RFLP and RAPD mapping of the sunflower Pl1 locus for resistance to Plasmopara halstedii race 1

The Pl1 locus in sunflower, Helianthus annuus L., conferring resistance to downy mildew, Plasmopara halstedii, race 1 has been located in linkage group 1 of the consensus RFLP map of the cultivated sunflower. Bulked segregant analyses were used on 135 plants of an F₂ progeny from a cross between a downy mildew susceptible line, GH, and RHA266, a line carrying Pl1. Two RFLP markers and one RAPD marker linked to the Pl1 locus have been identified. The RFLP markers are located at 5.6 cM and 7.1 cM on either side of Pl1. The RAPD marker is situated at 43.7 cM from Pl1. The significance and applications of these markers in sunflower breeding are discussed.     

Genomic organization of multiple genes coding for rhodotorucine A,a lipopeptide mating pheromone of the basidiomycetous yeast Rhodosporidium toruloides

Rhodotorucine A, a lipopeptide mating pheromone, is secreted from mating type A cells of Rhodosporidium toruloides and induces sexual differentiation of the opposite mating type a cells. Genome of A-type cells contains three homologous genes (RHA1, RHA2, and RHA3) encoding rhodotorucine A. Genomic Southern blot analysis using RHA1 DNA as a probe showed that RHA1 strongly hybridize with A-type genomic DNA but weakly with a-type, suggesting that the sequences of RHA genes were dissimilar in the opposite a-type genome. The range of dissimilar regions in a-type genome was searched using RHA-flanking DNA segments as probes. The result suggests that a-type genome lacks sequences coding for rhodotorucine A and its 5 upstream but contains its 3 non-coding sequences. The absence of mating pheromone genes in the opposite mating type genome suggests that the expression of mating-type-specific genes in R. toruloides is not controlled trans-criptionally, as shown in the yeast Saccharomyces cerevisiae.     

Antisense Expression of a NBS-LRR Sequence in Sunflower (Helianthus annuus L.) and Tobacco (Nicotiana tabacum L.): Evidence for a Dual Role in Plant Development and Fungal Resistance

A partial sunflower cDNA clone, PLFOR48, segregating with a resistance marker to Plasmopara halstedii, the causal agent of downy mildew, has been cloned from the mildew resistant sunflower line, RHA 266. PLFOR48 encodes a putative protein with a nucleotide-binding site and a leucine-rich repeat domain, showing significant homology with previously cloned resistance genes belonging to the TIR-NBS-LRR family. Southern blot analysis of non-transgenic sunflower suggests that PLFOR48 is part of a multigenic family. The potential role of PLFOR48 sequence in sunflower resistance to mildew was studied, by assessing loss of function, using expression of the antisense cDNA in RHA 266 sunflower line. Quite unexpectedly, transgenic sunflower lines displayed severe developmental abnormalities, and in particular, on the main meristems of homozygote T2 progeny, thus hampering any further challenge inoculation with Plasmopara halstedii. The presence of homologous sequences to PLFOR48 in Nicotiana tabacum var Samsun NN, as demonstrated by Southern blotting, drove us to consider tobacco as an additional model to investigate the potential role of this sequence in fungal resistance. Expression of the same antisense cDNA in transgenic tobacco lines gave rise to higher degree of susceptibility to Phytophthora parasitica, as well as to severe alterations in seed development. These results suggest that PLFOR48 and homologous sequences could be involved in both regulating developmental pathways and controlling resistance to fungal pathogens.     

