Conceived and designed the experiments: AHJW TAJVdL GHJK HJS. Performed the experiments: AHJW TAJVdL SBM SBW AK. Analyzed the data: AHJW TAJVdL SBM GHJK HJS. Contributed reagents/materials/analysis tools: AK. Wrote the paper: AHJW TAJVdL SBG RGFV GHJK HJS.
Current address: Keygene N.V., Wageningen, The Netherlands
The authors have declared that no competing interests exist.
Meiosis in the haploid plant-pathogenic fungus
Fungi provide attractive model systems to analyze processes that occur during meiosis. Many fungi are haploid, which greatly simplifies genetic studies. Furthermore, complete recovery of the meiotic products, or tetrads, is possible in ascomycete fungi, and these tetrads can be analyzed for the segregation of genetic markers. Tetrad analyses of
Analyses of a cross between two
The exact origin and maintenance of CNPs and CLPs are not known. A likely hypothesis is that they can be generated or lost during meiosis. Recombination between chromosomes that differ in length could give rise to derivatives with CLPs
We used three isolates of
Generation of genomic representations, library construction, target preparation and image analysis were essentially performed as described previously
AFLP markers were designated by the primer combination used for the amplification and the approximate length of the generated fragment
The binary scores of polymorphic markers were converted to the correct allelic phase based on the scores of the parents. A Perl script was written that grouped loci with identical segregation patterns after disregarding unknown scores. The marker with the highest call rate (percentage of scored individuals) was selected as a representative for each group. The script also calculated the call rate for each individual genotype and the global call rate for the whole dataset. Individual genotypes were incorporated into the scoring table when at least 95% of the grouped markers could be scored. In
The genetic linkage maps of the individual crosses as well as the bridge map were constructed with the software package JoinMap 3.0
We used graphical genotyping to compare the marker scores (A or B) and the phase (A or B) of the markers, which enabled us to identify whether each marker was present or absent in a particular progeny isolate. In cases where a linkage group (LG) was constructed from both marker types and a specific progeny isolate lacked all of these markers, we concluded that the isolate missed that LG. In cases where a LG was constructed from both marker types and a specific progeny isolate was scored present for all markers, we concluded that the isolate had an extra copy, derived from the other parent, of that particular LG. Hence, chromosome polymorphisms in progeny isolates were determined
DNA samples of the parental isolates (IPO323, IPO94269 and IPO95052), progeny isolates that showed absence of specific LGs by graphical genotyping (
PCR reactions were performed in a total volume of 20 µl containing 20 ng of genomic DNA, 1×PCR buffer (Roche), 1 µl of each of the forward and reverse primers used as a control (2 µM), 2 µl of each forward and reverse primer (2 µM), 0.8 µl of dNTPs (5 mM) and 0.2 µl of Taq DNA polymerase (5 U/µl). Amplification conditions were as follows: 94°C for 2 min, 12 cycles of 94°C for 30 sec, 66°C for 30 sec minus 1°C per cycle, 72°C for 30 sec; 27 cycles of 94°C for 30 sec, 53°C for 30 sec, 72°C for 30 sec; 72°C for 7 min, followed by a cooling-down step to 10°C. The SSR amplicons were separated on 6% non-denaturating acrylamide gels using a Mega-Gel Dual High-Throughput Vertical Electrophoresis Unit (CBS Scientific, Del Mar, California, USA). Amplicons based on the DArT sequences were separated on 2.5% agarose gels.
Among the 68 progeny isolates from the
The combined genetic linkage maps contain 2078 markers comprising 1793 DArT, 258 AFLP, and 25 SSR DNA markers, plus the two markers that co-segregate with the biological traits
The new genetic linkage map of the IPO323×IPO94269 cross is 638 cM longer than the first linkage map, and spans 1854 cM with 1317 markers on 451 unique map positions, with an average distance of 4.1 cM between the markers (
We also constructed a bridge map to compare the individual linkage maps using markers that segregated in both mapping populations. The resulting integrated map spans 1435 cM (∼75% of both individual maps) and contains 372 markers on 251 unique map positions. A total of 22 LGs from each of the individual crosses was aligned with the bridge map, and the marker order was similar to those on the two individual genetic maps (
Common markers are shown in bold and start with the prefix C, SSR markers are shown in blue and markers that are translocated in red. DArT markers were named according to phase of the marker (A = IPO323, B = IPO95052 or IPO94269), complexity reduction method used (BMR or HMR), and location in the spotting plate (e.g. BBMR_15L11). LG and AFLP nomenclature is according to Kema et al., 2002. Segregation distortion of the markers is indicated with * (P<0.05), ** (P<0.01), *** (P<0.005) or **** (P<0.001).
