ABSTRACT
Fusarium verticillioides is an agriculturally important fungus because of its association with maize and its propensity to contaminate grain with toxic compounds. Some isolates of the fungus harbor a meiotic drive element known as Spore killer (SkK) that causes nearly all surviving meiotic progeny from an SkK × Spore killer-susceptible (SkS) cross to inherit the SkK allele. SkK has been mapped to chromosome V but the genetic element responsible for meiotic drive and spore killing has yet to be identified. In this study, we used cleaved amplified polymorphic sequence markers to genotype individual progeny from an SkK × SkS mapping population. We also sequenced the genomes of three progeny from the mapping population to determine their single nucleotide polymorphisms. These techniques allowed us to refine the location of SkK to a contiguous 102-kb region of chromosome V, herein referred to as the Sk locus. Relative to SkS genotypes, SkK genotypes have one extra gene within this locus for a total of 42 genes. The additional gene in SkK genotypes, named SKL1 for Spore Killer Locus 1, is the most highly expressed gene from the Sk locus during early stages of sexual development. The Sk locus also has three hypervariable regions, the longest of which includes SKL1. The possibility that SKL1, or another gene from the Sk locus, is an essential component of meiotic drive and spore killing is discussed.
INTRODUCTION
Fusarium verticillioides is an ascomycete fungus that can exhibit both endophytic and pathogenic growth on maize (Bacon and Hinton 1996; White 1998). The fungus is of agriculture concern because of its ability to cause maize ear and stalk rot, and also because it can contaminate maize kernels with a group of mycotoxins known as fumonisins (Marasas 2001; Brown and Proctor 2016). The problems caused by F. verticillioides and its myoctoxins were noticed as early as 1970, when the fungus was correlated with an outbreak of Equine Leukoencephalomalacia (ELEM) in South Africa (Kellerman et al. 1972). Since then, fumonisins have been confirmed to cause ELEM in horses (Marasas et al. 1988; Kellerman et al. 1990), pulmonary edema in pigs (Harrison et al. 1990), and liver cancer in laboratory rats (Gelderblom et al. 1988, 1991). Fumonisin-contaminated grain has also been linked to neural tube defects in humans (Missmer et al. 2006). Thus, the potential presence of fumonisins, even in apparently healthy grain, makes it necessary to aggressively screen grain for these mycotoxins before it is used in food or feed. Contaminated grain must be destroyed, resulting in crop losses worth millions of dollars per year (Wu, 2007).
The primary sources of inoculum for endophytic and pathogenic growth of F. verticillioides on maize are asexual spores, such as macroconidia and microconidia (Munkvold 2003). Sexual spores, called ascospores, may also be important to the F. verticillioides life cycle (Reynoso et al. 2009). Sexual reproduction in this heterothallic fungus begins when the immature fruiting bodies of one strain are fertilized by a strain of opposite mating type (Martin et al. 2011). Fruiting bodies are called perithecia and meiosis occurs in spore sacs, called asci, within the perithecia. At the start of meiosis, a single diploid nucleus is formed from the haploid genomes of both parents. This diploid nucleus separates into four haploid nuclei through the standard reductional and equational division stages of meiosis, and subsequently, each of these four haploid nuclei undergo a post-meiotic mitosis to form eight nuclei. Each nucleus is then incorporated into a separate ascospore through a process known as ascospore delimitation. A phenotypically normal ascus should thus contain eight ascospores at maturity in F. verticillioides.
During normal development, asci break down and ascospores are exuded from perithecia in gelatinous masses. However, it is possible to examine ascospores within intact asci by perithecial dissection. Kathariou and Spieth used this procedure to discover an allele named Spore killer (SkK). In SkK × SkS crosses, where the latter stands for Spore killer-susceptible, asci contain four instead of eight ascospores (Kathariou and Spieth 1982). Early stages of ascus development are cytologically-normal in these crosses, but four ascospores degenerate shortly after ascospore delimitation (Raju 1994). The four surviving ascospores are almost always of the SkK genotype (Kathariou and Spieth 1982; Xu and Leslie 1996). Unlike SkK × SkS crosses, SkK × SkK crosses produce asci with eight ascospores (Kathariou and Spieth 1982). Thus, the SkK allele is sufficient for spore killing and resistance to spore killing.
