Genomic And Epigenomic Stability In Early Generations Of Synthetic Rice Inter-subspecific (Japonica-indica) Hybrids And Tetraploids

Polyploidization,or whole genome duplication（WGD）,is the pervasive driving force for different flowering plants.Polyploidization is often accompanied by emergent genetic and epigenetic instabilities.Nevertheless,to date,most studies on polyploidization have been accomplished based on limited genes or genetic loci,rendering situations at the genome-wide scale remain contentious.This study is designed to assess genome-scale genetic and epigenetic stabilities in synthetic（human-made）tetraploid rice plants between the two subspecies of rice（Oryza sativa L.）,i.e.,japonica and indica.Rice is one of the important cereals feeding more than half of the world population.The two subspecies of Asian cultivated rice share highly homologous and syntenic genomes but also habor substantial genetic and epigenetic differentiations due to domestication and/or human selection under cultivation.Thus,according to the authority definition by Ledyard G.Stebbins,the synthesized tetraploids between these two subspecies represent segmental allopolyploidy.The plant materials used in this study are the 5^thselfed generation（S5）of artificially made（synthetic）tetraploids（designated as NN99 and 99NN,in which the first letter or number denoting the maternal parent）.NN99 and 99NN were produced by colchicine-induced WGD of reciprocal F1 hybrids between Nipponbare and 9311,which are model laboratory cultivars for the japonica and indica subspecies,respectively.By taking advantage of the next generation sequencing（NGS）technology,leaf tissue of tetraploids,hybrids,and their diploid parental plants were sequenced.Genomic,epigenomic and transcriptomic data were obtained and anlyzed for interpreting the impact of genome-wide genetic and epigenetic stabilities,as well as gene expression,in different hybrids and tetraploids.The main conclusions are as follows:（1）Massive recombinations occurred between chromosomes of Nipponbare and9311 subgenomes in the S5 tetraploids,which led to five types of japonica-indica homolog/homeolog constitution in the tetraploids.These are 4 copies of Nipponbare and 0 copy of 9311（4N09）;3 copies of Nipponbare and 1 copy of 9311（3N19）;2copies of Nipponbare and 2 copies of 9311（2N29）;1 copy of Nipponbare and 3copies of 9311（1N39）and 0 copy of Nipponbare and 4 copies of 9311（0N49）.Frequencies for regional homozygosity,which is chromosomal segments containing copies of only one of the parents only（4N09 or 0N49）are ranging from 47%to 63%.This result means that ca.50%of the tetraploid genomes constituted of 4 copies of Nipponbare or 9311,while the rest 50%are still heterozygous.Physical sequence elimination events（loss of segments from both parental subgenomes）were detected in the tetraploids,with cumulative lengths ranging from 2.5 to 5 Mb and involving 1,126genes in four tetraploid individuals.（2）Methylation levels of tetraploids were similar to the parents and hybrids.We constructed in silico"hybrids"by mixing the methylome data of Nipponbare and 9311.Compared to the in silico"hybrids",lots of differentially methylated cytosines（DMCs）,especially DMCs of the CG dinucleotides（CG-DMCs）,markedly different methylation variations were observed between the tetraploid individuals of the reciprocal cross directions.Prominently,while more hyper CG-DMCs were observed in 99NN,the opposite trend（i.e.,mainly hypo CG-DMCs）was true in NN99.We found that the methylation variations were mainly observed from the repeat sequences.Interestingly,methylation levels of some homozygous segments（0N49 or 4N09）are different from their counterparts in the corresponding diploid parental genomes.This result suggests that the methylation variations which occured in the hybrids or earlier generations of tetraploids were inherited by following generations rather than being reverted back.As expected,the heterozygous segments（2N29,1N39 or 3N19）contained most of the polymorphisms of DNA methylation.（3）It has been reported that hybridization and WGD induces global changes in genes expression,a phenomenon collected termed as"transcriptomic shock"（sensu"genome shock"first put forth by Barbara Mc Clintockin 1984）.The primary manifestation of gene expression alteration in a hybrid or allopolyploid is nonadditive expression,which level is not equal to the averaged parental values.We found that the tetraploids showed substantially greater numbers of genes showing nonadditive expression than that of the hybrids.One possible explanation is that the repeat-containing genes are likely to have been impacted by the aforementioned methylation variations in the tetraploids.Similar to the situation of changed methylation,we found some of the genes landing on homozygous segments（0N49 or4N09）also showed altered expression compared to genes in corresponding parents,indicating inheritance of altered gene expression.In conclusion,large-scale genetic variants,largely attributable to meiotic recombination between the two parental subgenomes,occurred in the rice inter-subspecific tetraploid plants.Moreover,moderate changes in DNA methylation and corresponding gene expression changes occurred,too.These genetic and epigenetic variations,coupled with original parental differences,render the tetraploid plant to show phenotypic novelties that might be useful for rice genetic improvements.By analogy,under natural settings,the novelties rapidly generated by hybridization and WGD may serve as raw material for evolutionary selection.