Comparative genomics reveals mobile pathogenicity chromosomes in Fusarium.

Fusarium species are among the most important phytopathogenic and toxigenic fungi. To understand the molecular underpinnings of pathogenicity in the genus Fusarium, we compared the genomes of three phenotypically diverse species: Fusarium graminearum, Fusarium verticillioides and Fusarium oxysporum f. sp. lycopersici. Our analysis revealed lineage-specific (LS) genomic regions in F. oxysporum that include four entire chromosomes and account for more than one-quarter of the genome. LS regions are rich in transposons and genes with distinct evolutionary profiles but related to pathogenicity, indicative of horizontal acquisition. Experimentally, we demonstrate the transfer of two LS chromosomes between strains of F. oxysporum, converting a non-pathogenic strain into a pathogen. Transfer of LS chromosomes between otherwise genetically isolated strains explains the polyphyletic origin of host specificity and the emergence of new pathogenic lineages in F. oxysporum. These findings put the evolution of fungal pathogenicity into a new perspective.

Variation in the fumonisin biosynthetic gene cluster in fumonisin-producing and nonproducing black aspergilli.

The ability to produce fumonisin mycotoxins varies among members of the black aspergilli. Previously, analyses of selected genes in the fumonisin biosynthetic gene (fum) cluster in black aspergilli from California grapes indicated that fumonisin-nonproducing isolates of Aspergillus welwitschiae lack six fum genes, but nonproducing isolates of Aspergillus niger do not. In the current study, analyses of black aspergilli from grapes from the Mediterranean Basin indicate that the genomic context of the fum cluster is the same in isolates of A. niger and A. welwitschiae regardless of fumonisin-production ability and that full-length clusters occur in producing isolates of both species and nonproducing isolates of A. niger. In contrast, the cluster has undergone an eight-gene deletion in fumonisin-nonproducing isolates of A. welwitschiae. Phylogenetic analyses suggest each species consists of a mixed population of fumonisin-producing and nonproducing individuals, and that existence of both production phenotypes may provide a selective advantage to these species. Differences in gene content of fum cluster homologues and phylogenetic relationships of fum genes suggest that the mutation(s) responsible for the nonproduction phenotype differs, and therefore arose independently, in the two species. Partial fum cluster homologues were also identified in genome sequences of four other black Aspergillus species. Gene content of these partial clusters and phylogenetic relationships of fum sequences indicate that non-random partial deletion of the cluster has occurred multiple times among the species. This in turn suggests that an intact cluster and fumonisin production were once more widespread among black aspergilli.

Identification of gene clusters associated with fusaric acid, fusarin, and perithecial pigment production in Fusarium verticillioides.

The genus Fusarium is of concern to agricultural production and food/feed safety because of its ability to cause crop disease and to produce mycotoxins. Understanding the genetic basis for production of mycotoxins and other secondary metabolites (SMs) has the potential to limit crop disease and mycotoxin contamination. In fungi, SM biosynthetic genes are typically located adjacent to one another in clusters of co-expressed genes. Such clusters typically include a core gene, responsible for synthesis of an initial chemical, and several genes responsible for chemical modifications, transport, and/or regulation. Fusarium verticillioides is one of the most common pathogens of maize and produces a variety of SMs of concern. Here, we employed whole genome expression analysis and utilized existing knowledge of polyketide synthase (PKS) genes, a common cluster core gene, to identify three novel clusters of co-expressed genes in F. verticillioides. Functional analysis of the PKS genes linked the clusters to production of three known Fusarium SMs, a violet pigment in sexual fruiting bodies (perithecia) and the mycotoxins fusarin C and fusaric acid. The results indicate that microarray analysis of RNA derived from culture conditions that induce differential gene expression can be an effective tool for identifying SM biosynthetic gene clusters.

Lae1 regulates expression of multiple secondary metabolite gene clusters in Fusarium verticillioides.

