David R. Huff

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

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 [Buchloë 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.

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

Buffalograss, Buchloë 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.

Endophyte-mediated suppression of dollar spot disease in fine fescues

In 1989, a close association was found between single-plant progenies of strong creeping red fescue infected with the endophyte Epichloë 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.

Random amplified polymorphic DNA (RAPD) variation among native little bluestem [Schizachyrium scoparium (Michx.) Nash] populations from sites of high and low fertility in forest and grassland biomes

Random amplified polymorphic DNA (RAPD) markers were used to provide estimates of the comparative genetic variation within and among four native populations of Schizachyrium scoparium. Genotypes were collected from high‐ and low‐fertility sites in both New Jersey (forest biome) and in Oklahoma (grassland biome), USA, and propagated in the greenhouse. Four oligonucleotide primers, 10 bp in length, produced a total of 60 RAPD markers, with the minimum marker difference between any two individuals being 14 markers. Euclidean metric distances were calculated among all individuals, and the analysis of molecular variance (AMOVA) technique was used to apportion the total genetic variation among individuals within populations, populations within fertility levels, populations within biomes, fertility levels, and biomes. Even though most genetic variation resided within populations, statistically significant differences were detected between populations within each biome. Furthermore, genetic distances between high and low fertility levels within biomes were equal to or greater than biome distances. Therefore, in this wide‐ranging and highly variable species, RAPD analysis suggests that local site differences in fertility and ecological history can promote genetic differentiation equal to or greater than geographical differentiation.

Determining genetic origins of aberrant progeny from facultative apomictic Kentucky bluegrass using a combination of flow cytometry and silver-stained RAPD markers

Seeded plants that reproduce through facultative apomixis produce two types of progeny: (1) apomictic progeny genetically identical to the maternal genotype, and (2) aberrant progeny genetically different from the maternal genotype. Aberrant progeny have at least nine different genetic origins depending on gametic ploidy level and whether fertilization was self, cross, or absent. Multiple genetic origins of aberrant progeny complicate the results of basic and applied genetic studies. Determining the genetic origin of progeny plants using traditional techniques, such as cytology, embryology, and segregational studies, is technically difficult in Kentucky bluegrass. We have found that two relatively new techniques, flow cytometry and silver-stained RAPD (ssRAPD) markers, are powerful tools for rapidly determining the genetic origins of aberrant Kentucky bluegrass progeny. Our application of these techniques demonstrate that (1) flow cytometry accurately distinguishes progeny ploidy levels, and (2) ssRAPD markers distinguish progeny resulting from cross-fertilization. Therefore, a combination of flow cytometry and ss-RAPD data would be useful for most genetic studies of aberrant individuals. Moreover, ssRAPD s were found to be of value for measuring the loss of genetic markers from polyhaploids and quantifying the inheritance of parental genomes in polydiploid Bn (n+n) and polytriploid BIII (2n+n) hybrids. Quantifying shared ss-RAPD markers may also be useful for determining genetic relatedness between varieties and germplasm sources.

A Fungal Parasite Regulates a Putative Female-Suppressor Gene Homologous to Maize Tasselseed2 and Causes Induced Hermaphroditism in Male Buffalograss

Parasitically induced hermaphroditism is a fascinating illustration of floral sex organ modification; however, knowledge of how parasites induce hermaphroditism in plants is limited. Here, we show the fungal parasite pistil smut induces development of female sex organs (pistils) in flowers of male buffalograss, potentially by downregulating a putative female-suppressor gene, BdTs2, homologous to maize Tasselseed2 (ZmTs2). Full-length BdTs2, isolated using rapid amplification of cDNA ends, exhibits 89% nucleotide sequence similarity with ZmTs2 and 85% amino acid sequence homology with ZmTs2 protein. Scanning electron micrographs demonstrate that unisexual buffalograss flowers develop through a process of selective abortion of opposite sex organs within hermaphroditic floral primordia. Quantitative real-time polymerase chain reaction showed that high expression levels of BdTs2 within male inflorescences correlate with the selective abortion of gynoecium, leading to the development of unisexual male flowers. RNA in situ hybridization confirmed the expression of BdTs2 precisely within vestigial gynoeciums of male flowers and not in other floral organs of the inflorescence. Furthermore, we show that BdTs2 expression is downregulated by pistil smut infection, which corresponds to the presence of pistils in flowers otherwise destined to become unisexual male. This study provides a potential molecular basis for pistil smut-induced hermaphroditism in male buffalograss.