We identified eight DArT markers that were positioned very differently in the two maps, which is indicative of translocations. They represented five translocations between isolate IPO323 and either IPO94269 or IPO95052 and involved four inter-LG and one intra-LG translocations (CBBMR_14G17 in LG 6) (
LGs 21 and C in the
Graphical genotyping allows the tracing of the genetic make up of progeny isolates. Among the progeny of the
A. Meiosis starts with the merging of nuclei from two different strains, leading to a transitory diploid cell. Karyogamy is followed immediately by meiosis I and II, resulting in four haploid cells. These four cells are duplicated during a subsequent mitotic step, leading to eight ascospores per ascus. Each ascospore is genetically identical to one other ascospore within the same ascus. Such pairs of identical ascospores are called twins. We identified several twins in progenies of M. graminicola. When a strain of a descendant lacked one or more chromosomes, the twins originating from the first mitotic cell division after meiosis always appeared to lack the same chromosomes. This indicates that chromosomes are stable during mitosis but can be lost during meiosis. B. Chromosome loss during meiosis can be a result of failure of separation of homologous chromosomes during meiosis I, or C. of the failure of separation of sister chromatids during meiosis II. D. Graphical genotyping of LG 8. The chromosomal segments descending from IPO323 are rendered in red, and the segments from IPO95052 in blue. Markers are scored as present (black) or absent (white). As the marker scores on all linkage groups were identical for these two isolates, we concluded that the descendants 2137 and 2139 are twins. However, both isolates lack all markers located on LG 8. This is a clear indication of absence of this linkage group in these isolates. Strikingly, this linkage group is present in both parents. For further verification, seven DArT markers spanning the length of LG 8 were converted into simple PCR markers. In addition, one SSR marker was used. All markers appeared to be absent in the twin isolates 2137 and 2139. This confirms the absence of LG 8 in these twins and indicates nondisjunction during meiosis as the cause. E. Nondisjunction not only results in loss of a chromosome in one twin but also to disomy for that chromosome in another twin from the same ascus. The graphical genotyping of isolate #51 illustrates heterozygous disomy for LG 1, which was confirmed by a PCR screen for deletion markers that unequivocally showed the presence of two copies of this chromosome in this haploid fungus.
The large number of markers permitted easy identification of identical progeny and allowed determination of the stage at which CNPs were generated. In total, we detected 31 twins in the
The observed aberrations and graphical genotyping analyses were confirmed by PCR assays (
To confirm the disomy for LG 1 in progeny isolate #51, we performed PCR assays based on deletion polymorphisms (
In summary, the high-density mapping enabled the detection of meiotically driven and frequently occurring CNPs and CLPs in sexual progenies of the haploid plant pathogen
The genome of
Dispensable chromosomes have been found in other fungi but they usually occur at a low frequency and typically represent single or a few chromosomes. For example the plant-pathogenic fungi
Genome instability is a major cause of disorders, and a range of genes has been identified that have a role in maintaining genome integrity
It is very clear that meiosis not only maintains but also drives novel CNPs in
The high number of markers on the current linkage map enabled accurate identification of twin isolates. These originate from the mitotic division after meiosis and provided a unique opportunity to test the meiotic origin of CNPs. If CNPs resulted from aberrations during mitosis, twin isolates would show differences in chromosome number and could not have been identified. In
Meiotic processing of CNPs in other fungi varies. For the related ascomycete
Apart from these differences and the fact that
In contrast, all LGs in the entire progeny set of the IPO323×IPO95052 cross contain markers from both parents, indicating that all parental chromosomes have homologous partners. Hence, in this respect the differences between the two Dutch bread wheat isolates (IPO323 and IPO94269) seem to be larger than were those between IPO323 and the Algerian durum wheat isolate IPO95052, underscoring the extraordinarily large genetic differences within local populations of
CLPs have been observed in at least 37 fungal species and hence seem to be a common feature of fungal genomes
Compared to CLPs, CNPs in other fungi are observed less frequently, have not been analyzed through a map-based approach, and are generally highly unstable. For instance, a minichromosome in
In contrast to other species, CNPs in
In summary, our map-based approach is unique in analyses of genomic plasticity and demonstrates that CNPs in
Supplementary text
(0.06 MB DOC)
Co-linearity of genetic linkage maps for Mycosphaerella graminicola crosses IPO323×IPO95052 (left) and IPO323×IPO94269 (right) with a bridge map (middle) generated with markers that segregated in both crosses. Common markers are shown in bold and start with the prefix C, SSR markers are shown in blue and markers that are translocated in red. DArT markers were named according to phase of the marker (A = IPO323, B = IPO95052 or IPO94269), complexity reduction method used (BMR or HMR), and location in the spotting plate (e.g. BBMR_15L11). LG and AFLP nomenclature is according to Kema et al., 2002. Segregation distortion of the markers is indicated with * (P<0.05), ** (P<0.01), *** (P<0.005) or **** (P<0.001).
