The Phenotypic, Genetic And Epigenetic Variations During Wide Hybridisation And Polyploidisation Between Brassica Rapa And Brassica Nigra

During wide hybridisation and polyploidisation, various genomic responses occurred. Genomic responses involved genetic and epigenetic alterations. Genetic alterations refer to sequence elimination, gene conversion events, rDNA loci changes and chromosomal rearrangements, while, epigenetic alterations include DNA methylation changes, histone modifications, and small RNAs. The genetic and epigenetic alterations have important effect on the characters of hybrids, such as heterosis. In the present study, different phenotypes between the parents, hybrids and polyploids were observed. So, firstly, SRAP (sequence related amplified polymorphism) was used to analyze the difference in genetic changes during wide hybridisation and polyploidisation. Secondly, MSAP (methylation-sensitive amplification polymorphism) was used to analyze the different patterns and levels of DNA methylation during wide hybridisation and polyploidisation. Thirdly, the difference of small RNAs between the parents, hybrids and polyploids were systematically analyzed by high-throughput sequencing of small RNAs, to explore the effect of small RNAs on the wide hybrids and ployploids. The study explored the phenotypic, genetic and epigenetic variation during wide hybridisation and polyploidisation between Brassica rapa and Brassica nigra. It provided new theoretical basis for the genetic improvement and the creation of new variety of horticultural crops. The main results were summarized as follows:1. Wide hybridisations between Brassica rapa (♀, genome:AA) and Brassica nigra (♂, genome:BB) were performed. The allotetraploids (genome:AABB) and alloaneuploids could be induced by treating allodiploids (genome:AB) with colchicine. When allodiploids, T-allodiploids (allodiploids after treating with colchicine), allotetraploids and alloaneuploids were transferred to the field, the allotetraploids exhibited more vigorous growth as compared to the allodiploids.2. The genetic alterations during wide hybridisation and polyploidisation were analyzed by SRAP. These results showed that allotetraploids displayed21.07%polymorphic fragments, which was the doubled comparasion to the allodiploids. Among these polymorphic fragments, the novel fragments in allotetraploids were more than twice the number of that in allodiploids. The novel fragments matched homologous functional genes when blasted against genome databases. When the allotetraploids (F1) were self-crossed, the next generation (F2) exhibited the same phenotypic and genetic pattern. These results indicated that the degree of genetic changes were positively related with the polyploidy level during the process of heterogeneous chromosomes combinations and chromosome doubling, and both wide hybridisation and genome doubling could affect the genomic architecture in newly formed allopolyploids. Moreover, novel fragments may be correlated with novel phenotypes.3. The analysis of the pattern and level of DNA methylation by MSAP during wide hybridisation and polyploidisation showed that the proportion of changes of methylation pattern in hybrid was significantly higher than ployploid. However, the DNA methylation levels were low in allodiploid and allotetraploids when compared to parents. Thus, the DNA methylation levels were decreased with the increased polyploidy levels.4. The high-throughput sequencing results obtained for the small RNAs showed that the expression levels of miRNAs increased in the allodiploid and allotetraploid compared with the parents, and the expression levels of siRNAs increased and decreased compared with the parents B. rapa and B. nigra, respectively. Moreover, the percentages of miRNAs increased with an increase in the polyploidy levels, but the percentages of siRNAs alterations decreased with an increase in the polyploidy levels. Furthermore,320known and52novel miRNAs were obtained from the parents, allodiploid and allotetraploid. Moreover, Quantitative real-time polymerase chain reaction (qRT-PCR) analysis showed that the expression levels of the targets genes were negatively corrected with the expressed miRNAs. Thus, the miRNAs regulate gene expression and induce the phenotype variation, which may play an important role in the occurrence of heterosis in the allotetraploid.