Genetic diversity and relationships in native Hawaiian Saccharum officinarum sugarcane.

Commercial sugarcane hybrid cultivars currently in production are high-yielding, disease-resistant, millable canes and are the result of years of breeding work. In Hawaii, these commercial hybrids are quite distinct from many Saccharum officinarum canes still in existence that were brought to the islands and cultivated by the native Polynesians. The actual genetic relationships among the native canes and the extent to which they contributed to the commercial hybrid germplasm has been the subject of speculation over the years. Genetic analysis of 43 presumed native Hawaiian S. officinarum clones using 228 DNA markers confirmed them to be a group distinct from the modern hybrid cultivars. The resulting dendrogram tended to confirm that there were several separate S. officinarum introductions that, owing to selections of somatic mutations, diverged into a number of cluster groups. When the "Sandwich Isles" were discovered by Captain James Cook in 1778, the Hawaiians were found to be growing sugarcane, S. officinarum ( Cook 1785). Sugarcane (ko, in the Hawaiian language) appeared in a variety of stalk and leaf colors, often with stripes (the "ribbon canes"). In the interest of preserving this historic germplasm, a collection was assembled in the 1920s by Edward L. Caum of the Hawaiian Sugar Planters' Association and W. W. G. Moir of American Factors. Histories and descriptions of the canes were reported by Moir (1932).

Green fluorescent protein as a visual selection marker for papaya (Carica papaya L.) transformation.

Chemical-based selection for plant transformation is associated with a number of real and perceived problems that might be avoided through visual selection. We have used green fluorescent protein (GFP), as a visual selectable marker to produce transformed papaya ( Carica papaya) plants following microprojectile bombardment of embryogenic callus. GFP selection reduced the selection time from 3 months on a geneticin (G418) antibiotic-containing medium to 3-4 weeks. Moreover, GFP selection increased the number of transformed papaya plants by five-to eightfold compared to selection in the presence of antibiotics. Overall, the use of GFP for selecting transgenic papaya lines improved our throughput for transformation by 15- to 24-fold while avoiding the drawbacks associated with the use of antibiotic resistance-based selection markers.

Construction of a genetic map for arabica coffee.

We have used AFLPs to construct a genetic linkage map on a pseudo-F(2) population of arabica coffee ( Coffea arabica L.) derived from a cross between the cultivars Mokka hybrid and Catimor. Sixty trees from this population were selected on the basis of plant height distribution to construct a linkage map. A total of 456 dominant markers and eight co-dominant markers were generated from 288 AFLP primer combinations. Of the total number of markers generated, 68% were from cv. Catimor, 30% from cv. Mokka hybrid, and 2% were co-dominant. This distribution suggests that the heterozygosity within the cv. Catimor sub-genomes was twice that within the cv. Mokka hybrid sub-genomes. Linkage groups were constructed using MAPMAKER version 3.0, resulting in 16 major linkage groups containing 4-21 markers, and 15 small linkage groups consisting of 2-3 linked markers each. The total length of the map was 1,802.8 cM, with an average distance of 10.2 cM between adjacent markers. This genetic map will serve as the framework for mapping QTL controlling source-sink traits in the same population.

Molecular Diversity of Ralstonia solanacearum Isolated from Ginger in Hawaii.

ABSTRACT The genetic diversity of Ralstonia solanacearum strains isolated from ginger (Zingiber officinale) growing on the island of Hawaii was determined by analysis of amplified fragment length polymorphisms (AFLPs). Initially 28 strains of R. solanacearum collected from five host plant species worldwide were analyzed by AFLP. A second analysis was conducted on 55 R. solanacearum strains collected from three ginger farms along the Hamakua Coast of Hawaii, the principle area of ginger cultivation in the state. From the initial analysis, R. solanacearum strains from ginger in Hawaii showed a high degree of similarity at 0.853. In contrast, the average genetic similarity between R. solanacearum strains from heliconia and ginger was only 0.165, and strains from ginger showed little similarity with strains from all other hosts. The second analysis of 55 strains from ginger on different Hawaiian farms confirmed that they were distinct from race 1 strains from tomato. Strains from ginger also showed greater diversity among themselves in the second analysis, and the greatest diversity occurred among strains from a farm where ginger is frequently imported and maintained. Our results provide evidence that R. solanacearum strains from ginger in Hawaii are genetically distinct from local strains from tomato (race 1) and heliconia (race 2).