The filamentous fungus Fusarium verticillioides can cause disease of maize and is capable of producing fumonisins, a family of toxic secondary metabolites linked to esophageal cancer and neural tube defects in humans and lung edema in swine and leukoencephalomalacia in equines. The expression of fumonisin biosynthetic genes is influenced by broad-domain transcription factors (global regulators) and Fum21, a pathway-specific transcription factor. LaeA is a global regulator that in Aspergillus nidulans, affects the expression of multiple secondary metabolite gene clusters by modifying heterochromatin structure. Here, we employed gene deletion analysis to assess the effect of loss of a F. verticillioides laeA orthologue, LAE1, on genome-wide gene expression and secondary metabolite production. Loss of Lae1 resulted in reduced expression of gene clusters responsible for synthesis of the secondary metabolites bikaverin, fumonisins, fusaric acid and fusarins as well as two clusters for which the corresponding secondary metabolite is unknown. Analysis of secondary metabolites revealed that, in contrast to a previously described Fusarium fujikuroi lae1 mutant, bikaverin production is reduced. Fumonisin production is unchanged in the F. verticillioides lae1 mutant. Complementation of the F. verticillioides mutant resulted in increased fumonisin production.

Revitalization of a Forward Genetic Screen Identifies Three New Regulators of Fungal Secondary Metabolism in the Genus Aspergillus.

The study of aflatoxin in Aspergillus spp. has garnered the attention of many researchers due to aflatoxin's carcinogenic properties and frequency as a food and feed contaminant. Significant progress has been made by utilizing the model organism Aspergillus nidulans to characterize the regulation of sterigmatocystin (ST), the penultimate precursor of aflatoxin. A previous forward genetic screen identified 23 A. nidulans mutants involved in regulating ST production. Six mutants were characterized from this screen using classical mapping (five mutations in mcsA) and complementation with a cosmid library (one mutation in laeA). The remaining mutants were backcrossed and sequenced using Illumina and Ion Torrent sequencing platforms. All but one mutant contained one or more sequence variants in predicted open reading frames. Deletion of these genes resulted in identification of mutant alleles responsible for the loss of ST production in 12 of the 17 remaining mutants. Eight of these mutations were in genes already known to affect ST synthesis (laeA, mcsA, fluG, and stcA), while the remaining four mutations (in laeB, sntB, and hamI) were in previously uncharacterized genes not known to be involved in ST production. Deletion of laeB, sntB, and hamI in A. flavus results in loss of aflatoxin production, confirming that these regulators are conserved in the aflatoxigenic aspergilli. This report highlights the multifaceted regulatory mechanisms governing secondary metabolism in Aspergillus Additionally, these data contribute to the increasing number of studies showing that forward genetic screens of fungi coupled with whole-genome resequencing is a robust and cost-effective technique.IMPORTANCE In a postgenomic world, reverse genetic approaches have displaced their forward genetic counterparts. The techniques used in forward genetics to identify loci of interest were typically very cumbersome and time-consuming, relying on Mendelian traits in model organisms. The current work was pursued not only to identify alleles involved in regulation of secondary metabolism but also to demonstrate a return to forward genetics to track phenotypes and to discover genetic pathways that could not be predicted through a reverse genetics approach. While identification of mutant alleles from whole-genome sequencing has been done before, here we illustrate the possibility of coupling this strategy with a genetic screen to identify multiple alleles of interest. Sequencing of classically derived mutants revealed several uncharacterized genes, which represent novel pathways to regulate and control the biosynthesis of sterigmatocystin and of aflatoxin, a societally and medically important mycotoxin.

Deletion analysis of FUM genes involved in tricarballylic ester formation during fumonisin biosynthesis.

Fumonisins are carcinogenic mycotoxins produced by the maize ear rot pathogen Gibberella moniliformis (anamorph Fusarium verticillioides). These toxins consist of a linear polyketide-derived backbone substituted at various positions with an amine, one to four hydroxyl, two methyl, and two tricarballylic ester functions. In this study, we generated and characterized deletion mutants of G. moniliformis for five genes, FUM7, FUM10, FUM11, FUM14, and FUM16 in the fumonisin biosynthetic gene cluster. Functional analysis of mutants in four genes, predicted to encode unrelated proteins, affected formation of the tricarballylic esters. FUM7 deletion mutants produced a previously undescribed homologue of fumonisin B1 with an alkene function in both tricarballylic esters, FUM10 and FUM14 deletion mutants produced homologues of fumonisin B3 and fumonisin B4 that lack tricarballylic ester functions, and FUM11 deletion mutants produced fumonisins that lack one of the tricarballylic ester functions. These phenotypes indicated specific roles for FUM7, FUM10, FUM11, and FUM14 in fumonisin biosynthesis that are consistent with the predicted proteins encoded by each gene. Deletion of FUM16 had no apparent effect on fumonisin production. The phenotypes of the deletion mutants provide further insight into the order of steps in fumonisin biosynthesis.

