Analysis Of Sea-Island Cotton(Gossypium Barbadense L.)in Response To Fusarium Wilt Infection By RNA Sequencing

Sea-island cotton(Gossypium barbadense L.)is a cultivar with high quality cotton fiber in the world.Xinjiang is the main sea-island cotton cultivation area in China.Fusarium wilt seriously affects the yield and fiber quality of island cotton in Xinjiang.Studying the mechanism of resistance wilt in sea-island cotton will provide a basis for cultivating the disease-resistant varieties of sea-island cotton and solving the damage of wilt.In this study,Fusarium wilt resistant variety 06-146,Fusarium wilt sensitive variety Xinhai 14(XH 14),and the four F2:6 recombinant inbred lines with different disease resistances constructed from them were used as materials.These materials infected by Fusarium oxysporum,high-throughput sequenced by RNA-Seq technology.We used qPCR experiments to verify the accuracy of sequencing data,and analized sequencing data with bioinformatics method.Further,RNA-Seq sequencing data was used to carry out studies on lncRNA identification,classification,expression analysis,and functional analysis.The specific results are as follows:1.By RNA-Seq sequencing,400 millions raw reads of 12 samples of the control group and the treatment group were obtained.After filtering,a high-quality 350 million clean reads were obtained,and an average of 3G data per sample was obtained.The sample correlation analysis showed that the inoculation treatment group and the control group were clustered into two groups.The qPCR experiment showed that the Fusarium oxysporum FOTG gene was detected in the inoculation group.The gene expression level of the group showed that the known disease resistance-related genes were highly expressed in each sample The qPCR results of the 7 randomly selected genes were consistent with the transcriptome results.All of the above indicate that the island cotton hypocotyl has been infected with pathogens after 40 hours of inoculation.2.Analysis of differential expressed genes showed that the response trends of Fusarium oxysporum infection were similar,but the number and type of genes up-regulated and down-regulated among different varieties were still very different.After the materials were infected,the number of genes down-regulated was higher than that of the up-regulated genes,and the number of differential genes in susceptible materials was larger.Up-regulation and down-regulation were consistent in all infection and control samples,indicating that the trend of mRNA transcription response to Fov infection is conserved among the six breeds.480 co-up DEGs were obtained from the two resistance groups,and 510 co-up DEGs were obtained in the two susceptible groups;983 co-down DEGs were obtained in the two resistance groups.(gene),and 1008 co-down DEGs were obtained in two susceptible groups.The enrichment annotations of these differential gene showed that the genes involved in disease-resistant toxin catabolism,defense response,immune response,ABC transporter,and defense mechanisms were up-regulated expression in all samples,and the expression levels were consistent with material resistance;The enrichment of genes involved in metabolic processes such as flavonoid biosynthesis genes and glycosyltransferase-related genes was up-regulated expression in the samples,and the significance of enrichment was consistent with the resistance of the materials;the photosynthesis-related genes in the susceptible group were significantly down-regulated expression,but no differential expression of photosynthesis-related genes was detected in the resistant group,indicating that the photosynthetic system in the resistant group was protected,ensuring energy supply.3.Based on the above transcriptome sequencing data,using the upland cotton genome as a reference sequence,11336 lncRNAs were predicted,81.0%were long intergenic non-coding RNAs(lincRNA),14.4%were intron lncRNAs,and 4.6%were antisense lncRNAs.The lncRNA structural analysis showed an average length of 1042 bp,and 69.2%of lncRNA had only one exon.The qRT-PCR results of 16 randomly selected lncRNAs were consistent with the transcriptome results,indicating that the transcriptome also responded well to the expression of lncRNA.There were differences in the expression of lncRNA between the two parents,between the anti-inductive materials group and before and after the inoculation,but the number of DE lncRNA was not consistent with the material resistance;the number of lncRNA down-regulated in the susceptible group was significantly higher than that in the up-regulated group.DE lncRNA is unevenly distributed on the two subgenomic chromosomes of the corresponding upland cotton.The chromosomes A05,A07,A11,D05 and D06 contain obvious clusters,and the corresponding positions can be used as genomic hotspots in response to fungal infection.The functional enrichment analysis of DE lncRNA showed that the up-regulated DE lncRNA was co-expressed with glutathione metabolism,phytohormone signal transduction and vesicle transport in cotton in a cis-acting manner,indicating that the island cotton is resistant to disease.Related lncRNA may be involved in the response of the cotton wool to Fusarium wilt by regulating these metabolic or signaling pathways.4.Based on the above transcriptome sequencing data,the upland cotton transcription factor database was used as a reference sequence,and the differentially expressed genes of each group were subjected to transcription factor function annotation,and the transcription factor genes in the differentially expressed genes of each group were screened.A total of 1315 transcription factors from 48 transcription factor families in each group changed their expression levels after infection treatment,among which 503 transcription factors from 39 families were up-regulated,and there were 44 transcription factor families.812 transcription factors were down-regulated.The number of down-regulated expressions and family types were higher than those of up-regulation.Further analysis of the six alignment groups revealed that the six transcription factor families bHLH,ERF,G2-like,NAC,MYB,and S1 Fa-like were up-regulated in the six materials,and bHLH,ERF,G2-like,NAC,MYB,S1Fa-like,and WRKY transcription factor families have the highest number of transcription factors down-regulated in six materials.Analysis of overlapping differentially expressed transcription factors between the groups revealed that 17 transcription factors from 11 transcription factor families were up-regulated in all groups,and 12 transcription factors from 8 transcription factor families were down-regulated in all groups.The overlapping and transcription factors were mainly from ARF,ERF,MYB,S1Fa-like and other families.Four genes from CAMTA,LBD,WRKY and Trihelix families were up-regulated in 6 groups.Seven species-specific differentially expressed transcription factors were screened,and six transcription factors derived from NAC,G2-like,C2H2,HD-ZIP,AP2,and S1Fa-like families were differentially expressed only in resistant materials.One transcription factor from the G2-like family was down-regulated only in susceptible materials.