Genetic Variability in Populations of the Southern Chinch Bug, Blissus insularis, Assessed using AFLP Analysis

Abstract Southern chinch bug, Blissus insularis Barber (Heteroptera: Blissidae), is the most destructive insect pest of St. Augustine grass, Stenotaphrum secundatum Waltz (Kuntze), in the southern United States. The present study is focused on assessing genetic variability in five populations of B. insularis collected from Texas and Florida where St. Augustine grass is widely grown. The amplified fragment length polymorphism technique was used to DNA fingerprint individuals from each population (a total of 46 individuals) using five primer combinations (EcoRI/MSeI). Analysis of molecular variance results show no evidence to support significant genetic variability among Texas and Florida populations of B. insularis. Nearly all genetic variation was found to reside within populations (95%), with only approximately 3% residing among populations between regions. Low GST values obtained from POPGENE and low FST values obtained from the analysis of molecular variance both support the conclusion for high levels of gene flow resulting from interbreeding and/or migratory events among the populations. A Mantel test of Euclidean squared distances showed no correlation between the genetic distance and geographic distance matrices of tested populations of B. insularis. The results of the present study suggests that gene flow is occurring among populations of B. insularis and, therefore, breeders need to be aware that this resistance will most likely not remain localized, and it has the potential to spread as a result of migratory events.

Genetic Characterization of Heterogeneous Plant Populations in Forage, Turf and Native Grasses

Genetically characterizing plant populations is important for many plant scientists, including plant breeders and ecologists. Grass breeders not only have to show their cultivars are distinct for purposes of plant variety protection or seed certification but also may soon need to characterize their cultivars to accommodate new laws governing essentially derived variety (EDV). Restoration ecologists are interested in using genetic characterizations to replace native habits with “ecologically correct” populations. Because most of our forage, turf, and native grasses are cross-pollinated, their high amount of within-population variation often obscures our ability to easily detect between-population differences. Two sets of statistical analysis tools will be discussed that, when applied, offer useful interpretation and resolution of population structure of turf, forage, and native plant species. NTSYS, numerical taxonomy and multivariate analysis system, is a package of statistical programs that enables one to perform a wide range of user-friendly applications. The other is a technique known as analysis of molecular variance (AMOVA) that is an easy to use, yet powerful, tool for examining population differences using molecular markers. AMOVA estimates and partitions the observed total molecular variance into components that are analogous to F-statistic parameters. Finally, issues relating to sample size, breeding history and the use of discriminate analysis as applied to essentially derived varieties (EDVs) will be discussed.

Pistil Smut Infection Increases Ovary Production, Seed Yield Components, and Pseudosexual Reproductive Allocation in Buffalograss

Sex expression of dioecious buffalograss [Bouteloua dactyloides Columbus (syn. Buchloë dactyloides (Nutt.) Engelm.)] is known to be environmentally stable with approximate 1:1, male to female, sex ratios. Here we show that infection by the pistil smut fungus [Salmacisia buchloëana Huff & Chandra (syn. Tilletia buchloëana Kellerman and Swingle)] shifts sex ratios of buffalograss to be nearly 100% phenotypically hermaphroditic. In addition, pistil smut infection decreased vegetative reproductive allocation, increased most seed yield components, and increased pseudosexual reproductive allocation in both sex forms compared to uninfected clones. In female sex forms, pistil smut infection resulted in a 26 fold increase in ovary production and a 35 fold increase in potential harvest index. In male sex forms, pistil smut infection resulted in 2.37 fold increase in floret number and over 95% of these florets contained a well-developed pistil. Although all ovaries of infected plants are filled with fungal teliospores and hence reproductively sterile, an average male-female pair of infected plants exhibited an 87 fold increase in potential harvest index compared to their uninfected clones. Acquiring an ability to mimic the effects of pistil smut infection would enhance our understanding of the flowering process in grasses and our efforts to increase seed yield of buffalograss and perhaps other grasses.