Fumonisin production in the maize pathogen Fusarium verticillioides: genetic basis of naturally occurring chemical variation.

Fumonisins are polyketide-derived mycotoxins produced by the maize pathogen Fusarium verticillioides. Previous analyses identified naturally occurring variants of the fungus that are deficient in fumonisin C-10 hydroxylation or that do not produce any fumonisins. In the current study, gene deletion and genetic complementation analyses localized the C-10 hydroxylation deficiency to a cytochrome P450 monooxygenase gene in the fumonisin biosynthetic gene (FUM) cluster. Sequence analysis indicated that the hydroxylation deficiency resulted from a single nucleotide insertion that caused a frame shift in the coding region of the gene. Genetic complementation localized the fumonisin-nonproduction phenotype to the polyketide synthase gene in the FUM cluster, and sequence analysis indicated that the nonproduction phenotype resulted from a nucleotide substitution, which introduced a premature stop codon in the coding region. These results provide the first direct evidence that altered fumonisin production phenotypes of naturally occurring F. verticillioides variants can result from single point mutations in the FUM cluster.

FUM13 encodes a short chain dehydrogenase/reductase required for C-3 carbonyl reduction during fumonisin biosynthesis in Gibberella moniliformis.

Fumonisins are polyketide-derived mycotoxins produced by the filamentous fungus Gibberella moniliformis (anamorph Fusarium verticillioides). Wild-type strains of the fungus produce predominantly four B-series fumonisins, designated FB(1), FB(2), FB(3), and FB(4). Recently, a cluster of 15 putative fumonisin biosynthetic genes (FUM) was described in G. moniliformis. We have now conducted a functional analysis of FUM13, a gene in the cluster that is predicted by amino acid sequence similarity to encode a short chain dehydrogenase/reductase (SDR). Mass spectrometric analysis of metabolites from FUM13 deletion mutants revealed that they produce approximately 10% of wild-type levels of B-series fumonisins as well as two previously uncharacterized compounds. NMR analysis revealed that the new compounds are similar in structure to FB(3) and FB(4) but that they have a carbonyl function rather than a hydroxyl function at carbon atom 3 (C-3). These results indicate that the FUM13 protein catalyzes the reduction of the C-3 carbonyl to a hydroxyl group and are the first biochemical evidence directly linking a FUM gene to a specific reaction during fumonisin biosynthesis. The production of low levels of FB(1), FB(2), FB(3), and FB(4), which have a C-3 hydroxyl, by the FUM13 mutants suggests that G. moniliformis has an additional C-3 carbonyl reductase activity but that this enzyme functions less efficiently than the FUM13 protein.

Functional roles of FgLaeA in controlling secondary metabolism, sexual development, and virulence in Fusarium graminearum.

Fusarium graminearum, the causal agent of Fusarium head blight in cereal crops, produces mycotoxins such as trichothecenes and zearalenone in infected plants. Here, we focused on the function of FgLaeA in F. graminearum, a homolog of Aspergillus nidulans LaeA encoding the global regulator for both secondary metabolism and sexual development. Prior to gene analysis, we constructed a novel luciferase reporter system consisting of a transgenic F. graminearum strain expressing a firefly luciferase gene under control of the promoter for either TRI6 or ZEB2 controlling the biosynthesis of these mycotoxins. Targeted deletion of FgLaeA led to a dramatic reduction of luminescence in reporter strains, indicating that FgLaeA controls the expression of these transcription factors in F. graminearum; reduced toxin accumulation was further confirmed by GC-MS analysis. Overexpression of FgLaeA caused the increased production of trichothecenes and additional metabolites. RNA seq-analysis revealed that gene member(s) belonging to ~70% of total tentative gene clusters, which were previously proposed, were differentially expressed in the ΔFgLaeA strain. In addition, ΔFgLaeA strains exhibited an earlier induction of sexual fruiting body (perithecia) formation and drastically reduced disease symptoms in wheat, indicating that FgLaeA seems to negatively control perithecial induction, but positively control virulence toward the host plant. FgLaeA was constitutively expressed under both mycotoxin production and sexual development conditions. Overexpression of a GFP-FgLaeA fusion construct in the ΔFgLaeA strain restored all phenotypic changes to wild-type levels and led to constitutive expression of GFP in both nuclei and cytoplasm at different developmental stages. A split luciferase assay demonstrated that FgLaeA was able to interact with FgVeA, a homolog of A. nidulans veA. Taken together, these results demonstrate that FgLaeA, a member of putative FgVeA complex, controls secondary metabolism, sexual development, and virulence in F. graminearum, although the specific regulation pattern differs from that of LaeA in A. nidulans.

