Studies On The Molecular Mechanism Of Genome Diploidization In Ancient Polyploidy Vertebrate

Recent phylogenomic studies and extensive comparative analysis on complete genomic sequences of model organisms confirmed the proposition that today's typical diploid vertebrate species have evolved from ancient polyploids.However,little is known on how the ancient polyploids overcame the disadvantages of genome duplication,the molecular mechanism for the ancient polyploids returned to diploids again, and how the ancient polyploids acquired the advantages during the severe struggle for existence and further developed so many vertebrate species in nowadays.These topics are not only fascinating myths of life science but also the core of species evolution studies.In order to understand the molecular mechanism underlying the diploidization of the duplicated genes via gene silence and loss after the genome duplication in the ancient polyploid vertebrates,we investigated vsxl gene loci,the expression difference among the various loci and the causes which resulting in the expression difference in goldfish,the animal which might still be in the process of diploidization.The main results and conclusions of this research are as follows:1.By analyzing the 5' flanking sequence of goldfish vsxl,and further gene-specific PCR assay in single haploid embryos and various embryos at different developmental stages,two different gene loci of goldfish vsxl were determined,which were named vsxl A1 and vsxl A2 respectively.2.Both 5' RACE analysis and specific RT-PCR analysis showed that vsxl A1 is the transcriptional locus,while vsxl A2 is a non-functional locus as it cannot be transcribed and it has accumulated lots of mutations.These findings indicated that goldfish vsxl genes have been diploidized since the latest genome duplication,and that the duplicated genes are diploidized by silencing the redundant gene copy,rather than by function differentiation.The non-functionalization of the redundant copy is caused by the loss of transcriptional activity,rather than by producing non-functional transcripts.3.By comparing the expression pattern of GFP driven by vsxl A1 proximal promoter with the normal expression pattern of vsxl mRNA detected by whole mount in situ hybridization,and by analyzing the evolutionary conservation of vsxl promoter sequences from different species,the key region which is essential for the regulation of vsxl transcription has been identified.4.Bisulfate sequencing examination on the promoter CpG islands of vsxl A1 and vsxl A2 in sperm,unfertilized egg,embryos at different developmental stages and various adult tissues indicated that the proximal promoter region of both vsxl A1 and vsxl A2 were unmethylated and there was no epigenetic difference between vsxl A1 and vsxl A2.Such results indicated that the silence of vsxl A2 is unrelated to the methylation of the CpG islands within its promoter. Unlike in artificial polyploid plants,in which epigenetic modification plays crucial role in controlling dosage effects and diploidization, obviously,DNA methylation is not involved in the diploidization of goldfish polyploid ancestor.5.Compared with vsxl A1,the transcription regulation region of vsxl A2 promoter accumulated many nucleotide mutations,which leaded to the loss of one active transcription factor binding site and the emergence of two repressive transcription factor binding site.This result suggests that the nucleotide mutations might be the cause of the silence of vsxl A2.These results suggested that,during the evolution of the ancient polyploid vertebrates,the genome diploidization is not a rapid and short process accomplished by epigenetic modifications,but rather a very slow and long process accomplished by accumulation of mutations. Accumulation of nucleotide mutations might be the key cause of the redundant copies loss in the ancient duplicated vertebrate genome. However,the mutation is a relatively very slow process,which is obviously not the rapid primary adaptive step for the newly formed polyploids to adjust to the sudden genome duplication and to acquire advantages for existence.The vertebrate polyploid ancestors have survived in the struggle for existence and given rise to so many diploid species on nowadays,might be attribute to the reproductive style changes companying the genome duplication,which enable the vertebrate polyploidy ancestor to produce their offspring independent of sexual reproduction and the meiotic mechanism.