Diverged subpopulations in tropical Urochloa (Brachiaria) forage species indicate a role for facultative apomixis and varying ploidy in their population structure and evolution.

BACKGROUND: Urochloa (syn. Brachiaria) is a genus of tropical grasses sown as forage feedstock, particularly in marginal soils. Here we aimed to clarify the genetic diversity and population structure in Urochloa species to understand better how population evolution relates to ploidy level and occurrence of apomictic reproduction. METHODS: We explored the genetic diversity of 111 accessions from the five Urochloa species used to develop commercial cultivars. These accessions were conserved from wild materials collected at their centre of origin in Africa, and they tentatively represent the complete Urochloa gene pool used in breeding programmes. We used RNA-sequencing to generate 1.1 million single nucleotide polymorphism loci. We employed genetic admixture, principal component and phylogenetic analyses to define subpopulations. RESULTS: We observed three highly differentiated subpopulations in U. brizantha, which were unrelated to ploidy: one intermixed with U. decumbens, and two diverged from the former and the other species in the complex. We also observed two subpopulations in U. humidicola, unrelated to ploidy; one subpopulation had fewer accessions but included the only characterized sexual accession in the species. Our results also supported a division of U. decumbens between diploids and polyploids, and no subpopulations within U. ruziziensis and U. maxima. CONCLUSIONS: Polyploid U. decumbens are more closely related to polyploid U. brizantha than to diploid U. decumbens, which supports the divergence of both polyploid groups from a common tetraploid ancestor and provides evidence for the hybridization barrier of ploidy. The three differentiated subpopulations of apomictic polyploid U. brizantha accessions constitute diverged ecotypes, which can probably be utilized in hybrid breeding. Subpopulations were not observed in non-apomictic U. ruziziensis. Sexual Urochloa polyploids were not found (U. brizantha, U. decumbens) or were limited to small subpopulations (U. humidicola). The subpopulation structure observed in the Urochloa sexual-apomictic multiploidy complexes supports geographical parthenogenesis, where the polyploid genotypes exploit the evolutionary advantage of apomixis, i.e. uniparental reproduction and clonality, to occupy extensive geographical areas.

Comparative analysis of two emerging rice seed bacterial pathogens.

Seed sterility and grain discoloration limit rice production in Colombia and several Central American countries. In samples of discolored rice seed grown in Colombian fields, the species Burkholderia glumae and B. gladioli were isolated, and field isolates were compared phenotypically. An artificial inoculation assay was used to determine that, although both bacterial species cause symptoms on rice grains, B. glumae is a more aggressive pathogen, causing yield reduction and higher levels of grain sterility. To identify putative virulence genes differing between B. glumae and B. gladioli, four previously sequenced genomes of Asian and U.S. strains of the two pathogens were compared with each other and with two draft genomes of Colombian B. glumae and B. gladioli isolates generated for this study. Whereas previously characterized Burkholderia virulence factors are highly conserved between the two species, B. glumae and B. gladioli strains are predicted to encode distinct groups of genes encoding type VI secretion systems, transcriptional regulators, and membrane-sensing proteins. This study shows that both B. glumae and B. gladioli can threaten grain quality, although only one species affects yield. Furthermore, genotypic differences between the two strains are identified that could contribute to disease phenotypic differences.

Expression pattern conferred by a glutamic acid-rich protein gene promoter in field-grown transgenic cassava (Manihot esculenta Crantz).

A major constraint for incorporating new traits into cassava using biotechnology is the limited list of known/tested promoters that encourage the expression of transgenes in the cassava's starchy roots. Based on a previous report on the glutamic-acid-rich protein Pt2L4, indicating a preferential expression in roots, we cloned the corresponding gene including promoter sequence. A promoter fragment (CP2; 731 bp) was evaluated for its potential to regulate the expression of the reporter gene GUSPlus in transgenic cassava plants grown in the field. Intense GUS staining was observed in storage roots and vascular stem tissues; less intense staining in leaves; and none in the pith. Consistent with determined mRNA levels of the GUSPlus gene, fluorometric analyses revealed equal activities in root pulp and stems, but 3.5 times less in leaves. In a second approach, the activity of a longer promoter fragment (CP1) including an intrinsic intron was evaluated in carrot plants. CP1 exhibited a pronounced tissue preference, conferring high expression in the secondary phloem and vascular cambium of roots, but six times lower expression levels in leaf vascular tissues. Thus, CP1 and CP2 may be useful tools to improve nutritional and agronomical traits of cassava by genetic engineering. To date, this is the first study presenting field data on the specificity and potential of promoters for transgenic cassava.

