Whole genome analysis of the koa wilt pathogen (Fusarium oxysporum f. sp. koae) and the development of molecular tools for early detection and monitoring

- koae [ Fo koae.
- Results: This study presents whole genome analyses of one highly virulent Fo koae isolate and one non-.
- Using putative chromosomes and predicted gene comparisons, Fo koae -exclusive, virulence genes were identified.
- Unique genes from Fo koae were used to develop pathogen-specific PCR primers.
- These diagnostic primers allowed target amplification in the characterized highly virulent Fo koae isolates but did not allow product amplification in low-virulence or non-pathogenic isolates of Fo .
- Thus, primers developed in this study will be useful for early detection and monitoring of highly virulent strains of Fo koae .
- Isolate verification is also important for disease resistance-breeding programs that require a diverse set of highly virulent Fo koae isolates for their disease-screening assays to develop disease-resistant koa..
- Conclusions: These results provide the framework for understanding the pathogen genes necessary for koa wilt disease and the genetic variation of Fo koae populations across the Hawaiian Islands..
- These screening trials are costly and require diverse pathogenic Fo koae strains to ensure that the disease resistance is robust in the selected germplasm.
- Consequently, these disease resistance- screening programs would greatly benefit from faster, more cost-effective strategies for screening pathogenic Fo koae isolates.
- Objectives for this study were to 1) conduct and analyze whole genome sequences of one highly virulent Fo koae isolate and one non-pathogenic F.
- 3) use the identified differences to develop Fo koae-specific PCR primers.
- and 4) test the robustness of the developed primers to identify highly virulent Fo koae isolates among uncharacterized F.
- To conduct whole genome comparisons, one pathogenic Fo koae isolate (44) and one non-pathogenic Fo isolate (170) were whole genome sequenced using Illumina TruSeq short-read sequencing (150 bp).
- Libraries re- sulted in reads with 1245x coverage for pathogenic isolate Fo koae 44 and reads with 1150x coverage for non-pathogenic isolate Fo 170..
- De novo assembly of both Fo koae 44 and Fo 170 identi- fied genome sizes of 48 and 50 Mb, respectfully.
- A whole-genome maximum likelihood phylogeny was con- structed to elucidate the evolutionary relationships of Fo koae 44 and Fo 170 compared with other Fusarium spp..
- The phylogeny showed that both Fo koae 44 and Fo 170 grouped into a well-supported [Bootstrap (BS.
- Fo koae 44 clus- tered in a sub-clade (BS =100) that included F.
- oxysporum and that Fo koae 44 and Fo 170 were sufficiently, genetically differ- ent to allow comparison for identifying lineage-specific DNA necessary for pathogenicity in koa..
- Genomic analyses of Fo koae 44 and Fo 170.
- Genomic sequences of Fo koae 44 and Fo 170 were aligned with reference genome sequences of a well- characterized F.
- chromosomes shared synteny with Fo koae 44 and Fo 170.
- The average nucleotide identity (ANIm) between Fo koae 44 and Fo 170 was 96.5%.
- Further analyses comparing the putative chromosomes of Fo koae 44 and Fo 170 indicated that variants (SNPs and indels) were lo- calized on specific chromosomes (LSX_44 and LSX_170) and at chromosome ends (Fig.
- oxysporum transposable elements (e.g., Foxy, Skippy, Impala, Hop, Mariner, Hat, and Helitron) occurred on the LSX of both Fo koae 44 and Fo 170 [29].
- The average core chromosome was comprised of 3 and 4% transposable elements and repeats in Fo koae 44 and Fo 170, respectively.
- In contrast, the transposable el- ements and repeats comprised 19 and 20% of LSX for Fo koae 44 and Fo 170, respectively (Fig.
- This localization of transposable elements provides evidence that differences within the LSX are likely attributable to one or more conditionally dispensable chromosome(s) in both Fo koae 44 and Fo 170..
- Using the Maker pipeline, 15,380 and 15,763 transcripts and corresponding proteins were predicted for Fo koae 44 and Fo 170, respectively, for use in identifying genes associated with pathogenicity in koa wilt disease.
- Using OrthoVenn2, 1760 non-orthologous proteins from 78 gene clusters and 1576 single genes, were identified as exclusive to Fo koae 44 when compared to Fo 170.
- Fo koae 44 had enrichment of gene ontology (GO) terms for nucleic acid binding (Additional File 2), whereas Fo Table 1 QUAST assembly statistics of the SPAdes de novo.