Genetic diversity of Carica papaya as revealed by AFLP markers.

Genetic relationships among Carica papaya cultivars, breeding lines, unimproved germplasm, and related species were established using amplified fragment length polymorphism (AFLP) markers. Seventy-one papaya accessions and related species were analyzed with nine EcoRI-MseI primer combinations. A total of 186 informative AFLP markers was generated and analyzed. Cluster analysis suggested limited genetic variation in papaya, with an average genetic similarity among 63 papaya accessions of 0.880. Genetic diversity among cultivars derived from the same or similar gene pools was smaller, such as Hawaiian Solo hermaphrodite cultivars and Australian dioecious cultivars with genetic similarity at 0.921 and 0.912, respectively. The results indicated that self-pollinated hermaphrodite cultivars were as variable as open-pollinated dioecious cultivars. Genetic diversity between C. papaya and six other Carica species was also evaluated. Carica papaya shared the least genetic similarity with these species, with an average genetic similarity of 0.432; the average genetic similarity among the six other species was 0.729. The results from AFLP markers provided detailed estimates of the genetic variation within and among papaya cultivars, and supported the notion that C. papaya diverged from the rest of Carica species early in the evolution of this genus.

QTL analysis in a complex autopolyploid: genetic control of sugar content in sugarcane.

QTL mapping in autopolyploids is complicated by the possibility of segregation for three or more alleles at a locus and by a lack of preferential pairing, however the subset of polymorphic alleles that show simplex segregation ratios can be used to locate QTLs. In autopolyploid Saccharum, 36 significant associations between variation in sugar content and unlinked loci detected by 31 different probes were found in two interspecific F(1) populations. Most QTL alleles showed phenotypic effects consistent with the parental phenotypes, but occasional transgressive QTLs revealed opportunities to purge unfavorable alleles from cultivars or introgress valuable alleles from exotics. Several QTLs on homologous chromosomes appeared to correspond to one another-multiple doses of favorable 'alleles' at such chromosomal region(s) yielded diminishing returns-such negative epistasis may contribute to phenotypic buffering. Fewer sugar content QTLs were discovered from the highest-sugar genotype than from lower-sugar genotypes, perhaps suggesting that many favorable alleles have been fixed by prior selection, i.e. that the genes for which allelic variants (QTLs) persist in improved sugarcanes may be a biased subset of the population of genes controlling sugar content. Comparison of these data to mutations and QTLs previously mapped in maize hinted that seed and biomass crops may share a partly-overlapping basis for genetic variation in carbohydrate deposition. However, many QTLs do not correspond to known candidate genes, suggesting that other approaches will be necessary to isolate the genetic determinants of high sugar content of vegetative tissues.

Vocational training: the case scenario workshop.

This article describes the procedure for conducting a workshop for a vocational training group, using six clinical case scenarios harvested from the vocational training group's own experience. During the session, the main group (which includes trainers) breaks up into smaller groups to discuss the management of each case, then a spokesperson presents the management solutions back to the re-assembled larger group: further interaction and discussion then takes place. This article discusses six typical case scenarios from a recent workshop. The case scenario workshop is an exciting way to encourage discussion and interaction with all parties involved.

Quantitative chromosome map of the polyploid Saccharum spontaneum by multicolor fluorescence in situ hybridization and imaging methods.

