| Retrotransposons are the most abundant and widespread class of transposable elements (TEs) in plant kingdom. Tyl-copia-like retrotransposon is one of the most deeply studied types, which transpose through a RNA intermediate by a "copy-paste" mechanism. They play an important role in plant genome size, structure, evolution, and so on. Allopolyploidy is a prominent process for plant speciation, and plays a significant role in the origin of many species. Newly formed allopolyploid experiences a huge "genomic shock", which can activate the expression of retrotransposons. In contrast, the transcription of retrotransposons can affect genetic and epigenetic changes of allopolyploid. Therefore, in this work, we studied the heterogeneity, copy numbers, genomic and chromosomal distribution of Tyl-copia retrotransposons in Cucumis hystrix (2n=2x=24), a wild species of Cucumis. And then researched the effect of allopolyploidization on transcriptional activation of retrotransposons in the newly formed allotetraploid C. hytivus (2n=4x=38). Finally, the effect of retrotransposons on genetic and epigenetic changes in early generations of Cucumis allotetraploid was investigated to explore the genetic and epigenetic mechanisms.1. The isolation, distribution and genetic diversity analysis of Tyl-copia retrotransposons in CucumisThe reverse transcriptase sequences of Tyl-copia retrotransposons were isolated from genome of Cucumis sativus using degenerate oligonucleotide primers. The amplified production was recovered and sequenced,17 different sequences of reverse transcriptase were obtained. These sequences showed high heterogeneity with length varied from 207bp-266bp and homology ranged from 44.1%-98.1%. Among them,10 were intact with potential transcriptional activity, whereas the remaining sequences had a stop codon, a frameshift, or both. Phylogenetic analysis showed that these sequences were divided into four families and had high homology with other species.In addition,24 highly heterogeneous reverse transcriptase sequences of Tyl-copia retrotransposon were isolated from C. hystrix. Southern hybridization analysis showed that retrotransposons existed in C. hystrix by high copies. Southern dot-blot analysis revealed that the copy number was about 5460, which constituted approximately 8.5% of the C hystrix genome. Phylogenetic analysis showed that these sequences were divided into 5 families and had high homology with other species. Fluorescence in situ hybridization (FISH) revealed the retrotransposons of this kind were widely dispersed over all the chromosomes with clusters in terminal heterochromatin regions.2. The influence of allopolyploidization on expression of Tyl-copia retrotransposons in CucumisTo investigate the effect of allopolyploidy on the expression of Tyl-copia retrotransposons in Cucumis newly synthesized allotetraploid, C. hytivus, RT-PCR strategy was carried out to amplify reverse transcriptase (RT) genes from C. hytivus and its diploid parents (C hystrix and C. sativus). Only the allotetraploid yielded the expected product. When recovered and sequenced,18 unique clones with significantly high heterogeneity were obtained. The synonymous (dS) and nonsynonymous (dN) substitution analysis suggested that the RT sequences had been under purifying selection. RT-PCR and gPCR analysis of the cloned reverse transcriptase in the first four generations of Cucumis allotetraploid further proved that the activation of retrotransposons was induced by allopolyploidization.3. Effect of retrotransposons on genomic changes in early generations of Cucumis allotetraploidIn order to study the genetic variation of early generations of Cucumis allotetraploid, genomic DNAs from the reciprocal F1 hybrids of C. hystrix and C. sativus, early generations of the newly synthesized allotetraploid and their diploid parents were used for SSAP (Sequence specific amplification polymorphism) analysis. The results showed that loss of parental fragments and gaining of novel fragments were detected in the reciprocal F1 hybrids and allotetraploid of interspecific hybridization. No significant differences with regards to the number of fragments lost or gained between reciprocal crosses were noted. Both loss and gaining of fragments are mainly occurred in F1, S1 and S2 generations, indicating the variation in early generations is a rapid process.In the meantime, two retrotransposon-based markers IRAP (Inter-retrotransposon amplified polymorphism) and REMAP (Retrotransposon-microsatellite amplified polymorphism) and a microsatellite-based marker ISSR (Inter-simple sequence repeat) were employed to investigate genomic changes in early generations of C. hytivus. Twenty-eight fragments were examined, included 24 loss of parent fragment and 4 gaining of novel fragment, suggesting that sequence loss is a relatively frequent event. Among the 24 lost fragments,18 were of C. hystrix origin,4 were C. sativus-specific, and the remaining 2 were shared by both species, implying that fragment loss may be correlated with the genomic constitution of diploid parents. Moreover, most changes were observed in the S1 generation and stably inherited in the subsequent three generations (S2-S4), indicating that genomic changes were rapid driving force for the stabilization of allotetraploid.Further, the altered DNA fragments were recovered and sequenced, and finally 11 sequences were obtained. Sequence analysis showed that genomic changes in the allotetraploid occurred in both coding and non-coding regions, which might suggest that retrotransposons inserted in genome randomly. Subsequently, FISH analysis revealed a unique chromosomal distribution of altered fragments, where the preferential FISH signals occurred in the centromeric and telomeric regions, implying that these regions were the possible hotspots for genomic changes.4. Effect of retrotransposons on epigenetic changes in early generations of Cucumis allotetraploidFISH technique was employed to study the chromosomal distribution of LTR retrotransposons in Cucumis allotetraploid. The result revealed that LTR retrotransposons distributed throughout all the chromosomes, with clusters on terminal regions. Most chromosomes showed clusters only on one terminal whereas a few had clusters on both terminals, indicating their high copy numbers and uneven distribution in C. hytivus.cDNA-SSAP strategy was used to investigate the effect of retrotransposons on epigenetic changes in the first four generations of newly formed Cucumis allotetraploid. Extensive epigenetic changes were detected, including gene silencing and gene activation. The transcripts that exhibited gene expression alteration were recovered and sequenced, and 20 sequences were obtained, containing 12 gene silencing and 8 gene activation. Sequence analysis showed that the silenced/activated genes consisted of known and unknown genes. Further RT-PCR and gPCR analysis validated the above result. Both gene silencing and activation mainly occurred in early generations of the allotetraploid, and could be stably inherited in subsequent generations. These results implied the transcriptional interference of retrotransposons on their adjacent genes, which contributed to the stabilization of newly formed allotetraploid. |
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