| Fuasrium wilt causal organism.F.oxysporum.f.sp.batatas fob has been hindering sweet potato cultivation,using resistant cultivar has been found to control it efficiently.Genetic studies relating to sweet potato has been limited,facilitating its functional genomic at the molecular level will better improve the crop.To acquire more knowledge into the resistant mechanism of sweet potato against fob,de no votranscriptome assembly and digital gene expression(DGE)was performed using illuminaHiseq technology.In the findings,a total of 89,944,188 clean reads was found and were assembled into 101,988 unigenes with an average length of 666bp,62,605(61.38%)of them were functional annotated in the non-redundant(nr)protein database from NCBI by using BLASTX with a cut-off E-value of 10-5,and COG,GO and KEGG annotations were examined to understand their functions more.Five DGE libraries were constructed from the sweet potato cultivar JinShan57(JS57,highly resistance)and XinZhongHua(XZH,highly susceptible)challenged with pathogenic and Nonpathogenic Fob.Understanding the mechanism of the two cultivars used,.Deep-sequencing produced 91,366,006 sequence reads with a length of 100bp each,and average GC content of 45.10%.After the removal of adaptors,primer sequences,poly-A tails as well as short and low quality sequences,a total of 89,944,188(98.44%,11.24Gbp)high quality reads with Q>20 were retained and used for assembly.Using Trinity to align and assembled yield a set of 146,859 transcripts sequences longer than 201 bp,with a N50 length of 1371 bp,and 101,988 unigenes with average length of 666bp and N50 length of 1079bp.The length of assembled unigenes varied from 201 to 15576 bp,more than 36.6%of these unigenes were longer than 501 bp,and ABlASTX search against KEGG protein database with a cutoff E value of e-5 was made on the assembled gene database.Total of 19,578 distinct gene sequences were assigned to 276 KEGG pathways.The pathways most represented by unique sequences were translation(2560 genes),signal transduction(2089 genes)and carbohydrate metabolism(1963 genes).In order to confirm the quality of illumine solexa sequencing data and the DGE results,five unigenesc47391g1,c51550g2,c52297g1,c54420g2 and c56184gl were selected for quantitative RT-PCR assays,result revealed the same expression tendency as that of the DGE already analyzed suggesting the high reliability of IlluminaSoIexa sequencing used for the experiment.A further study was done to compare the gene expression of highly resistant and highly susceptible after colonization with fob.Expression of genes pattern involved in plant pathogen interaction was observed in both cultivars.The gene level was found by normalizing the number of unambiguous tags in each library to expected number fragments per kilobase of transcript sequence per millions base pairs sequence(FPKM)biological samples differential expression analysis was done by DESeq R package(1.10.1),Results shows a lot of genes were up-regulated or down-regulated in both of the two cultivars,the number of up-regulated were more than those down-regulated.WRKY genes were identified in the transcription data base,out of 123,10 WRKY genes(8.1%)were differentially expressed significantly at least in one comparison.Unigenesunigenes c54613_gl and c55532_gl were found and annotated to WRKY 70 and WRKY50,they were both up-regulated in Js57 after inoculated with NPF Fo-04.unigenes(c54420_g2,c56135_g1,c57247_g1,c60589_g1,c64728_g1)were exclusively up-regulated in XZH after inoculated with Fo-07,they were annotated to WRKY61,WRKY75,WRKY22,WRKY71 and WRKY6,respectively,all of them are related to defense response.c46250gl was annotated to WRKY 56,which were down-regulated exclusively in XZH after inoculated with Fo-07,however,the function of WRKY56 gene was hitherto keep unknown.The unigenes c50623_g1 and c33599_g2 were differentially expressed in the comparison of JS57_CK vs XZH CK.c50623_g1 was annotated to WRKY48-like gene,its expression level was significant lower in JS57 CK than that in XZH CK.We identified 20 NAC transcription factor genes in the transcriptome database,only two of them(10%)were differentially expressed significantly at least in one of the comparisonsTheunigene c51550_g2 was annotated to NAC29 and its expression level increased in XZH after inoculated with Fo-07.The other unigene c52292_g3 was annotated to NAC7-like gene,in JS57CK its expression level was 3.7 fold lower than that in XZH,indicating it is also a negative regulator of defense response.The c52634_g1 was annotated to ERF5;its expression level was lower in JS57 than that in XZH,indicating that it was a negative regulator of disease resistance in sweet potato.We found the unigene c5221_g1 was annotated to IbMYB1 and in Js57 it expressed 4.6 fold higher than that in XZH,indicating its potential function in defense response.We identified 57 PR genes in the transcriptome,in which four were differentially expressed at least in one of the comparisons.c55964_g1 was annotated to PR-10 and was up-regulated in XZH after inoculated with Fo-07,PR10 proteins are considered to participate in the defense of plants against fungi.We indentified 20 genes associated with SA pathway in the transcriptome.Three of them were differentially expressed at least in one of the comparisons.We found the two unigenes c21763-gl and c39777_g1 were up-regulated in XZH after inoculated with Fo-07 and both of them were annotated to S-adenosyl-L-methionine:salicylic acid carboxyl methyltransferase(SAMT).The differentially expressed genes including up-and down-regulation in five libraries were identified and calculated based on comparisons of transcriptomes,showing differences in gene expression profiles among the samples.A set of differentially expressed genes involved in defense response were identified,including WRKY,NAC,MYB,and ERF transcription factors,resistance genes,pathogenesis-related genes,and genes involved in SA signal pathway.Five selected genes were validated using quantitative real time PCR(qRT-PCR)to confirm the DGE results.The differences in their expressed genes explain the resistant in the resistance cultivar and will give more idea in understanding relationship between sweet potato and Fob. |
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