QTL analysis of agronomic traits in recombinant inbred lines of sunflower under partial irrigation

The objective of the present research was to map QTLs associated with agronomic traits such as days from sowing to flowering, plant height, yield and leaf-related traits in a population of recombinant inbred lines (RILs) of sunflower (Helianthus annuus). Two field experiments were conducted with well-irrigated and partially irrigated conditions in randomized complete block design with three replications. A map with 304 AFLP and 191 SSR markers with a mean density of 1 marker per 3.7 cM was used to identify QTLs related to the studied traits. The difference among RILs was significant for all studied traits in both conditions. Three to seven QTLs were found for each studied trait in both conditions. The percentage of phenotypic variance (R ²) explained by QTLs ranged from 4 to 49%. Three to six QTLs were found for each yield-related trait in both conditions. The most important QTL for grain yield per plant on linkage group 13 (GYP-P-13-1) under partial-irrigated condition controls 49% of phenotypic variance (R ²). The most important QTL for 1,000-grain weight (TGW-P-11-1) was identified on linkage group 11. Favorable alleles for this QTL come from RHA266. The major QTL for days from sowing to flowering (DSF-P-14-1) were observed on linkage group 14 and explained 38% of the phenotypic variance. The positive alleles for this QTL come from RHA266. The major QTL for HD (HD-P-13-1) was also identified on linkage group 13 and explained 37% of the phenotypic variance. Both parents (PAC2 and RHA266) contributed to QTLs controlling leaf-related traits in both conditions. Common QTL for leaf area at flowering (LAF-P-12-1, LAF-W-12-1) was detected in linkage group 12. The results emphasise the importance of the role of linkage groups 2, 10 and 13 for studied traits. Genomic regions on the linkage groups 9 and 12 are specific for QTLs of leaf-related traits in sunflower.     

Symbiotic seed germination and evidence for <Emphasis Type="Italic">in vitro</Emphasis> mycobiont specificity in <Emphasis Type="Italic">Spiranthes brevilabris</Emphasis> (Orchidaceae) and its implications for species-level conservation

Orchid–mycobiont specificity in the Orchidaceae was considered controversial and not well understood for many years. Differences in mycobiont specificity during germination in vitro vs in situ have lead some to consider orchid–mycobiont specificity as being generally low; however, others have suggested that specificity, especially in vitro, is surprisingly high. Mycobiont specificity may be genus or species specific. An in vitro symbiotic seed germination experiment was designed to examine mycobiont specificity of the endangered Florida terrestrial orchid Spiranthes brevilabris using mycobionts isolated from both the study species and the endemic congener Spiranthes floridana. In a screen of mycobionts, isolates Sflo-305 (99.5%), Sflo-306 (99.5%), and Sflo-308 (89.9%) (originating from S. floridana) supported higher initial (stage 1) seed germination than isolate Sbrev-266 (32.4%) (originating from S. brevilabris) after 3 wk culture. However, only isolate Sbrev-266 supported advanced germination and protocorm development to stage 5 (53.1%) after 12 wk culture. These findings suggest that S. brevilabris maintains a high degree of mycobiont specificity under in vitro symbiotic seed germination conditions. High orchid–mycobiont specificity in S. brevilabris may be indicative of the rare status of this orchid in Florida.     

Genetic mapping of regulatory mutations of Bacillus subtilis riboflavin operon

Summary Seven mutations leading to riboflavin overproduction inBacillus subtilis were found to be linked to the markerdnaF133 (145° on theB. subtilis genetic map) by transformation. Cotransfer indexes (42.5%–61.7%) suggest that theribC mutations are alleles of the same locus. Results of transduction and transformation crosses suggest the following order of markers:pyrD26 –ts-6 –dnaF133 –ribC –recA1.     

Genetic analysis and molecular mapping of a wheat gene conferring tolerance to the greenbug (<Emphasis Type="Italic">Schizaphis graminum</Emphasis> Rondani)

The greenbug, Schizaphis graminum (Rondani), is one of the major pests of wheat worldwide. The efficient utilization of wheat genes expressing resistance to greenbug infestation is highly dependent on a clear understanding of their relationships. The use of such genes will be further facilitated through the use of molecular markers linked to resistance genes. The present study involved several F₂ wheat populations derived from crosses between susceptible cultivars and resistant germplasm carrying different greenbug resistance genes. These populations were used to characterize the inheritance of a wheat gene (Gbz) conferring tolerance to greenbug biotype I, to identify molecular markers linked to Gbz, and to investigate the relationship between Gbz and Gb3, a previously identified greenbug resistance gene. Our results indicated that Gbz is inherited as a single dominant gene. Microsatellite marker Xwmc157 is completely linked to Gbz, and Xbarc53 and Xgdm46 flank Gbz at distances of 5.1 and 9.5 cM, respectively. Selection of Gbz using marker Xwmc157 alone gives breeders 100% selection accuracy. Gbz may be placed in the distal region of the long arm of the wheat chromosome 7D. The results of allelism tests indicated that Gbz is either allelic or tightly linked to Gb3.Communicated by D.A. Hoisington     

