Alternative Splicing And Novel TU Analysis Of Maize CMS-C Anther Based On RNA-seq

Maize is the first crop to world,maize is widely used as food, feed and biofuels in the world. CMS was firstly used to produce hybrid seeds. In this study, the RNA-sequence technology was used to explore the alternative splicing (AS) and Novel TU, for using CMS C48-2 line and maintainer line N48-2 with anther, B73 were used the reference genome. The main results are as follows:1.125965 AS events of 5712 genes were detected in C48-2 (18.85% of all B73 genes), 8747 AS events of 4554 genes were detected in N48-2 (14.67% of all B73 genes) based on RNA-Seq data. Retained intron (RI) and Alternative 3’splice site(A3SS) accounted for the larges number of AS events in C48-2 and N48-2, mutually exclusive exons (MXE) type splicing events was not detected in this study, and other four type of (A5SS,ES,AFE and (?) ALE) alternative splicing were detected in this study. Alternative splicing of exon length distribution statistics show that alternative splicing of exon are smaller exons. Splice site sequence indicate splice site sequence is very conservative. Based on the comparison of the amount and the length distribution between RIs and cassette exons (CEs), the results suggested that maize might perform splice site recognition predominantly by the intron definition (ID) and exon definition(ED) mechanism. Two kinds of alternative splicing events were done by RT-PCR for intron retention (RI) and exon skipping (Exon skipping, ES),the results show that GRMZM2G076526 intron retention and GRMZM2G056442 exon skipping gene splicing events was reliable.2.2668 Novel TU were detected in C48-2,2376 novel TU were detected in N48-2 based on RNA-seq data. CPC software was used for predicting coding capability of novel TU.1711 novel TU have coding capacity (64.13% of all novel TU) in C48-2, and 1573 novel TU have coding capacity(64.68% of all novel TU) in N48-2. Coding region (CDS) of novel TU was predicted by ESTscan software. The result show that most of the novel TU had shorter length and 1892 novel TU had less than 1000bp (70.1% all novel TU in C48-2), and 1770 in N48-2(74.49% all novel TU in N48-2). Three novel TU (TU850, TU855, TU1062) were randomly selected from coding capacity in C48-2, and two novel TU (TU751, TU1313) from coding capacity in N48-2. The results showed that TU850, TU855 and TU1062 are the transcriptional expression of the novel TU in C48-2, TU1062 may be one of a new gene in C48-2, TU751 and TU1313 are the expression transcription of the novel TU in N48-2, and TU751 may be one fusion transcript.3. Difference in expression genes, difference in splicing type or splice sites between C48-2 and N48-2 were based on Venn diagram,562 candidate genes were obtained from the Venn diagram,and BLAST were done with Gene ontology (GO) Database, in which 964 transcripts were identified and were annotated with significant GO terms by using Blast2go software. It showed that Cellular components contains 9 functional categories,Molecular function contains 11 functional categories,Biological processt contains 24 functional categories.4. Novel TU (1711 in C48-2,1537 in N48-2) having coding capability were searched by BLAST in Nr Database. Species distribution statistics show that corn is the most and sorghum more. All of novel TU were identified and annotated with significant GO terms by using Blast2go software. It showed that Cellular components contains 10 functional categories,Molecular function contains 11 functional categories,Biological processt contains 24 functional categories.