Distant hybridization occurs under both natural and artificial conditions. After genome duplication, allopolyploids can be obtained, and these polyploids play a significant role in the origin and evolution of many plant species. In crop improvement, allopolyploids are also valuable as bridge materials for breeding. During the course of hybridization, it is difficult to get F1 plants due to sterility. The F1 hybrid could be the real hybrid containing whole or partial genetic material from both of the parents; the false hybrid resulting from female parthenogenesis or self-crossing; or an introgression where most of the male parent DNA was digested by the female nuclease and a few segments combined with the genome of the female parent.
Allopolyploidy has been intensively studied in naturally evolved allopolyploids of wheat, cotton, rapeseed, Arabidopsis, and tomato. Genetic changes are best evaluated by comparing the parental lines and their progeny; however, it is difficult to ascertain the parents in naturally evolved allopolyploids since genetic changes have occurred in subsequent generations. Artificial allopolyploids have defined parents and genetic lineages, making them suitable for elucidating changes that occur during the formation of allopolyploids.
The hybridization between Raphanus and Brassica was first reported by Sageret in 1862 . About 100 years later, Karpechenko identified an F1 hybrid between Raphanus and Brassica [31, 32]. To transfer the nucleic genes of Brassica tournefortii (TT) into B. carinata (BBCC), Mukhopadhyay et al. produced a bridge species (TCBB) by protoplast fusion between the F1 hybrid (TC) of B. tournefortii×B. oleracea and B. nigra (BB) and RAPD primers were used to show that all the hybrids had specific bands from the genomes of the parents. These results indicated that the T, B, and C genomes may coexist in a hybrid state. RFLP molecular markers confirmed that these hybrids contained chloroplast and mitochondrion genomes of Brassica tournefortii and B. nigra . Chrungu et al. (1999) synthesized allotetraploids of B. maurorum-B. napus, B. maurorum-B. carinata, and B. maurorum-B. nigra through interspecific hybridization and genomic doubling and proved the reality of the hybrids through RAPD and RFLP . In the present study, we synthesized F1 hybrids through sexual hybridization followed by embryo rescue and identified its origin with RAPD. The results showed that F1 hybrids lost some of the specific or common bands of the parents. The probable reason was that hybridization resulted in a genomic recombination and changed the primer binding sites of some segments, leading to the appearance or disappearance of some DNA bands.
Our results showed that, DNA methylation patterns differed substantially between the parents and F1 hybrids, indicating its possible key role in transgenerational stability. During the process of hybrid formation, DNA methylation patterns may adjust to some extent to coordinate the interactions among nuclear genes or those between nuclear genes and cytoplasmic genes. Both hypomethylation and hypermethylation events may affect gene expression patterns. In our work, hypomethylation was a more frequent than hypermethylation.
Polyploidy has played a fundamental role in the evolution of higher plants. Plant breeders have been routinely producing allopolyploids with interesting agronomic traits to be used for breeding programs. However, the poor genetic stability of allopolyploids in early generations is a challenge for plant breeding programs.
Intergeneric hybridization between a long genetic distance may cause mixoploidy, leading to genetic instability. Hybrids between Orychophragmus violaceus (2n = 24) and cultivated Brassica species, including tetraploids (B. carinata and B. juncea) and diploids (B. campestris and B. nigra), led to mixoploids [18, 19]. For example, the hybrid with B.
campestris (2n = 20, AA) was mixoploid (2n = 23-42), and cells with 2n = 34 were most frequent. Partial separation of parental genomes during mitosis, leading to the addition of O. violaceus chromosomes to the B. campestris complement, was proposed to explain the findings in the mitotic and meiotic cells of the hybrid and its progeny. In crosses with B. nigra (2n = 16, BB), a small fraction consisted of mixoploids (2n = 16-18), predominantly with 2n = 16 cells, and three plants, each with a specific morphology, were mixoploids consisting of cells with varying ranges of chromosome numbers (2n = 17-26, 11-17 and 14-17). The origin of these different types of plants was inferred to be the result of complete and partial separation of parental genomes and the loss of O. violaceus chromosomes.
In the present study, our intergeneric hybrid of R. sativus L. and B. alboglabra Bailey was a mixoploid (2n = 23-42) in the early generations (F4-F8). After several generations, mixoploids gradually turned to euploids through formation of neo-chromosomes or chromosome elimination. The possible cytological mechanisms pertaining to these hybrid generations and the genetic motifs from unstable to stable generations are unknown
We suggest that the stability of the F10 generation is a result of epigenesist. Epigenetic changes were mainly materialized by covalent modifications of DNA methylation and protein modifications (methylation, acetylation, phosphorylation). In plants, newly acquired epigenetic states of transcriptional gene activity can be readily transmitted to the progeny through meiosis. Epigenetic reconfiguration after hybridization between diploid members of the same species may be an important mechanism for reconciling two non-identical genomes in the same nucleus as allopolyploid formation occurs [5, 35, 36].
Our results show that methylation patterns and methylation states changed in the intergeneric hybrid both in the process of F1 hybrid formation and through the formation of generations of genetically unstable and stable progenies. DNA methylation levels of F4 and F10 (29.17% and 31.64%) did not show a great difference, indicating the similar status of DNA methylation between stable and unstable generations. These levels were similar to that of Arabidopsis seedlings (35%-43%) , but these were much higher than that of rice leaves (16.30%) . These differences in DNA methylation among different species may be caused by detection methods (primer numbers, amplification conditions, time of electrophoresis, and staining methods), material differences (seed, seedling, mature leaves), or genetic control. For DNA methylation patterns, hemi-methylation levels of the external cytosine at CCGG sites of F4 and F10 are 20.14% and 20.05%, respectively, while full methylation of the internal cytosine at CCGG sites of F4 and F10 were 9.03% and 11.59%, respectively. These results showed that hemi-methylation was the main DNA methylation pattern in both F4 and F10 generations. The full-methylation level of the F10 was higher than that of the F4, indicating that methylation changes from an unstable to a stable generation happened mainly on full-methylation sites. The relationship between the changes in genetics and epigenetics remains elusive. We suggest that the inheritance of allopolyploids seems to be governed by multiple mechanisms, including both genetic and epigenetic mechanisms. The contribution of these different mechanisms to inheritance is largely unknown, as many of these interact with each other.