Analysis Of Genetic And Epigenetic Variation Of The Resynthesized Brassica Napus

Hybridization and chromosome doubling usually occur in the allopolyploid evolutionary process,during which a new genome forms.Some genetic and epigenetic variations may also appear.These variations include gene rearrangement,DNA structural variation,and transposon activation,as well as changes in gene-expression level,expression patterns,and DNA methylation.Therefore,exploring the possible mechanism of mutation in the early stage of allopolyploid formation is very crucial to elucidating the polyploid evolution theory.The allopolyploid Brassica napus L.has been generated from the interspecific hybridization and chromosome doubling between the diploid parents Brassica rapa and B.oleracea.It has currently become one of the most important plant in the study of plant polyploidization because it contains clear pedigree information.In this study,the differences in morphological and cytological characteristics between the synthetic B.napus and its two diploid parents during the early developmental stage were investigated using the experimental materials of synthetic B.napus and its diploid parents(B.rapa and B.oleracea)generated by our research group.Meanwhile,the genomic variation during the early stage of genome formation of B.napus was analyzed in molecular level using the RNA-Seq analysis of gene and transposon expression variation in the genome of allopolyploidization B.napus.In addition,the variations of DNA methylation and transposon polymorphism and their possible mechanism were also investigated using the molecular marker technology.The main results are as follows:1.Comparative analysis of phenotypic traits of resynthesized B.napus and two diploid parentsThe plant morphological features including plant type,leaf and flower organs,and seed cytological characteristics of s resynthesized B.napus and its diploid parents were compared and investigated in this study.Results showed that a series of phenotypic changes occurred in the synthetic B.napus.These changes are shown as follows:? The whole plant presented large and loose type,many effective branches and low branches.Meanwhile,many siliques appeared in the first effective branch and the number of grains per silique was more than that in synthetic B.napusand its diploid parents.?The leaf epidermis was not smooth with a small amount of leaf trichome in veins,and remarkable auricles,trichomes,and serrated margin were found in the leaf surface.? The petal shape was similar to that of B.oleracea,and the petal color was close to that of B.rapa.Furthermore,the pollen viability of synthetic B.napus exhibited stronger features than that of the two diploid parents(97.1%,92.1%and 75.09%,respectively).? The increased number of stomata on the leaf surface and large stomata density were found in synthetic B.napus.However,the length and width of stomata were smaller than that of B.rapa,? Light microscopy and electronic microscopy were applied to analyze the microstructure and ultrastructure of seeds of diploid parents and their allotetraploid progeny.Results showed that the morphology of the seed coat and the ridge of the B.rapa and B.oleracea were obviously different,whereas that of B.napus were characterized as reticulate or reticulate-foveate and close to that of B.rapa.The area index of the protein body was higher in central meristematic cells than in parenchyma cells.By contrast,the area index of the oil body was the lowest in central meristematic cells.Protein bodies were found to be heterogeneous with crystal globoids in two diploid parents and resynthesized allotetraploid progenies.Oil bodies consisted of large and small oil bodies,the sizes of which differed between two parents and allotetraploid progenies.Small oil bodies were spheroid,whereas large oil bodies were ovoid in shape.2.Comparative studies on the transcriptome of resynthesized B.napus and two diploid parentsThe transcriptome analysis of the leaves of synthetic B.napus and its two diploid parents was conducted using RNA sequencing(RNA-Seq)technology,and the main results are shown as follows:? 21,491,21,947,and 40,544 expressed genes were detected in B.rapa,B.oleracea,and resynthesized B.napus,respectively.Compared with B.rapa,synthetic B.napus contains 183 up-regulated and 2,192 down-regulated genes.Moreover,compared with B.oleracea,resynthesized B.napus consisted of 548 up-regulated and 1,894 down-regulated genes.The gene functional analysis showed that these differentially expressed genes between the progenies and their parents were mainly related to some biological processes,such as material synthesis,signal transduction,and stress response.? The analysis of the expression characteristics of 17,823 homologous gene pairs in hybrid progenies indicated that the expression patterns of most of the homologous genes(63.77%)were consistent with those of their two parents.Thus,the expression pattern of the homologous gene pairs is conservative during the process of polyploidization.The expression preference analysis of some homologous gene pairs showed that no difference was observed in the expression level of 12,221 genes indicating a non-expression preference.Simultaneously,2,322 and 3,280 partial homologous genes pairs exhibited biased expression characteristic toward the A and C genomes,respectively.?The analysis of some homologous gene pairs that were differentially expressed showed that 713 genes were expressed in additive expression form,and 2,628 and 3,162 were expressed in C-expression level dominance(C-ELD)and A-expression level dominance(A-ELD),respectively.The numbers of down-regulated and up-regulated transgressive genes were 54 and 1,056,respectively.Gene functional annotation analysis showed that the C-ELD and A-ELD genes were related to salicylic acid synthesis,biological stress defense,MAPK pathway,RNA methylation,nucleotide biosynthesis,rRNA processing,and protein transport,and up-regulated genes with transgressive characteristic were involved in the biosynthesis of pigments,flavonoids,and organic matter.3.Characteristic analysis of transposon in the transcriptome