(0.21 MB PDF)
Alignment of linkage group 21 between the IPO323×IPO95052 cross (left) and the IPO323×IPO94269 cross (right) shows recombination in the former but not in the latter. This indicates absence of this linkage group in isolate IPO94269. For IPO323×IPO94269, only markers from IPO323 could be mapped on this linkage group, and no markers from IPO94269, confirming that IPO94269 lacks this linkage group. Lines are drawn between markers that segregated in both populations. Stars next to the markers for the IPO323×IPO94269 cross indicate segregation distortion of the markers; * (P<0.05), ** (P<0.01), *** (P<0.005) or **** (P<0.001).
(0.03 MB PDF)
Confirmation of chromosome loss by PCR amplification. A. Confirmation of loss of LG 8 and LG 12 by SSR amplification. Loci ac-0007 (LG 8) and gga-0001 (LG 12) confirm that these linkage groups are absent in the underlined progeny isolates from the crosses IPO323×IPO94269 and IPO323×IPO95052 as neither of the parental alleles are amplified. Isolates 1158 and 1179 are positive controls and SSR ag-0003 (LG 2) is a positive PCR control in all duplex reactions. B. Confirmation of loss of LGs 13, 15, A and C by PCR with primers developed from DArT marker sequence data in the underlined progeny isolates derived from crosses between M. graminicola IPO323×IPO94269 and IPO323×IPO95052. Isolates 1158 and 1179 are positive control isolates, except in LGs C and 13 that have isolates 1158/2026 and 2032/2033, respectively, as positive checks. For LG 15* the CABMR_07D07 DArT fragment (129 bp) was used as a positive PCR control, while for the other linkage groups DArT fragment AHMR_08O09 (728 bp) was used. C. Confirmation of loss of LG 8 by PCR with primers developed from DArT marker sequence data in underlined progeny isolates derived from crosses between M. graminicola IPO323×IPO94269 and IPO323×IPO95052. This figure is composed of eight panels that are individually divided by a central marker lane. The left part of each panel represents the three parental isolates of the mapping populations (IPO323, IPO94269 and IPO95052), two positive control isolates (1158/1179), and seven progeny isolates that lack LG 8. The right part of each panel links to
(1.60 MB PDF)
Mycosphaerella graminicola progeny isolates (n = 76) from the IPO323×IPO94269 in planta cross, that was made on the susceptible bread wheat cultivar Obelisk, that were used for hybridization to the DArT arrays.
(0.05 MB DOC)
Mycosphaerella graminicola progeny isolates (n = 164) from the IPO323×IPO95052 in planta crosses that were made on the bread wheat cultivar Obelisk and the durum wheat cultivar Inbar. Sixteen isolates (gray-shaded) were not used, leaving a total of 148 that were used in the construction of the genetic linkage map. The first two numbers indicate the year of isolation and the next three numbers the order of isolation.
(0.08 MB DOC)
The adapter and primer oligonucleotide sequences used for generation of the genomic representation (cloning) from Mycosphaerella graminicola isolates IPO323 and IPO95052 and for hybridization to the micro-arrays (genotyping) of parental and progeny isolates.
(0.04 MB DOC)
Overview of Mycosphaerella graminicola F1 isolates that lack one or more linkage groups compared to the parental isolates IPO323, IPO94269 and IPO95052.
(0.03 MB DOC)
Primer sequences used to verify the absence of several linkage groups in some progeny isolates of the two crosses. The primers were developed using the sequences of the DArT markers located on these linkage groups.
(0.07 MB DOC)
Primer sequences used to verify the disomy for linkage group 1, isolate #51. The primers were developed around InDels obtained by comparison of BAC-end sequences from parental isolate IPO94269 with the genome sequence of isolate IPO323.
(0.04 MB DOC)
Overview of type and number of molecular markers that were scored in the progeny of the cross between Mycosphaerella graminicola isolates IPO323 and IPO94269 before and after grouping.
(0.03 MB DOC)
Overview of type and number of molecular markers that were scored in the progeny of the cross between Mycosphaerella graminicola isolates IPO323 and IPO95052 before and after grouping.
(0.04 MB DOC)
Identified twin isolates in the two progenies derived from crosses between either Mycosphaerella graminicola isolates IPO323 and IPO94269 or IPO323 and IPO95052.
(0.04 MB DOC)
Scoring tables
(3.33 MB XLS)
Overview of the number of markers for both crosses. Mapping was performed using the software package JoinMap 3.0.
(0.03 MB DOC)
Graphical genotyping
(5.15 MB XLS)
Alignment of the identified linkage groups in the Mycosphaerella graminicola IPO323×IPO94269 and IPO323×IPO95052 mapping populations with the identified chromosomes in the Mycosphaerella graminicola genome sequence.
(0.05 MB DOC)
DArT and SSR markers that showed translocations between two genetic linkage maps derived from crosses between either Mycosphaerella graminicola isolates IPO323 and IPO94269 or IPO95052.
(0.03 MB DOC)
Back crosses and intercrosses of M. graminicola IPO323×IPO94269 progeny isolates with isolates that either lost or gained specific chromosomes.
(0.05 MB DOC)
We thank Els C.P. Verstappen for generating the