The ability of SkK-ascospores to kill SkS-ascospores suggests that SkK could be the predominant allele in some F. verticillioides populations. Although the literature is limited on this subject, a screen of 225 F. verticillioides isolates from 24 fields across Europe and North America found the SkK allele to be present in 81% of isolates (Kathariou and Spieth 1982). Shortly after the discovery of SkK in F. verticillioides, a spore killing allele was also discovered in F. subglutinans. This allele was also named Spore killer, but it was given the slightly different notation of SKk. Along with the report of its discovery, Sidhu (1984) presented evidence that the SKk allele was present in 10 of 15 F. subglutinans isolates obtained from a single maize field. Therefore, SkK and SKk appear to be the predominant alleles in at least some populations of F. verticillioides and F. subglutinans.
The relationship between SkK and SKk is still unclear despite both being discovered more than 30 years ago. To our knowledge, the only other significant research involving either allele was performed by Xu and Leslie (1996) during construction of a genetic map for F. verticillioides. During this study, SkK was mapped between two restriction fragment length polymorphism (RFLP) markers on chromosome V. These markers, named RFLP1 and 11p18, are located 2.5 cM and 8.6 cM from SkK, respectively (Xu and Leslie 1996). Xu and Leslie's mapping population was derived from an SkK × SkS cross, and interestingly, only one progeny from over 100 did not inherit the SkK allele (Xu and Leslie 1996). This evidence demonstrates that SkK can achieve transmission rates of over 99 percent in laboratory crosses.
An F. verticillioides reference genome, derived from an SkS strain known as Fv149-SkS, was published 14 years after the initial mapping of SkK (Ma et al. 2010). Here, we advance Fusarium Spore killer research by delineating physical borders for SkK with respect to the F. verticillioides reference genome. Our data place SkK within a 102-kb contiguous sequence of DNA, which we refer to as the Sk locus. Notable differences exist between the Sk locus in Fv999-SkK and Fv149-SkS strains. These are described and discussed below.
MATERIAL AND METHODS
Strains, media and culture conditions
Key strains are listed in Table 1. Vegetative propagation was performed on V8 Juice Agar (VJA) (Tuite, 1969) in 16 mm test tubes at room temperature on a laboratory benchtop. Carrot agar (CA), which was originally described by Klittich and Leslie (1988), was prepared as follows: 200 g of organic peeled baby cut carrots were autoclaved in 200 ml of water, pureed with a blender, then adjusted to 500 ml with sterile water to create a 1× stock. Diluted CA (e.g., 0.1× and 0.25×) was prepared by mixing the appropriate volumes of 1× CA stock and sterile water, before adding agar to a final concentration of 2% and autoclaving. Liquid Vogel’s Minimal Medium (VMM, Vogel 1956) or GYP medium (2% glucose, 1% peptone, and 0.3% yeast extract) were used to produce mycelia for genomic DNA isolation. Liquid cultures for genomic DNA isolation were incubated at 28 °C in the dark without agitation.
Sexual crosses
Crosses were performed on CA in a similar manner to previously described methods (Klittich and Leslie 1988). Asexual spores (conidia) from the female parent were transferred to the center of a 60 mm petri dish containing 20 ml of CA or diluted CA. The female parent was then cultured for 10-14 days before fertilization with a suspension of conidia from the male parent. Conidial suspensions were prepared by adding 2.0 ml of 0.001% Tween-20 to a 10-14 day old test-tube culture of the male parent and dislodging the conidia with a pipette tip. Fertilization was performed by transferring 1.0 ml of a conidial suspension of the male parent to the surface of a culture of the female parent. The conidial suspension was spread over the surface of the female culture with a glass rod. Crosses were incubated in a culture chamber that alternated between 23.0 °C (12 hours, light) and 22.5 °C (12 hours, dark). Light was provided by white (Philips F34T12/CW/RS/EW/ALTO) and black (General Electric F40T12BL) fluorescent lamps.
