| Polyploidiation is a common natural phenomenon in nature,and plays an important role in plant evolution and speeiation.It is believed that at least 70%angiosperms underwent at least one round of polyploidization during their evolution.The newly formed allopolyploid must establish a compatible relationship between the outer cytoplasm and the nucleus between the two different genomes,which leads to rapid changes in genomic structure,gene expression and developmental characteristics.It showed that polyploidization evolution patterns were different among different plants falilies.Asteraceae is one of the largest families in the angiosperms,includes a number of polyploidy species,and the hybridization-polyploidization of Chrysanthemum and their related species are more common,which provides favorable material for the study of plant polyploids.In the present study,we obtained allopolyploids after colchicines treatment of a synthesized Chrysanthemum.nankingense × Tanacetum.vulgare hybrid.To further clarify the genome evolution process of these plalts,we detected genomic and epigenetic changes induced by intergeneric hybridization and polyploidization using SRAP and MSAP marker technologies and the relative transcript impacts of hybridization and polyploidization by applying RNA-Seq to compare the transcriptome of hybrid,allopolyploid,and its parents.According to the transcriptome library of C.nankingense,we also cloned flfferentially genes CnTCP4 and CnTCP13.The main contents and conclusions are as follows:1.A series of allopolyploids were generated with colchicine treatment of a C.nankingense × T.vulgare hybrid.The leaf length and width of the allopolyploids were significantly greater than those of the hybrid.Inflorescence diameter and florets(both ligulate and tubular)size were larger in the allopolyploids.The tubular flower quantity increased between the hybrid and allopolyploid plants.The genomic alterations in the hybrid and allopolyploid mainly involved the loss of parental fragments and the gain of novel fragments.Analyze the variation fragments,we found that allopolyploid lines 1/2/3 respecitively had 84/84/85 fragments(84.0%/85.7%/85.0%of total alterations)alterations were induced by hybridization,6/4/6(6.0%/4.1%/6.0%)fragments alterations were induced by polyploidization,and 10/10/9(10.0%/10.2%/9.0%)fragments alterations were induced by both hybridization and polyploidization,which indicate that hybridization may be the major force for genomic changes.There were 84/84/85 fragments altered in both the hybrid and allopolyploids.When compared to the 46/46/47 that disappeared from T.vulgare,while only 23/24/24 disappeared from C.nankingense.The maternal parent C.nankingense was domination in hybrid and allopolyploids.DNA methylation level of hybrid was reduced by the wide hybridization but somewhat restored after polyploidization,and fragments of increased DNA methylation in the allopolyploid were twice(51.5/25.7)those of decreased methylation.2.RNA-Seq technology was used to investigate transcriptome of hybrid,allopolyploid,and its parents.A total of 73,990,84,846,81,603,and 81,107 unigenes were yielded from libraries C.nankingense(Jhn),T.vulgare(Jh),hybrid(JJ),and allopolyploid(JJD),respectively.Most differentially expressed genes(DEGs)showed down-regulation in hybrid/allopolyploid compared with its parents.Among those nonadditive genes,transgressive patterms appeared to be dominant,specifically repression pattern.The genes co-expressed in C.nankingense and the hybrid were more abundant than the genes co-expressed in T.vulgare and the hybrid(15.7%vs 3.8%).Similarly,the genes co-expressed in C.nankingense and the allopolyploid were more abundant than the genes co-expressed in T.vulgare and the allopolyploid(16.4%vs 4.1%).The DEGs between the hybrid and C.nankingense accounted for 32.4%,while between the hybrid and T.vulgare the value was 40.6%.The DEGs between the allopolyploid and C.nankingense accounted for 32.6%,while between the allopolyploid and T.vulgare the value was 40.8%.These findings suggested directional gene expression change deviations from the paternal parent in the hybrid and the allopolyploid.The