Expression of pigmentation genes following electroporation of albino Monascus purpureus.

A UV-induced albino strain of Monascus purpureus was subjected to electroporation in the presence of genomic DNA from a wild-type red strain of the fungus. Eight colonies expressed color after several weeks of growth. The growth rates of all eight color variants were significantly greater than the recipient and donor strains under some culture conditions. Spectrophotometric analysis of the pigments extracted from the color variants revealed the pigments had absorbance spectra different from the DNA donor strain. These color variants may have resulted from transformation with wild-type DNA, mutation reversion, or activation of alternative pathway(s)-i.e., new mutations-that resulted in pigment production.

Agrobacterium-mediated transformation of creeping bentgrass using GFP as a reporter gene.

Creeping bentgrass (Agrostis palustris Huds.) is a cool season grass widely used on putting greens in golf courses. Transformation of creeping bentgrass has been conducted using microprojectile bombardment and protoplast electroporation. The objective of our study is to develop an alternative and more efficient approach in transforming the grass using Agrobacterium (strain EHA 101). This technique was effective in transforming 40-day old calli derived from mature seeds cultured on MS medium supplemented with 2,4-D, kinetin, and sucrose. Dozens of transgenic plants have been produced from two independent transformed calli. Presence of functional green fluorescence protein (GFP) was detected in leaves, stems, and roots of transgenic seedlings. Four putative transgenic plants and two control plants were randomly chosen and analyzed by Southern blot analysis. Bands corresponding to the GFP gene were clearly shown in transgenic plants. These results indicated that Agrobacterium transformation can successfully be applied to creeping bentgrass.

Phylogenetic relationships of sorghum taxa inferred from mitochondrial DNA restriction fragment analysis.

To elucidate the evolutionary history and affinity of sorghum species, 41 sorghum taxa were analyzed using variability in mitochondrial DNA. Analysis of species relationships at the molecular level can provide additional data to supplement the existing classification based on morphological characters and may also furnish unexpected but useful information. Total DNA extracted from each of the sorghum accessions was digested with each of five restriction enzymes, BamHI, HindIII, EcoRI, EcoRV, and XbaI, and probed with five mitochondrial DNAs cloned from Sorghumbicolor. A total of 180 restriction fragments was detected by the 25 probe-enzyme combinations. Forty-three fragment bands were phylogenetically informative. Multiple correspondence analysis was performed to visualize associations among the accessions and suggested that section Eusorghum species may be divided into four groups, with Sorghumlaxiflorum (section Heterosorghum) and Sorghumnitidum (section Parasorghum) appearing as outliers. A phylogenetic tree was assembled from mitochondrial restriction fragment data. The taxa analyzed formed three major groups comprising section Heterosorghum (group I), section Parasorghum (group II), and all accessions in section Eusorghum (group III). Group III is further divided into four groups: (i) two sweet sorghums and shattercane; (ii) Sorghumhalepense, Sorghummiliaceum, Sorghumhewisonii, Sorghumaethiopicum, Sorghumverticilliflorum, and S. bicolor, including Sorghumsudanense (sudangrass), the Chinese Kaoliangs, and a number of commercial sorghum inbreds from the U.S.A.; (iii) Sorghumpropinquum; and (iv) Sorghumarundinaceum, Sorghumniloticum, Sorghumalmum, Sorghumcontroversum, and the Chinese material C-401 and 5-27. Results indicate that the analysis of fragmented mitochondrial DNA was diagnostic and useful in sorghum phylogenetic and taxonomic research at the species, subspecies, and race levels, and can complement results from those analyses using nuclear ribosomal DNA and chloroplast DNA that effectively distinguish taxa at species and genus levels. Key words : Sorghum, mitochondrial DNA, phylogeny, restriction fragment.

Genetic Diversity of Xanthomonas oryzae pv. oryzae in Asia.