The Fv999-SkK × Fv149-SkS mapping population
In F. verticillioides, ascospores are exuded from mature perithecia in a hair-like structure called a cirrus. To obtain an SkK mapping population, Fv999-SkK was crossed with Fv149-SkS and cirri were isolated from the tops of a few perithecia with a sterile needle, dispersed in sterile water, and spread onto a plate of 4% water agar. Germinating ascospores were transferred to VJA in 16 mm test tubes.
Microscopy
Asci were dissected from perithecia in 25% glycerol under magnification. A Vanguard 1433Phi light microscope and attached digital camera (Amscope MU1000) were used for imaging. The condenser and aperture diaphragm were set for high contrast, which allowed for the number of ascospores in mature asci to be determined without tissue staining.
DNA methods
Genomic DNA was prepared using one of three methods. In method one, strains were cultured in 25 ml liquid VMM at 28 °C in the dark for three days. Mycelia were washed with 0.9% NaCl and dried by lyophilization before extraction with IBI Scientific’s Genomic DNA Mini Kit for Plants. Method two, based on Henderson et al. (2005), was used as an inexpensive and time-efficient alternative to method one. A 6” plain-tipped wood applicator was used to transfer ≤ 10 mg of conidia to 200 μl of TE buffer (10 mM Tris, 1 mM EDTA, pH 8.0). The suspension was boiled at 105 °C in a heat block for 12 minutes, incubated on ice for 2 minutes, then vortexed for 5 seconds. Insoluble material was pelleted at 15,000 × g for 10 minutes at room temperature, after which 25 μl of supernatant was transferred to a new vial and frozen at −20 °C for storage.In our hands, this method works well for the amplification of PCR products shorter than 1 kb. Method three was used for the preparation of genomic DNA for high-throughput sequencing. Strains were cultured in 25 ml of liquid GYP at 28 °C for 48 hrs. Mycelia was washed with water and DNA was extracted with the Zymo Research Fungal DNA Miniprep Kit.
Polymerase chain reaction (PCR) assays were performed with MidSci Bullseye Taq DNA Polymerase or New England Biolabs Phusion High-Fidelity DNA Polymerase.
Genome sequencing
DNA libraries for MiSeq sequencing (Illumina) were constructed from 1 ng of genomic DNA using the Illumina Nextera XT DNA Library Preparation Kit. Sequencing was performed with MiSeq Reagent Kit Version 3. Adapters were removed and low-quality reads were trimmed with CLC Genomics Workbench (Version 8.0). The datasets were deposited in the National Center for Biotechnology Information’s (NCBI) Sequence Read Archive (Leinonen et al. 2011). They can be obtained with the following accession numbers: SRR3271586 (Fv999-SkK), SRR3273544 (JP98.75-SkK), SRR3273545 (JP98.111-SkK), and SRR3273546 (JP98.118-SkK). The data sets are of high-quality. For example, draft genomes were assembled with CLC Genomics Workbench and all assemblies had N50 values over 90 kb with coverage levels between 49 and 73 fold (Table 2).
