Genetic And Epigenetic Changes In Early Generations Of Amphidiploid 'Cucumis Hytivus'

Polyploidy is a prominent process in higher plants and has played an important role in the evolutionary history of plants.Recent studies indicate that extensive genetic and epigenetic changes often occur at the early stage of allopolyploid's genome formation,these changes have played a significant role in stabilization of newly formed genome of species. Though,many investigations were focused on the characters and reasons for allopolyploid induced genomic changes,the mechanisms remain elusive,because of the lack of information during allopolyploid evolution.Although,the development of molecular markers and comparative genomics have provide important basis for understanding of hybridization and allopolyploidization,the mechanisms still remain poorly understood, because of the complicated molecular evolutionary mechanism.Synthesized allopolyploids can be a very useful model system to study polyploidization, as the information at the early stage can be obtained exactly.With the newly formed allopolyploids,the genomic changes can be studied accurately compared to their diploid parents,which might help enriching the evolutionary theory.In this work,the chromosome behavior,genomic changes and DNA methylation pattem changes of a newly formed Cucumis amphidiploid were studied,in order to investigate the mechanisms of genetic and epigenetic changes.The detailed results as follows:1.Meiotic chromosome behavior and pollen fertility of the interspecific hybrid F₁ and amphidiploidThe meiotic chromosome behavior and pollen fertility of the F₁ hybrid(2n = 2X = 19) and self-cross progenies(S_1-4) of amphidiploid(2n = 4X = 38) from Cucumis hystrix chakr(2n = 2X = 24)×C.sativus L(2n = 2X = 14) were studied.The results showed that at metaphase I(MI),the chromosome configuration of F₁ hybrid was 16.75â… +0.5â…¡+0.25â…¢+0.13â…£.Most chromosomes in the F₁ were univalent,while in the amphidiploid were bivalent.At metaphaseâ…¡,the F₁ could not produce normal tetrads but mostly polyads,while the amphidiploid mainly produced tetrads.Moreover,the frequency of bivalent at MI,tetrads at anphaseâ…¡and fertility of pollen increased during the self-cross course that indicated the improved cytogenetic stability of the amphidiploid.It was also found that at anaphase I(AI) and Aâ…¡the PMCs contained lagging chromosome,bridge fragment,unequal disjunctions and nonsynchronized disjunctions in amphidiploid,which might cause the low pollen fertility.2.Studies on sequence changes in the early genomes of interspecific F₁ hybrid and amphidiploid in CucumisIn the present study,extensive genomic changes were detected in newly obtained allotetraploid of Cucumis,namely Cucumis hytivus Chen and Kirkbride(C.hytivus),by AFLP analysis using 12 pairs of EcoR-I/Mse-I primers.The results indicated that extensive genomic changes occurred at the early stage of allopolyploid genome.The genomic changes occurred form F₁ and the greatest changes of sequence was occurred from S₀ generation to S₁ generation,while,there were miner changes from S₁ to S₂, which indicated that the allopolyploid genome became more stable following the selfing process and the sequence change was a rapid event.Most genomic changes involved loss of parental restriction fragments and appearance of novel fragments,with some fragments disappearing in one generation and reappearing in others.Further investigation suggested that most of the changed sequences were related to repetitive sequence.In addition,it was observed that some variable bands were inherited preferentially from the male parent.During diploidization,sequence loss in reciprocal alloployploids often appears variable in different species,suggesting that the nuclear-cytoplasmic interactions play different roles in different polyploid plants.In this study,Amplified fragment length polymorphism(AFLP) analysis was carried out by using 23 pairs of EcoRI-NN/MseI-NNN selective primers in amplification of the DNAs from reciprocal F₁ hybrids of Cucumis hystrix Chakr.and C. sativus L.,the synthetic amphidiploids and the diploid parents.The results indicated that extensive DNA sequence loss was occurred in the reciprocal F₁ hybrids and amphidiploids. The frequency of loss of parental sequences was not statistically significant between the reciprocal crosses;moreover,the time(both started in the F₁) and the type of loss were also the same,suggesting that the nuclear-cytoplasmic interactions might not be the main factor causing sequence loss.In addition,the frequency of lost sequence from the C.sativus genome was more than from the C.hystrix,indicating that the sequence loss was more common from the parent,which has fewer chromosomes.3.Changes of cytosine methylation induced by wide hybridization and allopolyploidy in CucumisTo explore the molecular involvement of epigenetic phenomena,cytosine methylation of interspecific F₁ hybrids,allotetraploid and parental genome was investigated by using methylation-sensitive amplified polymorphism in Cucumis.Results from analysis of the methylation pattern found that methylation pattern changed in both the F₁ hybrids and in the allotetraploid genome compared to their corresponding parents,suggesting that wide hybridization and allopolyploidization induced cytosine methylation changes.Furthermore, these methylation changes could be classfied into five types according to the changed pattern,mainly including hyper-methylation and de-methylation,moreover,the hyper methylated sites was 2 to 4 fold more than the de-methylated sites,indicating that most of the changed sites showed an increase in cytosine methylation.The results also found that a majority of the methylated sites were from the maternal parent,which suggested that DNA methylation have played a significant role in protecting the maternal genome.Observations in different generations of the allopolyploids found that the extent of cytosine methylation pattern changes between the S₁ and S₂ were significantly higher than that between the S₂ and S₃,suggesting stability in advanced generations which further supported the hypothesis that DNA methylation plays a significant role in the diploidization.Analysis of the methylaion sensitive sequences induced by wide hybridization and allopolyploidization indicated that among 15 cloned sequences,seven of them showed high similarity to the known functional genes or genes involved in regulating gene expression. Specific primers were designed and the results from PCR confirmed that the polymorphisms were from the DNA modification.In addition,the reverse transcription-polymerase chain reaction analysis suggested that cytosine methylation might be related to regulation of gene expression,which further supported the hypothesis that DNA methylation plays a significant role in the allopolyploidization.Southern blot analysis indicated that the altered sequences referred to both single-copy and multi-copy sequence.Plantlets and seedlings of randomly selected common cucumber 'Changchun mici' were studied to investigate the cytosine methylation changes induced by tissue culture using methylation-sensitive amplified polymorphism.Results revealed that there were no significant differences in the level of cytosine methylation in the plantlets and seedlings. Analysis of the methylation pattern found that only 0.6%of total sites changed in plantlets compared to the seedlings,suggesting that tissue culture could not induce a large array of cytosine methylation changes.