| Allopolyploidization is one of the important driving forces in plant evolution.Investigation of the transcriptional changes in the process of plant polyploidization facilitated our understanding of the adaptability,domestication and evolution of poliploid species.The Triticum durum-Haynaldia villosa amphiploid was derived from the distant hybridization between T.durum(AABB)and H.villosa(VV),followed by the natural genome duplication.Compared with its parents,the amphiploid showed distinct plant morphology and adaptability.The amphiploid has taller in plant height,longer spike,larger grain size,and larger cell size in grain and glume,especially at 6 days after pollination(DAP).In order to explore the molecular mechanisms of the superior adaptability and growth vigor after allopolyploidization,in this study RNA-seq was used to compare the gene expression profile in the 6 DAP caryopsis(without embryo)of T.durum-H.villosa amphiploid and its two parents,T.durum and H.villosa and amphiploid.The main results are as follows:1.Phenotypic comparison between T.durum-H.villosa amphiploid and its parentsThe plant height,spike and grain length,as well as the grain and glume cell size at 6 DAP were investigated.Significant difference between the amphiploidy and its two parents were observed for all the examined characters.The plant height of the amphiploid was 111.2 cm,while the T.durum and H.villosa were 58.6 cm and 80.0 cm,respectively.The spike length of the amphiploid was 10.6 cm,while the T.durum and H.villosa were 7.6 cm and 8.5 cm,respectively.Mature grain length of the amphiploid was 8.6 cm,while the T.durum and H.villosa were 4.7 cm and 7.7 cm,respectively.The grain cell size at 6 DAP of the amphiploid was 1224.5 um2,and the T.durum and H.villosa were 446.9 um2 and 938.5 um2,respectively.The glume cell size of the amphiploid was 213.6 um2,and the T.durum and H.villosa were 119.1 um2 and 169.2 um2,respectively.Difference in the starch accumulation of the mature grains was also observed.2.The dosage effect played dominant role of the regulation of gene expression in the amphiploidy.Using RNA-seq,the gene expression patterns in the caryopsis(without embryo)at 6 DAP of H.villosa,T.durum and amphiploid were compared.By analyzing the homoeologous allelic expression from different genomes,it was found that during polyploidization the change of gene expression can be defined as dosage-dependent expression(in which the expression level of an allele is correlated with the dosage)and dosage-independent expression(expression levels do not correlate with the dosage).The dosage-independent expression can be further classified as dosage-induced expression or dosage-repressed expression.Our results showed that most of the expressed genes in amphiploid belong to the dosage-dependent type(5418,72.5%).Among the remained dosage-independent type genes,1500(20.1%)were dosage-induced and 553(7.4%)were dosage-repressed.3.Distribution of the dosage-independent genes and the association with histone modificationThe synteny of dosage-independent genes was well conserved among different genomes.The homeologous group 1 has the least number of dosage-induced type genes and the largest number of dosage-repressed type genes,compare to other homeologous groups.For the three subgenomes,7789 expressed genes were from subgenome A,in which 572(7.3%)were upregulated and 241(3.1%)were down-regulated;7711 expressed genes were from subgenome B,in which 834(10.8%)were up-regulated and 413(5.4%)were down-regulated;A total of 8130 expressed genes were from subgenome V,in which 525(6.5%)and 594(7.3%)were up-regulated and down-regulated,respectively.The subgenome V had the most dosageindependent type genes,indicating in the polyploid they were more induced or repressed by dosage effect.H3K4me3 and H3K27me3 modifications,in which the former represents the methylation at the 4th lysine and is related to gene activation,and the latter represents the methylation at the 27th lysine and is related to gene repression.To investigate the role of the two histone modifications in gene expression during polyploidization of the amphiploid,immunostaining was conducted using antibodies of H3K4me3 and H3K27me3.The average signal intensity for each single chromosome was calculated.Immunostaining using antibody H3K4me3 showed that,the signal intensity in the amphiploid(127.9)was similar to that in T.durum(131.8),and they were both higher than that in H.villosa(78.2).Immunostaining using antibody H3K27me3 showed that,the signal intensity in the amphiploid(104.2)was also similar to that in T.durum(102.6)and both were higher than in H.villosa(80.8).No statistical difference was observed between T.durum and the amphiploid,but they were both significantly different when compared with H.villosa.The chromosomal distributed of H3K27me3 modification was different among the three materials.The signal was evenly distributed along each chromosome of H.villosa,mainly at the centromeric region in T.durum,and mainly at the chromosome ends in the amphiploid.4.Gene Ontology(GO)and pathway enrichment of dosage-independent genesGO enrichment showed that,the dosage-independent genes were mainly related to biological process,generation of precursor metabolites and energy,DNA metabolic process,carbohydrate metabolic process,catabolic process and protein metabolic process.The pollenpistil interaction was enriched in dosage-induced type genes,and cell cycles was enriched in dosage-repressed genes.Pathway enrichment using MapMan showed that pathways related to grain development and plant hormone signaling(Auxin,BR,CTK,ABA,JA)were enriched.This indicated that hormone pathways were involved in the allopolyploidy and may play important role in regulating grain development.Motifs in the promoter regions of dosage-independent genes were enriched by MEME.Two transcription factor families,Dof and MIKC_MADS were identified.They may participated in the auxin pathway and regulated grain development.These genes were verified by qRT-PCR,and the expression patterns were in consistent to the dosage-induced and dosage-repressed type expression patterns revealed by RNA-seq. |
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