Clint W. Magill

A RFLP linkage map of Sorghum bicolor (L.) Moench

A RFLP linkage map of sorghum composed principally of markers detected with sorghum low-copy-number nuclear DNA clones has been constructed. The map spans 1789 cMs and consists of 190 loci grouped into 14 linkage groups. The 10 largest linkage groups consist of from 10 to 24 markers and from 103 to 237 cMs, and the other 4 linkage groups consist of from 2 to 5 markers and from 7 to 62 cMs. The map was derived in Sorghum bicolor ssp. bicolor by analysis of a F2 population composed of 50 plants derived from a cross of IS 3620C, a guinea line, and BTx 623, an agronomically important inbred line derived from a cross between a zera zera (a caudatum-like sorghum) and an established kafir line. The restriction fragment length polymorphism (RFLP) frequency detected in this population using polymerase chain reaction (PCR)-amplifiable low-copy-number sorghum clones and five restriction enzymes was 51%. A minimal estimate of the number of clones that detect duplicate sequences is 11 %. Null alleles occurred at 13% of the mapped RFLP loci.

RFLP-based assay of Sorghum bicolor (L.) Moench genetic diversity

Sixty-two single-copy sorghum DNA clones were used to compare restriction fragment patterns of 53 sorghum accessions from Africa, Asia and the United States. Included were accessions from five morphological races of the cultivated subspecies bicolor, and four races of the wild subspecies verticilliflorum. From two to twelve alleles were detected with each probe. There was greater nuclear diversity in the wild subspecies (255 alleles in ten accessions) than in the domestic accessions (236 alleles in 37 accessions). Overall, 204 of the 340 alleles (60%) that were detected occurred in both subspecies. Phylogenetic analysis using parsimony separated the subspecies into separate clusters, with one group of intermediate accessions. Though exceptions were common, especially for the race bicolor, accessions classified as the same morphological race tended to group together on the basis of RFLP similarities. Selection for traits such as forage quality may have led to accessions genetically more similar to other races being classified as bicolors, which have a loose, small-grained panicle similar to wild races. Population statistics, calculated using four nuclear and four cytoplasmic probes that detect two alleles each, revealed a low but significant amount of heterozygosity, and showed little differentiation in alleles in the wild and cultivated subspecies. Outcrossing with foreign pollen appears to have been more important than migration via seed dispersal as a mechanism for gene flow between the wild and domestic accessions included in this study.

Stereoselective coupling of hemigossypol to form (+)-gossypol in moco cotton is mediated by a dirigent protein

The terpenoid gossypol, a secondary metabolite found in the cotton plant, is synthesized by a free radical dimerization of hemigossypol. Gossypol exists as an atropisomeric mixture because of restricted rotation around the central binaphthyl bond. The dimerization of hemigossypol is regiospecific in cotton. In the case of some moco cotton, the dimerization also exhibits a high level of stereoselectivity. The mechanism that controls this stereoselective dimerization is poorly understood. In this paper, we demonstrate that a dirigent protein controls this stereoselective dimerization process. A partially purified protein preparation from cotton flower petals, which by itself is unable to convert hemigossypol to gossypol, converts hemigossypol with a 30% atropisomeric excess into (+)-gossypol when combined with an exogenous laccase, which by itself produces racemic gossypol.

The enzymatic cyclization of nerolidyl diphosphate by δ-cadinene synthase from cotton stele tissue infected with verticillium dahliae

Abstract Soluble preparations of cotton stele tissue infected with Verticillium dahliae containing δ-cadinene synthase convert (1- RS )-[1- 2 H]- E,E -farnesyl diphosphate to [5- 2 H]- and [11- 2 H]-δ-cadinene and convert [4,4,13,13,13- 2 H 5 ]-nerolidyl diphosphate to [8,8,15,15,15- 2 H 5 ]-δ-cadinene. These data imply that nerolidyl diphosphate is an intermediate in the enzymatic cyclization of the natural substrate E,E -farnesyl diphosphate to δ-cadinene by δ-cadinene synthase and involves the conversion of E,E -farnesyl diphosphate to nerolidyl diphosphate followed by cyclization to cis -germacradienyl cation, a 1,3-hydride shift, a second cyclization to a cadinanyl cation and deprotonation to δ-cadinene. Kinetic analyses of induced δ-cadinene synthase mRNA, δ-cadinene synthase activity and formation of sesquiterpenoid phytoalexins in cotton stele tissue infected with Verticillium dahliae show that 12 hr after fungal inoculation the δ-cadinene synthase mRNA was at a maximum level. The tissue injected with H 2 O in place of fungal inoculation showed no detectable δ-cadinene synthase mRNA or δ-cadinene synthase activity after 12 to 96 hr. After 12 hr, 54% of the δ-cadinene synthase activity had developed, but no phytoalexins were detected, the midpoint in the formation of the phytoalexins was 48 hr. These data, together with the enzyme analyses, support the conclusion that Verticillium dahliae initiates a signal in the stele tissue that results in an increased steady-state level of δ-cadinene synthase mRNA and an increased activity of δ-cadinene synthase which functions in the conversion of E,E -farnesyl diphosphate → nerolidyl diphosphate → δ-cadinene that is metabolically converted to desoxyhemigossypol, desoxyhemigossypol-6-methyl ether, hemigossypol and hemigossypol-6-methyl ether.