Tricarballylic ester formation during biosynthesis of fumonisin mycotoxins in Fusarium verticillioides.

Fumonisins are agriculturally important mycotoxins produced by the maize pathogen Fusarium verticillioides. The chemical structure of fumonisins contains two tricarballylic esters, which are rare structural moieties and important for toxicity. The mechanism for the tricarballylic ester formation is not well understood. FUM7 gene of F. verticillioides was predicted to encode a dehydrogenase/reductase, and when it was deleted, the mutant produced tetradehydro fumonisins (DH4-FB). MS and NMR analysis of DH4-FB1 indicated that the esters consist of aconitate with a 3'-alkene function, rather than a 2'-alkene function. Interestingly, the purified DH4-FB1 eventually yielded three chromatographic peaks in HPLC. However, MS revealed that the metabolites of the three peaks all had the same mass as the initial single-peak DH4-FB1. The results suggest that DH4-FB1 can undergo spontaneous isomerization, probably including both cis-trans stereoisomerization and 3'- to 2'-ene regioisomerization. In addition, when FUM7 was expressed in Escherichia coli and the resulting enzyme, Fum7p, was incubated with DH4-FB, no fumonisin with typical tricarballylic esters was formed. Instead, new fumonisin analogs that probably contained isocitrate and/or oxalosuccinate esters were formed, which reveals new insight into fumonisin biosynthesis. Together, the data provided both genetic and biochemical evidence for the mechanism of tricarballylic ester formation in fumonisin biosynthesis.

Proteomic comparison of Gibberella moniliformis in limited-nitrogen (fumonisin-inducing) and excess-nitrogen (fumonisin-repressing) conditions.

The maize pathogen Gibberella moniliformis produces fumonisins, a group of mycotoxins associated with several disorders in animals and humans, including cancer. The current focus of our research is to understand the regulatory mechanisms involved in fumonisin biosynthesis. In this study, we employed a proteomics approach to identify novel genes involved in the fumonisin biosynthesis under nitrogen stress. The combination of genome sequence, mutant strains, EST database, microarrays, and proteomics offers an opportunity to advance our understanding of this process. We investigated the response of the G. moniliformis proteome in limited nitrogen (N0, fumonisininducing) and excess nitrogen (N+, fumonisin-repressing) conditions by one- and two-dimensional electrophoresis. We selected 11 differentially expressed proteins, six from limited nitrogen conditions and five from excess nitrogen conditions, and determined the sequences by peptide mass fingerprinting and MS/MS spectrophotometry. Subsequently, we identified the EST sequences corresponding to the proteins and studied their expression profiles in different culture conditions. Through the comparative analysis of gene and protein expression data, we identified three candidate genes for functional analysis and our results provided valuable clues regarding the regulatory mechanisms of fumonisin biosynthesis.

Phylogenomic and functional domain analysis of polyketide synthases in Fusarium.