CAPS markers
Cleaved amplified polymorphic sequence (CAPS) markers (Konieczny and Ausubel 1993) were used to help refine the location of SkK (Table 3). CAPS markers were identified by first aligning genome sequencing reads from strain Fv999-SkK to the Fv149-SkS reference genome (NCBI, ASM14955v1) and then visually scanning aligned reads with Tablet (Milne et al. 2010) for polymorphisms in GGCC sites. This four-base sequence is cleaved by the restriction endonuclease HaeIII. Six CAPS markers on chromosome V were chosen for this study, along with one CAPS marker on each of chromosomes I, VII, and XI (Table 3). PCR-primers for each CAPS marker were designed to amplify a short product (< 500 bp) from both Fv999-SkK and Fv149-SkS sequences. The PCR primers used for amplification of each CAPS marker are described in Table S1.
The following protocol was used to analyze the segregation patterns of CAPS markers in each individual of the Fv999-SkK × Fv149-SkS mapping population. Genomic DNA was isolated from each progeny, CAPS markers were amplified by PCR, and PCR products were digested with HaeIII. The digested products were then examined for Fv999-SkK or Fv149-SkS cleavage patterns by gel electrophoresis on standard 1% agarose-TAE (40mM Tris, 20mM acetic acid, and 1mM EDTA) gels. The HaeIII-based DNA digest patterns of each CAPS marker for Fv999-SkK and Fv149-SkS alleles are listed in Table S2.
Single nucleotide polymorphisms (SNPs)
Reads from each MiSeq dataset were aligned to the reference genome of strain Fv149-SkS with Bowtie 2 (Langmead and Salzberg 2012). SAMtools 1.3 and BCFtools 1.3 were then used to report SNPs in variant call format (VCF) (Li et al. 2009; Danecek et al. 2011). Only reads that matched to a single region of Fv149-SkS with less than 10 mismatches were used to produce the VCF files. Custom Perl scripts were then used to extract significant SNPs from the VCF files. Significant SNPs were defined as those which were supported by at least 90% of reads at each position. Only positions covered by more than nine reads were considered. Polymorphisms caused by insertions or deletions were ignored.
Gene predictions
Protein-coding genes within the Sk locus of Fv999-SkK were predicted as follows: first, the sequence of the Sk locus in Fv999-SkK was obtained by de novo assembly of MiSeq reads (Table 2); second, the coding sequences of annotated genes from the Sk locus of Fv149-SkS were obtained from GenBank (CM000582.1); third, the Fv149-SkS coding sequences were aligned to the Sk locus of Fv999-SkK with Clustal Omega (Sievers et al. 2011; Li et al. 2015); and fourth, the alignments were used to manually annotate protein-coding sequences within the sequence of the Fv999-SkK Sk locus. These steps identified 41 putative protein-coding genes within the Sk locus of Fv999-SkK. Augustus 3.2.1 (Stanke and Waack 2003) was then used for de novo prediction of protein-coding genes. Augustus predictions matched our manual annotation with the notable exception of an additional identified gene (SKL1, described below) within the Sk locus of Fv999-SkK. The complete sequence and annotation of the 102-kb SkK locus from Fv999-SkK can be obtained from GenBank with accession number KU963213.
RESULTS
Increased production of fruiting bodies by strain Fv999-SkK on diluted CA
Directional crosses are often used to investigate sexual processes in heterothallic ascomycete fungi. In this type of cross, one parent is designated as the female and the other is designated as the male. The female provides protoperithecia, i.e., immature fruiting bodies, which are fertilized by conidia from the male. After fertilization, protoperithecia develop into perithecia.
Carrot Agar (CA) is commonly used as a growth medium to study sexual processes in F. verticillioides. Its preparation essentially involves purchasing carrots from a local market, followed by peeling, blending, and autoclaving a predetermined weight of carrots within a specified volume of water (Leslie and Summerell 2006). Because we were curious about how the amount of carrot in CA medium influences the productivity of Fv999-SkK × Fv149-SkS crosses, Fv999-SkK was cultured on 0.001×, 0.01×, 0.1×, 0.25×, 0.5×, and 1.0× CA, then fertilized with Fv149-SkS conidia. The crosses performed on 0.1× and 0.25× CA resulted in nearly three to six fold more perithecia than the crosses performed on 0.5× and 1.0× CA (Figure 1). No perithecia were produced when crosses were performed on 0.001× and 0.01× CA (Figure 1). Additionally, less conidia were produced on 0.1× CA than on 0.25× CA (data not shown). Because conidia can be a source of cross-contamination, 0.1× CA was used as the crossing medium for the remainder of this study.