Expression in Escherichia coli of Biphenyl 2,3-Dioxygenase Genes from a Gram-Positive Polychlorinated Biphenyl Degrader,Rhodococcus jostii RHA1

Rhodococcus jostii RHA1 is a polychlorinated biphenyl degrader. Multi-component biphenyl 2,3-dioxygenase (BphA) genes of RHA1 encode large and small subunits of oxygenase component and ferredoxin and reductase components. They did not express enzyme activity in Escherichia coli. To obtain BphA activity in E. coli, hybrid BphA gene derivatives were constructed by replacing ferredoxin and/or reductase component genes of RHA1 with those of Pseudomonas pseudoalcaligenes KF707. The results obtained indicate a lack of catalytic activity of the RHA1 ferredoxin component gene, bphAc in E. coli. To determine the cause of inability of RHA1 bphAc to express in E. coli, the bphAc gene was introduced into Rosetta (DE3) pLacI, which has extra tRNA genes for rare codons in E. coli. The resulting strain abundantly produced the bphAc product, and showed activity. These results suggest that codon usage bias is involved in inability of RHA1 bphAc to express its catalytic activity in E. coli.     

Variability of sunflower inbred lines to iron deficiency stress

Sunflower (Helianthus annuus L.) has been classified as a Fe-efficient species; however differences have been reported in susceptibility to Fe deficiency stress among cultivars and inbred lines. This paper reports research on responses of inbred lines to Fe deficiency stress (release of protons and root capacity to reduce Fe). When plants were grown individually in aerated nutrient solution without Fe the new selected inbred lines were classified as: a) Lines with good Fe deficiency stress response (RHA 271, RHA 273 and RHA 274); b) Lines that did not lower root external pH (HA 89 and RHA 299), one with very low reducing capacity (HA 89) and the other with reducing capacity (RHA 299); and c) One segregating line (RHA 276) from which two sister lines were selected. When a buffer (5 mM MES, 2-(N- morpholino) ethanesulfonic acid) was added to the root nutrient solution without Fe during growth, the reducing capacity of Fe-inefficient lines was higher for buffered than for unbuffered roots. Therefore, differences among lines for reducing capacity depend on experimental conditions.     

<Emphasis Type="Italic">Lr41, Lr39,</Emphasis> and a leaf rust resistance gene from<Emphasis Type="Italic"> Aegilops cylindrica</Emphasis> may be allelic and are located on wheat chromosome 2DS

The leaf rust resistance gene Lr41 in wheat germplasm KS90WGRC10 and a resistance gene in wheat breeding line WX93D246-R-1 were transferred to Triticum aestivum from Aegilops tauschii and Ae. cylindrica, respectively. The leaf rust resistance gene in WX93D246-R-1 was located on wheat chromosome 2D by monosomic analysis. Molecular marker analysis of F₂ plants from non-critical crosses determined that this gene is 11.2 cM distal to marker Xgwm210 on the short arm of 2D. No susceptible plants were detected in a population of 300 F₂ plants from a cross between WX93D246-R-1 and TA 4186 (Lr39), suggesting that the gene in WX93D246-R-1 is the same as, or closely linked to, Lr39. In addition, no susceptible plants were detected in a population of 180 F₂ plants from the cross between KS90WGRC10 and WX93D246-R-1. The resistance gene in KS90WGRC10, Lr41, was previously reported to be located on wheat chromosome 1D. In this study, no genetic association was found between Lr41 and 51 markers located on chromosome 1D. A population of 110 F₃ lines from a cross between KS90WGRC10 and TAM 107 was evaluated with polymorphic SSR markers from chromosome 2D and marker Xgdm35 was found to be 1.9 cM proximal to Lr41. When evaluated with diverse isolates of Puccinia triticina, similar reactions were observed on WX93D246-R-1, KS90WGRC10, and TA 4186. The results of mapping, allelism, and race specificity test indicate that these germplasms likely have the same gene for resistance to leaf rust.Contribution number 03-348-J from the Kansas Agricultural Experimental Station, Manhattan, KansasCommunicated by J. Dvorak     

