Transcriptome Sequencing And Differental Expression Genes Analysis Of Spike Developmental Stage From Wheat

Wheat(Triticum aestivum L.)is one of the most important food crops in the world.The main objectives of wheat breeding include higher yield,better quality,stronger resistance to the diseases,and better adaptation.The spike traits of wheat,which are mostly controlled by multi-genes,directly influence wheat yield.For now,there were few reports about the development of young spike of wheat.Therefore,it is necessary to find and evaluate the genes relative to the development of young spike of wheat.In this research,we applied the transcriptomic sequencing technology(RNA-Seq)in sequence of filling-staged seeds,spike and leaf of floret differentiation stage which belong to three ordinary hexaploid wheat to find and validate the spike-typical genes through RT-qPCR.The main results are as follow:(1)We analyzed eight samples separately through Illumina RNA-Seq.The correct rate of base recognition was More than 99%in each sample,and the mapping rates of the reads in 8 samples are about 70%which met the experimental requirements;Mapping the reads to reference genomes,we get 51610490,52559616,52559616 reads and 91051,97652,99786 genes from spike and leaf of floret differentiating stage,and filling-staged seeds in 10-A;53941731,51320211,50601900 reads and 91948,93681,93243 genes from spike and leaf of floret differentiation stage and filling-staged seeds in BE89;53024625,5072760 reads and 98379,96422 genes from spike of floret differentiating stage and filling-staged seeds in Chuannong 16;(2)The PC A principal component analysis was performed on TPM(Transcripts per Million)of eight samples.The picture show that spike,leaf and seed of three materials are gathered as group separately.It was found that the genetic differences between the same organs of the three materials were smaller than those between the materials,that is,10-A and BE89 had a relatively small difference between the spike genes despite the significant difference between their spike traits.(3)In order to screen the genes related to spike development in different wheat materials,TPM(Transcripts per Million)was used to standardize the data of the expression in the transcripts sequencing within samples,and the log FC threshold was 6.65(the difference was 100 times).161 spike specific expressed genes(Up-regulated expression of 74 and down-regulated expression of 87)were screened out from 10-A and BE89,leaves and seeds of three materials were used as control.(4)We get the DEGs in database.Through GO(Gene Ontology)gene enrichment analysis,the gene function consist of cellular component(2 genes),molecular function(13 genes)and biological process(24 genes)in up-DEGs.Through KEGG(Kyoto Encyclopedia of Genes and Genomes)enrichment analysis,4 genes of up-DEGs can be assigned to 4 categories.Through GO gene enrichment analysis,the gene function consist of cellular component(2 genes),molecular function(13 genes)and biological process(20 genes)in down-DEGs.Through KEGG enrichment analysis,6 genes of down-DEGs can be assigned to 4 categories.(5)According to the annotation information of DEGs on Internet,identifying the reported/unreported genes related to the spike development in other species,a total 22 genes were obtained.The 22 genes were denominated TaST1-22(spike-typical).TaST13 and TaST15 were found to be homologous gene PAP27 and OSH15 in arabidopsis and rice.PAP27 is considered as relative to absorb of Fe and Mn.OSH15 first expresses in seeding of meristem in embryonic development,then stop expressing,finally,becomes a flower-typical gene.(6)We verify the expressing pattern of 22 genes in the 36 materials with RT-qPCR.36 materials are the root,stem,leaf and spike of 3 growth stages(tillering stage,jointing stage,flowering stage)in 10-A,BE89,chuannong16.Exons region of 22 genes were used as templates,primers were designed according to the principle of RT-qPCR primer.Primers were prime by Blast to testify it's specificity.After that.RT-PCR experiment to verify whether the primers are available.We found that the transcriptome differential expression analysis of 8 genes during floret differentiation stage in spikes(control for leaf of floret differentiating stage and filling-staged seed)expressed specifically though RT-qPCR verification experiment.However,according to the analysis of RT-qPCR.The 8 genes in all of the 36 materials perform nonspecific expression(control for root,stem and leaf).Only 15 genes were confirmed specific expression in the spike by experiment.The expression of these genes in the flowering stage of each material is very low.But In each material of tillering stage and jointing stage,the expression of spike was higher than other organs.As far as spike of different varieties of Wheat.TaSTl?TaST2?TaST3?TaST4?TaST5?TaST8?TaST9?TaST10?TaST11?TaST12?TaST14?TaPAP27?TaOSH15 at tillering stage.The expression of 10-A was lower than BE89.At jointing stage,the expression of 10-A was higher than BE89.TaST6?TaST7?TaST9 at tillering stage.The expression of 10-A was higher than BE89.While in jointing stage.The expression of 10-A was higher than BE89 too.For the same variety of wheat ear,The spike expression of TaST1?TaST2?TaST3?TaST4?TaST5?TaST9?TaST10?TaST11?TaST12?TaST14?TaPAP27?TaOSH15 increased during tillering and jointing stage of 10-A.The spike expression of TaST6?TaST7 decreased in the tillering and jointing stage of 10-A and also decreased in the same stage of BE89.The spike expression of TaST8 increased in the tillering and jointing stage of 10-A and also increased in BE89.The change law of above-mentioned expression quantity of the first expression pattern is most obvious in material's spike traits,which should be taken as the focus of the subsequent experiment,so as to discuss the function of the gene and the spatiotemporal expression characteristics.