Genetic Structure of Mycosphaerella graminicola Populations from Iran, Argentina and Australia

SUMMARY Sterol demethylation inhibitors (DMIs) represent one of the largest groups of systemic fungicides that have been used to control agriculturally important fungal pathogens. Knowledge regarding the evolution of fungicide resistance in agricultural ecosystems is fragmentary and a better understanding of the processes driving the development of DMI resistance in populations of fungal pathogens is needed by plant pathologists and the agrochemical industry. We considered some of these processes using approaches based on molecular population and quantitative genetics. Five Mycosphaerella graminicola populations sampled from unsprayed wheat fields on four continents were assayed for eight restriction fragment length polymorphism (RFLP) markers and their level of tolerance to cyproconazole. DMI fungicides such as cyproconazole inhibit the enzyme eburicol 14-alpha-demethylase. The gene encoding this target, CYP51, was sequenced for all isolates. We found unimodal, continuous variations in cyproconazole tolerance among the M. graminicola isolates sampled from individual fields, consistent with a polygenic mode of inheritance. We also found that population differentiation for cyproconazole tolerance (Q(ST)) among the five M. graminicola populations was significantly higher than the corresponding population differentiation for neutral RFLP markers (G(ST)), suggesting that selection for cyproconazole tolerance in the Swiss population has already led to local adaptation that can be seen even in an unsprayed population. The Swiss population displayed the highest level of tolerance to cyproconazole, in addition to a lower than expected quantitative variation in fungicide tolerance and a skewed distribution, indicating that selection had increased the overall tolerance of this population. Further analysis with DNA sequencing showed that the population from Switzerland was dominated by isolates with several point mutations and a 6-bp deletion in CYP51. This deletion and one of the point mutations were previously related to increased resistance in field isolates. The fungal population from Oregon sampled from an unsprayed resistant host cultivar displayed the same gene diversity in RFLP loci but higher cyproconazole tolerance and quantitative variation in tolerance than the fungal population from the same field sampled from an unsprayed susceptible host cultivar.

Restriction fragment length polymorphism (RFLP) markers were used to compare the genetic structure of field populations of Rhynchosporium secalis from barley. A total of 543 isolates representing 8 field populations were sampled from Australia, California, Finland, and Norway. Gene and genotype diversity were high in all populations. Nei's average gene diversity across seven RFLP loci was 0.513. Hierarchical gene diversity analysis showed that 9% of the total genetic variability was distributed among continents, 4% was distributed among fields within continents, and 13% was distributed among collection stations within a field. The majority (74%) of genetic variability was distributed within collection areas of approximately 1 m(2) within fields. Gene flow appears to be significant on a regional scale but more restricted among continents. Allele frequencies were significantly different at several RFLP loci. Genetic distances were small among populations within regions and large between regions. Pairwise comparisons of genotype diversity in the populations revealed significant differences among populations that were related mainly to differences in sampling strategies. Isolates from Norway and Finland showed a lower copy hybridization pattern with probe pRS26. This probe functioned as a fingerprint probe for the California and Australian isolates. Seven out of the eight populations studied were at gametic equilibrium for RFLP loci, suggesting that R. secalis populations in Norway, Finland, and Australia undergo regular recombination, although a teleomorph has not yet been recognized.

Genetic comparisons between North American and European populations of Lumbricus terrestris L.

The global genetic structure of the wheat pathogen Mycosphaerella graminicola is characterized by high nuclear diversity, low mitochondrial diversity, regular recombination, and gene flow