Cytochrome P450 125 (CYP125) catalyses C26‐hydroxylation to initiate sterol side‐chain degradation in Rhodococcus jostii RHA1

The cyp125 gene of Rhodococcus jostii RHA1 was previously found to be highly upregulated during growth on cholesterol and the orthologue in Mycobacterium tuberculosis (rv3545c) has been implicated in pathogenesis. Here we show that cyp125 is essential for R. jostii RHA1 to grow on 3‐hydroxysterols such as cholesterol, but not on 3‐oxo sterol derivatives, and that CYP125 performs an obligate first step in cholesterol degradation. The involvement of cyp125 in sterol side‐chain degradation was confirmed by disrupting the homologous gene in Rhodococcus rhodochrous RG32, a strain that selectively degrades the cholesterol side‐chain. The RG32Ωcyp125 mutant failed to transform the side‐chain of cholesterol, but degraded that of 5‐cholestene‐26‐oic acid‐3β‐ol, a cholesterol catabolite. Spectral analysis revealed that while purified ferric CYP125_RHA1 was < 10% in the low‐spin state, cholesterol (K_D^app = 0.20 ± 0.08 μM), 5α‐cholestanol (K_D^app = 0.15 ± 0.03 μM) and 4‐cholestene‐3‐one (K_D^app = 0.20 ± 0.03 μM) further reduced the low spin character of the haem iron consistent with substrate binding. Our data indicate that CYP125 is involved in steroid C26‐carboxylic acid formation, catalysing the oxidation of C26 either to the corresponding carboxylic acid or to an intermediate state.     

Quantitation of loperamide and N-demethyl-loperamide in human plasma using electrospray ionization with selected reaction ion monitoring liquid chromatography–mass spectrometry

We report here the development and validation of an LC–MS method for quantitation of loperamide (LOP) and its N-demethyl metabolite (DMLOP) in human plasma. O-Acetyl-loperamide (A-LOP) was synthesized by us for use as an internal standard in the assay. After addition of the internal standard, the compounds of interest were extracted with methyl tert.-butylether and separated by HPLC on a C₁₈ reversed-phase column using an acetonitrile–water gradient containing 20 mM ammonium acetate. The three compounds were well separated by HPLC and no interfering peaks were detected at the usual concentrations found in plasma. Analytes were quantitated using positive electrospray ionization in a triple quadrupole mass spectrometer operating in the MS–MS mode. Selected reaction monitoring was used to quantify LOP (m/z 477→266), DMLOP (m/z 463→252) and A-LOP (m/z 519→266) on ions formed by loss of the 4-(p-chlorophenyl)-4-hydroxy-piperidyl group upon low energy collision-induced dissociation. Calibration curves, which were linear over the range 1.04 to 41.7 pmol/ml (LOP) and 1.55 to 41.9 pmol/ml (DMLOP), were run contemporaneously with each batch of samples, along with low (4.2 pmol/ml), medium (16.7 pmol/ml) and high (33.4 pmol/ml) quality control samples. The lower limit of quantitation (LLQ) of LOP and DMLOP was about 0.25 pmol/ml in plasma. The extraction efficiency of LOP and DMLOP from human plasma was 72.3±1.50% (range: 70.7–73.7%) and 79.4±12.8% (64.9–88.8%), respectively. The intra- and inter-assay variability of LOP and DMLOP ranged from 2.1 to 14.5% for the low, medium and high quality control samples. The method has been used successfully to study loperamide pharmacokinetics in adult humans.     