Genetic mitigation strategies to tackle agricultural GHG emissions: The case for biological nitrification inhibition technology.

Accelerated soil-nitrifier activity and rapid nitrification are the cause of declining nitrogen-use efficiency (NUE) and enhanced nitrous oxide (N2O) emissions from farming. Biological nitrification inhibition (BNI) is the ability of certain plant roots to suppress soil-nitrifier activity, through production and release of nitrification inhibitors. The power of phytochemicals with BNI-function needs to be harnessed to control soil-nitrifier activity and improve nitrogen-cycling in agricultural systems. Transformative biological technologies designed for genetic mitigation are needed, so that BNI-enabled crop-livestock and cropping systems can rein in soil-nitrifier activity, to help reduce greenhouse gas (GHG) emissions and globally make farming nitrogen efficient and less harmful to environment. This will reinforce the adaptation or mitigation impact of other climate-smart agriculture technologies.

Isolation and characterisation of a cassava catalase expressed during post-harvest physiological deterioration.

Storage roots of cassava undergo a rapid, endogenous, post-harvest deterioration response that is thought to involve oxidative processes. A cassava catalase (MecCAT1) was isolated from a root cDNA library. The transcript is expressed predominantly in roots with little expression in leaves. Catalase enzyme activity and MecCAT1 transcript expression during the post-harvest period were compared in highly susceptible and less susceptible cultivars and suggest that high levels of catalase activity may play a role in delaying the deterioration response.

Tracing evolutionary and developmental implications of mitochondrial stoichiometric shifting in the common bean.

The recombination and copy number shifting activities of the plant mitochondrial genome are widely documented across plant genera, but these genome processes have not been as well examined with regard to their roles in plant evolution. Because of the extensive plant collections of Phaseolus spp and the degree to which cytoplasmic male sterility (cms) has been characterized in the common bean, this system would be valuable for investigating mitochondrial genome dynamics in natural populations. We have used the cms-associated sequence pvs-orf239 as a mitochondrial genetic marker for these studies and have demonstrated its universal presence throughout a diversity of undomesticated Phaseolus lines. Within these populations, the pvs-orf239 sequence is present in high copy number in approximately 10% of the lines, but substoichiometric in all others. This mitochondrial sequence, derived apparently by at least two recombination events, is well conserved with two point mutations identified that are both apparently silent with regard to the sterility phenotype. A putative progenitor sequence was identified in Phaseolus glabelus in substoichiometric levels, suggesting that the present-day pvs-orf239 sequence was likely introduced substoichiometrically. Copy number shifting within the mitochondrial genome results in a 1000- to 2000-fold change, so that substoichiometric forms are estimated at less than one copy per every 100 cells. On the basis of PCR analysis of root tips, we postulate that a mitochondrial "transmitted form" resides within the meristem to assure transmission of a complete genetic complement to progeny.

Reserpine in propranolol-resistant thyroid storm.

beta-Adrenergic blockers are considered the drugs of choice in the management of the hyperadrenergic state in hyperthyroidism. However, we observed a patient in thyroid storm and coma who failed to respond to large doses of oral and intravenous propranolol hydrochloride but who responded promptly to intramuscular reserpine. Reserpine may have been lifesaving and should be considered in propranolol-resistant hyperthyroidism and in hyperthyroid patients in whom propranolol is contraindicated.

Tumor-induced osteomalacia: clinical and basic studies.

A patient with classic clinical and biochemical features of tumor-induced osteomalacia (hypophosphatemia, phosphaturia, and undetectable serum concentrations of 1,25-dihydroxyvitamin D [1,25(OH)2D]) was studied before and after resection of a benign extraskeletal chondroma from the plantar surface of the foot. Presurgical laboratory evaluation was notable for normal serum concentrations of calcium, intact parathyroid hormone (PTH), parathyroid hormone-related protein (PTHrP), and osteocalcin, increased serum alkaline phosphate activity, and frankly elevated urinary cyclic adenosine monophosphate (cAMP) and pyridinium cross-link excretion. Quantitative histomorphometry showed severe osteomalacia and deep erosions of the cancellous surface by active osteoclasts. After resection, serum 1,25(OH)2D normalized within 24 h, while renal tubular phosphorus reabsorption and serum phosphorus did not normalized until days 2 and 3, respectively; serum Ca declined slightly, and serum intact PTH, osteocalcin, and urinary pyridinium cross-link excretion increased dramatically. Urinary cAMP excretion declined immediately after resection and then began to increase concomitant with the increase in serum intact PTH. A second bone biopsy taken 3 months after resection demonstrated complete resolution of the osteomalacia, increased mineral apposition rate (1.09 mu/day), resorption surface (9.2%), mineralizing surface (71%), and bone formation rate (0.83 mm3/mm2/day), and marked decrease in cancellous bone volume (13.1%) and trabecular connectivity compared with first biopsy. Tumor extracts did not affect phosphate transport in renal epithelial cell lines or 1 alpha-hydroxylase activity in a myelomonocytic cell line. The patient's course suggests that the normal 1,25(OH)2D and phosphorus metabolism is due to a tumor product that may be acting via stimulation of adenylate activity. Increased bone resorption prior to surgical resection suggests that the tumor may also produce an osteoclast activator. The rise in resorption surface and pyridinium cross-link excretion, increase in serum osteocalcin and bone mineralization, normalization of osteoid width, and fall in cancellous bone volume after resection are consistent with healing of osteomalacia by rapid remodeling.

