomal localization and molecular organization of rDNA clusters in allotetraploidy、autotetraploidy and autotriploidy were analyzed in our research.The main research contents were presented as following:1.The structural units and chromosomal location of 5S rRNA gene in allotetraploid hybirds（4nRB）and allopentaploid hybrids（5nH）from4nRB（♀）×BSB（♂）were analyzed.The results showed that the structural units of 5S rRNA gene from RCC were found in 4nRB and 5nH,and the structural units of 5S rRNA gene from BSB were not found.These results indicated that the newly established allotetraploid genome occurred a large number of genomic changes.With 5S rRNA gene as probe,the results of FISH indicated that chromosomal locus deletions and chromosome recombinations were detected in 5nH.These data showed that the newly established allotetraploid genome was instability.2.The structural units and chromosomal location of 5S rRNA gene in autotetraploid lineages were comparatively studied.The results indicated that the structural units of 5S rRNA gene from RCC were found in 4nRR.The variation of 5S rRNA gene structural units was not found in them.Analyzing the 5S rRNA gene sequence,the results showed that obvious base variations in NTS and array recombination of repeat units were found in 4nRR.According to the genome of 4nRR,there should be four large 5S rDNA gene loci located on two pairs of submetacentric chromosomes,respectively.However,the FISH results with 5S rRNA gene（340bp）as probe presented that two large 5S rDNA gene loci located on a pair of submetacentric chromosomes and two small 5S rDNA gene loci located on a pair of submetacentric chromosomes in4nRR（F₁）.In 4nRR（F₇）,two small 5S rDNA gene loci were not found.The results of Ag-NOR staining also showed that the loss of rDNA gene loci were occurred in 4nRR.These observations suggested that the genome of 4nRR occurred obvious variation and recombination during autopolyplodization process.3.In order to investigate the pairing behavior of homologous chromosomes in 4nRR during meiotic,we applied FISH to performe hybridisation on 4nRR somatic and gamete with 5S rRNA gene（340bp）and species-specific centromere as probe.The results showed that the number of chromosomal loci（5S rRNA gene and centromeric）were halving in their gamete.These data indicated that the coexistence of four homologous chromosome sets not occurred multivalent pairing formation during meiosis in autotetraploid lineages,and diploid-like pairing behavior was restored.This pairing behavior might contribute to the successful establishment of autotetraploid lineages.4.We comparatively analyzed the sequences and chromosomal locus of 5S rRNA gene in natural autotriploid Carassius auratus（3nCC）and artificial autotriploid gynogenetic（3nG）as well as the related diploid ancestors,diploid Carassius auratus（2nCC）and Carassius auratus red var.（RCC）.The similarities of the 5S rDNA coding region（class I,class II and class III）between RCC and 3nG were 98.3%,100%and 99.1%,respectively,and the similarities of the 5S rDNA coding region（class IV,class V and class VI）between 2nCC and 3nCC were 97.5%,100%and100%,respectively.The phylogenetic tree was constructed with the 5S rRNA gene sequence.The results showed that a remarkably close relationship existed among 3nG,3nCC,RCC and 2nCC.Fluorescence in situ hybridization（FISH）indicated that the expected numbers of maternal chromosomal loci were found in 3nG and 3nCC,respectively.These results indicatied that autotriploidization has little influence on the divergence of the 5S rDNA family in Carassius auratus.Additionally,The similar genetic characteristics and phylogenetic relationship of the 5S rRNA gene in 3nCC and 3nG revealed that 3nCC was hybrid origin with the similar diploid ancestors as occurred in 3nG,which further confirmed the viewpoint that 3nCC was derived from the hybridization of a female2nCC and a male of a different species.5.We comparatively analyzed the internal transcribed regions（ITS）sequences of 45S rRNA in 4nRB,4nRR and their parents,RCC and BSB.The results showed that the genetic traits and expression of ITS in 4nRB and 4nRR were basically consistent with that of RCC.And the ITS region sequence showed high similarity between 4nRB and 4nRR individuals.Although the ITS1 sequence shows differences among 4nRB individuals and 4nRR individuals,the entire ITS region still has a high similarity.