genes co-expressed in C.nankingense and the hybrid showed no greater difference than genes co-expressed in C.nankingense and the allopolyploid(15.7%vs 16.4%);T.vulgare and the hybrid also showed no larger differences than T.vulgare and the allopolyploid(3.8%vs 4.1%).The DEGs percentage between the hybrid and C.nankingense was 32.4%,and that between the allopolyploid and C.nankingense was 32.6%;the DEGs percentage between the hybrid and T.vulgare was 40.6%,and that between the allopolyploid and T.vulgare was 40.8%.The percentage of co-expressed genes and DEGs between the allopolyploid and its parents had the same profile as that between the hybrid and its parents;these findings indicated that the hybridization triggered the majority of the transcriptomic changes.The formation of allopolyploids may not simply be the sum of hybridization and polyploidization changes but may also involve an interaction between these processes.The expression levels of transcription factors related to leaf and flower development had changed in the procee of allopolyploidization.3.CnTCP4 and CnTCP13 were cloned from C.nankingense,which both were CIN genes belonged to Class Ⅱ,and contained an atypical basis-helix-loop-helix domain.CnTCP4 and CnTCP13 were both located in nucleus.CnTCP4 had transcriptional activation activity,and it was further shown that the transcriptional activation region was in the C-terminal region of 201-300 polypeptide;CnTCP13 had no transcriptional activation activity.CnTCP4 had the highest expression level in leaf,and the following were ligulate flower and tubular flower;CnTCP13 had the highest expression level in ligulate flower,and the following were tubular flower and leaf.The transcription level of CnTCP4 and Cn TCP13 were both significantly inhibited after exogenous 6-BA treatment.4.Compared with the transgenic vector pESPM yeast,the growth potential of transgenic CnTCP4 yeast was significantly decreased and the appearance was abnormal,with 25.65%of the cells showed double nuclei,indicating that CnTCP4 inhibited yeast proliferation and promoted yeast growth.In Arabidopsis thaliana,over-expression CnTCP4 and CnTCP13 plants both had smaller plants and leaves,more rosette leaves,larger flowers,longer petals,and the bolting times were delayed.This indicated that the over-expression of CnTCP4 and CnTCP13 genes both had a significant inhibitory effect on the growth of leaf,and had a obvious promote effect on the growth of petal.RT-PCR analysis showed that the expression level of the cell division-related gene AtCDKD;2 was downregulated,the expression level of AtCDKC;1 was upregulated in CnTCP4 transgenic Arabidopsis thaliana;the expression levels of the cell division-related genes AtCDKC;1,AtCYCL;1,AtCDKG;2 and AtDPB were upregulated in CnTCP13 transgenic Arabidopsis thaliana.5.CnTCP2 and CnF-box interacting proteins were screened from the yeast cDNA library of chrysanthemum ’Jinba’ top bud by CnTCP13.CnTCP2 and CnF-box were both located in nucleus,and had no transcriptional activation activity.Yeast two-hybrid experiment and bimolecular fluorescence complementaton experiment proved that CnTCP2 interacted witih CnTCP4,CnTCP2 interacted with CnTCP13,and CnTCP4 interacted with CnTCP13.CnF-box interacted with CnTCP13,but not interacted with CnTCP2 and CnTCP4.6.In Arabidopsis thaliana,over-expression CnTCP2 plants had similar phenotype to over-expression CnTCP13 plants.RT-PCR analysis showed that the expression levels of the cell division-related genes AtCDKD;1 and AtCYCB2;4 were downregulated,the expression levels of AtCDKC;1、AtKRP5 and AtE2F3 were upregulated in CnTCP2 transgenic Arabidopsis thaliana.Over-expression CnF-box plants had smaller plants,smaller leaves with curled downwards edges.This indicated that the over-expression of CnF-box gene also had a significant inhibitory effect on the growth of leaf.RT-PCR analysis showed that the expression levels of the cell division-related genes AtCDKB2;2,AtCDC25 and AtCYCB2;4 were downregulated in CnF-box transgenic Arabidopsis thaliana.CnTCP2,CnTCP4 and CnTCP13 could form protein complex,then promoted the expression of AtCDKC;1 to inhibit leaf development. |