Recent development of DNA markers provides powerful tools for population genetic analyses. Amplified fragment length polymorphism (AFLP) markers result from a polymerase chain reaction (PCR)-based DNA fingerprinting technique that can detect multiple restriction fragments in a single polyacrylamide gel, and thus are potentially useful for population genetic studies. Because AFLP markers have to be analysed as dominant loci in order to estimate population genetic diversity and genetic structure parameters, one must assume that dominant (amplified) alleles are identical in state, recessive (unamplified) alleles are identical in state, AFLP fragments segregate according to Mendelian expectations and that the genotypes of an AFLP locus are in Hardy-Weinberg equilibrium (HWE). The HWE assumption is untestable for natural populations using dominant markers. Restriction fragment length polymorphism (RFLP) markers segregate as codominant alleles, and can therefore be used to test the HWE assumption that is critical for analysing AFLP data. This study examined whether the dominant AFLP markers could provide accurate estimates of genetic variability for the Aedes aegypti mosquito populations of Trinidad, West Indies, by comparing genetic structure parameters using AFLP and RFLP markers. For AFLP markers, we tested a total of five primer combinations and scored 137 putative loci. For RFLP, we examined a total of eight mapped markers that provide a broad coverage of mosquito genome. The estimated average heterozygosity with AFLP markers was similar among the populations (0.39), and the observed average heterozygosity with RFLP markers varied from 0.44 to 0.58. The average FST (standardized among-population genetic variance) estimates were 0.033 for AFLP and 0.063 for RFLP markers. The genotypes at several RFLP loci were not in HWE, suggesting that the assumption critical for analysing AFLP data was invalid for some loci of the mosquito populations in Trinidad. Therefore, the results suggest that, compared with dominant molecular markers, codominant DNA markers provide better estimates of population genetic variability, and offer more statistical power for detecting population genetic structure.

Resistance to Plasmodiophora brassicae Wor. race 7, the causal agent of the disease clubroot, was examined in an F2 population of a cross between a clubroot resistant broccoli (Brassica oleracea var. italica) and a susceptible cauliflower (B. oleracea var. botrytis). A genetic linkage map was constructed in the same population based on the segregation of 58 dispersed restriction fragment length polymorphism (RFLP) markers. Associations between the inheritance of RFLP marker genotypes and segregation for disease resistance, morphological and maturity characteristics were examined. For each triat examined, several chromosomal regions marked by RFLP probes appeared to contain trait loci, suggesting that each trait was under polygenic control. RFLP marker linkage to a major factor imparting dominance for clubroot resistance from the broccoli parent was observed in this population. Additionally, RFLP marker linkage to an independently segregating factor contributing clubroot resistance from the cauliflower parent was observed, indicating that it should be possible to use RFLP markers to facilitate selection of transgressive segregants having the combined resistance from both parental sources. In some instances, RFLP markers from the same or closely linked chromosomal regions were associated with both clubroot resistance and morphological traits. Analysis of RFLP marker genotypes at linked loci should facilitate the selection of desired disease resistant morphotypes.

A total of 243 individuals from Red Jungle Fowl (Gallus gallus spadiceus), Rugao, Anka, Wenchang and Silikes chicken populations were used for polymorphism analysis in functional apoVLDL-II gene by Restriction fragment length polymorphism and single strand conformation polymorphism markers. The results show that Anka population has highest gene diversity and Shannon information index, while Red jungle fowl shows highest effective number of allele. In addition, the higher coefficient of genetic differentiation (Gst) across all loci in apoVLDL-II was indicating that high variation is proportioned among populations. As expected total gene diversity (Ht) has upper estimate compared with within population genetic diversity (Hs) across all loci. The mean Gst value across all loci was (0.194) indicating about 19.4% of total genetic variation could be explained by breeds differences, while the remaining 80.6% was accounted for differences among individuals. The average apoVLDL-II gene flow across all loci in five chicken populations was 1.189. The estimates of genetic identity and distance confirm that these genes are significantly different between genetically fat and lean population, because fat type breed Anka shows highest distance with the other Silikes and Rugao whish are genetically lean. In addition, Wenchang and Red jungle fowl were found more closely and genetically related than the other breeds with 49.4% bootstrapping percentages, then they were related to Silikes by 100% bootstrapping percentages followed by Rugao and finally all of them are related with exotic fat breed Anka.

