genalex is a user-friendly cross-platform package that runs within Microsoft Excel, enabling population genetic analyses of codominant, haploid and binary data. Allele frequency-based analyses include heterozygosity, F statistics, Nei's genetic distance, population assignment, probabilities of identity and pairwise relatedness. Distance-based calculations include amova, principal coordinates analysis (PCA), Mantel tests, multivariate and 2D spatial autocorrelation and twogener. More than 20 different graphs summarize data and aid exploration. Sequence and genotype data can be imported from automated sequencers, and exported to other software. Initially designed as tool for teaching, genalex 6 now offers features for researchers as well. Documentation and the program are available at http://www.anu.edu.au/BoZo/GenAlEx/

genalex 6: genetic analysis in Excel. Population genetic software for teaching and research

The utility of RFLP (restriction fragment length polymorphism), RAPD (random-amplified polymorphic DNA), AFLP (amplified fragment length polymorphism) and SSR (simple sequence repeat, microsatellite) markers in soybean germplasm analysis was determined by evaluating information content (expected heterozygosity), number of loci simultaneously analyzed per experiment (multiplex ratio) and effectiveness in assessing relationships between accessions. SSR markers have the highest expected heterozygosity (0.60), while AFLP markers have the highest effective multiplex ratio (19). A single parameter, defined as the marker index, which is the product of expected heterozygosity and multiplex ratio, may be used to evaluate overall utility of a marker system. A comparison of genetic similarity matrices revealed that, if the comparison involved both cultivated (Glycine max) and wild soybean (Glycine soja) accessions, estimates based on RFLPs, AFLPs and SSRs are highly correlated, indicating congruence between these assays. However, correlations of RAPD marker data with those obtained using other marker systems were lower. This is because RAPDs produce higher estimates of interspecific similarities. If the comparisons involvedG. max only, then overall correlations between marker systems are significantly lower. WithinG. max, RAPD and AFLP similarity estimates are more closely correlated than those involving other marker systems.

The comparison of RFLP, RAPD, AFLP and SSR (microsatellite) markers for germplasm analysis

Summary 1 Summary All plants in natural ecosystems appear to be symbiotic with fungal endophytes. This highly diverse group of fungi can have profound impacts on plant communities through increasing fitness by conferring abiotic and biotic stress tolerance, increasing biomass and decreasing water consumption, or decreasing fitness by altering resource allocation. Despite more than 100 yr of research resulting in thousands of journal articles, the ecological significance of these fungi remains poorly characterized. Historically, two endophytic groups (clavicipitaceous (C) and nonclavicipitaceous (NC)) have been discriminated based on phylogeny and life history traits. Here, we show that NC-endophytes represent three distinct functional groups based on host colonization and transmission, in planta biodiversity and fitness benefits conferred to hosts. Using this framework, we contrast the life histories, interactions with hosts and potential roles in plant ecophysiology of C- and NC-endophytes, and highlight several key questions for future work in endophyte biology.

https://courses.washington.edu/cfr523/documents/2009TansleyReview.pdf

Fungal endophytes: diversity and functional roles.

A compilation was made of 307 studies using nuclear DNA markers for evaluating among- and within-population diversity in wild angiosperms and gymnosperms. Estimates derived by the dominantly inherited markers (RAPD, AFLP, ISSR) are very similar and may be directly comparable. STMS analysis yields almost three times higher values for within-population diversity whereas among-population diversity estimates are similar to those derived by the dominantly inherited markers. Number of sampled plants per population and number of scored microsatellite DNA alleles are correlated with some of the population genetics parameters. In addition, maximum geographical distance between sampled populations has a strong positive effect on among-population diversity. As previously verified with allozyme data, RAPD- and STMS-based analyses show that long-lived, outcrossing, late successional taxa retain most of their genetic variability within populations. By contrast, annual, selfing and/or early successional taxa allocate most of the genetic variability among populations. Estimates for among- and within-population diversity, respectively, were negatively correlated. The only major discrepancy between allozymes and STMS on the one hand, and RAPD on the other hand, concerns geographical range; within-population diversity was strongly affected when the former methods were used but not so in the RAPD-based studies. Direct comparisons between the different methods, when applied to the same plant material, indicate large similarities between the dominant markers and somewhat lower similarity with the STMS-based data, presumably due to insufficient number of analysed microsatellite DNA loci in many studies.