Fusarium species are ubiquitous in nature, cause a range of plant diseases, and produce a variety of chemicals often referred to as secondary metabolites. Although some fungal secondary metabolites affect plant growth or protect plants from other fungi and bacteria, their presence in grain-based food and feed is more often associated with a variety of diseases in plants and in animals. Many of these structurally diverse metabolites are derived from a family of related enzymes called polyketide synthases (PKSs). A search of genomic sequence of Fusarium verticillioides, Fusarium graminearum, Fusarium oxysporum, and Fusarium solani identified a total of 58 PKS genes. To gain insight into how this gene family evolved and to guide future studies, we conducted phylogenomic and functional domain analyses. The resulting geneaology suggested that Fusarium PKSs represent 34 different groups responsible for synthesis of different core metabolites. The analyses indicate that variation in the Fusarium PKS gene family is due to gene duplication and loss events as well as enzyme gain-of-function due to the acquisition of new domains or of loss-of-function due to nucleotide mutations. Transcriptional analysis indicates that the 16 F. verticillioides PKS genes are expressed under a range of conditions, further evidence that they are functional genes that confer the ability to produce secondary metabolites.

FvVE1 regulates biosynthesis of the mycotoxins fumonisins and fusarins in Fusarium verticillioides.

The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus . Whether veA homologues have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologue, FvVE1, on the production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen Fusarium verticillioides . We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 plays an important role in regulating mycotoxin production in F. verticillioides .

The Fusarium verticillioides FUM gene cluster encodes a Zn(II)2Cys6 protein that affects FUM gene expression and fumonisin production.

Fumonisins are mycotoxins produced by some Fusarium species and can contaminate maize or maize products. Ingestion of fumonisins is associated with diseases, including cancer and neural tube defects, in humans and animals. In fungi, genes involved in the synthesis of mycotoxins and other secondary metabolites are often located adjacent to each other in gene clusters. Such genes can encode structural enzymes, regulatory proteins, and/or proteins that provide self-protection. The fumonisin biosynthetic gene cluster includes 16 genes, none of which appear to play a role in regulation. In this study, we identified a previously undescribed gene (FUM21) located adjacent to the fumonisin polyketide synthase gene, FUM1. The presence of a Zn(II)2Cys6 DNA-binding domain in the predicted protein suggested that FUM21 was involved in transcriptional regulation. FUM21 deletion (Deltafum21) mutants produce little to no fumonisin in cracked maize cultures but some FUM1 and FUM8 transcripts in a liquid GYAM medium. Complementation of a Deltafum21 mutant with a wild-type copy of the gene restored fumonisin production. Analysis of FUM21 cDNAs identified four alternative splice forms (ASFs), and microarray analysis indicated the ASFs were differentially expressed. Based on these data, we present a model for how FUM21 ASFs may regulate fumonisin biosynthesis.

Fusarium genomic resources: tools to limit crop diseases and mycotoxin contamination.

It has been almost 10 years since Joan Bennett suggested that fungal biologists create a "wish list" for fungal genome sequences (Bennett JW. White paper: Genomics for filamentous fungi. Fungal Genet Biol 1997; 21: 3-7). The availability of over 200 review papers concerning fungal genomics is a reflection of significant progress with a diversity of fungal species. Although much progress has been made, the use of genomic data to study mycotoxin synthesis and function, pathogenesis and other aspects of fungal biology is in its infancy. Here, we briefly present the status of publicly available genomic resources for Fusarium, a genus of important plant pathogenic and mycotoxin-producing fungi of worldwide concern. Preliminary examination of microarray data collected from F. verticillioides liquid cultures provides evidence of widespread differential gene expression over time.

The putative monomeric G-protein GBP1 is negatively associated with fumonisin B production in Fusarium verticillioides.

SUMMARY Fumonisin B(1) (FB(1)) is a mycotoxin produced by Fusarium verticillioides that contaminates maize. FB(1) has been linked to a number of human and animal mycotoxicoses worldwide. Despite its significance, our understanding of the FB(1) biosynthesis regulatory mechanisms is limited. Here, we describe F. verticillioides GBP1, encoding a monomeric G-protein, and its role in FB(1) biosynthesis. GBP1 was discovered as an expressed sequence tag (EST) up-regulated in the F. verticillioides fcc1 mutant that showed reduced conidiation and no FB(1) biosynthesis when grown on maize kernels. Sequence analysis showed that GBP1 encodes a putative 368-amino-acid protein with similarity to DRG and Obg subclasses of G-proteins that are involved in development and stress responses. A GBP1 knockout mutant (Deltagbp1) exhibited normal growth, but increased FB(1) production (> 58%) compared with the wild-type when grown on corn kernels. Complementation of Deltagbp1 with the wild-type GBP1 gene restored FB(1) production levels to that of the wild-type. Our data indicate that GBP1 is negatively associated with FB(1) biosynthesis but not with conidiation in F. verticillioides. The deletion of GBP1 led to up-regulation of key FB(1) biosynthetic genes, FUM1 and FUM8, suggesting that the increased FB(1) production in Deltagbp1 is due to over-expression of FUM genes.