Bruchids (genus Callosobruchus) are among the most destructive insect pests of mungbeans and other members of the genus, Vigna. Genetic resistance to bruchids was previously identified in a wild mungbean relative, TC1966. To analyze the underlying genetics, accelerate breeding, and provide a basis for map-based cloning of this gene, we have mapped the TC1966 bruchid resistance gene using restriction fragment length polymorphism (RFLP) markers. Fifty-eight F2 progeny from a cross between TC1966 and a susceptible mungbean cultivar were analyzed with 153 RFLP markers. Resistance mapped to a single locus on linkage group VIII, approximately 3.6 centimorgans from the nearest RFLP marker. Because the genome of mungbean is relatively small (estimated to be between 470 and 560 million base pairs), this RFLP marker may be suitable as a starting point for chromosome walking. Based on RFLP analysis, an individual was also identified in the F2 population that retained the bruchid resistance gene within a tightly linked double crossover. This individual will be valuable in developing resistant mungbean lines free of linkage drag.

Random amplified polymorphic DNA (RAPD) and restriction fragment length polymorphism (RFLP) markers were used to estimate intra- and inter-specific variation in three species of an exclusive mangrove genus, Avicennia. Intrapopulation polymorphism among the 10 populations of Avicennia marina, as measured by percentage of polymorphic RAPDs, varied between 17.8 and 38.9%, with a standard deviation of 7.28, and the coefficient of variation was 26.5%. Polymorphism in Avicennia officinalis (Pichavaram population, 32.3%) and Avicennia alba (Coringa population, 37.8%) was greater than the intrapopulation variation observed in the populations of A. marina from each of the respective locations. It was greater than the average percentage of polymorphism at the intrapopulation level (27.47%) but far less than the variation measured at the interpopulation level in A. marina. Interpopulation variation in A. marina (76.7% for RAPDs and 66% for RFLPs) was greater than the variation in any individual population of this species, indicating a high degree of divergence between the populations. Interpopulation variation as revealed by RAPD and RFLP markers did not indicate the existence of more than one distinct entity in this species in India. The implications of these observations in genetic sampling and conservation are discussed. Statistical analysis of 109 RAPDs and 84 RFLPs observed in one representative genotype from each species showed that the widely distributed A. marina was more closely related to A. alba (genetic distance (1 − F) = 0.22) than to A. officinalis (genetic distance (1 − F) = 0.37). RAPD analysis of six randomly selected genotypes in each species and principal component analysis of the data also favoured this observation.

Molecular markers are being used to tag genes conferring resistance to bacterial blight of rice. Near-isogenic lines (NILs) for the resistance genes were compared with the susceptible recurrent parent, and polymorphic markers were used for linkage analysis on segregating populations. The genes Xa-1, Xa-2 and Xa-10 were located on restriction fragment length polymorphism (RFLP) linkage map. Xa-1and Xa-2, on chromosome 4, were linked to the RFLP marker XNpb264 at distances 1.97 cM and 2.04 cM, respectively. Xa-10 was located on chromosome 11, 15.7 cM from RFLP marker XNpb78. Random amplified polymorphic DNA (RAPD) markers flanking Xa-1 and Xa-10 were also identified using NILs. One marker was tightly linked to Xa-1. No recombinant was observed between Xa-1 and this marker in 84 F2 plants. Two markers were linked to Xa-10 at distances of 6.3 cM and 19.0 cM, respectively. IR-BB3 and IR-BB4, NILs with Xa-3 and Xa-4, respectively, were analyzed by using RFLP markers. In both lines, introgressed segments containing Xa-3 and Xa-4 were retained at the same end of the RFLP map of chromosome 11.