Suppression of parathyroid hormone secretion with oral calcium in normal subjects and patients with primary hyperparathyroidism.

Exquisite sensitivity of normal parathyroid glands to small changes in ambient calcium concentrations and impaired sensitivity in primary hyperparathyroidism have been shown in vitro. Using an assay for PTH that detects rapid changes in PTH secretion (N-terminal-specific RIA; normal range, less than 3-33 pg/mL), we determined PTH suppressibility in response to a standardized dose of oral calcium in normal subjects and patients with primary hyperparathyroidism. Nine normal subjects were given oral calcium (25 mg/kg), and blood was analyzed half-hourly for 3 h for calcium and N-terminal PTH (N-PTH). Serum calcium rose by 0.34 +/- 0.06 mg/dL (0.085 +/- 0.015 mmol/L), and N-PTH levels declined rapidly from 15.3 +/- 1.4 to 4.2 +/- 1.1 pg/mL (-73 +/- 6%; P less than 0.01). In six subjects N-PTH concentrations became undetectable. Nine patients with primary hyperparathyroidism were tested in the same manner. Serum calcium rose by 0.53 +/- 0.1 mg/dL (0.13 +/- 0.025 mmol/L), and N-PTH levels declined less, from 66 +/- 14 to 52 +/- 12 pg/mL (-21 +/- 4%; P less than 0.05). In none of the patients was the PTH reduced to less than 20 pg/mL. These results illustrate in vivo that the PTH response to oral calcium in primary hyperparathyroidism is markedly different from that in normal subjects.

Hypocalcemic emergencies.

The approach to the hypocalcemic patient is best considered with due regard to the underlying etiology and the extent to which features of hypocalcemia are present. When hypocalcemia is a medical emergency, aggressive but judicious measures must be taken immediately to correct, in part, the hypocalcemia. Parenteral therapy of hypocalcemia is advisable only under these conditions. The aim of acute management is not to return the serum calcium to normal but rather to ameliorate the acute manifestations of hypocalcemia. If the hypocalcemic state is owing to a chronic condition that will not remit, a plan for long-term management with a vitamin D preparation and calcium supplementation is implemented after the emergency therapy is provided.

Estrogen treatment of patients with established postmenopausal osteoporosis.

We conducted a controlled study of the effects of oral estrogen therapy in postmenopausal women with established postmenopausal osteoporosis. Bone mass was measured in the lumbar vertebrae and hip using dual photon absorptiometry. Both estrogen-treated women and the control group received calcium supplements to bring total intake to approximately 1500 mg/day. For those women with an intact uterus in the estrogen wing of the study, a progestin was added to the therapy for 12-14 days each calendar month. The number of years from menopause was 14.6 +/- 0.9 in the estrogen-treated group and 13.7 +/- 1.1 in the calcium-treated group. Estrogen treatment was associated with increased vertebral bone mass by dual photon absorptiometry during the 2 years of the study (+10.6%; P less than .01). There was also an increase in bone density at the femoral neck (+5.5%; P less than .1), but the difference from the initial value was not statistically significant. The group given calcium alone lost bone at both sites, although the loss was not statistically significant at either site. The response to estrogen was greatest in those who were furthest from menopause (r = 0.38, P less than .05) and consequently among those who had the lowest bone mass (r = -0.34, P less than .05). Estrogen therapy appears to be an effective therapy for patients with established osteoporosis. Intervention is associated with a significant increase in bone mass compatible with reduced skeletal turnover and activation frequency.

Toward the construction of a molecular map of cassava (Manihot esculenta Crantz): comparison of restriction enzymes and probe sources in detecting RFLPs.