RFLP 11p18 overlaps with FVEG_02851 on chromosome V
Although the sequence of RFLP1 was not available, we obtained the sequence of RFLP 11p18 from the Fusarium Comparative Database. We used this sequence as the query in a BLASTn (Altschul et al. 1990) search of the F. verticillioides reference genome. This search identified positions 1672753 to 1673602 on chromosome V (CM000582.1) as a match to 11p18. This region overlaps with most of the predicted coding sequence of gene FVEG_02851, which spans positions 1672886 to 1673769 and encodes a protein of unknown function. Its position on chromosome V was used to choose CAPS markers near the Sk locus (Figure 2A).
Loci linked to SkK drive through meiosis
To begin refining the location of SkK on chromosome V, Fv999-SkK was crossed with Fv149-SkS and the segregation patterns of nine CAPS markers were examined in 60 progeny. Both Fv999-SkK and Fv149-SkS patterns were inherited in a 1:1 ratio for chromosomes XI, VII, and I. In contrast, Fv999-SkK patterns of CAPS markers on chromosome V were inherited more frequently (>= 79.6%) than expected (Figure 2B and Table S3). The only exception was CAPS-5, which is located approximately 20 kb from a telomere (Figure 2A and Table 3). Overall, these results mark the first independent confirmation of Xu and Leslie’s (1996) original findings on the biased recovery of molecular markers linked to SkK in SkK × SkS crosses.
Genome sequencing refines the Sk locus to a 102-kb region of chromosome V
To delineate the position of SkK on chromosome V, we narrowed our focus to progeny JP98.75-SkK, JP98.111-SkK, and JP98.118-SkK. Even though CAPS marker analysis failed to identify an Fv999-SkK region of chromosome V that was inherited by all three progeny (Figure 2C, black shaded regions), all were determined to carry the SkK allele by phenotypic analysis (Figure 3 and data not shown). Therefore, their genomes were sequenced (Table 2) and their SNPs along chromosome V were examined. This analysis allowed us to identify two additional recombination events. Both of these recombination events occurred between CAPS markers CAPS-3 and CAPS-4 in the ascus producing progeny JP98.111-SkK (Figure 2D), explaining why they were not revealed by our CAPS marker analysis. More importantly, the SNP profiles of all three progeny require that SkK be located between positions 665,669 and 767,411 (Figure 2D, red lines) with respect to the reference sequence (GenBank, CM000582.1). We refer to this 102-kb contiguous sequence as the Sk locus (GenBank, KU963213).
SkK strains carry a unique gene in the Sk locus
The Sk locus spans 102,256 bases in Fv999-SkK, but only 101,743 bases in Fv149-SkS. A ClustalW-based alignment (Thompson et al. 1994) of the sequences from both strains is 102,557 bases long, with 301 gaps in the Fv999-SkK sequence and 814 gaps in the Fv149-SkS sequence (Figure 4). To identify specific regions in SkK and SkS strains with high levels of SNPs or gaps, the number of SNPs or gaps were calculated for each 100-base window across the alignment. A qualitative analysis of these results finds at least three hypervariable regions (Figure 4). One of the shorter hypervariable regions spans genes FVEG_03180 to FVEG_03175, while another spans genes FVEG_03174 and FVEG_03173 (Figure 4). The longest hypervariable region is approximately 14 kb long, spans gene FVEG_03167, and extends to the right border of the Sk locus (Figure 4).
