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Wageningen University and Research Centre, Plant Research International B.V., P.O. Box 16, 6700 AA, Wageningen, the Netherlands
Masatoki Taga 1
Department of Biology, Faculty of Science, Okayama University, Tsushima-naka, Okayama 700-8530, Japan
Gert H.J. Kema 1
Plant Research International B.V., P.O. Box 16, 6700 AA, Wageningen, the Netherlands
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ABSTRACT |
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The karyotypes of three isolates of Mycosphaerella graminicola, the septoria tritici blotch pathogen of wheat, were analyzed with both pulsed field gel electrophoresis (PFGE) and the cytological technique called germ tube burst method (GTBM). These analyses revealed a chromosome length polymorphism among these isolates. The estimated genome size was 31–40 Mb depending on the isolates, indicating 17–22% redundancy in the genome of the standard strain IPO323 because such differences do not affect development, pathogenicity and sexual reproduction of the other isolates. The chromosome numbers in the three isolates were 18–20 and the chromosome size was 0.3–6 Mb. These data show that M. graminicola has the highest chromosome number and the smallest autosomes (A chromosomes) in filamentous ascomycetes. Our data also confirmed a large (
6 Mb) chromosome that was assembled recently in the IPO323 genome sequence. GTBM analyses revealed the mitotic metaphase chromosomes, enabling chromosome quantification, which was fully congruent with the PFGE analyses. These data will be instrumental in the final assembly of the M. graminicola genome.
Key words: CHEF, contour clamped homogenous field, cytology, electrophoresis, genome size, protoplast preparation, septoria tritici blotch
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INTRODUCTION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, Dean et al 2005, Tyler et al 2006, Lim et al 2004). Despite the advanced resolution of these studies the minute nature of fungal chromosomes significantly delimits our understanding of genome structure and organization in terms of chromosome complements (Wieloch 2006). Some genomes have few chromosomes that can be deduced from genetic linkage studies, but their size may hamper visualization while others have many that complicate precise genetic mapping (Taga et al 2003, Kuhn et al 2006). Moreover aneuploidy, usually deleterious in higher organisms but common in fungi, complicates genome analysis (Selmecki et al 2006).
The ascomycete Mycosphaerella graminicola (Fuckel) J. Schröt. causes the major foliar septoria tritici blotch disease of wheat and is a major representative of the genus Mycosphaerella that is responsible for a range of foliar diseases in many crops (Farr et al 1995). M. graminicola has become an important model for the order Dothideales due to its unique way of living (Goodwin et al 2004, Mehrabi et al 2006, 2006b). The genome of M. graminicola currently is being sequenced at the US Department of Energy Joint Genome Institute (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home.html for public release and http://www.jgi.doe.gov for information). The final sequence assembly of the M. graminicola genome requires accurate karyotypic data, including chromosome number and relative size. Karyotyping of this fungus so far has been carried out with pulsed field gel electrophoresis (PFGE) to obtain electrophoretic karyotypes with transverse alternating field electrophoresis (TAFE) resulting in 17–18 chromosomes, measuring ca. 330–3500 kb, for seven field isolates (McDonald and Martinez 1991). Kema et al (1999, 2002) resolved 13–15 chromosomal DNA bands for the M. graminicola mating type tester strains IPO323 and IPO94269 with contour-clamped homogeneous electric field electrophoresis (CHEF). However, the resolution limitations of PFGE have significantly hampered the establishment of a definitive karyotype of M. graminicola.
In this study we combined PFGE and the germ tube burst method (GTBM; Shirane et al 1988, Taga et al 1994) to analyze the chromosomal complement of M. graminicola isolates that were used in genetic mapping and genomic sequencing programs (Kema et al 2002, Goodwin et al 2007). We conclude that M. graminicola has an unusually wide chromosome size range and the largest chromosome number observed in ascomycetes (see reviews by Beadle et al 2003, Walz 2004).