Somatic chromosomes of a wild relative of sugarcane (Saccharum spontaneum L.) anther culture-derived clone (AP 85-361, 2n = 32) were identified and characterized by computer-aided imaging technology and molecular cytological methods. The presence of four satellite chromosomes and four nearly identical chromosome sets suggests that the clone is a tetrahaploid with the basic number x = 8. A quantitative chromosome map, or idiogram, was developed using image analysis of the condensation pattern (CP) at the prometaphase stage of somatic chromosomes. The 45S and 5S ribosomal RNA gene (rDNA) loci were simultaneously visualized by multi-color fluorescence in situ hybridization (McFISH) and precisely localized to the regions of 3p3.1 and 6q1.3 on the idiogram. The simultaneous visualization of two sets of four ribosomal RNA genes confirms tetraploidy of this clone. This conclusion is consistent with results of molecular marker mapping. The quantitative chromosome map produced will become the foundation for genome analyses based on chromosome identity and structure. Previously impossible identification of small chromosomes and untestable hypotheses about the polyploid nature of plants can now be settled with these two approaches of quantitative karyotyping and FISH.

Detailed alignment of saccharum and sorghum chromosomes: comparative organization of closely related diploid and polyploid genomes.

The complex polyploid genomes of three Saccharum species have been aligned with the compact diploid genome of Sorghum (2n = 2x = 20). A set of 428 DNA probes from different Poaceae (grasses) detected 2460 loci in F1 progeny of the crosses Saccharum officinarum Green German x S. spontaneum IND 81-146, and S. spontaneum PIN 84-1 x S. officinarum Muntok Java. Thirty-one DNA probes detected 226 loci in S. officinarum LA Purple x S. robustum Molokai 5829. Genetic maps of the six Saccharum genotypes, including up to 72 linkage groups, were assembled into "homologous groups" based on parallel arrangements of duplicated loci. About 84% of the loci mapped by 242 common probes were homologous between Saccharum and Sorghum. Only one interchromosomal and two intrachromosomal rearrangements differentiated both S. officinarum and S. spontaneum from Sorghum, but 11 additional cases of chromosome structural polymorphism were found within Saccharum. Diploidization was advanced in S. robustum, incipient in S. officinarum, and absent in S. spontaneum, consistent with biogeographic data suggesting that S. robustum is the ancestor of S. officinarum, but raising new questions about the antiquity of S. spontaneum. The densely mapped Sorghum genome will be a valuable tool in ongoing molecular analysis of the complex Saccharum genome.

Comparative efficacy evaluation of dicationic carbazole compounds, nitazoxanide, and paromomycin against Cryptosporidium parvum infections in a neonatal mouse model.

The efficacies of dicationic carbazole compounds, nitazoxanide (NTZ), and paromomycin were evaluated against the AUCp1 isolate of Cryptosporidium parvum by using a neonatal mouse model. Compounds were solubilized or suspended in deionized water and administered orally by gavage to neonatal mice at a constant dose rate on days 0 to 5 (treatment started on day 0). Dose rates varied for individual carbazole compounds but ranged from 0.65 to 20 mg/kg of body weight. NTZ was tested at 100 and 150 mg/kg, and paromomycin was tested at 50 mg/kg. Efficacies were determined by comparing numbers of oocysts present in treated versus control mice at necropsy examination on day 6. Demonstrable efficacy was observed for several carbazole compounds, based on significant reductions in the numbers of oocysts recovered from treated mice versus control mice. Compounds 1, 7, and 10 (19.0 mg/kg) reduced oocyst passage in treated mice to less than 5% of that in control mice. Treatment with compounds 6, 8, and 9 (17.0 mg/kg) resulted in reductions of oocyst output to less than 10% of that in controls. Although they were not comparable in efficacy to compounds 1, 6, 7, 8, 9, and 10, treatment with other carbazole compounds resulted in statistically significant reductions in oocyst output in treated versus control mice. Compound 1 retained efficacy resulted in reduction of oocyst output to approximately 6% of that in controls when the dose was reduced to 5 mg/kg. Further reductions in the dose rate resulted in considerable reductions in anticryposporidial activity. Likewise, the efficacies of compounds 9 and 10 were reduced substantially when the doses were lowered to one-half the screening dose. Paromomycin yielded excellent activity (reduction of oocyst output to <2% of that in controls) at a dose of 50 mg/kg. NTZ yielded moderate efficacy as powder and injectable formulations administered at 100 mg/kg orally (reduction of oocyst output to 42 and 26% of that in controls, respectively). Oral administration of the injectable formulation of NTZ at a dose of 150 mg/kg resulted in improved efficacy (oocyst output, <5% of that in controls).

