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Population genetics of Septoria pathogens. Isolates of the wheat pathogens M. graminicola and P. nodorum and of the barley pathogen Septoria passerinii were collected in the Midwestern and North Central states. Isozymes and RAPDs were tested for their relative abilities to reveal polymorphisms. An abstract of this work has been published:

Enzyme polymorphism among isolates of Mycosphaerella graminicola (anamorph Septoria tritici) and Septoria passerinii. Guodong Zhang and Stephen B. Goodwin, USDA-ARS, Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, IN 47907-1155. Phytopathology 86:S90.
Cellulose-acetate electrophoresis was used to investigate isoenzyme polymorpohism among 59 isolates of Mycosphaerella graminicola collected from wheat in four states (North Dakota, Ohio, Minnesota and Indiana), and 22 isolates of Septoria passerinii collected from barley in two states (Minnesota and North Dakota) Among 29 enzymes tested, eight gave clear, repeatable results for M. graminicola and nine putative loci were identified. All enzymes but one were polymorphic, although the frequency of the most commion isozyme was over 94% at six of the nine putative loci. The same allele predominated in populations from all four states. Among the eight enzymes that gave the best results for M. graminicola, only seven worked well for S. passerinii, revealing seven putative loci. Six of these were polymorphic, but only two of the six enzymes had the frequency of the most common isozyme greater than 90%. Single-spore isolates from the same leaf seldom had the same zymogram, indicating that usually more than one genotype infected each leaf. The above two species were different at all but one enzyme locus and thus were easily distinguished by isozyme analysis.

In a collaborative project with Gert Kema (Wageningen, The Netherlands), genetic analyses of more than 100 RAPD loci were performed using progeny from a cross between two Dutch isolates of M. graminicola. Four loci were identified that appeared to have "co-dominant" alleles. The putative alleles were cloned, sequenced and converted to Sequence Tagged Sites (aka SCARs). An poster about this work will be presented at the International Congress of Plant Pathology (August 1998). The abstract is presented below. Come see the poster for details.

POPULATION GENETICS OF RAPD VARIATION AND THE DEVELOPMENT OF SCARS IN MYCOSPHAERELLA GRAMINICOLA

STEPHEN B. GOODWIN¹ and GERT H. J. KEMA²

¹USDA-ARS, Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, IN 47907-1155, USA; ²IPO-DLO, P.O. Box 9060, 6700 GW Wageningen, Netherlands

Background and objectives

Septoria tritici blotch, caused by Mycosphaerella graminicola, is an important disease of wheat worldwide. Recently, populations of M. graminicola were analyzed for genetic variation using restriction fragment length polymorphism (RFLP) markers [1]. These studies showed that most populations of M. graminicola worldwide contained high levels of genetic diversity, and that sexual reproduction probably occurs commonly.

Although these studies provided an excellent initial picture of global genetic diversity, many important gaps remain. The major wheat-growing regions of the central United States have not been sampled extensively, and it is not known whether results from other parts of the world are representative of the central United States. The different market classes of wheat in the central United States (hard red spring, hard red winter, soft red winter, durum wheat) might select for different pathogen populations, and differences in the growing seasons might affect the frequency of sexual recombination.

Addressing these questions will require more extensive samples and faster markers. The RFLP technology used in previous studies provides excellent resolution, but is slow and labor intensive compared to other markers. The goal of this research was to develop additional markers and perform a preliminary analysis of genetic diversity within and among populations of M. graminicola in the central United States.

Materials and methods

Isolates of M. graminicola were obtained from infected leaves of hard red spring and soft red winter wheat kindly provided by D. Long (USDA-ARS, Cereal Rust Laboratory). RAPD analysis was performed according to standard protocols. Genetic analyses of RAPD bands were done using progeny of a cross between two Dutch isolates of M. graminicola [2]. Genetic data were analyzed using MAPMAKER and HAPMAP. Specific polymorphic RAPD bands were cloned using a TA Cloning Kit and sequenced by automatic sequencer. DNA sequences were aligned using DNASIS and primers designed using OLIGO.

