| Stripe rust,caused by Puccinia striiformis f.sp.tritici(Pst),is one of the most destructive diseases of wheat,which can cause a great reduction in grain yield.Both the domestic and foreign researches showed that the breakdown of disease resistance due to virulence changes occurred in the pathogen population of wheat.The genomic variation is the root of virulence variation in rust fungi.Whole genome sequencing analysis is an efficient way to illuminate the evolvement rule and the molecular mechanism of virulence variation in rust fungi.This paper synthetically utilized the respective advantages of the third generation sequencing(TGS)and the second generation sequencing(NGS)to re-assemble the genome of the finished CY32 genome draft in order to get a brand new genome.We further conducted the qRT-PCR experiments for the predicted secreted protein genes which showed polymorphism after whole genome re-sequencing analysis.This study mainly includes four aspects:1.In this study,we got a significantly improved draft genome of a Chinese Pst isolate,CY32,using the Pac Bio TGS technology that was combined with the NGS data to repair the errors and gaps of the single DNA bases insertions and deletions(INDELs).We utilized the Pac Bio sequencing platform to construct 20 Kb SMRTbell library,which produced 7.44 G raw data after quality control and achieved 61.97 times sequence coverage.Finally,we got 3.5G corrected data after quality control with the sequence coverage of 30 times.The libraty of 200 bp,300bp,500 bp,700bp,2K and 6K were constructed using the Illumina-Solexa sequencing platform to produce 29.55 G raw data after quality control with the sequence coverage of 246.08 times.The 30 times Pac Bio data was used to construct contig,and combined with the 246.08 times Illumina data to construct scaffold.The errors and gaps of the data were repaired using Ilummina-Pacbio data.The result of assembly statistics showed that the contig N50 was 390,736 bp,scaffold N50 was 532,961 bp,and GC content was 44.37%.Our results showed that the number of scaffold decreased by 3.6 times from 4,283 to 1,191,the size of contig N50 increased by 21.7 times from 18,007 bp to 390,736 bp,the size of Scaffold N50 increased by 4.3 times from 125,324 bp to 532,961 bp,and the size of gaps decreased by 182 times from 15,119,423 bp to 83,089 bp.The quality of CY32 genome has been greatly enhanced after re-assembling,which will lay a solid foundation for large-scale re-sequencing variation analysis as a standard genome.2.The assembled genome sequence of CY32 was 120,898,252 bp using the IlumminaPacbio data.Global analysis of the DNA sequence revealed that the repetitive elements and primarily retrotransposons account for 43.5% of the genome using repeatmasker and TRF software.The Pst genome contains 22,287 protein-coding genes were annotated using softwares(Genemarkes,SNAP,Augustus,Homology,Glean).Using software(Signalp,Targetp,Tmhmm,Pred GPI)predictions,we identified 2,100 putative secretory protein genes,of which the pst-specific putative secretory protein genes were 499 through homology searches.Based on the analysis,the genes annotated by Gene Ontology(GO)can be classified into several parts,among which the gene numbers(1,761)participating in genetic information processes such as replication recombination and repair are more than the other biological processes,which was also consistent with the characteristics of rapid virulence variation.Illuminating the function of these genes and the polymorphism positions among different Pst isolates will give us better ways to understand the relationship between genomic variation and virulence variation.3.Verifying the candidate gene predicted by re-sequencing.The result of sequence variation analysis by re-sequencing include a mass of Single Nucleotide Polymorphisms(SNPs)and short insertions and deletions(In Dels).5 of the 15 candidate genes which have significantly polymorphic loci were selected by cloning,and sequencing analysis.More details were as follows:1)There were 5 amino acid replacements in striiformis_Gene4629 because of the SNP variation.2)There were short insertions and deletions in striiformis_Gene16278 because of the repetitive sequence and transposons.3)There were 7 amino acid replacements in striiformis_Gene2933 because of the SNP.4)There were short insertions and deletions from 279 to 331 amino acid site in striiformis_Gene28648 because of the repetitive sequence and transposons.5)There were 9 amino acid replacements in striiformis_Gene23763 because of the SNP.4.qRT-PCR experiments for the candidate putative secretory protein genes.20 candidate putative secretory protein genes were selected to analyze the relative expression of Pst development during plant infection.Finally,we found that 14 genes reveal peaks of expression for the selected effector candidates during plant infection.1)The qRT-PCR analysis showed that the relative expression of striiformis_Gene4096,striiformis_Gene6071 and striiformis_Gene23763 in urediniospore was much higher than the other stages,which indicated that these genes may play an important role in the development of urediniospore.2)The qRT-PCR analysis showed that the relative expression of striiformis_Gene4096 was at a higher level during the whole infection.This gene has a Dna J conserved motif which has a heat shock protein 40(HSP40)binding site.3)The qRT-PCR analysis showed that the relative expression of striiformis_Gene28555,striiformis_Gene6785,striiformis_Gene12678 and striiformis_Gene 28338 was increased and maintained at a high level at later infection stage of post-inoculation(5d-14d),which indicated that those genes may play an important role in the development during the hypha expansion and spore production.4)The qRT-PCR analysis showed that the relative expression of striiformis_Gene4112,striiformis_Gene4629,striiformis_Gene29175,striiformis_Gene1506 and striiformis_Gene 2933 maintained at a low level during the whole infection,and further studies will be needed to study the function of these genes. |
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