Dicationic furans inhibit development of Cryptosporidium parvum in HSD/ICR suckling Swiss mice.

The efficacy of dicationic diarylfurans was evaluated against Cryptosporidium parvum by a suckling murine model. Candidate drugs were solubilized or suspended in deionized water and administered orally at a constant dose rate on days 0-5 (treatment day 0) to suckling ICR Swiss mice experimentally inoculated with oocysts of C. parvum. Efficacy was based on numbers of oocysts recovered from the intestinal tracts of mice subjected to necropsy examination on day 6. Numerous candidate furans significantly reduced the numbers of oocysts recovered from treated mice compared with control mice. Compounds 1, 2, 4, and 9 demonstrated superior efficacies (10% of controls or better) against C. parvum. Compounds 3, 5, 6, 7, 8, 11, 17, 18, and 19 also significantly reduced the numbers of oocysts recovered from treated mice but demonstrated efficacies ranging from 17 to 65% of controls. Compound 4 was particularly efficacious against C. parvum at a dosage as low as 8.5 mg/kg of body weight. Compound 4 is identified as a lead compound for additional studies in other animal models.

Synthesis of new immunogens for estrone and estradiol-17 beta and antisera evaluation.

Syntheses of the 6 alpha-O-carboxymethyl ether derivatives of estrone and estradiol-17 beta and the preparation of their bovine serum albumin conjugates are described. The generation and evaluation of antisera produced from these conjugates is discussed.

Machinery-related fatalities in the construction industry.

The National Traumatic Occupational Fatalities (NTOF) surveillance system identified machinery-related incidents as the fourth leading cause of traumatic occupational fatalities in the U.S. construction industry between 1980 and 1992, resulting in 1,901 deaths and 2.13 deaths per 100,000 workers. Fatality rates declined 50% over the study period. Workers in three occupation divisions-precision production, craft, and repair; transportation and material moving; and handlers, equipment cleaners, helpers, and laborers-had both the highest frequency and rate of fatalities. Cranes, excavating machinery, and tractors were the machines most frequently involved. The most common incident types were: struck by a mobile machine; overturn; and struck by a boom. Further delineation of groups at highest risk for machinery-related injuries is complicated by a lack of data on exposure to machinery. The findings suggest that injury prevention programs should focus not only on machine operators, but on those who work on foot around machines.

Characterization of a composite gradient gel for the electrophoretic separation of lipoproteins.

We describe a protocol for making a new type of gradient gel, the Composite gradient gel, that was designed to resolve plasma lipoproteins using nondenaturing gradient gel electrophoresis. The new gel format allows analysis both of high density lipoproteins (HDLs) and low density lipoproteins (LDLs) on the same gel. The gel gave highly repeatable (r2 = 0.999) size estimates. We compared lipoprotein phenotypes determined from the new gradient gel with those obtained using specialized HDL and LDL gradient gels. The comparisons indicated that the Composite gel gave lipoprotein particle size estimates for HDLs and LDLs that were virtually identical to those obtained, respectively, from the specialized HDL and LDL gradient gels. We measured median diameters, which reflect the distributions of absorbance, for LDLs and for HDLs and found that the Composite gel gave lipoprotein size distributions that were virtually identical to those measured using the specialized LDL and HDL gels. Finally, comparison of fractional absorbance for six lipoprotein size intervals obtained from the Composite and specialized gels revealed a close correlation (r2 = 0.828). Thus, it appears that both LDL and HDL size phenotypes may be evaluated simultaneously using a single gradient gel format.

Sucrose Accumulation in the Sugarcane Stem Is Regulated by the Difference between the Activities of Soluble Acid Invertase and Sucrose Phosphate Synthase.