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MATERIALS AND METHODS |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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, were used in PFGE and GTBM analyses. The Dutch strains are mating type testers and are the parents of the first M. graminicola mapping population (Kema et al 2002, Goodwin et al 2007). The genome of IPO323 currently is sequenced at US Department of Energy Joint Genome Institute, and IPO94241 was included as a reference from earlier electrophoretic karyotypic analysis (McDonald and Martinez 1991). Conditions for growing and storage of the strains were as described by Kema and van Silfhout (1997). Spores used for PFGE and GTBM were produced in yeast glucose broth (YG; 1% yeast extract, 3% glucose) at 18 C by shaking 5 d on an orbital incubated shaker at a speed of 120 rpm. Preparation of chromosomal DNA.— – Spores were collected from YG by centrifugation and washed three times with sterile water and once in 600 mM MgSO4 (pH 5.8). They were partially digested in 1.2 M MgSO4 containing 1 mg/mL Novozyme 234 (Novo Biolabs, Bagsvaecd, Denmark) by incubating 30 min or longer at 30 C. The spores were collected by centrifugation, suspended in STC (1M Sorbitol, 50 mM Tris, pH 8, 50 mM CaCl2) at a concentration of 5 x 108 spore/mL and mixed with an equal volume of 1% low melting agarose (Seakem® gold agarose) prepared with STC and solidified in a mold to make agarose plugs. These were incubated in lysing solution (500 mM EDTA, 10 mM Tris, pH 8.0, and 1% (w/v) N-lauroylsarcosinate, 1 mg/mL proteinase K) for 48 h at 50 C, rinsed in 50 mM EDTA (pH 8.0) and stored in 50 mM EDTA (pH 8.0) at 4 C until use.
Pulsed field gel electrophoresis.— – CHEF equipment (Bio-Rad DRII, Veenendaal, the Netherlands) was used to analyze electrophoretic karyotypes of the isolates. Small chromosomes were separated in 1% SeaKem® gold agarose under 200 V at 11 C with a 60–120 s switching interval for 24 h, while separation of large chromosomes was performed in 0.8% SeaKem® gold agarose using the running conditions, 50 V with a ramped 3600–1800 s switching interval for 115 h, 1800–1300 s for 24 h; 60 V with a 1300–800 s interval for 30 h and 80 V with a 800–600 s interval for 27 h. After electrophoresis gels were stained with ethidium bromide (0.5 µ g/mL) and destained in water 1 h. We used Saccharomyces cerevisiae YPH80, Hansenula wingei YB-4662-VIA and Schizosaccharomyces pombe 972 h size standards for small, medium and large chromosomes (Bio-Rad DRII, Veenendaal, the Netherlands), respectively.
Southern hybridization.— –
Southern analysis was performed by hybridization of alkaline transferred chromosomal DNA from PFGE gels to a nylon membrane (Sambrook et al 1989) with the M. graminicola 32P random prime labeled (Roche Applied Science) PCR-amplified ITS region of rDNA as a probe. PCR was performed with universal primers ITS1 (5'-TCCGTAGGTGAACCTGCGG-3') and ITS4 (5'-TCCTCCGCTTATTGATATGC-3') with these conditions: 3 min at 94 C, followed by 35 cycles of 94 C for 30 s, 55 C for 30 s and 72 C for 90 s with a final extension of 5 min at 72 C. Hybridization was performed at 65 C overnight followed by three washing steps at 65 C for 20 minutes each, with decreasing stringencies at 2 x SSC, 0.2 x SSC and 0.1 x SSC, respectively. Hybridization signals were retrieved with a phosphor-imager (BAS Fuji, Tokyo, Japan).
Cytology.— –
GTBM was performed according to Taga et al (1998) with minor modifications. Spores of IPO323 and IPO94241 produced in YG were harvested by centrifugation and resuspended in fresh YG at a concentration of 2–5 x 105 spores/mL. A drop of 200 µ L spore suspension was placed on a poly-L-lysine-coated glass slide and incubated at 18 C overnight for germination in a humid chamber. YG was replaced with 200 µ L of fresh YG containing 50 µ g/mL thiabendazole (TBZ) to arrest mitosis at metaphase. After an additional incubation for 2.5 h at 22 C the slides were dipped in water to wash off YG, gently immersed in a fixative solution (methanol:acetic acid 17:3) for 30 min, flame-dried and stained with 1 µ g/mL DAPI (4',6-diamidino-2-phenylindole) and dissolved in antifade mounting solution (Johnson and Araujo 1981). Chromosomes were observed under an epifluorescence microscope (Olympus, BH2-RFCA, Tokyo, Japan) by UV excitation. Photographs were taken on 400 ASA/ISO negative color film (Fujicolor Super HG400, Fuji Film, Tokyo, Japan), and the images were digitalized by a film scanner (CoolScan IV, Nikon) and processed with Photoshop version 7 (Adobe).