Reduced levels of cadinane sesquiterpenoids in cotton plants expressing antisense (+)-δ-cadinene synthase

Cotton plants were transformed with an antisense construct of cdn1-Cl, a member of a complex gene family of delta-(+)cadinene (CDN) synthase. This synthase catalyzes the cyclization of (E,E)-farnesyl diphosphate to form CDN, and in cotton, it occupies the committed step in the biosynthesis of cadinane sesquiterpenoids and heliocides (sesterterpenoids). Southern analyses of the digestion of leaf DNA from R(o), T(o), and T(1) plants with Hind III, Pst I and Kpn I restriction enzymes show the integration of antisense cdn1-C1 cDNA driven by the CaMV 35S promoter into the cotton genome. Northern blots demonstrate the appearance of cdn synthase mRNA preceding CDN synthase activity and the formation of gossypol in developing cottonseed. T(2) cottonseed show a reduced CDN synthase activity and up to a 70% reduction in gossypol. In T(1) leaves the accumulated amounts of gossypol, hemigossypolone and heliocides are reduced 92.4, 83.3 and 68.4%, respectively. These data demonstrate that the integration of antisense cdn1-C1 cDNA into the cotton genome leads to a reduction of CDN synthase activity and negatively impacts on the biosynthesis of cadinane sesquiterpenoids and heliocides in cotton plants.

Length heterogeneity in ITS 2 and the methylation status of CCGG and GCGC sites in the rRNA genes of the genus Peronosclerospora.

SummaryThe polymerase chain reaction (PCR) was used with primers complementary to conserved flanking sequences to amplify the internal transcribed spacer 2 (ITS 2) of the rDNA repeat units of five Peronoscleropora isolates, one each of P. sorghi, P. maydis, P. sacchari and tow of P. zeae. In contrast to the situation found in mostfungi that have been examined, length heterogeneity was evident in each sample. The rDNA composition of the amplified bands was confirmed by Southern hybridizations using an ITS 2 amplified from P. sorghi and cloned rDNA from Neurospora crassa as probes. Length heterogeneity was also detected in genomic DNA digests using the same probes. In addition to one dominant fragment for each isolate, there were several less frequent fragments of different sizes, and the isolate(s) for each species had a unique banding pattern for ITS 2. The absence of 5-methylcytosine residues in CCGG and GCGC sequences in the ribosomal genes of these four Peronosclerospora species was demonstrated by the production of identical banding patterns with ribosomal DNA probes following digestion of genomic DNA with MspI and HpaII, and by complete digestion with CfoI.

DNA methylation and expression of NPT II in transgenic petunias and progeny

The expression and inheritance of the NPT II (neomycin phosphotransferase II) gene was studied in four transgenic petunia (Petunia hybrida Vilm.) plants and their progeny. The four transgenic plants, each of which had more than one site of insertion, were different from each other in the level of foreign gene expression. Transmission of one or more NPT II alleles to progeny as deteceted by DNA hybridization did not lead to consistant or predictable patterns of NPT II expression. All transgenic plants and their progeny displaying NPT II enzyme activity contained unmethylated SstII (methylation-sensitive restriction enzyme) sites in the nopaline synthase (NOS) promoter (controlling NPT II gene transcription); whereas, 13 of 17 plants which contained the NPT II gene and which showed no NPT II activity had methylated SstII sites. Two progeny of 1 transgenic plant appeared to have some unmethylated SstII sites, but no NPT II enzyme activity was found in leaf tissue samples. DNA methylation of the SstII site in the NOS promoter is strongly correlated with a decrease in NPT II gene expression in transgenic petunia plants and their progeny. However, DNA methylation alone could not account for the variability seen in NPT II gene expression.

