Molecular Mechanisms For Genetic Imprinting In Rice And Subgenomes Interaction In Allopolyploid Wheat That Regulate Endosperm And Seed Development

Seeds of cereals are important to food supply.A typical seed consists of the endosperm,embryo,and seed coat.Endosperms like rice and wheat can occupy-80%of the seed mass,and they provide most calories consumed by humans.Biologically,endosperm is a triploid that contains 2m:1p(maternal-to-paternal)genomes and is essential for embryonic and seed development.Using diploid rice and allopolyploid wheat materials,analyzed the molecular mechanisms for genetic imprinting in rice and subgenomic interactions in wheat that regulate endosperm and seed development.1.Molecular mechanisms for genetic imprinting in the rice endosperm.Imprinting is an epigenetic phenomenon that refers to unequal expression of paternal and maternal alleles in sexually reproducing organisms.The imprinting genes are only expressed maternal alleles or paternal alleles.Although many imprinted genes are identified in plants,the role for imprinting in seed development is largely unknown.In this study,two inbred lines of Oryza saliva L.susp.indica cv.Longtefu(LTF)and O.sativa L.susp.japonica cv.02428 were maintained by self-pollination,which were cross-pollinated to make F1(LTFX02428)and the reciprocal F1(F1r,02428XLTF)crosses.Endosperm at 7 days after pollination(DAP)was used to make libraries for small RNA,transcriptome and methylC sequencing.Imprinting genes,small RNA loci,and DNA methylation regions were identify to reveal their roles in endosperm and seed development.1)Genome-wide analysis of imprinted genes in rice endosperm:According to the published standards for identifying imprinted genes(expression cut-off values of 80%for maternal and 70%for paternal alleles)in the endosperm,identified 162 maternally expressed genes(MEGs),94 paternally expressed genes(PEGs).PEGs were expressed not only in the endosperm but also in many other tissues at similarly high levels,whereas MEGs were expressed primarily in the endosperm and seed.2)Analysis of imprinting DNA methylation regions:To understrand the relationship between DNA methylation and imprinting,we performed bisulfite methylC-seq of the endosperm(7 DAP).Except for 20 CHH iDMRs(H=A,T,or C),most iDMRs were in CG(18,382)and CHG(861)sites,which accounted for 5.8%and 0.5%of all methylation regions in the genome.Consistent with previous findings,the imprinting genes were more hypomethylated in the maternal alleles than in the paternal alleles and more hypermethylated in the seedling than in the endosperm.3)Miniature inverted-repeat transposable elements(MITE)are associated with imprinted genes and iDMRs:MITEs were present in～77.8%of MEGs and-83%of PEGs,which were significantly higher than the genome-wide average(-59%)of MITEs within or near the genes.And polymorphic MITEs are enriched in 5’ and 3’ flanking sequences of imprinting genes.The imprinted genes associated with polymorphic MITEs evolve faster than non-imprinted genes,suggesting co-evolution of imprinted genes with MITEs in rice.4)Imprinted non-coding RNAs in the endosperm:We identified 27 maternally expressed siRNA loci(MESRL)and 29 paternally expressed siRNA loci(PESRL).Interestingly,some maternal 21-nt siRNAs correlated with MEGs,and imprinted long non-coding RNAs(lncRNAs)were located in the upstream regions of the same alleles of imprinting genes,suggesting roles for these IncRNAs and 21-nt siRNAs in imprinting.5)Imprinted genes are associated with grain weight quantitative trait loci(QTLs)and regulate seed development:Remarkably,one-third of MEGs and nearly half of PEGs were co-localized with rice grain weight QTLs.Allelic expression for a subset of imprinted genes was validated;these imprinted genes encode storage proteins and transcription factors that regulate grain filling and starch deposition during seed development.2.Mechanisms for subgenomic interactions in the endosperm of allopolyploid wheat.The development of wheat seeds is not only regulated by genetic imprinting,but also by subgenomic interactions between progenitor species.Kerber crossed common wheat and a tetraploid wheat,followed by repeated backcrossing to the hexaploid wheat(TAA10)as the recurrent parent,to construct a ploidy-reversed "extracted" tetraploid wheat(ETW)with a genomic composition of AABB that is virtually identical to the AABB subgenomes of itsbread wheat donor.In this study,we performed transcriptome sequencing in endosperm of extracted allotetraploid wheat(ETW)of the allohexaploid bread wheat(Triticum aestivum cv Canthach;designated as TAA10),natural allotetraploid wheat(T.durum,AABB),Aegilops tauschii(DD),and resynthesized allohexaploid wheat(AABBDD).We tested the effects of intergenomic interactions on gene expression and endosperm development in wheat.1)Transcriptome changes in AB subgenomes of the common wheat:We found that 11,217(21%)genes were upregulated in ETW,and 10,286(19%)genes were downregulated in ETW.The down-regulated genes were enriched in ovule development,DNA methylation,sucrose biosynthetic process,and mitotic cell cycle,while the upregulated genes were enriched in programmed cell death and response to abiotic stimulus.We randomly selected a subset of genes for expression validation.All genes tested showed consistent allelic expression levels between RNAs-seq and quantitative RT-PCR(qRT-PCR)data.2)Expression of homoeologous genes between AB and D subgenomes:Among all genes analyzed,45%-50%of them were homoeologous between AB and D genomes,and 78%genes of homoeologous genes are additively expressed.3)Interactions between AB and D subgenomes maintain genome and gene-expression stability:Most alleles were upregulated in the D subgenome than in the DD progenitor,whereas AB alleles were downregulated in the AB subgenomes than in ETW.Half of these up-and down-regulated genes were homoeologous.These genes were additively expressed and enriched in protein transport,signal transduction,GTP binding,cellular homeostasis,and developmental regulation.4)The genes that are highly expressed in the endosperm are involved in seed development:Highly expressed genes in the wheat endosperm were homologous to the genes that are shown to affect seed development in rice.A larger number of genes that are involved in seed development were expressed at higher levels in the D subgenome than AB subgenomes in resynthesized allohexaploid wheat.In this study,I provided genome-wide data for genetic imprinting in the rice endosperm and gene expression dynamics for seed development in extracted tetraploid wheat and resynthesized hexaploid wheat.Gene expression changes in allopolyploid wheat are dependent on subgenomic interactions during polyploid formation and evolution.The homoeologous genes involved in seed development could be used to improve grain production of cereal crops through conventional breeding and biotechnological applications.