Results and conclusions

Analyses of 59 isolates of M. graminicola from Minnesota, North Dakota, Indiana and Ohio with 20 RAPD primers revealed that almost every isolate had a unique genotype. Thus, sexual reproduction probably occurs commonly in these populations. The only exceptions were isolates from different lesions on the same leaf, which usually had identical genotypes. This pattern is consistent with epidemics initiated by ascospores, with subsequent spread on the same leaf by asexual pycnidiospores.

Genetic analyses of more than 100 putative RAPD loci on 99 progeny isolates revealed that most behaved as simple Mendelian markers. The loci associated into several loose linkage groups that will be integrated into a more complete genetic map of M. graminicola. Most of the loci segregated for the presence or absence of a band. Knowledge of the genetic basis for each RAPD phenotype simplified scoring and ensured that only reliable bands were retained.

Four loci segregated as if they had "codominant" alleles, i.e., each isolate possessed exactly one band within a certain size range. Putative alternative alleles at each locus were cloned and sequenced. In each case tested, the different bands were identical except for an insertion or deletion of 20-60 base pairs. Thus, they were alternative alleles at single genetic loci. These loci were converted to sequence characterized amplified regions (SCARs) by designing specific primer pairs that amplified the variable regions. The longer primer lengths and increased specificity of SCARs eliminate the problems with RAPDs. Scoring is more reliable because the alleles differ in size rather than varying plus or minus. These markers are now available for analyzing genetic variability within populations of M. graminicola.

References

1. McDonald BA, Zhan J, Yarden O, Hogan K, Garton J, Pettway RE, 1998. Proceedings of the Long Ashton Septoria Conference (in press).
2. Kema GHJ, Verstappen ECP, Todorova M, Waalwijk C, 1996. Current Genetics 30, 251-258.
   

Septoria resistance genes. Recently we have worked with segregating progenies of wheat lines that differ for seporia resistance to find molecular markers linked to the resistance genes. So far, one linked AFLP marker has been identified. Work is in progress to clone this marker and develop it into a sequence tagged site, and to find additional linked markers. A related project involved cloning and characterizing resistance gene analogs from wheat. Two posters on these projects were presented recently. The abstracts are below:

MOLECULAR MAPPING OF A GENE FOR RESISTANCE TO SEPTORIA TRITICI BLOTCH IN WHEAT

Hu, Xueyi (1), Stephen B. Goodwin (1), and Gregory Shaner (2)

1. USDA-ARS, Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, Indiana 47907-1155
2. Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, Indiana 47907-1155

Septoria leaf blotch caused by Mycosphaerella graminicola (aka Septoria tritici) is an important disease of wheat. A population of recombinant inbred lines (RILs) was developed from a cross between the resistant line 72626E2 and the susceptible cultivar Arthur to analyze the inheritance of Septoria resistance and to identify molecular markers linked to the resistance gene. Genetic analysis of RILs indicated that the resistance in line 72626E2 is controlled by a single dominant gene. Bulked segregant analysis of resistant and susceptible RILs was performed with amplified fragment length polymorphism (AFLP) analysis and simple sequence repeat (SSR) or microsatellite markers. Potentially linked AFLP and SSR markers identified from the bulked segregant analysis were scored on the complete RIL population to determine the linkage relationships. Two AFLP markers were loosely linked to the resistance gene at map distances of approximately 14 and 16 cM. A genetic map of the resistance gene will be constructed with additional AFLP and SSR markers using the complete population of recombinant inbred lines.

 

CLONING AND CHARACTERIZATION OF RESISTANCE GENE ANALOGS FROM WHEAT

Hu, Xueyi, and Stephen B. Goodwin

USDA-ARS, Department of Botany and Plant Pathology, 1155 Lilly Hall, Purdue University, West Lafayette, Indiana 47907-1155

Degenerate primers based on the conserved nucleotide binding sequence (NBS) of the cloned plant disease resistance genes N, L6 and RPS2, were used to amplify genomic DNA of wheat lines that were near-isogenic for a powdery mildew resistance gene. Amplification products in the anticipated 500 base-pair size range were extracted from agarose gels and cloned using a TA cloning kit. Following color selection, clones containing inserts of approximately 500 base pairs were identified by digesting plasmid DNA with the restriction enzyme Eco RI and separating the fragments by gel electrophoresis. Restriction analysis of approximately 300 randomly chosen clones using four enzymes with 4- or 6-base recognition sequences separated the putative NBS sequences into at least 10 different classes. Preliminary sequencing of some clones and analyses of the probable translation products revealed that all encoded amino acid sequences similar to protein kinases 2(?) and 3(?) found in the cloned plant disease resistance genes N, L6 and RPS2. Representative clones from each of the 10 resistance gene analog classes are currently being sequenced, and tested for linkage to known powdery mildew resistance genes by Southern analysis of near-isogenic wheat lines.