The 102-kb Sk locus in strain Fv149-SkS includes 41 putative protein coding genes (Figure 4 and Table 4). The Sk locus in Fv999-SkK carries the same 41 genes as well as one additional protein coding gene, SKL1 (for Spore Killer Locus 1), that is located between FVEG_03165 and FVEG_03164 (Figure 4). BLASTp analysis (Altschul et al. 1997) of the NCBI non-redundant protein database with the predicted 70-amino acid sequence of the putative Skl1 protein identified hypothetical proteins with Expect values ranging from 2 × 10-49 to 8 ×10-04 from various formae speciales of the F. oxysporum species complex. No significant hits were identified in other species (data not shown). A search of the NCBI Conserved Domain Database (Marchler-Bauer et al. 2010) failed to identify a domain within SKl1.
To confirm that SKL1 is absent in Fv149-SkS, the sequences corresponding to the FVEG_03165-FVEG_03164 intergenic region from Fv999-SkK and Fv149-SkS were aligned and examined. This intergenic region consists of 1,234 and 755 bases in Fv999-SkK and Fv149-SkS respectively (Figure S1). Examination of the alignment revealed that this difference in length was due to the presence of SKL1 in Fv999-SkK and its absence in Fv149-SkS (Figure S1). To confirm that the absence of SKL1 in Fv149-SkS was not due to an error in the reference genome sequence, we examined the lengths of the FVEG_03165-FVEG_03164 intergenic region by PCR in our laboratory stocks of Fv999-SkK and Fv149-SkS. The PCR product lengths were consistent with the presence of SKL1 in Fv999-SkK and its absence in Fv149-SkS (Figure 5).
ClustalW alignments were used to investigate the level of polymorphism between proteins encoded by the Sk locus in Fv999-SkK and Fv149-SkS strains (Table 4). Analysis of these pairwise alignments revealed that Fveg_15199 is the most polymorphic protein of the locus. Only 80% of the 133 amino acids of Fveg_15199 are identical between the two strains. This is a remarkably high level of polymorphism given that 34 of the 41 shared proteins within the Sk locus of Fv999-SkK and Fv149-SkS are greater than 94% identical, and 40 of 41 are greater than 88% identical (Table 4). A BLASTp search of the NCBI non-redundant protein database identified homologs of Fveg_15199 in several Fusarium, Neonectria, Neosartorya, and Aspergillus species, among others (data not shown). A search of the NCBI Conserved Domain Database failed to identify a domain within Fveg_15199.
To shed light upon the transcriptional profile of protein-coding genes within the Sk locus, we analyzed five F. verticillioides RNAseq datasets from NCBI’s Sequence Read Archive. These datasets were for five time points following the induction of sexual development in a cross of Fv999-SkK × Fv149-SkS (Sikhakolli et al. 2012). Of the genes within the Sk locus, FVEG_03171 exhibited the greatest increase in expression from the first to last time point (Table 5), suggesting that this gene may have an important role during later stages of sexual development. The corresponding protein, Fveg_03171, includes a WSC-domain (data not shown), which has been linked to carbohydrate-binding, cell wall integrity, and stress response. The gene FVEG_03194 exhibited the greatest fold change over all five time points (>1050×) (Table 5). The Fveg_03194 protein does not have any recognized domains, although it is conserved in Fusarium, Nectria, Trichoderma, Colletotrichum, and a few other fungi (data not shown). Interestingly, SKL1 reached the highest level of expression of all examined genes 2 hours after fertilization (Table 5). Furthermore, SKL1 transcript levels were second only to FVEG_03197 at the last time point (144 h, Table 5). The latter gene appears to be widely conserved among bacteria and eukaryotes (data not shown), but a function for it or its homologs is unknown.