Comparison of different nuclear DNA markers for estimating intraspecific genetic diversity in plants.

Population genetic theory predicts that plant populations will exhibit internal spatial autocorrelation when propagule flow is restricted, but as an empirical reality, spatial structure is rarely consistent across loci or sites, and is generally weak. A lack of sensitivity in the statistical procedures may explain the discrepancy. Most work to date, based on allozymes, has involved pattern analysis for individual alleles, but new PCR-based genetic markers are coming into vogue, with vastly increased numbers of alleles. The field is badly in need of an explicitly multivariate approach to autocorrelation analysis, and our purpose here is to introduce a new approach that is applicable to multiallelic codominant, multilocus arrays. The procedure treats the genetic data set as a whole, strengthening the spatial signal and reducing the stochastic (allele-to-allele, and locus-to-locus) noise. We (i) develop a very general multivariate method, based on genetic distance methods, (ii) illustrate it for multiallelic codominant loci, and (iii) provide nonparametric permutational testing procedures for the full correlogram. We illustrate the new method with an example data set from the orchid Caladenia tentaculata, for which we show (iv) how the multivariate treatment compares with the single-allele treatment, (v) that intermediate frequency alleles from highly polymorphic loci perform well and rare alleles poorly, (vi) that a multilocus treatment provides clearer answers than separate single-locus treatments, and (vii) that weighting alleles differentially improves our resolution minimally. The results, though specific to Caladenia, offer encouragement for wider application.

/pdf/spatial-autocorrelation-analysis-of-individual-multiallele-286x81x1b5.pdf

Spatial autocorrelation analysis of individual multiallele and multilocus genetic structure.

RAPD markers provide a powerful tool for the investigation of genetic variation in natural and domesticated populations. Recent studies of strain/cultivar identification have shown extensive RAPD divergence among, but little variation within, inbred species or cultivars. In contrast, little is known about the pattern and extent of RAPD variation in heterogeneous, outcrossing species. We describe the population genetic variation of RAPD markers in natural, diploid sources of dioecious buffalograss [Buchloe dactyloides (Nutt.) Engelm.]. Buffalograss is native to the semi-arid regions of the Great Plains of North America, where it is important for rangeland forage, soil conservation, and as turfgrass. Most sources of buffalograss germplasm are polyploid; diploid populations are previously known only from semi-arid Central Mexico. This is the first report of diploids from humid Gulf Coastal Texas. These two diploid sources represent divergent adaptive ecotypes. Seven 10-mer primers produced 98 polymorphic banding sites. Based on the presence/ absence of bands, a genetic distance matrix was calculated. The new Analysis of Molecular Variance (AMOVA) technique was used to apportion the variation among individuals within populations, among populations within adaptive regions, and among regions. There was considerable variation within each of the four populations, and every individual was genetically distinct. Even so, genetic divergence was found among local populations. Within-population variation was larger and among-population variation smaller in Mexico than in Texas. The largest observed genetic differences were those between the two regional ecotypes. These patterns of genetic variation were very different from those reported for inbred species and provide important baseline data for cultivar identification and continuing studies of the evolution of polyploid races in this species.

RAPD variation within and among natural populations of outcrossing buffalograss [Buchloë dactyloides (Nutt.) Engelm.]

Buffalograss, Buchloe dactyloides, is widely distributed throughout the Great Plains of North America, where it is an important species for rangeland forage and soil conservation. The species consists of two widespread polyploid races, with narrowly endemic diploid populations known from two regions: central Mexico and Gulf Coast Texas. We describe and compare the patterns of allozyme and RAPD variation in the two diploid races, using a set of 48 individuals from Texas and Mexico (four population samples of 12 individuals each). Twelve of 22 allozyme loci were polymorphic, exhibiting 35 alleles, while seven 10-mer RAPD primers revealed 98 polymorphic bands. Strong regional differences were detected in the extent of allozyme polymorphism: Mexican populations exhibited more internal gene diversity (He= 0.20, 0.19) than did the Texan populations (He= 0.08, 0.06), although the number of RAPD bands in Texas (n= 62) was only marginally smaller than in Mexico (n= 68). F-statistics for the allozyme data, averaged over loci, revealed strong regional differentiation (mean FRT=+ 0.30), as well as some differentiation among populations within regions (mean FPR=+ 0.09). In order to describe and compare the partitioning of genetic variation for multiple allozyme and RAPD loci, we performed an Analysis of Molecular Variance (AMOVA). AMOVA for both allozyme and RAPD data revealed similar qualitative patterns: large regional differences and smaller (but significant) population differences within regions. RAPDs revealed greater variation among regions (58.4% of total variance) than allozymes (45.2%), but less variation among individuals within populations (31.9% for RAPDs vs. 45.2% for allozymes); the proportion of genetic variance among populations within regions was similar (9.7% for RAPDs vs. 9.6% for allozymes). Despite this large-scale concordance of allozyme and RAPD variation patterns, multiple correlation Mantel techniques revealed that the correlations were low on an individual by individual basis. Our findings of strong regional differences among the diploid races will facilitate further study of polyploid evolution in buffalograss.

Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloë dactyloides

In 1989, a close association was found between single-plant progenies of strong creeping red fescue infected with the endophyte Epichloe festucae and enhanced suppression of dollar spot, a widespread foliar disease of turfgrass caused by Sclerotinia homoeocarpa. From this limited observation, extensive field evaluations were conducted on a wide range of fine fescue germplasm obtained throughout the United States and Europe to determine the frequency and magnitude of this association. In five field trials established between 1985 and 1991, endophyte-infected Chewings, hard, blue, and strong creeping red fescue cultivars, selections, and crosses consistently exhibited endophyte-mediated suppression of dollar spot, when compared with closely related endophyte-free entries. Endophyte-infected Chewings and hard fescue cultivars and selections also had greater turf density and supported less foliar mycelium of S. homoeocarpa than endophyte-free entries.

https://apsjournals.apsnet.org/doi/pdf/10.1094/PD-90-0994

Endophyte-Mediated Suppression of Dollar Spot Disease in Fine Fescues.

The number of cultivars of perennial ryegrass has steadily increased since the early 1960s. Perennial ryegrass has a self-incompatible, cross-pollinated breeding system and thus, each cultivar is a heterogeneous population of individual genotypes. The ability to accurately distinguish large numbers of genetically heterogeneous populations from one another, based solely on morphological data and agronomic performance, is limited. Random amplified polymorphic DNA (RAPD) markers offer a nearly unlimited supply of molecular traits on which to base distinctive characterizations of plant populations. Analysis of molecular variance (AMOVA) provides a statistical tool which partitions molecular marker variation within and among populations and performs significance testing of differences. The objective of this research was to survey RAPD marker variation within and among populations of perennial ryegrass to characterize breeding germplasm and commercial cultivars using AMOVA. A range of genetic diversity was represented by choosing 18 grass populations that included related crosses, derived selections, and unrelated ecotypes. For each population, 10 individuals were examined for 33 RAPD markers produced with two primers. Markers present in all individuals of a particular population but not present in any other individual of another population (fixed marker difference) were not observed among the 18 grass populations, so characterizations were based on marker frequency differences among populations. RAPD markers analyzed with AMOVA closely reflected the breeding histories of the perennial ryegrass germplasm or commercial cultivars. This was particularly true for relationships structured on a population basis. Results indicated that high selection intensity substantially altered the distribution of RAPD markers between parent and progeny populations thereby obscuring measurements of genetic relatedness. Furthermore, this study observed a limited ability to separate closely related populations. Evidence for the narrow germplasm base of turfgrass cultivars was observed; although, many perennial ryegrass populations appeared to retain levels of within population variability comparable with that of land race ecotypes. The methods of analysis presented should provide a supplement to traditional morphological and agronomic data for plant variety protection and for measuring genetic diversity within breeding programs.

RAPD Characterization of Heterogenous Perennial Ryegrass Cultivars

Random amplified polymorphic DNA-PCR (RAPD-PCR) analysis was used to investigate genetic variation among 35 isolates of Alternaria solani, the causal agent of early blight on solanaceous crops, and...

David R. Huff

Papers

RAPD variation within and among natural populations of outcrossing buffalograss [Buchloë dactyloides (Nutt.) Engelm.]

Evolutionary implications of allozyme and RAPD variation in diploid populations of dioecious buffalograss Buchloë dactyloides

Endophyte-Mediated Suppression of Dollar Spot Disease in Fine Fescues.

RAPD Characterization of Heterogenous Perennial Ryegrass Cultivars

RAPD-PCR analysis of genetic variation among isolates of Alternaria solani and Alternaria alternata from potato and tomato