Transcriptome Analysis Of Spike And Profiling Of Alternative Splicing In Wheat (Triticum Asetivum L.) Based On RNA-Seq

Wheat(Triticum aestivum L.)is one of the most important food crops in China.Increasing production has always been the main goal of wheat breeding.The spike traits of wheat are closely related to its yield.However,the study on the genome-scale genes expression profile of wheat spike is relatively shallow.The release of the hexaploid wheat genome sequence provides a solid foundation for wheat transcriptome research.Therefore,it is important for wheat molecular or genome-selection breeding to identify the gene targets related to the spike traits by the transcriptome profiles based on the wheat fine genome reference.In addition to transcriptional regulation,alternative splicing(AS),as an important post-transcriptional regulation,plays a pivotal role in plant growth and development.However,at present,reports about AS in plants are mostly concentrated on response to various stress,while there are few studies on AS during plant normal growth and development,especially in wheat.In this study,the transcriptome analysis of wheat spike development and construction of AS profiles for different wheat tissues and development periods in normal condition were performed in “Chinese Spring” lines using RNA-Seq technology.The analysis of 6 RNA-Seq samples from 5,10 and 15 days post anthesis in wheat(Chinese Spring)spikes were performed.A total of 57,003 expressed genes were identified;And 18,711 differentially expressed genes were identified by differential expression analysis;Gene onconology enrichment analysis revealed that these genes are mainly enriched in biological processes related to cell wall organization,mitosis,organic substance metabolism and transport,photosynthesis,and plant hormones signal pathway.The AS analysis in combination with 19 RNA-Seq samples from four different tissues and ten different developmental stages of wheat(Chinese spring)from public database was performed.Finally,a total of 210,014 alternative splicing events were identified,corresponding to 27,919 genes,accounting for 31.8% of the multiple exon genes in wheat,which includes 6500 “Low Confidence” genes.The intron retention(IR)type has the largest number of alternative splicing events,followed by the alternative exon ends(AE)type,and the exon skip(SKIP)type is the least.Since the bread wheat is hexaploid,statistical analysis of the distribution of alternative splicing genes on the wheat homologous subgenome revealed that the number of alternative splicing genes on the B subgenome was slightly higher than that of the A and D subgenomes.The results of AS analysis in four wheat tissues showed that the number of AS genes in the leaf was the highest,the spike was slightly lower than the leaf,and the grain was the least.The number of four tissue-specific expressed AS genes is consistent with this result.The results of AS analysis at ten wheat developmental stages from seedling to maturity showed that there was a significant V-shaped trend for the AS genes number change during wheat growth,in which the two peaks were at 14 days post emergence and 5 days post anthesis,and the bottom was at the booting stage.The trend of the number of stage-specific expressed AS genes is consistent with this result.This study further quantitatively analyzed the differential expression of AS genes in wheat spike.Finally,72 AS genes differentially expressed during wheat spike development were identified.The clustering analysis of the 72 genes expression patterns showed that the expression levels of 21 genes were continuously up-regulated;19 genes were continuously down-regulated,and the remaining 32 genes were firstly up-regulated and then down-regulated.Gene functional enrichment analysis indicated that these alternative splicing genes differentially expressed are mainly involved in biological processes such as metabolic processes,cellular processes,biological regulation,response to stimulus and growth.In this study,the transcriptome data during wheat spike development were analyzed for differential genes expression and functional enrichment;The 19 wheat(Chinese spring)transcriptome data from public database were used to construct the wheat alternative splicing landscape in normal growth condition,and the results showed that 31.8% of the multiple exon genes were alternatively spliced,including 6500 "Low Confidence" genes.Then,the distribution of alternative splicing genes in 4 tissues and 10 developmental stages of wheat was further analyzed.Finally,the quantitative analysis of alternative splicing genes during wheat spike development was performed.In this study,the differentially expressed genes during the development in spike were revealed at the transcriptome level,and the AS profiling of wheat in different tissues and stages under normal growth conditions were constructed,which laid a foundation for subsequent molecular breeding and related basic research.