Two classes of molecular markers, RFLPs (restriction fragment length polymorphisms) and RAPDs (random amplified polymorphic DNA) were used to assess genetic diversity among Australian bean rust isolates (Uromyces appendiculatus (Pers.) Unger var. appendiculatus) collected from diverse geographic locations spanning the period 1973-1990. Initially we screened 22 isolates using WLPs from five DNA probes. This was followed by analysis of a subset of 12 of these isolates using RFLPs from 10 cDNA probes and RAPDs from 10 arbitrary primers for a comprehensive evaluation of background genotype. Polymorphic bands revealed the existence of two divergent clusters of isolates, representing genotypes designated A and B. The RFLP markers showed 30% band dissimilarity between A and B, and RAPDs 16% dissimilarity. Isolates in a third cluster (genotype AB) exhibited most of the polymorphic bands present in A and B, but no unique polymorphic bands of their own, indicating that they had most probably arisen from recent hybridisation between isolates of genotype A and B. The subset of 12 isolates included 10 race phenotypes, but no clear correlation between background genotype (as assessed by RFLP and RAPD markers) and race phenotype was evident. We suggest that Australian races of bean rust have most probably evolved by a combination of mutation to virulence from common A and B background genotypes, and genetic recombination.

Paleontological evidence and current patterns of angiosperm species richness suggest that European biota experienced more severe bottlenecks than North American ones during the last glacial maximum. How well this pattern fits other plant species is less clear. Bryophytes offer a unique opportunity to contrast the impact of the last glacial maximum in North America and Europe because about 60% of the European bryoflora is shared with North America. Here, we use population genetic analyses based on approximate Bayesian computation on eight amphi-Atlantic species to test the hypothesis that North American populations were less impacted by the last glacial maximum, exhibiting higher levels of genetic diversity than European ones and ultimately serving as a refugium for the postglacial recolonization of Europe. In contrast with this hypothesis, the best-fit demographic model involved similar patterns of population size contractions, comparable levels of genetic diversity and balanced migration rates between European and North American populations. Our results thus suggest that bryophytes have experienced comparable demographic glacial histories on both sides of the Atlantic. Although a weak, but significant genetic structure was systematically recovered between European and North American populations, evidence for migration from and towards both continents suggests that amphi-Atlantic bryophyte population may function as a metapopulation network. Reconstructing the biogeographic history of either North American or European bryophyte populations therefore requires a large, trans-Atlantic geographic framework.

Genetic structure of <i>Mycosphaerella fijiensis</i> populations from Australia, Papua New Guinea and the Pacific Islands

Restriction fragment length polymorphism (RFLP) markers were used to determine the genetic structure of Australian field populations of the barley scald pathogen Rhynchosporium secalis. Fungal isolates were collected by hierarchical sampling from five naturally infected barley fields in different geographic locations during a single growing season. Genetic variation was high in Australian R. secalis populations. Among the 265 fungal isolates analyzed, 214 distinct genotypes were identified. Average genotype diversity within a field population was 65% of its theoretical maximum. Nei's average gene diversity across seven RFLP loci was 0.54. The majority (76%) of gene diversity was distributed within sampling site areas measuring 1 m(2); 19% of gene diversity was distributed among sampling sites within fields; and 5% of gene diversity was distributed among fields. Fungal populations from different locations differed significantly both in allele frequencies and genotype diversities. The degree of genetic differentiation was significantly correlated with geographic distance between populations. Our results suggest that the R. secalis population in Western Australia has a different genetic structure than populations in Victoria and South Australia.

Restriction fragment length polymorphism (RFLP) markers derived from the yellow fever mosquito, Aedes aegypti, were used in hybridizations to genomic DNA of the following mosquito species: Ae. albopictus, Ae. togoi, Armigeres subalbatus, Culex pipiens, and Anopheles gambiae. Interspecific hybridization with Ae. aegypti probes varied from 50% (An. gambiae) to 100% (Ae. albopictus) under high stringency conditions. We demonstrated the usefulness of using RFLP profiles to examine genetic diversity between mosquito populations; Ae. aegypti RFLP markers were used to examine genetic relatedness between 10 laboratory strains of Ae. aegypti as well as between nine populations representing four Cx. pipiens subspecies. These results indicate that many Ae. aegypti RFLP markers should have direct applicability in gaining a better understanding of genome structure in other mosquito species, including RFLP linkage mapping and determinations of genetic relatedness among field populations.