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RESULTS |
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). We assumed that migration distance of chromosomal bands is a linear function of chromosome size and that more intensely stained bands within one lane contained two similar size chromosomes. With the short running time, 7–9 chromosomes were counted in size range 0.3–0.8 Mb (FIG. 1a), while 13, 10 and 11 chromosomes were estimated for IPO323, IPO94269 and IPO94241, respectively, in size range 1.15–6 Mb with the long running time (FIG. 1b). The summarized data of the electrophoretic karyotypes are shown (TABLE I). We estimate the chromosome numbers for M. graminicola isolates IPO323, IPO94241 and IPO94269 at 20, 19 and 18, respectively. Of note, the three strains hardly shared chromosomes of the same size and we observed 2–3 chromosomal bands in sizes larger than 3.5 Mb that were not reported previously (McDonald and Martinez 1991, Kema et al 2002). The total estimated genome size of the studied isolates varied 31–40 Mb (TABLE I).
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). Because we did not observe hybridization with the 6 Mb bands (FIG. 1
) we conclude that these represent large individual chromosomes.
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), which substantially increased the metaphase frequency in the growing germ tubes due to its antimicrotubular function. Condensed metaphase chromosomes were clumped in the limited nuclear space in intact cells, hence minute chromosomes were difficult to discern in such specimens (see the middle cell denoted as 2 in FIG. 3c). In contrast chromosomes were released discretely from burst germ tubes, which enabled the viewing and counting of even minute chromosomes (FIG. 3a, b). We selected 27 and 35 specimens of isolates IPO323 and IPO94241, respectively, that were of good quality to estimate chromosome numbers (TABLE II). The chromosome counts were not constant and, similar to observations by Tsuchiya and Taga (2001), we frequently encountered smaller numbers than the most frequent count. Such unambiguous observations probably were due to the occurrence of the minute chromosomes that may be easily overlapped by or attached to larger chromosomes. However because the most frequent count represented almost exclusively (except the count 20–21 in IPO94241) the largest chromosome number we decided to adopt this as the best estimate of the cytological chromosome number in isolates IPO323 (N = 20) and IPO94241 (N = 19). The size differences were significant among the chromosomes in each specimen and the largest 6 Mb chromosome was unambiguously identifiable (FIG. 3), which was in accord with the results of PFGE (TABLE I). In rare cases centromere-like constrictions were visible in the large chromosomes (arrow-head in FIG. 3a), while sister chromatids never were distinguished in any of the chromosomes. Overall the results of GTBM were consistent with the PFGE analyses and contributed to a precise estimation of the electrophoretic karyotype of M. graminicola.
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DISCUSSION |
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TOPABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONLITERATURE CITED |
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). Kema et al (2002) observed a large DNA band of ca. 5.7 Mb in M. graminicola isolates IPO323 and IPO94269, which they interpreted as a compressed mass of chromosome fragments derived from multiple chromosomes. This interpretation however was not confirmed by this study because the rDNA probe did not hybridize to the large bands in our Southern analysis. Based on our experience with other filamentous fungi (Taga et al 1998, Tsuchiya and Taga 2001, Suga et al 2002) as well as numerous reports of karyotyping of other filamentous fungi containing > 3.9 Mb chromosome bands (Beadle et al 2003) with CHEF, we concluded that the > 3.5 Mb bands in this study represent individual chromosomes. Moreover the recently released genome sequence of M. graminicola IPO323 revealed a large ( < 6 Mb) scaffold (http://genome.jgi-psf.org/Mycgr1/Mycgr1.home.html) supporting the current observation. Our data also differed from McDonald and Martinez (1991) for the middle and small size chromosomes as shown for M. graminicola isolate IPO94241. The migration of DNA molecules in a PFGE gel depends not only on their size but also on the amount of DNA molecules to be separated in a well. In addition many other parameters, such as running temperature and concentration of agarose and buffer strength, can influence the migration (Beadle et al 2003). Therefore a direct comparison of our current data and that obtained by TAFE (McDonald and Martinez 1991) is difficult. However the use of H. wingei as an additional size marker in addition to S. cerevisiae and S. pombe ascertains our size measurements and analyses.