A bidomain nonribosomal peptide synthetase encoded by FUM14 catalyzes the formation of tricarballylic esters in the biosynthesis of fumonisins.

Fumonisins are a group of polyketide-derived mycotoxins produced by Fusarium verticillioides, a filamentous fungus infecting corn and contaminating food and feeds. Fumonisins contain two tricarballylic esters that are critical for toxicity. Here, we present genetic and biochemical data for the esterification mechanism. FUM14 in F. verticillioides has been deleted by homologous recombination, and the resultant mutant lost the ability to produce fumonisins. Two new metabolites, HFB(3) and HFB(4), which are biosynthetic precursors of fumonisins lacking the tricarballylic esters, were detected in the mutant. The results suggest that FUM14 is required for the esterification of fumonisins. FUM14 was predicted to encode a nonribosomal peptide synthetase (NRPS) containing two domains, peptidyl carrier protein and condensation domain. Both the intact Fum14p and the condensation domain have been expressed in Escherichia coli and purified for activity assays. Fum14p was able to convert HFB(3) and HFB(4) to the tricarballylic esters-containing fumonisins, FB(3) and FB(4), respectively, when incubated with tricarballylic thioester of N-acetylcysteamine. In addition, the condensation domain was able to convert HFB(1) to FB(1). These data provide direct evidence for the role of Fum14p in the esterification of fumonisins. More interestingly, the results are the first example of an NRPS condensation domain catalyzing a C-O bond (ester) formation, instead of the typical C-N bond (amide) formation in nonribosomal peptides. The understanding of the esterification mechanism provides useful knowledge for mycotoxin reduction and elimination. The study also provides new insight into the reactions catalyzed by NRPS.

Comparative analysis of 87,000 expressed sequence tags from the fumonisin-producing fungus Fusarium verticillioides.

Fusarium verticillioides (teleomorph Gibberella moniliformis) is a pathogen of maize worldwide and produces fumonisins, a family of mycotoxins that have been associated with several animal diseases as well as cancer in humans. In this study, we sought to identify fungal genes that affect fumonisin production and/or the plant-fungal interaction. We generated over 87,000 expressed sequence tags from nine different cDNA libraries that correspond to 11,119 unique sequences and are estimated to represent 80% of the genomic complement of genes. A comparative analysis of the libraries showed that all 15 genes in the fumonisin gene cluster were differentially expressed. In addition, nine candidate fumonisin regulatory genes and a number of genes that may play a role in plant-fungal interaction were identified. Analysis of over 700 FUM gene transcripts from five different libraries provided evidence for transcripts with unspliced introns and spliced introns with alternative 3' splice sites. The abundance of the alternative splice forms and the frequency with which they were found for genes involved in the biosynthesis of a single family of metabolites as well as their differential expression suggest they may have a biological function. Finally, analysis of an EST that aligns to genomic sequence between FUM12 and FUM13 provided evidence for a previously unidentified gene (FUM20) in the FUM gene cluster.

FUM9 is required for C-5 hydroxylation of fumonisins and complements the meitotically defined Fum3 locus in Gibberella moniliformis.

Deletion of the Gibberella moniliformis FUM9 gene resulted in mutants that produce only fumonisins that lack a C-5 hydroxyl group. This phenotype is identical to that of previously described mutants with defective alleles at the meiotically defined Fum3 locus. Transformation with a wild-type FUM9 gene into a Fum3-defective mutant restored wild-type fumonisin production. These results indicate that the FUM9 protein catalyzes the C-5 hydroxylation of fumonisins and that FUM9 and the Fum3 locus are the same gene.