The Escherichia coli cell division proteins FtsY, FtsE and FtsX are inner membrane-associated

Summary The cell division genes ftsY, ftsE and ftsX form an operon mapping at 76 min on the Escherichia coli chromosome. The protein products of these genes have been indentified previously. We have studied the cellular location of the radiolabelled Fts proteins using maxicells and standard fractionation procedures. Previous protein sequence homologies suggested an inner membrane location for FtsE. We have confirmed this predicted location and have shown that FtsY and FtsX are also inner membrane-associated. These results are igreement with the hypothesis that FtsE may act at the inner membrane, in a septalsome complex, by coupling ATP hydrolysis to the process of bacterial cell division.     

Identification and characterization of dominant lactic acid bacteria isolated from traditional fermented milk <Emphasis Type="Italic">Dahi</Emphasis> in Bangladesh

We isolated a total of 266 strains of lactic acid bacteria (LAB) from 28 dahi samples that were collected from different areas in Bangladesh. The isolated strains were identified on basis of their morphological, physiological and biochemical characteristics, the lactic acid isomer produced, the ability to ferment sugars and 16S rDNA analysis. Among the isolates, the cocci (73%) were dominant over the rods (27%). The distribution of the isolates by genus was as follows: Streptococcus (50%), Lactobacillus (27%), Enterococcus (9%), Leuconostoc (5%), Lactococcus (5%) and Pediococcus (4%). In this study, S. bovis was the most predominant species as this species represents 47.0% of the total isolates in dahi. The other species we isolated were identified as Lb. fermentum, Lb. delbrueckii ssp. bulgaricus, Lb. delbrueckii ssp. lactis, Lb. sp., Ec. faecium, S. thermophilus, Leuc. mesenteroides ssp. mesenteroides, Leuc. mesenteroides ssp. dextranicum, Lc. lactis ssp. lactis, Lc. raffinolactis and P. pentosaceus.     

Rates of movement of transposable elements inDrosophila melanogaster

Mobilization rates of nine families of transposable elements (P, hobo, FB, gypsy, 412, copia, blood, 297, andjockey) were estimated by using 182 lines. Lines were started from a completely isogenic population ofDrosophila melanogaster, carrying the markersepia as an indicator of possible contamination, and have been accumulating spontaneous mutations independently for 80 generations of brother-sister (or two double-first-cousin) matings. Transposable element movements have been analyzed in complete genomes by the Southern technique. Mobilization was a rare event, with an average rate of 10^–5 per site per generation. The most active element wasFB. In contrast, the retroelementsgypsy andblood did not move at all. Most changes in restriction patterns were consistent with rearrangements rather than with true transposition. The euchromatic or heterochromatic location of elements was tested by comparing insertion patterns from adults and salivary glands. Certain putative rearrangements involved heterochromatic copies of the retroelements412, copia or297. Clustering of movement across families was observed, suggesting that movement of different families may be non-independent. An association between modified insertion patterns and mutant effects on quantitative traits shows that spontaneous transposition events cause continuous variation.     

Environmentally induced nuclear 2C DNA content instability in Helianthus annuus (Asteraceae)

Experiments were conducted to detect developmental and environmental factors that influence nuclear DNA content in H. annuus inbred lines RHA 271 and RHA 299. DNA content (2C) was determined by laser flow cytometry of nuclei isolated from the first leaf pair of seedlings grown in a greenhouse and in growth chambers. DNA content of greenhouse grown seedlings was highly variable, ranging from 3.2 to 8.0 pg for RHA 299 and 5.2 to 8.2 pg for RHA 271. DNA content only weakly correlated to the position of the achene in the head from which the seedlings derived, and not at all to the position of the head on the plant. Experimentally varied environmental parameters of heat stress and water deficit, phosphate fertilizer levels in the substrate, and pH had little or no effect on the DNA content of seedlings. Seedlings grown with increased levels of substrate nitrogen in the form of NH₄NO₃ showed a significant increase in the mean DNA content. Plants grown in one of two types of growth chambers possessed less variability in DNA content, 6.2–8.4 pg for RHA 299 and 6.8–7.4 pg for RHA 271. Plants grown in a second growth chamber were highly variable with DNA content ranging from 3.0 to 8.6 pg for RHA 299 and 3.0 to 7.8 pg for RHA 271. Measurable physical differences between the growth chambers were irradiance level and the ratio of red to far red light. The hypothesis is presented that DNA stability of sunflowers is influenced by light quantity and/or quality.     

