Toward integration of comparative genetic, physical, diversity, and cytomolecular maps for grasses and grains, using the sorghum genome as a foundation.

The small genome of sorghum (Sorghum bicolor L. Moench.) provides an important template for study of closely related large-genome crops such as maize (Zea mays) and sugarcane (Saccharum spp.), and is a logical complement to distantly related rice (Oryza sativa) as a "grass genome model." Using a high-density RFLP map as a framework, a robust physical map of sorghum is being assembled by integrating hybridization and fingerprint data with comparative data from related taxa such as rice and using new methods to resolve genomic duplications into locus-specific groups. By taking advantage of allelic variation revealed by heterologous probes, the positions of corresponding loci on the wheat (Triticum aestivum), rice, maize, sugarcane, and Arabidopsis genomes are being interpolated on the sorghum physical map. Bacterial artificial chromosomes for the small genome of rice are shown to close several gaps in the sorghum contigs; the emerging rice physical map and assembled sequence will further accelerate progress. An important motivation for developing genomic tools is to relate molecular level variation to phenotypic diversity. "Diversity maps," which depict the levels and patterns of variation in different gene pools, shed light on relationships of allelic diversity with chromosome organization, and suggest possible locations of genomic regions that are under selection due to major gene effects (some of which may be revealed by quantitative trait locus mapping). Both physical maps and diversity maps suggest interesting features that may be integrally related to the chromosomal context of DNA-progress in cytology promises to provide a means to elucidate such relationships. We seek to provide a detailed picture of the structure, function, and evolution of the genome of sorghum and its relatives, together with molecular tools such as locus-specific sequence-tagged site DNA markers and bacterial artificial chromosome contigs that will have enduring value for many aspects of genome analysis.

A peanut seed lipoxygenase responsive to Aspergillus colonization.

Several lines of evidence have indicated that lipoxygenase enzymes (LOX) and their products, especially 9S- and 13S-hydroperoxy fatty acids, could play a role in the Aspergillus/seed interaction. Both hydroperoxides exhibit sporogenic effects on Aspergillus spp. (Calvo, A., Hinze, L., Gardner, H.W. and Keller, N.P. 1999. Appl. Environ. Microbiol. 65: 3668-3673) and differentially modulate aflatoxin pathway gene transcription (Burow, G.B., Nesbitt, T.C., Dunlap, J. and Keller, N.P. 1997. Mol. Plant-Microbe Interact. 10: 380-387). To examine the role of seed LOXs at the molecular level, a peanut (Arachis hypogaea L.) seed gene, PnLOX1, was cloned and characterized. Analysis of nucleotide sequence suggests that PnLOX1 encodes a predicted 98 kDa protein highly similar in sequence and biochemical properties to soybean LOX2. The full-length PnLOX1 cDNA was subcloned into an expression vector to determine the type(s) of hydroperoxide products the enzyme produces. Analysis of the oxidation products of PnLOX1 revealed that it produced a mixture of 30% 9S-HPODE (9S-hydroperoxy-10E, 12Z-octadecadienoic acid) and 70% 13S-HPODE (13S-hydroperoxy-9Z, 11E-octadecadienoic acid) at pH 7. PnLOX1 is an organ-specific gene which is constitutively expressed in immature cotyledons but is highly induced by methyl jasmonate, wounding and Aspergillus infections in mature cotyledons. Examination of HPODE production in infected cotyledons suggests PnLOX1 expression may lead to an increase in 9S-HPODE in the seed.

pH Regulation of Sterigmatocystin and Aflatoxin Biosynthesis in Aspergillus spp.

ABSTRACT Aflatoxin (AF) and sterigmatocystin (ST) are toxic secondary metabolites produced by the same biochemical pathway found in several Aspergillus spp. The expression of the homologous ST/AF structural gene, stcU in A. nidulans and ver-1 in A. parasiticus, was affected by external pH of liquid growth media. Both stcU and ver-1 mRNAs appeared earlier and were expressed at higher levels in cultures grown in acidic media (pH 4 to 6) versus neutral (pH 7) and alkali (pH 8) media. Transcript levels correlated with ST/AF production. Visual and spectrophotometric analysis of production of the orange ST/AF intermediate, norsolorinic acid (NOR), also paralleled transcript patterns and indicated that the pH effects were operative in different nitrogen- and carbon-based solid growth media. Five- to 10-fold increases in ST, AF, and NOR were measured in cultures grown in pH 4 or 5 versus pH 8 media. An A. nidulans strain carrying a mutation resulting in constitutive activity of the pH regulatory factor, PacC, produced 10-fold less ST than did wild type. The stcU transcript was not noticeably affected by pH in this strain. The results suggest a general pattern of pH regulation of ST/AF biosynthesis that may override previously noted carbon and nitrogen effects.