| Sclerotinia sclerotiorum is a devastating plant fungal pathogen with a wide host range.Previous researches proved that DNA double-strand break(DBS)and repair of DNA damage are activated in S.sclerotiorum when invaded by Sclerotinia sclerotiorum hypovirulence-associated DNA virus 1(Ss HADV-1),including nonhomologous end joining(NHEJ)and homologous recombination(HR).NHEJ always leads to mismatch in process of DNA repair.Therefore,it is presumed that the invasion of mycoviruse Ss HADV-1 may promote the genomic variation among S.sclerotiorum.In order to monitor the influence of Ss HADV-1 to the genome of S.sclerotiorum,strain PB4 collected from a pinto bean(Phaseolus vulgaris cv.Pinto)in Washington State,USA and strain PB4-V infected with Ss HADV-1,along with their single-ascospore-isolation offspring,were chosen to perform bioinformatics analyses.In this study,we isolated S.sclerotiorum strains from sclerotium samples in Quincy,Washington State,USA,and found that strains PB4 had almost identical genome to the reference strain 1980 isolated in the west of Nebraska 40 years ago.Next-generation sequencing(NGS)technology was used to detect SNPs in 15 strains from Quincy,USA,14 strains from Xinyang,China,and4 strains from other regions in China.Based on data generated,The SNPs of in three strains(PB273,PB615 and PB623)were much less than in strain PB4 to compared with the genome of strain 1980.Comparing the strains from China and the USA,it was found that Chinese strains,even from a single small rapeseed field,have much richer SNPs than the USA strains.The results suggest that there are two types of genome in S.sclerotiorum,one is very stable and the other tends to variate constantly.The main results were as follows:Using both NGS and Pacific Biosciences single molecule real time sequencing(Pac Bio SMRT)hybrid and hierarchical assembly strategies,a complete draft genome of S.sclerotiorum strain PB4 was assembled to 26 contigs with N50 length of 2.08 Mb,a guanine-cytosine(GC)content of 41.52% and a total assembly size of 39.09 Mb.11,248 genes,32,363 CDS,32,358 exons and 11,033 m RNA were predicted in the genome.Based on databases and functional domains,476 genes of arbohydrate-Active enzymes(CAZymes),102 genes of Cytochrome P450,63 genes of effector,2924 genes of pathogen–host interaction(PHI)protein,and 33 secondary metabolite gene clusters were predicted in S.sclerotiorum.The genome of strain PB4 sequenced by the NGS and Pac Bio SMRT sequencing carried only 135 single nucleotide polymorphisms(SNPs)and 18 structural variations(SVs)compared with the genome of strain 1980.Based on data generated by NGS,other three strains PB4,PB273,PB615 and PB623 had 532,256,275,and 262 SNPs,respectively,against strain 1980.The other strains of PB are rich in SNPs with a range of34,391-47,264 when compared with strain 1980.18 Chinese strains(14 from a single rapeseed field at Xinyang city and 4 from different locations)were much more SNPs(65,317-77,618)in all Chinese strains than in the PB strains.A phylogenetic tree based on SNPs was constructed and the strains were divided into two large groups.There was obvious genomic diversity among Chinese strains while the SNPs-based genome diversity of strains from the USA was not rich.The SNPs in the 31 offspring were divided into two groups: occurred on Contig＿20 and occurred on other contigs.Fisher’s exact test showed that there was a significant difference between the frequency of SNPs on Contig＿20 and among the whole genomes.Contig＿20 is a hotspot mutation regions for strain PB4.Protein sequences of strain PB4 and 1980 were comparative analyzed.Twenty-six genes encoding proteins with conserved domains were found unique to strain PB4.Sixtythree genes were predicted to encode effectors in strain PB4,16 of which were first detected in S.sclerotiorum.The 16 genes mentioned above were not predicted to encode effectors in strain 1980.Compared with Sclerotinia borealis;Sclerotinia trifoliorum,Sclerotinia.glacialis,Botrytis.cinerea,Coniothyrium.minitans,Cryptonectria.parasitica,Fusarium.graminearum,Trichoderma.atroviride and Trichoderma.harzianum,there was a significant increase in the number of non-ribosomal peptide synthases(NRPS)secondary metabolite gene clustres and intron RNAs in S.sclerotiorum.The genomes of 31 single-ascospore-isolation offspring of strain PB4 were comparative analyzed.A total of 1778 SNPs were found at 405 loci with average 57 SNPs in each offspring;A total of 835 In Dels(insertion/deletion)were found at 219 loci with average 27 In Dels in each offspring;Totally 147 SVs were found at 101 loci including 74 deletions,39 inversions and 34 duplications,on average,2.4 deletions,1.25 inversions,and 1.1 duplications in each offspring.The prediction to the genes of variation based on functional domain revealed that they might be involved in the regulation of p H response,stress response and metabolism of life cycle.GO(GO ontology)analysis showed that genes with SNPs and In Dels produced by sexual reproduction were enriched in growth and development,biosynthesis and metabolism of S.sclerotiorum.Genes with SVs were mainly enriched in the adapted to the environment,ascospores sporulation and development and methylation.Combined with the colony morphology,sclerotia formed,and growth rate of all the single-ascospore-isolation offspring,the variations produced by sexual reproduction might not influence the biological characteristics.To explore the influence of Ss HADV-1,the single-ascospore-isolation offspring of strain PB4 and PB4-V were comparative analyzed.26% of strain PB4-V offspring showed slower growth rate,29% of strain PB4-V offspring showed smaller less sclerotia,19% of strain PB4-V offspring showed reduced virulence.Ss HADV-1 was more stable in offspring of strain PB4-V.After 3 times successive transfer culturing,the positive rate of Ss HADV-1 in offspring of strain PB4-V was 77%,much higher than in offspring of strain PB4 by 9%.The genomes of 31 single-ascospore-isolation offspring of strain PB4-V were comparative analyzed.A total of 1904 SNPs were found at 432 loci with average 61 SNPs in each offspring;A total of 890 In Dels were found at 223 loci with average 29 In Dels in each offspring;Totally 95 SVs were found at 78 loci including50 deletions,26 inversions and 19 duplications,on average,1.6 deletions,0.84 inversions,and 0.61 duplications in each offspring.Five of single-ascospore-isolation offspring of strain PB4-V with reduced growth rate were found had two SNPs.Six single-ascospore-isolation offspring of strain PB4-V with more sclerotia had two SNPs and In Del.Comparing the single-ascospore-isolation offspring of strain PB4,there were more SNPs and In Dels in those of strain PB4-V.There were significant differences among genes with SNPs and In Dels and GO terms enriched among offspring of strain PB4 and PB4-V.Most genes with SNPs and In Dels in offspring of PB4-V were enriched in homeostasis and response to toxic substance.Genes with SVs in offspring of PB4-V were mainly enriched in reacts to threats in external environment,and several negative regulation of cell life activities,while the GO terms mentioned above were not enriched in offspring of strian PB4.In conclusion,infection of Ss HADV-1 influenced the number and loci of variations in the genome of S.sclerotiorum.Different from those in offspring non-infected with Ss HADV-1,genes with viriations were mainly enriched in environment reaction and homeostasis of cell.In this study,we assembled genome sequence of S.sclerotiorum strian PB4.compared the genetic diversity in different regions to provided the basis for evolution pattern of S.sclerotiorum.The analyses of the genome among single-ascospore-isolation offspring supplied important clues about the variation.We further measured the promotion to genomic variation by Ss HADV-1,which might be a driver for genome evolution. |