of resynthesized B.napus and two diploid parentsAfter RNA-Seq data were explored,the content,type,quantity distribution,expression pattern,and expression level of synthetic B.napus and its two diploid parents(B.oleracea and B.napus)in the whole genome level were then analyzed using comparative genomics method,and the results are shown as follows:? The proportions of transposon in transcriptomes of two diploid parents were 32.56%and 14.76%,respectively,and that of synthetic B.napus was 18.09%.In the transcriptomes of these three species,12 types of transposons were identified,including hAT,CACTA,PIF-Harbinger,Mutator,Pong,Tc1/Mariner,Helitron,LTR/Copia,LTR/Gypsy,LTR/Unclassified,SINE,and LINE.Among those,CACTA(2.02%to 5.44%),Pong(2.66%to 6.71%),LTR/Copia(4.32%to 10.90%),and LTR/Gypsy(2.20%to 4.75%)accounted for the largest proportion,and other types of transposons only accounted for a small proportion(approximately 0.09%to 0.94%)in the transcriptomes.? According to the value of RPKM,the difference of the transposon expression level was determined.Compared with B.rapa,the resynthesized B.napus presented 154 up-regulated and 162 down-regulated transposons,and compared with B.oleracea,the resynthesized B.napus presented 147 up-regulated and 190 down-regulated transposons.Among those,the differentially expressed transposons with largest number were CACTA,Pong,LTR/Copia,LTR/Gypsy,and LTR/Unclassified.? The expression pattern of the transposon in resynthesized B.napus was mainly in the form of emergence,silence,and expression in only one parent and the hybrid progeny.? The preference analysis of transposon inserting in gene showed that the intron region of the gene is easier to be inserted by transposon than the exon.The annotation analysis of the genes with transposon insertion indicated that these genes were involved in cell components,biological processes,and molecular functions.The cell component item was mainly related to cytoplasm,nucleus,chloroplast,plasma membrane,and pigment.Biological process item was mainly related to protein metabolism,stress resistance,signal transduction,cell composition and biotransformation,cell processes,and other metabolic pathways.Molecular function item was mainly related to nucleotide binding,DNA(or RNA)binding,protein binding,transferase activity,hydrolase,and other enzyme activities.These results suggested that transposons play an important role in the formation of genome structure and the function of gene.4.Analysis of retrotransposons and reverse transcriptase sequences in the genome of resynthesized B.napusThe changes and possible mechanisms of retrotransposons in the whole genome of synthetic B.napus were analyzed using inter-retrotransposon amplified polymorphism(IRAP)method,and the sequence composition and evolutionary relationship of retrotransposon reverse transcriptase were also studied.The main results are shown as follows:? A total of 1,729 bands were amplified under the random combination of 377 pairs of retrotransposon primers.Among those,parental bands accounted for 71.13%,the unknown bands accounted for 15.50%,and the disappeared and newly increased bands in the hybrids accounted for 5.73%and 7.63%,respectively.The activation frequency of retrotransposons was approximately 0.134 in the process of polyploidization.? A total of 70 sequences containing the retrotransposon were successfully cloned in this study and their functional prediction showed that the gene with transposon insertion were related to molecular function,biological process,and cellular components.Among them,the genes with largest proportion were enriched in catalysis or binding,special macromolecular complex,inner membrane system,and other metabolic processes.This result suggests that a close relationship existed between genes and retrotransposons,and this relationship might be significant to the adaptation of polyploid to the new environment.? The amplification of retrotransposons was performed using oligo-nucleotide primer,and a total of 32 reverse transcriptase sequences were isolated and cloned successfully.The nucleotide sequence length varied from 275-284 bp.Three conserved regions(TAFLHG,LYGLKQ,and YVDDM)appeared in the amino acid sequence.Results indicated that the reverse transcriptase sequences of the retrotransposons are characterized in homologous and highly heterogeneous.Cluster analysis on the cloned sequence and the amino acid sequence of reverse transcriptase of other species showed that these sequences are homologous,which suggested that retrotransposons in different species may have a common ancestor before speciation.5.Analysis of cytosine methylation in early generations of resynthesized B.napusDNA methylation is an important epigenetic modification and serves as a key function in the polyploidization of numerous crops.In this study,early generations of resynthesized B.napus(F1,S1-S3),and ancestral parents B.rapa and B.oleracea were analyzed to characterize their DNA methylation status during polyploidization,applying DNA methylation-sensitive amplification polymorphism(MSAP)and high-performance liquid chromatography(HPLC)methods.In F1,53.4%fragments were inherited from both A-and C-genomes.Besides,5.04%and 8.87%fragments in F1 were inherited from A-and C-genomes,respectively.5.85%and 0.8%fragments newly appeared and disappeared in resynthesized B.napus,respectively.13.1%of these gene sites were identified with methylation changes in F1,namely,hypermethylation(7.86%)and hypomethylation(5.24%).F1(38.7%)showed the lowest methylation status compared with that in S1-S3.In S3,40.32%genes were methylated according to MSAP analysis.Sequencing of methylated fragments indicated that genes involved in multiple biological processes were modified,including transcription factors,protein modification,and transporters.The expression analysis of DNA methyltransferasel and DNA methyltransferase chromomethylase3 in different materials was consistent to the DNA methylation status.These results can generally facilitate the investigation on how DNA methylation contributes to the genetic stability and improvement of B.napus during polyploidization.