| The Alternative Splicing(AS)process occurs during the conversion of precursor messenger RNA to mature messenger RNA,and can significantly increase transcriptome and proteome diversity by allowing one gene to produce multiple mRNA matures.Currently,researchers have used high-throughput sequencing technology to uncover the content and complexity of AS in model organisms.However,we still know very little about the alternative splicing phenomenon in the polyploid plant Brassica napus.In this study,we extracted high-quality RNA from 14 different developmental stages/tissues of Brassica napus,and mixed these RNAs in equal amounts,using the Pacbio RSII sequencing system to perform single-molecule real-time on mixed RNA samples(Single Molecule Real-Time,SMRT)sequencing and analysis.RNA from mixed samples was extracted and cDNA libraries of different lengths were constructed:1-2 kb,2-3 kb and 3 kb.Among them,two SMRT cells were measured in the library above 3 kb,and three SMRT cells were measured in the other two libraries.In addition,21 different developmental stages/tissues of Brassica napus.were subjected to Next-generation sequencing(NGS)using Illumina HiSeqTM 2000 platform,with 2 biological replicates per sampling point.We combined SMRT sequencing and NGS sequencing to identify and analyze the alternative splicing phenomenon in Brassica napus on a genome-wide scale.The main results obtained in this study are as follows:(1)Using SMRT sequencing technology,we identified 70,815 full-length transcripts covering 20,898 annotation-encoding genes.A total of 1,360 new loci and 1,821 mis-annotated genes were identified,which in turn updated the existing gene annotation of Brassica napus.(2)At the same time,71,889 annotated genes were identified by SMRT and NGS sequencing,and 39,908(55.51%)of the multiple exon genes had AS phenomenon,A total of 80,598 AS events occurred in these genes.Among the different types of alternative splicing,Intron retention(IR)occurs most frequently,accounting for more than 41.06%,and variable receptor end site type(17.67%)is about variable donor end site.The type(10.97%)was 1.6 times,and the exon exclusion event(1.73%)accounted for the lowest proportion.(3)AS analysis of different tissues/developmental stages of Brassica napus,showed that AS events showed dynamic changes between different tissues/developmental stages.For the same tissue type,the AS event in the early developmental stage is basically more than the late developmental stage.For example,in leaf tissue,the total number of AS events in young leaves was 47,017,which was significantly higher than in cotyledons(37,722).In flower buds,seeds and cuticle tissues,the total number of splicing events increased with maturity.The tendency to increase after the increase.This unequal distribution may be related to the total number of genes expressed in different tissues,and the AS gene ratio(AS gene number/total expression gene number)is higher in younger tissues.(4)Different AS types have different characteristics,and these characteristics may have different influences on each AS type.Among them,the results of this study indicate that in Brassica napus,the increase in the number of exons can promote the frequency of IR;increase the length of introns,increase the proportion of exon skipping(ES),and decrease the proportion of IR.On the other hand,as the level of gene expression increases,IR events show a decreasing trend and the frequency of ES events increases significantly.Through alternative splicing analysis of the polyploid plant Brassica napus,we identified a large number of alternative splicing events,and the full-length transcript sequence obtained by SMRT sequencing updated the existing Brassica napus genome annotations,which are different.Subsequent functional studies of the construct provide abundant data resources and further enhance the understanding of alternative splicing in polyploid plants.in polyploid plants. |
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