- koae 44 ( Fo koae 44) and non-pathogenic F.
- Fo koae 44 Fo 170.
- The 13,500 protein clusters that were shared between Fo koae 44 and Fo 170 had no identified GO enrichment..
- Using InterProScan, 16,429 and 16,487 putative pro- tein family domains were identified in Fo koae 44 and Fo 170, respectively.
- Of these putative proteins, 30 were unique to Fo koae 44, and 32 were unique to Fo 170..
- Interestingly, a sterigmatocystin biosynthesis gene was identified as unique in Fo koae 44.
- Using the antiSMASH database, the genes associated with secondary metabolites were similar in both ge- nomes of Fo koae 44 and Fo 170.
- Non- ribosomal peptide synthetases (NRPS), NRPS-like, and indoles had higher gene copy numbers for Fo koae 44..
- Using the PHI-base, shared virulence-associated genes that were identified in both Fo koae 44 and Fo 170 include the following: transport associated genes, such as the ATP-binding cassette (Gpabc1.
- Nine virulence-associated genes were identified as unique to Fo koae 44.
- The SIX1 and SIX6 genes, were identified on the LSX of Fo koae 44, but were absent in the Fo 170 genome..
- These Fo koae 44 virulence-associated genes showed sequence similarity to other F.
- The SIX1 gene of Fo koae 44 showed highest sequence similarity with F.
- b Distribution of variants (single nucleotide polymorphisms, SNPs) comparing the putative chromosomes of the koa wilt pathogen Fo koae 44 and non-pathogenic Fo 170.
- Fo koae -specific PCR primer development.
- To increase the likelihood of developing a pathogen- specific primer, two approaches were used to classify genomic regions and genes unique to Fo koae 44 for PCR primer development.
- First, unique genomic regions, that may be non-coding DNA sequences, were identified in Fo koae 44.
- Second, predicted transcripts unique to Fo koae 44 were identified..
- To identify unique genomic regions, Fo koae 44 chromo- somes were aligned to chromosomes of Fo 170, other Fusarium spp., and F.
- Analysis of these align- ments identified a total of 445 DNA sequences unique to Fo koae 44.
- To identify unique genes, Fo koae 44 and Fo 170 were analyzed using OrthoVenn2.
- One thousand seven hundred sixty-two predicted proteins were identified as exclusive to Fo koae 44.
- These primers were tested on characterized pathogenic Fo koae isolates and non-pathogenic F.
- koae ( Fo koae 44.
- Though Fo 170 had a larger genome, this study identified unique genes and DNA sequences, most notably the well-characterized secreted in xylem effectors (SIX genes), that were exclusive to Fo koae 44.
- Most of these unique genomic features of Fo koae 44 were identified amongst the LSX..
- The identified LSX of Fo koae 44 in this study was transposon- rich and contained important wilt-inducing effector genes, SIX1 and SIX6, providing evidence of one or more pathogen-specific, accessory chromosome(s).
- However, a portion of DNA found within the LSX likely represents one or more lineage-specific chromosome(s) for both Fo koae 44 and Fo 170.
- Virulence-associated genes within the LSX are likely the determining factors that distinguishes patho- genicity of Fo koae 44 on koa..
- Even though 1353 and 2369 predicted proteins were identified on the LSX of Fo koae 44 and Fo 170, re- spectively, the function of 58 and 50% of genes for Fo koae 44 and Fo 170, respectively, could not be deter- mined from the available databases used for this study (Additional File 8).
- Perhaps these genes are highly specific to Fo koae 44 and Fo 170 and are possibly important for pathogenicity but have yet to be described..
- Amongst the identified genes, most virulence-associated genes, secondary metabolites, and carbohydrate active enzymes were found to be similar for Fo koae 44 and Fo 170.
- (e.g., Gpabc1, GzmetE, Tom1, Ace1, Mocdip1, Ftf2, and SGE1) were identified in both Fo koae 44 and Fo 170..
- Interestingly, these genes suggest that Fo koae 44 and Fo 170 both have the capacity for host recognition and dis- ease development, even though Fo 170 does not cause wilt disease on koa [36]..
- Of the 57 and 59 unique predicted genes from the LSX of Fo koae 44 and Fo 170, respectively, the major distinc- tions found in Fo koae 44 were a mitogen-activated protein kinase pathway (associated with genes Fmk1, Ste11, and Ste7), which were previously identified as important for host penetration and pathogenicity, and the SIX1 and SIX6 [37].