DISCUSSION
The segregation of alternate alleles into separate gametes during meiosis encourages genetic conflict (Burt and Trivers 2008). Evidence for this is found in meiotic drive, which occurs when an allele is transmitted through meiosis in a biased manner. Meiotic drive elements are found in a diverse range of fungi, where they achieve biased transmission through sexual reproduction by killing meiospores carrying an alternate allele (Turner and Perkins 1979; Kathariou and Spieth 1982; Raju 1994; Dalstra et al. 2003; Grognet et al. 2014). Thus, fungal meiotic drive elements are often referred to as Spore killers. The molecular basis of meiotic drive by most Spore killers is unknown.
The existence of meiotic drive by spore killing in Fusarium was first recognized in 1982. More than three decades later, we still do not understand the molecular mechanism that mediates this process. This is similar to the situation in Neurospora, where three distinct spore killers, known as Sk-1, Sk-2, and Sk-3, were identified in 1979 (Turner and Perkins 1979). A breakthrough in Sk-2 research was recently made by refining the location of an Sk-2 resistance gene to a 52-kb sequence of DNA (Hammond, Rehard, Harris, et al. 2012). This provided the necessary foundation to clone and characterize the Sk-2 resistance gene (Hammond, Rehard, Xiao, et al. 2012), which, in turn, allowed for the isolation of killer-less Sk-2 mutants (Harvey et al. 2014). Here, we have taken a similar approach towards identifying the genetic basis for SkK in F. verticillioides by first refining the position of SkK to a 102-kb region of chromosome V.
As with previous work on SkK (Xu and Leslie 1996), we observed a biased transmission of SkK-linked molecular markers (Figure 2B). This is not unexpected since the driving ability of SkK should also affect the transmission of alleles linked to SkK. This phenomenon is referred to as genetic hitchhiking (Lyttle 1991). The closer a hitchhiker is to a meiotic driver, the more likely it is to be transmitted to the next generation through the sexual cycle. In our study, patterns of CAPS markers from the parent Fv999-SkK were inherited at a higher frequency than could be attributed to chance alone for five of six markers on chromosome V, and the transmission bias generally decreased with increasing distance from the Sk locus (Figure 2B). Surprisingly, CAPS-1 and CAPS-2 demonstrated higher levels of hitchhiking than CAPS-4 (Figure 2B), despite the latter being closer to the Sk locus (Figure 2D). One explanation for this could be the existence of a recombination hotspot between CAPS-4 and the Sk locus; however, we do not have additional data to support this hypothesis.
While a recombination hotspot could explain the unexpected decrease in hitchhiking for CAPS-4, the opposite phenomenon of recombination-suppression is often associated with meiotic drive elements (reviewed by Lyttle 1991). For example, Neurospora Sk-2 requires specific alleles of at least two genes to mediate drive, a resistance gene called rsk and a killer gene called rfk (Hammond, Rehard, Xiao, et al. 2012; Harvey et al. 2014). Each gene is located on a different arm of chromosome III (Harvey et al. 2014). For Sk-2 to succeed as a meiotic driver, it is imperative that an Sk-2 ascospore inherit both rsk and rfk alleles because loss of rsk from Sk-2 leads to a self-killing genotype (Hammond, Rehard, Xiao, et al. 2012). This helps explain why Sk-2 is associated with a 30 cM long “recombination-blocked” region of chromosome III (Campbell and Turner 1987; Harvey et al. 2014). By suppressing recombination between rsk and rfk, Sk-2 prevents these critical components of its meiotic drive mechanism from separating during meiosis. With respect to F. verticillioides SkK, recombination suppression, if it exists at all, does not appear to be a significant phenomenon. For example, our CAPS marker analysis identified twelve recombination events between CAPS-3 and CAPS-4 (n = 59, Table S3), and two recombination events between CAPS-2 and CAPS-3 (n = 59, Table S3). This relatively high number of recombination events near the Sk locus argues against an Sk-2-like recombination block for SkK.