Growth inhibition of Rhodococcus sp. strain RHA1 in the course of PCB transformation

Summary Growth of a PCB degrader Rhodococcus sp. RHA1 on biphenyl and ethylbenzene was inhibited by 100 g/ml PCB 48. A PCB tolerant derivative of RHA1 designated RCD1 was deficient in growth on biphenyl. Southern hybridization experiments suggested that RCD1 has the bphDE gene deletion in a 390-kb linear plasmid of RHA1. The bphD gene complementation restored growth deficiency on biphenyl and growth inhibition on ethylbenzene by PCB 48, indicating that PCB metabolites are the cause of growth inhibition.     

The production of podophyllotoxin and its 5-methoxy derivative through bioconversion of cyclodextrin-complexed desoxypodophyllotoxin by plant cell cultures

The bioconversion of the lignan desoxypodophyllotoxin by cell suspensions of Linum flavum and of Podophyllum hexandrum was investigated. The apolar substrate could be easily dissolved in the culture medium at a concentration of 2 mM by complexation with dimethyl--cyclodextrin. Growth parameters of the cell suspensions were not affected by either the addition of cyclodextrin itself, or when cyclodextrin-complexed desoxypodophyllotoxin was present in the medium. The complexed lignan disappeared from the medium within 7 days for both cell cultures. Cellularly only small amounts of desoxypodophyllotoxin were found. After feeding of desoxypodophyllotoxin, the cell culture of L. flavum accumulated 5-methoxypodophyllotoxin and 5-methoxypodophyllotoxin--D-glucoside. After 7 days a total maximal content of 2.38% on a dry weight basis of 5-methoxypodophyllotoxin was formed, corresponding with 249 mg l^-1 suspension. The highest bioconversion percentage of 52.3% was found at day 14. The desoxypodophyllotoxin-fed culture of P. hexandrum accumulated podophyllotoxin and its -D-glucoside with a maximal content of 2.87% on a dry weight basis after 9 days, corresponding with 192 mg 1^-1 suspension. The highest bioconversion percentage of 33.2% was also found at day 9.     

FadD3 is an acyl‐CoA synthetase that initiates catabolism of cholesterol rings C and D in actinobacteria

The cholesterol catabolic pathway occurs in most mycolic acid‐containing actinobacteria, such as Rhodococcus jostii RHA1, and is critical for Mycobacterium tuberculosis (Mtb) during infection. FadD3 is one of four predicted acyl‐CoA synthetases potentially involved in cholesterol catabolism. A ΔfadD3 mutant of RHA1 grew on cholesterol to half the yield of wild‐type and accumulated 3aα‐H‐4α(3′‐propanoate)‐7aβ‐methylhexahydro‐1,5‐indanedione (HIP), consistent with the catabolism of half the steroid molecule. This phenotype was rescued by fadD3 of Mtb. Moreover, RHA1 but not ΔfadD3 grew on HIP. Purified FadD3_Mtb catalysed the ATP‐dependent CoA thioesterification of HIP and its hydroxylated analogues, 5α‐OH HIP and 1β‐OH HIP. The apparent specificity constant (k_cat/K_m) of FadD3_Mtb for HIP was 7.3 ± 0.3 × 10⁵ M^?1 s^?1, 165 times higher than for 5α‐OH HIP, while the apparent K_m for CoASH was 110 ± 10 μM. In contrast to enzymes involved in the catabolism of rings A and B, FadD3_Mtb did not detectably transform a metabolite with a partially degraded C17 side‐chain. Overall, these results indicate that FadD3 is a HIP‐CoA synthetase that initiates catabolism of steroid rings C and D after side‐chain degradation is complete. These findings are consistent with the actinobacterial kstR2 regulon encoding ring C/D degradation enzymes.