To assess the relative importance of morphological and biochemical factors in the regulation of sucrose (Suc) accumulation in the sugarcane (Saccharum spp. hybrids) stem, we investigated morphological and biochemical correlates of Suc accumulation among parents and progeny of a family segregating for differences. In contrast to the parents, no relationship was observed between morphology and the level of Suc accumulation among the progeny. The level and timing of Suc accumulation in the whole stalk and within individual internodes was correlated with the down-regulation of soluble acid invertase (SAI) activity. High SAI activity prevented most, but not all, Suc accumulation. There was a critical threshold of SAI activity above which high concentrations of Suc did not accumulate. This low level of SAI activity was always exceeded in the internodes of the lower-Suc-storing genotypes. However, low activity of SAI was not sufficient by itself to account for the Suc accumulation in the higher-Suc-storing genotypes. Major differences in Suc accumulation among the population were attributed to the difference between activities of SAI and Suc phosphate synthase, provided SAI is below the critical threshold concentration. This result is not unexpected, since the pathway of Suc transport for storage involves Suc hydrolysis and resynthesis.

Genetic control of apolipoprotein A-I distribution among HDL subclasses.

We conducted genetic analyses to determine the components of variation for size distributions of apolipoprotein (apo) A-I among human plasma lipoproteins resolved on the basis of size. Analyses used data for 717 individuals in 26 pedigrees. Apo A-I distributions among lipoprotein size classes were measured by nondenaturing gradient gel electrophoresis (GGE) and immunoblotting procedures. Curves were fitted to apo A-I absorbance profiles to estimate fractional absorbance in each of five high-density lipoprotein (HDL) subclasses. Multivariate regression analyses revealed several covariates (sex, age, diabetes, and apo A-I concentrations) that were significantly associated with variation in one or more HDL subclasses. Female gender and elevated apo A-I concentrations were associated with increases in proportion of apo A-I in larger HDLs, while increasing age and diabetes were associated with decreases. The analyses showed significant heritabilities. h2, for each variable representing the different HDL subclasses. Both genetic and nongenetic effects on apo A-I size distributions were generally exerted across the range of lipoprotein sizes, as suggested by high genetic and environmental correlations between HDL subclass variables. Decomposition of total overall variance showed that unidentified environmental factors accounted for 48% of variation in apo A-I size distribution, while genetic factors explained about 36% and the identified covariates explained the remaining 16%. When considered separately, apo A-I concentration explained only 5% of the total variation in apo A-I size distribution, indicating that apo A-I concentration is a poor predictor of apo A-I size distribution. In summary, the data suggest that there are significant genetic and environmental effects on apo A-I size distribution in humans, and that they are general metabolic effects rather than effects on specific HDL subclasses.

Effect of diabetes on lipoprotein size.

The effects of diabetes on lipoprotein particle sizes were assessed using samples from 94 subjects with non-insulin-dependent diabetes mellitus. From a larger population of nondiabetic subjects who showed normal glucose tolerance, we selected an exact match in terms of age, sex, and menopausal status. We designed a protocol to make nondenaturing gradient gels for the resolution of LDL subfractions and generated two measures of LDL size: diameter of the predominant LDL species and proportion of LDL cholesterol (LDL-C) in particles larger than 25.5 nm (large LDL-C). Similarly, we made two measures of HDL size, large HDL cholesterol (HDL-C) and large HDL-apoAI, which represents the proportion of HDL-C and apoAI, respectively, occurring on particles larger than HDL-3. In pairwise comparisons, diabetes was associated with significantly (P < .004) smaller lipoprotein particles for all measures except large HDL-C. Each of the size measures was significantly and positively correlated with each of the others, suggesting that common metabolic mechanisms influence lipoprotein particle sizes across classes of lipoproteins. In addition, each of the size measures was correlated with a variety of measures of HDL and beta-lipoprotein concentrations, which included HDL-C, LDL-C, triglycerides, and apoAI, apoB, and apoE. We used stepwise regression analyses to select from the measures of lipoprotein concentrations those independently correlated with each of the lipoprotein size measures. After adjusting for these metabolic correlates of lipoprotein size measures, we found the effect of diabetes on lipoprotein size measures was no longer significant except for a modest effect (P = .027) on large HDL-apoAI.

