| Polyploidy is not only a typical characteristic of plant species,but can also be found in almost all organisms from protists to humans,which is especially a more common phenomenon in fishes.Duplication of the genome,and extra-copies of genes resulting from polyploidy,provide the necessary genetic materials for biological evolution and play an important role in the evolutionary process of the vertebrate.The allotetraploid produced by distant hybridization（red crucian carp♀X common carp♂）,is the first case artificial polyploid fish with the bisexual fertile and stable inheritance in vertebrate in the wold.It has been proved to have two chromosome sets of red crucian carp and two chromosome sets of common carp.In order to understand how allotetraploid genomes evolve after their formation,in this study,we investigated the genomic change and molecular evolution relationships of the allotetraploid and their original parents.The research results will have great significance of further understanding the formation and evolution mechanisms of the polyploid in the fish and even vertebrate.The major results in this paper were presented as follows:Population genetic structure and genomic change of the allotetraploid and their Original parents were revealed by ISSR-PCR and AFLP-PCR markers.The abundant genetic polymorphism were detected among the three populations.The genetic distances and cluster analysis indicated that the allotetraploids inherited the greater part of genetic materials from their original parents after 15 successive generations,and kept the stable genetic characteristics.But the allotetraploids had a higher sister relationships with their original maternal red crucian carp and were inclined to inherite more genetic information from the female parents;The analysis of DNA fingerprintings showed that the allotetraploids,derived from crossing of two original parents genomes,had undergone DNA sequence eliminations, the percentages of sequence elimination statisted from two sets of markers were 19%and 26.5%,respectively,comparing to the female parents,and 48.6%and 28.4%,respectively,comparing to the male parents.The ratio of sequence eliminatiuon from the male parents was larger than the from the female parents.At the same time,some non-parentage bands were founded in the allotetraploid hygbrids.The cyclin A and B gene are the key regulators ofcell cycle,which can regulate the cell from G2/M to an M phase.The cDNA encoding cyclin A and B were derived from the ovaries of the allotetraploid and their original parents,using RT-PCR and RACE.Based on the cDNA and amino acids sequences of cyclin Al and Bl genes,the sequence homology and nucleotide divergence loci were analyzed between the allotetraploid and its original parents,the results indicated the the homology of encoding cDNA sequences of allotetraploid and its original parents were more than 90%, especially,the homology of cyclin boxes sequences were more than 94%, and all the sequence homology between the allotetraploid and its female parent was higher than that between the allotetraploid and its male parent.The well-resolved phylogenetic trees showed that the allotetraploid had more close relative relationship with the original maternal red crucian carp in the evolution process.On the encoding regions of cyclin Al and Bl, the nucleotides mutation levels of allotetraploid were 1.8%and 0.9%, respectively,comparing with the original maternal red crucian carp,and which were 4.7%and 5.7%,respectively,in contrasting to the original paternal common carp,which suggesting that the mutation degrees of cyclin Al and Bl,in the genome of allotetraploid,the original paternal common carp was higher than that of the original maternal red crucian carp. The amino acids sequence alignments of cyclin Al and Bl revealed that there were 4 and 2 amino acids mutation loci between allotetraploids and their original parents,respectively,and the non-parentage loci were resulted from the nonsynonymous substitutions of single nucleotide of codon.According to the splicing ways of the zebrafish cyclin Bl gene,four pairs degenerate primers were designed to be specific for the exon regions of cyclin B in allotetraploid and its original parents,we amplified 8 introns of cycline B gene from allotetraploid and its original parents by PCR,and all the introns obeyed "GT-AG" rule.The combined intron sequence of allotetraploid shared 96.8%and 66.4%homology respectively,with those of the original maternal red crucian carp and original paternal common carp. Partial fragments of the cyclin B gene from triploid,tetraploid,and pentaploid hybrids of red crucian carpx blunt snout bream,blunt snout bream,grass carp,silver carp,and bighead carp were amplified by the primer pairs P3/P4.One DNA fragment was amplified from the blunt snout bream,grass carp,silver carp,and bighead carp（750,900,720,and 720 bp, respectively）.Two fragments（1200 and 900 bp） were amplified from the red crucian carp,common carp,and allotetraploids.The triploid and pentaploid hybrids yielded three DNA fragments（1200,900,and 750 bp）. The 1200 bp fragments of the allotetraploid crucian carp,triploid, tetraploid,pentaploid hybrids of red crucian carpx blunt snout bream shared 99.5,98.9,99.5,and 88.7%homology,respectively,with the maternal DNA.The 900 bp fragment shared 97.5,94.6,94.2 and 89.9% homology,respectively.Our results suggest that inheritance is maternally dominated.Furthermore,we observed preferential elimination of the paternal sequences in the allotetraploid hybrids.Based on these sequences analyses we constructed a phylogenetic tree to explain the relationships among the different ploidy levels.Real-time Quantitative RT-PCR was used to analyze the cyclin Al and Bl gene expression in ovary of the diploid gynogenetic progeny of allotetraploid hybrid,red crucian carp and common carp at the early stage. The results showed that the expression level of cyclin Al and Bl gene in the diploid gynogenesis hybrid was significantly lower than that in red crucian carp and common carp,at the same time,which was also lower of the diploid gynogenetic progeny of allotetraploid hybrid at the early stage than that at the mature stage.The low expression level of cyclin Al and Bl gene in ovary of the diploid gynogenesis may be correlative to the endoreduplication of oogonia at the early stage. |