- The same SIX1 and SIX6 genes reported here in Fo koae 44 have been previously reported in Fo cubense race 1 [5].
- Our results concur with these previous observations because we observed the localization of the two SIX genes amongst the transposon-rich LSX of Fo koae 44 [29].
- predicted genes of Fo koae 44 [43–45].
- oxysporum formae speciales isolated from koa, we chose to develop primers that would amplify genes unique to Fo koae 44 that did not have identified gene ontology terms..
- The LSX primer pair (P6) resulted in amplification of DNA from only one rpb2 haplotype which suggests that it may be too specific to amplify all highly virulent iso- lates within the Fo koae population.
- however, primer pair P6 produced amplified product for all pathogenic Fo koae isolates used in HARC’s disease resistance breeding program.
- The seven Fo koae isolates used in HARC’s disease resistance breeding program were collected on Hawai‘i (44 and 166), Kauai and 79), and Oahu (90).
- Targeted studies of the Fo koae population are needed to better understand the role of horizontal gene transfer in pathogenicity..
- Population genomic analyses are needed to determine genetic relationships among pathogenic Fo koae and non-pathogenic F.
- In addition, karyotyping could validate the presence and quantity of the putative lineage-specific chromosome(s) of Fo koae 44..
- This study used whole genome analyses to predict virulence-associated genes that may be necessary for the development of koa wilt disease by Fo koae.
- This information was used to develop PCR primers for distinguishing highly virulent Fo koae isolates from non-pathogenic F.
- Genomic compar- isons between Fo koae 44 and Fo 170 identified Fo 170- exclusive genes, including pathogenicity-related genes, which suggests that Fo 170 may be pathogenic to a different host without necessarily causing a wilt disease..
- Pathogen-specific primers were developed that only amplified DNA of character- ized, pathogenic isolates of Fo koae..
- These results provide the framework for under- standing the pathogen genes necessary for development of koa wilt disease and determining genetic variation of Fo koae populations across the Hawaiian Islands..
- De novo genome assemblies of both Fo koae 44 and Fo 170 were constructed using SPAdes using default parameters.
- A whole-genome, maximum likelihood phylogeny was constructed to elucidate evolutionary relationships among Fo koae 44, Fo 170, other Fusarium spp., and other F..
- To determine overall sequence similarity of Fo koae 44 and Fo 170 isolates, average nucleotide identity was calculated using Pyani [56].
- harvest, and TransposonPSI (http://transposonpsi.sour ceforge.net) and subsequently RepeatMasker with this library were used to identify transposable elements and repeat-rich regions for both Fo koae 44 and Fo .
- To identify unique sequences of Fo koae 44 and Fo 170, putative chromosomes were constructed using a closely related reference genome, F.
- Paired reads of the genomes of Fo koae 44 and Fo 170 were trimmed using BBDuk (decontamination using kmers.
- Variant calling format (VCF) was exported for use in comparison of SNPs and indels between Fo koae 44 and Fo 170.
- The de novo assembled genomes were annotated for both Fo koae 44 and Fo 170 using the MAKER 2.31.8 annotation pipeline [67] with RepeatMasker 4.0.8 [60].
- Putative virulence-associated proteins were tested for exclusivity to Fo koae 44 by aligning these sequences to sequences obtained from the NCBI database using MUSCLE in Geneious [75] (Additional File 10)..
- Using these alignments, unique sequences of Fo koae 44 were identified and selected..
- Also, predicted transcripts specific to Fo koae 44 were iden- tified by comparing the predicted transcriptomes of Fo koae 44 and Fo 170 from the Maker annotation identified from Orthovenn2.
- Fo koae 44 predicted transcripts were further identified as unique by using a local BLAST against Fo 170 and the NCBI database.
- Primers were tested for specificity using template DNA from four characterized highly virulent Fo koae isolates (isolates used in HARC greenhouse screening trials and 79), seven characterized moderately virulent Fo koae isolates (HARC isolates and 0540 K), two characterized non-pathogenic Fo isolates (HARC isolates 27 and 170), two F.
- These isolates were tested with the developed Fo koae-specific primers to identify haplotypes that may contain the putative lineage- specific DNA identified from Fo koae 44..
- koae 44.
- Fo koae 44 had a higher proportion of virulence associated genes and Fo 170 had a higher proportion of secondary metabolite genes on the LSX..
- koae 44..
- Fo koae : Fusarium oxysporum f

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