Restriction fragment length polymorphism and virulence analyses were used to evaluate the population structure of Xanthomonas oryzae pv. oryzae, the rice bacterial blight pathogen, from several rice-growing countries in Asia. Two DNA sequences from X. oryzae pv. oryzae, IS1112, an insertion sequence, and avrXa10, a member of a family of avirulence genes, were used as probes to analyze the genomes of 308 strains of X. oryzae pv. oryzae collected from China, India, Indonesia, Korea, Malaysia, Nepal, and the Philippines. On the basis of the consensus of three clustering statistics, the collection formed five clusters. Genetic distances within the five clusters ranged from 0.16 to 0.51, and distances between clusters ranged from 0.48 to 0.64. Three of the five clusters consisted of strains from a single country. Strains within two clusters, however, were found in more than one country, suggesting patterns of movement of the pathogen. The pathotype of X. oryzae pv. oryzae was determined for 226 strains by inoculating five rice differential cultivars. More than one pathotype was associated with each cluster; however, some pathotypes were associated with only one cluster. Most strains from South Asia (Nepal and India) were virulent to cultivars containing the bacterial blight resistance gene xa-5, while most strains from other countries were avirulent to xa-5. The regional differentiation of clusters of X. oryzae pv. oryzae in Asia and the association of some pathotypes of X. oryzae pv. oryzae with single clusters suggested that strategies that target regional resistance breeding and gene deployment are feasible.

Phylogenetic analysis of Sorghum and related taxa using internal transcribed spacers of nuclear ribosomal DNA.

The phylogenetic relationships of the genus Sorghum and related genera were studied by sequencing the nuclear ribosomal DNA (rDNA) internal transcribed spacer region (ITS). DNA was extracted from 15 Sorghum accessions, including one accession from each of the sections Chaetosorghum and Heterosorghum, four accessions from Parasorghum, two accessions from Stiposorghum, and seven representatives from three species of the section Sorghum (one accession from each of S. propinquum and S. halepense, and five races of S. bicolor). The maize (Zea mays) line, H95, and an accession from Cleistachne sorghoides were also included in the study. Variable nucleotides were used to construct a strict consensus phylogenetic tree. The analyses indicate that S. propinquum, S. halepense and S. bicolor subsp. arundinaceum race aethiopicum may be the closest wild relatives of cultivated sorghum; Sorghum nitidum may be the closest 2n=10 relative to S. bicolor, the sections Chaetosorghum and Heterosorghum appear closely related to each other and more closely related to the section Sorghum than Parasorghum; and the section Parasorghum is not monophyletic. The results also indicate that the genus Sorghum is a very ancient and diverse group.

Genome organization of Magnaporthe grisea: genetic map, electrophoretic karyotype, and occurrence of repeated DNAs.

A genetic map of Magnaporthe grisea (anamorph=Pyricularia oryzae and P. grisea), the causal agent of rice blast disease, was generated from segregation data utilizing 97 RFLP markers, two isoenzyme loci and the mating type locus among progeny of a cross between parental strains Guy 11 and 2539. Of the seven chromosomes of M. Grisea, three were resolved by contour-clamped homogeneous electric field (CHEF) electrophoresis, while the remaining four migrated as two doublet bands. By utilizing differences between CHEF mobilities of unresolved chromosomes from the parental strains, Southern analysis with selected markers allowed the chromosomal assignment of all linkage groups. A small translocation involving 1 marker was found in the parental strains used to produce the segregating population from which the map was constructed. Nine classes of repetitive DNA elements were found in the genome of a fungal isolate pathogenic to rice. These occurred only a few times or not at all in the genomes of isolates showing reduced virulence on rice. One repetitive DNA was shown to have structural similarity to the Alu sequences found in primates, a sequence similarity to the copia-like elements of Drosophila, and peptide similarity to transposable elements found in Drosophila, other fungi, and higher plants.

A PCR-based method of identifying species-specific repeated DNAs.

A PCR-based method is described to facilitate the identification of DNA fragments that are highly repeated and species-specific. The precision of the technique was demonstrated by cloning a fragment that occurred in a high number of copies in the plant species Medicago granadensis but in a low number of copies in 17 other Medicago species and Melilotus officinalis. This method should greatly accelerate the isolation and cloning of short and long interspersed nuclear elements with species-specific distributions. Such clones should prove useful in studies of phylogenetic relationships in the identification of interspecific hybrids, as in situ chromosome markers and other applications.