基于二代和三代转录组测序揭示甘蔗重要亲本对黑穗病菌侵染的响应机制

Comparative transcriptome analysis of elite‘ROC’sugarcane parents for exploring genes involved in Sporisorium scitamineum infection by using Illumina-and SMRT-based RNA-seq

黑穗病是甘蔗生产中的主要病害,严重影响甘蔗产量。解析甘蔗重要亲本与黑穗病菌相互作用的分子机制及筛选抗病基因对抗黑穗病优良品种的培育具有重要意义。本研究选用我国甘蔗育种中的重要亲本新台糖ROC25(抗黑穗病)及其姊妹系ROC22(感黑穗病)为研究对象,采用单分子实时测序技术(三代测序)和二代转录组测序技术分析和鉴定2个亲本感染黑穗病菌后的转录组谱及差异转录本。三代转录组测序分析共获得79,885条转录本序列,其中含60,115条完整开放阅读框、3692个可变剪接事件、1799个LncRNA、29,139个SSR和7794个转录因子,共有74,066个转录本得到注释,占总数的92.72%。通过对二代测序数据分析,在抗病亲本中筛选出9716个差异转录本,在感病亲本中筛选出2033个差异转录本。差异转录本的GO和KEGG富集分析结果表明抗病亲本中共富集到的GO条目和KEGG通路均要多于感病亲本,且植物MAPK信号通路、苯丙素生物合成、植物-病原互作、亚油酸代谢和蔗糖和淀粉代谢等代谢通路在抗感亲本中均被显著富集,为抗感亲本对黑穗病的共同抗病途径。进一步,对植物MAPK信号通路的分析表明,MAPK超家族基因成员在抗感亲本中呈现不同的表达方式,在抗病亲本中具有更多差异表达的MEKK1和MKK4的转录本,且MKK5、MPK10和MPK12基因仅在抗病品种中发生显著表达变化,推测其可能与亲本的抗病表型关联。此外,抗感亲本中众多WRKY、MYB、NAC和AP2/ERF-ERF等抗病相关转录因子响应了黑穗病菌胁迫,且主要表现为上调表达。与感病亲本相比,抗病亲本显示出更多的差异表达转录因子,推测这些抗病亲本特有的转录因子可能对防御黑穗病菌具有积极作用。本研究完善了新台糖亲本基因组注释信息,为解析新台糖优异亲本与黑穗病菌互作机制以及抗黑穗病基因资源的挖掘利用提供指导。

Sugarcane smut,caused by the fungus Sporisorium scitamineum,is the most challenging disease of sugarcane,causing significant losses in cane yield.There is a dearth of information on smut resistance mechanism in elite parents for the development of smut-resistant varieties.In the present study,we adopted joint Illumina-and Single Molecule Real-Time(SMRT)-based RNA-seq analysis to identify transcript expression in an smut-resistant and-susceptible parents(ROC25 and ROC22)infected with S.scitamineum.A total of 79,885 high-quality transcripts was obtained,including 60,115 open reading frames,3692 alternate splicing isoforms,1799 long non-coding RNAs,29,139 simple sequence repeats,and 7794 transcription factors.About 92.72%of the total transcripts were annotated,which should have increased the available data amount for transcriptome profile analysis.There were 2033 and 9716 differentially expressed transcripts(DETs)in ROC22 and ROC25,respectively.The analyses of GO and KEGG enrichment showed that more GO terms and KEGG pathways were observed in ROC25 than ROC22.It was found that MAPK signalling pathway-plant,phenylpropanoid biosynthesis,plant-pathogen interaction,linoleic acid metabolism,and starch and sucrose metabolism were enriched both in resistant and susceptible parents.In addition,MAPK superfamily genes were differentially regulated in different parents,more DETs of MEKK1 and MKK4 were detected in resistant parent,and the relative expression levels of MKK5,MPK10,and MPK12 genes were specifically altered in resistant parent.It suggested that MAPK superfamily genes might play the important roles in the regulation of sugarcane response to S.scitamineum infection.Moreover,lots of transcription factors(TFs)associated with plant disease resistance were found to respond to S.scitamineum infection in both ROC22 and ROC25 parents,including WRKY,MYB,NAC,and AP2/ERF-ERF.Majority of the TFs were up-regulated.Compared to the susceptible ROC22 parent,the number of activated transcription factors in the resistant ROC25 parent was higher,indicating that these extra TFs might have positive effects in the defense against S.scitamineum.This study provides a comprehensive set of reference transcripts for sugarcane and thus increases our understanding on the interactions between sugarcane and S.scitamineum,which should be helpful in guiding on exploitation and utilization of smut-resistance gene resources.