This study showed that M. graminicola has a distinctive karyotype. One of the characteristics is that the genome comprises many small chromosomes ( < 2 Mb) like budding yeasts but also large chromosomes ( > 3.5 Mb), including one 6 Mb, as in other filamentous ascomycetes. The smallest chromosome we observed was 320 kb in IPO94241, and 7–9 chromosomes smaller than 1 Mb were detected in the three strains. Chromosomes less than 1–2 Mb are called minichromosomes and often supposed to be supernumerary or B-like chromosomes in filamentous fungi (Beadle et al 2003, Covert 1998, Mills and McCluskey 1990). Of interest, most of the small chromosomes of M. graminicola are considered to be autosomes or A chromosomes because (i) they are common among other representatives of the species; (ii) the majority of 1–2 Mb chromosomes does not contain excessive volumes of repetitive sequences (150–200 per Mb) compared to the larger ( > 2 Mb) chromosomes, although those shorter than 1 Mb contain approximately twice this number (C. Crane and S.B. Goodwin, USDA-ARS, Purdue University, pers comm); (iii) they have a regular behavior during meiosis as reflected in karyotypes of isolates within a single ascus (Kema et al 1999). To our knowledge these minute chromosomes of M. graminicola are among the smallest A chromosomes so far found in filamentous ascomycetes (Beadle et al 2003). Thus the genome of M. graminicola combines features of both budding yeasts and most filamentous fungi in terms of chromosome complements. It is intriguing to link this feature to our observations that M. graminicola shows cultural dimorphism with both inducible blastic conidiogenesis and filamentous growth (Mehrabi et al 2006b). Of note, other filamentous genera with blastic conidiogenesis reproduction systems such as Tilletia and Ustilago also contain many small autosomes in their genomes (Kinscherf and Leong 1988, McCluskey and Mills 1990, Russell and Mills 1993).
Another characteristic of the M. graminicola genome is its large chromosome number. We estimated that the three M. graminicola strains have 18–20 chromosomes. Such high numbers also were reported in basidiomycete smuts including Tilletia controversa, T. caries (Russell and Mills 1993), Tilletiopsis washingtonensis (Boekhout et al 1992) and Ustilago maydis (Kinscherf and Leong 1988). However, with the exception of hybrid brewery yeast that contains 20 chromosomes (Jager and Philippsen 1989), M. graminicola has the largest reported chromosome number in the Ascomycota.
Cytology.— –
Chromosome counts in Cladosporium herbarum represent the first and only observation of mitotic chromosomes in the Dothideales (Crackower 1972). However these data resulted from the conventional squash technique that is known to yield erroneous results (Lu 1996) and hence should be reevaluated. Therefore our data provide the first reliable cytological visualization of mitotic chromosomes in the Dothideales. Although GTBM is potentially applicable to various fungi that produce conidial germ tubes, it has been applied so far to only a limited number of species (Chuma et al 2003, Shirane et al 1988, Shirane et al 1989; Taga and Murata 1994, Taga et al 1998, Taga et al 1999, Tsuchiya and Taga 2001, Wieloch 2006). Here we show the added value of cytological karyotyping with GTBM because it enables effective visualization of small mitotic chromosomes in M. graminicola, which supported PFGE karyotyping. To our knowledge the smallest eukaryotic chromosome ever observed under a microscope is the 245 kb chromosome of S. cerevisiae at metaphase I of meiosis (Kuroiwa et al 1986), while the smallest chromosome in M. graminicola IPO323 is 360 kb. Furthermore GTBM is a useful technology whenever chromosomes are too big for PFGE separation, as recently shown in Fusarium graminearum (Gale et al 2005). Taga et al (1998) discussed the merits and disadvantages of PFGE and cytology in fungal karyotyping and recommended the combined use of both PFGE and GTBM to obtain reliable karyotypes. Using both techniques our results were fully compatible and hence reinforced the reliability of our conclusions. Therefore both techniques should be explored whenever karyotyping of other species is considered.
Extensive genome differences among M. graminicola isolates.— –
Extensive variation of genome size in a single species has been observed in plant pathogenic fungi including F. oxysporum, U. hordei and Beauveria bassiana with approximately 13, 7.5 and 10 Mb difference among isolates respectively (Migheli et al 1995, McCluskey and Mills 1990, Viaud et al 1996). We estimated that the genome size for the three M. graminicola isolates is 31–40 Mb, which fits with data from several other fungi that were derived from detailed PFGE studies and genome sequencing projects (Beadle et al 2003, Galagan et al 2003, Dean et al 2005). The difference in genome size between IPO323 and IPO94269 is approximately 17% but does not preclude the generation of viable sexual offspring (Kema et al 2000, Kema et al 2002, Ware et al 2007). This suggests a substantial redundancy in the genome of IPO323 not affecting development, sexual reproduction and pathogenicity and that possibly can be attributed to the larger number and size of chromosomes in this isolate, which might originate from duplication and translocation during meiosis (Wittenberg et al 2007). McDonald and Martinez (1991) reported chromosome length polymorphisms of up to 20% among homologous chromosomes in the isolates they examined and one isolate was probably a partial diploid. The M. graminicola genome sequence will be instrumental in resolving processes generating these polymorphisms and their role in the life strategy of this important plant pathogen.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 The first and second author contributed equally to this work. 
2 Current address: Agricultural Research & Education Organization, Seed & Plant Improvement Institute, P.O. Box 31585-4119, Karaj, Iran.
3 Corresponding author. E-mail: gert.kema{at}wur.nl
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