Simple sequence repeat markers useful for sorghum downy mildew (Peronosclerospora sorghi) and related species

BackgroundA recent outbreak of sorghum downy mildew in Texas has led to the discovery of both metalaxyl resistance and a new pathotype in the causal organism, Peronosclerospora sorghi. These observations and the difficulty in resolving among phylogenetically related downy mildew pathogens dramatically point out the need for simply scored markers in order to differentiate among isolates and species, and to study the population structure within these obligate oomycetes. Here we present the initial results from the use of a biotin capture method to discover, clone and develop PCR primers that permit the use of simple sequence repeats (microsatellites) to detect differences at the DNA level.ResultsAmong the 55 primers pairs designed from clones from pathotype 3 of P. sorghi, 36 flanked microsatellite loci containing simple repeats, including 28 (55%) with dinucleotide repeats and 6 (11%) with trinucleotide repeats. A total of 22 microsatellites with CA/AC or GT/TG repeats were the most abundant (40%) and GA/AG or CT/TC types contribute 15% in our collection. When used to amplify DNA from 19 isolates from P. sorghi, as well as from 5 related species that cause downy mildew on other hosts, the number of different bands detected for each SSR primer pair using a LI-COR- DNA Analyzer ranged from two to eight. Successful cross-amplification for 12 primer pairs studied in detail using DNA from downy mildews that attack maize (P. maydis & P. philippinensis), sugar cane (P. sacchari), pearl millet (Sclerospora graminicola) and rose (Peronospora sparsa) indicate that the flanking regions are conserved in all these species. A total of 15 SSR amplicons unique to P. philippinensis (one of the potential threats to US maize production) were detected, and these have potential for development of diagnostic tests. A total of 260 alleles were obtained using 54 microsatellites primer combinations, with an average of 4.8 polymorphic markers per SSR across 34 Peronosclerospora, Peronospora and Sclerospora spp isolates studied. Cluster analysis by UPGMA as well as principal coordinate analysis (PCA) grouped the 34 isolates into three distinct groups (all 19 isolates of Peronosclerospora sorghi in cluster I, five isolates of P. maydis and three isolates of P. sacchari in cluster II and five isolates of Sclerospora graminicola in cluster III).ConclusionTo our knowledge, this is the first attempt to extensively develop SSR markers from Peronosclerospora genomic DNA. The newly developed SSR markers can be readily used to distinguish isolates within several species of the oomycetes that cause downy mildew diseases. Also, microsatellite fragments likely include retrotransposon regions of DNA and these sequences can serve as useful genetic markers for strain identification, due to their degree of variability and their widespread occurrence among sorghum, maize, sugarcane, pearl millet and rose downy mildew isolates.

A molecular marker that segregates with sorghum leaf blight resistance in one cross is maternally inherited in another.

Abstract Leaf blight-resistant sorghum accession SC326-6 was crossed to the susceptible cultivar BTx623 to analyze the genetic basis for resistance. Field scoring of inoculated F2 progeny revealed that resistance was transmitted as a dominant single-gene trait. By combining the random amplified polymorphic DNA (RAPD) technique with bulked-segregant analysis, it was possible to identify PCR amplification products that␣segregated with disease response. Primer OPD12 amplified a 323-bp band (D12R) that segregated with resistance. Creation of longer primers, or SCARs (sequence characterized amplified regions) for D12R resulted in the amplification of a single major band of the predicted size from all the resistant F2 progeny and the resistant parent SC326-6, but not from BTx623 or 24 of 29 susceptible F2 progeny. The SCAR primers also amplified a single band with DNA from IS3620C, the female parent in a cross with BTx623 that has been used to produce a recombinant inbred population for RFLP mapping. An equivalent band was amplified from all 137 recombinant inbred progeny, indicating that organelle DNA is the amplification target in this cross.

The essentiality of sulfhydryl groups to transport in Neurospora crassa

Abstract Three active transport systems for amino acids and another for glucose in germinated conidia of Neurospora were all inactivated during incubation with the sulfhydryl reagents N -ethylmaleimide, p -chloromercuribenzoate, and iodoacetamide. Where tested, the inactivation by p -chloromercuribenzoate was reversed by the action of dithiothreitol. However, two other sulfhydryl reagents, the chloromethyl ketone derivatives of leucine and lysine, were found to have differential effects on the various transport systems. Inactivation by these two compounds did not occur at the active site of the permeases by affinity labeling, suggesting differential availability of sulfhydryl groups among the transport systems. Also, inactivation by these reagents was greatly enhanced in the presence of p -chloromercuribenzoate.