If F. verticillioides SkK requires multiple genes to function as a meiotic drive element, all should be found within the 102-kb Sk locus defined by this study. For example, SNP-profiling of progeny JP98.75-SkK, JP98.111-SkK, and JP98.118-SkK indicated that only this region of chromosome V is common between them and the Fv999-Skk parent (Figures 2D and Figure 4). Assuming spore killing is mediated by two or more distinct genes within the Sk locus, the close proximity of these alleles may negate the requirement for a Neurospora Sk-2-like recombination block. Alternatively, meiotic drive and spore killing may be mediated by a single gene. A fungal precedent for this is seen in the het-s spore killing mechanism of Podospora anserina (Coustou et al. 1997; Saupe 2011). This system is controlled by two alternate alleles of a single gene, named het-S and het-s. The former, het-s, encodes the HET-s prion and is the meiotic driver, while the latter, het-S, encodes the HET-S protein. Interaction of the HET-s prion with HET-S causes HET-S to relocate to cell membranes, resulting in cell death, presumably through loss of membrane integrity (Seuring et al. 2012). The het-s ascospores escape cell death because they do not produce the HET-S protein, and thus the het-s prion is not toxic to them. This is one example of how meiotic drive and spore killing can be mediated by alternate alleles of a single gene in fungi. Grognet et al. (2014) have recently identified spok1 and spok2, two additional spore killing genes in P. anserina, both of which appear to be single-gene-based meiotic drive systems.
In the current study, comparative sequence analysis revealed multiple differences between the 102-kb Sk locus from SkK and SkS strains of F. verticillioides. Presumably, one or more of these differences is the genetic basis for the different Sk phenotypes exhibited by the strains. An analysis of SNPs and gaps identified at least three hypervariable regions within the locus (Figure 4). The interspersed nature of these regions is somewhat surprising and suggests that they could be under the influence of different selective pressures than the less variable regions of the Sk locus. The most striking difference between the Sk locus is the presence the SKL1 gene in SkK strains and its absence in SkS strains. Differences between the hypervariable regions and SKL1 in SkK versus SkS strains raise at least two key questions: 1) are the hypervariable regions and/or SKL1 responsible for spore killing, and 2) do they correspond to the SkK allele that was previously defined by phenotypic analysis (Kathariou and Spieth 1982). The high levels of expression of SKL1 throughout sexual development are consistent with its involvement in sexual reproduction. However, in addition to SKL1 and the hypervariable regions, there are also numerous less dramatic sequence differences between genes and intergenic regions of the Sk locus in SkK and SkS strains. The current data does not rule out the possibility that one or more of these differences is responsible for the SkK and SkS phenotypes. As a result, our efforts are now focused on targeted deletions of SKL1 and other regions of the Sk locus to determine the genetic basis of the spore killing phenotype in F. verticillioides.
ACKNOWLEDGEMENTS
We thank the Fungal Genetics Stock Center (McCluskey et al., 2010) for some of the strains used in this study and the Broad Institute of Harvard and MIT (www.broad.mit.edu) for access to the Fusarium Comparative Database. Dr. John Leslie (Kansas State University) and Dr. Frances Trail (Michigan State University) kindly donated Fv999-SkK and Fv149-SkS for this study. Chris McGovern and Nathane Orwig of NCAUR provided much appreciated technical assistance. We thank Dr. Sven Saupe (CNRS - Université de Bordeaux) for sharing his knowledge of the het-s spore killing mechanism. TMH was supported by a grant from the Eunice Kennedy Shriver National Institute of Child Health and Human Development of the National Institutes of Health (NICHD, 1R15HD076309-01). Mention of trade names or commercial products in this article is solely for the purpose of providing specific information and does not imply recommendation or endorsement by the US Department of Agriculture. USDA is an equal opportunity provider and employer.
Footnotes
Data deposition: Genome sequencing data are available through NCBI’s Sequence Read Archive under accession numbers SRR3271586, SRR3273544, SRR3273545, and SRR3273546. Other sequence data are available from GenBank (KU963213).