Resin-purified digoxigenin-labeled DNA probes: an efficient protocol for mapping and fingerprinting sugarcane.

Nonradioactive Southern blotting using digoxigenin (DIG) has become routinely applied to the analysis of single-copy genes for genetic mapping because it is fast and safe. Previous studies indicate that DIG-labeled probes are suitable for single-gene detection in less complex genomes, but their efficient application to mapping a large octoploid genome has not been discussed. We developed a stream-lined procedure for nonradioactive restriction fragment length polymorphism mapping and DNA fingerprinting of sugarcane that combines DIG-11-dUTP and anion-exchange chromatography. In this report, we show that anion-exchange chromatography provides a reliable and simple technique for the resin-purification of large numbers of DIG-labeled DNA fragments 0.3-3.0 kb in size, and it is essential in minimizing contaminants and nonspecific signal.

Circulating levels of anxiolytic steroids in the luteal phase in women with premenstrual syndrome and in control subjects.

The levels of 3 alpha-hydroxy-5 alpha-pregnan-20-one (allopregnanolone) and the epimeric 3 alpha-hydroxy-5 beta-pregnan-20-one (pregnanolone) were studied in women with prospectively confirmed premenstrual syndrome (n = 15) and in a group of asymptomatic control women (n = 12) during the luteal phase of the menstrual cycle. Single late luteal phase plasma samples were selected to make comparisons of plasma hormone levels between patients and controls in the following measures: allopregnanolone, pregnanolone, the ratio of allopregnanolone to pregnanolone, the ratio for each of these anxiolytic steroids to the parent compound progesterone, and the ratio of the sum of allopregnanolone and pregnanolone to progesterone. Differences in these measures were compared by analysis of variance. Additionally, correlations were performed among the various hormone measures and between the hormone measures and the symptom self-ratings. Analysis of variance showed no significant between group differences in the plasma levels of allopregnanolone, pregnanolone, and progesterone. Plasma levels of both allopregnanolone and pregnanolone were correlated with plasma progesterone levels. However, there were no significant correlations between the severity of mood and behavioral symptoms and plasma levels of progesterone, allopregnanolone, and pregnanolone. These data suggest that symptoms of premenstrual syndrome are not associated with a simple deficiency state of either progesterone or its anxiolytic steroid metabolites.

Effects of photodynamic therapy using mono-L-aspartyl chlorin e6 on vessel constriction, vessel leakage, and tumor response.

The effect of photodynamic therapy using mono-L-aspartyl chlorin e6 (NPe6) on both direct cytotoxicity and vascular damage was examined. Sprague-Dawley rats bearing chondrosarcoma tumor were given i.v. injections of 5 or 10 mg/kg NPe6 and exposed to 135-J/cm2 664-nm laser light either 4 or 24h after NPe6 injection. The percentage of viable tumor cells was estimated either immediately after the completion of light treatment or 24 h after treatment using a 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assay. Measurements of arteriole constriction and venule leakage in normal cremaster tissues were made during and 1 h after the light treatment. Tumor response was evaluated for the 4 different NPe6 dose and time combinations. Both direct tumor cytotoxicity and vascular stasis were observed during light treatment. Vessel leakage did not occur. Blood flow stasis was a result of platelet aggregation and the mechanical obstruction of flow rather than vessel constriction. The magnitude of direct cytotoxicity and vascular response was dependent on both the amount of NPe6 delivered and the delay between injection and light treatment. Tumor cure was found in animals either when given high NPe6 doses or when treated early after NPe6 injection. Treatment regimens which maximized the effect of both vascular stasis and direct tumor cytotoxicity were found to produce the best tumor response. Dose combinations which produced vascular stasis with minimal early cytotoxicity did not result in cure. The combined mechanisms of damage after photodynamic therapy using NPe6 suggests that this photosensitizer may have specific advantages for clinical use and provides a benchmark for the development of new photosensitizers.