Potato late blight disease. Although currently not working on late blight, the PI has unpublished data from his postdoctoral work that is still being written up. Recent abstracts from two of these studies are listed below:

Wright's fixation index analysis reveals the probable mating system for 16 species of Phytophthora. Stephen B. Goodwin, USDA-ARS/Purdue University, West Lafayette, Indiana.
Approximately half of the 67 species of Phytophthora are heterothallic; the rest are homothallic. If hetero- or homothallism dictates the mating system, there should be almost no heterozygosity in populations of homothallic Phytophthora species due to self fertilization. In contrast, heterothallic species should contain high levels of heterozygosity. However, levels of heterozygosity within species of Phytophthora so far have not been analyzed. To test whether there are differences in mating system, Wright's fixation index [F = 1 - (HObs / HExp), where HObs is the observed heterozygosity and HExp the expected heterozygosity assuming random mating] was calculated for 16 species of Phytophthora by reanalysis of previously published data. As expected, fixation indices were near 1.0 for four of the six homothallic species. Fixation indices for four of the ten heterothallic species were between zero and 0.3, as expected for random mating populations. The remaining species could be divided into three groups. One group, consisting of three hetero- and one homothallic species, probably had a mixed mating system with intermediate fixation index values near 0.5. A second group of heterothallic species had high fixation index values similar to those for homothallic species, probably due to asexual reproduction or inbreeding. A third group contained two heterothallic species with negative fixation index values. Deviations from expectation probably were due to asexual reproduction, incorrect scoring of some isozyme data, or possibly a Wahlund effect. Many Phytophthora species probably have a mixed mating system in nature that cannot be predicted on the basis of hetero- or homothallism. However, this conclusion is preliminary and must be confirmed by analyses of larger samples from carefully defined populations.

Origin of the A2 mating type of Phytophthora infestans outside Mexico. Stephen B. Goodwin, USDA-ARS/Purdue University, West Lafayette, Indiana. Phytopathology 87: S34.
Cluster analyses of genotypes of Phytophthora infestans from six locations where the A2 mating type was detected recently were used to explicitly test the mating type change hypothesis for the origin of the A2 outside Mexico. Origin by mating type change predicts that A1 and A2 genotypes should be very similar. However, in all six locations (Northwestern Mexico, South America/Costa Rica, Eastern Europe, East Asia, Western Europe, the United States/Canada), A2 genotypes did not cluster with the previously existing A1s and the mating type change hypothesis was falsified. Isolates in new populations worldwide were very different genetically from the predominant (US-1) clonal lineage in old populations. However, the mean number of genetic differences from the US-1 clonal lineage in new populations of P. infestans was not significantly different from that in Mexican populations. Migration is the only viable explanation for these results. Early reports of oospores of P. infestans also were evaluated to determine whether the isolates they described were homothallic. In all cases, oospores were produced only sporadically in old cultures under special conditions. The few oospores produced rarely had antheridia and usually were aborted. None of the isolates described in reports prior to the 1950s matched the characteristics of known self-fertile isolates and thus none could be classified as homothallic. A previous conclusion that A2 isolates were found in Japan during the 1930s also was evaluated and was not supported. There was no evidence for the occurrence of A2 or homothallic isolates of P. infestans in any location prior to their discovery in central Mexico during the 1950s.

The data set for the above analysis is available to anyone wishing to verify these results.

While writing up this work, I compiled a Phytophthora bibliography of papers on the population biology of Phytophthora species. These references are in Phytopathlogy format and correct as far as I know. However, neither the USDA nor I make any guarantees about the accuracy or completeness of this reference list.

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