The construction of a detailed genetic map of cassava (Manihot esculenta Crantz), classified as a tetraploid species, depends on the ability of cloned sequences to detect polymorphisms. As a first step in developing this map, 200 cloned nuclear sequences generated with different restriction enzymes were hybridized to total digested DNA from eleven cultivated lines and one wild Manihot species, M. aesculifolia. Polymorphism was detected less frequently with both BamHI and EcoRI genomic probes than with PstI, HindIII and XbaI genomic probes. DNA digested with HpaII, DraI and TaqI displayed less polymorphism, whereas DNA digested with EcoRI and EcoRV displayed more polymorphism like that found in lettuce, rice and tomato (Landry et al., 1987; McCouch et al., 1988; Miller and Tanksley, 1990). Four-cutter restriction enzymes displayed less frequency of polymorphism when compared with six-cutter restriction enzymes. Polymorphism displayed by DraI was extremely low, indicating that regions rich in adenine and thymine may not be hot spots for mutation in cassava. Polymorphism detected between cultivated genotypes and M. aesculifolia was dramatically higher than that found among cultivated genotypes.

The effect of oral contraceptive use on vertebral bone mass in pre- and post-menopausal women.

The effects of oral contraceptive use on bone mineral density in pre- and post-menopausal women were evaluated in two separate studies. First, in a population of young women carefully controlled for all risk factors known to be associated with osteoporosis, it was determined that vertebral bone mineral was increased by about 1% for each year of exposure to oral contraceptives. A similar result was obtained by examining vertebral mineral content of an unselected, but healthy premenopausal population. Effects of oral contraceptives on bone mass could not be found among postmenopausal women, unless perhaps in the initial year or two after loss of ovarian function.

PIP: The effects of oral contraceptive use on bone mineral density in pre- and post-menopausal women were evaluated in 2 separate studies. 1st, in a population of young women carfully controlled for all risk factors known to be associated with osteoporosis, it was determined that vertebral bone mineral was increased by about 1% for each year of exposure to oral contraceptives. On a rough calculation, 5-10 years of use of oral contraceptives could significantly reduce th incidence of osteoporosis if the trend were maintained and result in a 5-10% increase in bone mass in that population and if thses differences could be maintained after menopause. A similar result was obtained in a 2nd study by examining vertebral mineral content of an unselected, but healthy premenopausal population. However, the effect was not observed in the post-menopausal women, with perhaps the exception of the initial 2 years of post-menopausal period. This suggests that the onset of estrogen deficiency with ovarian failure is a sufficiently potent stimulus to the skeleton that the resulting loss of bone would be sufficient to negate the pre-menopausal oral contraceptive effect. Prospective data would be needed to substantiate this finding. Clearly, if oral contraceptive exposure can be expected to have a long-term effect on bone, prevention of post-menopausal bone loss will still be required after cessation of oral contraceptive treatment.

Hydrogen peroxide and flavan-3-ols in storage roots of cassava (Manihot esculenta crantz) during postharvest deterioration.

Cassava storage roots are an important staple food throughout the lowland humid tropics. However, cassava suffers from a poorly understood storage disorder, known as postharvest physiological deterioration (PPD), which constrains its exploitation. In an attempt to broaden the understanding of PPD, nine different cassava cultivars were analyzed for specific compounds accumulating during the process. The production of hydrogen peroxide (H(2)O(2)) is involved in the early stages of PPD in cassava roots. H(2)O(2) was quantified and localized histochemically at the tissue and cell level in deteriorating roots. This reactive oxygen species accumulated during the first 24 h after harvest, especially in the inner parenchymatic tissue. Three flavan-3-ols, (+)-catechin, (+)-catechin gallate, and (+)-gallocatechin, accumulated during the storage of cassava roots. However, these potential antioxidants cannot be related to early storage disorders or wound responses because they start to accumulate only after 4-6 days.

Using cDNA and genomic sequences as tools to develop SNP strategies in cassava (Manihot esculenta Crantz).

Single nucleotide polymorphisms (SNP) are the most abundant type of DNA polymorphism found in animal and plant genomes. They provide an important new source of molecular markers that are useful in genetic mapping, map-based positional cloning, quantitative trait locus mapping and the assessment of genetic distances between individuals. Very little is known on the frequency of SNPs in cassava. We have exploited the recently-developed collection of cassava expressed sequence tags (ESTs) to detect SNPs in the five cultivars of cassava used to generate the sequences. The frequency of intra-cultivar and inter-cultivar SNPs after analysis of 111 contigs was one polymorphism per 905 and one per 1,032 bp, respectively; totaling 1 each 509 bp. We have obtained further information on the frequency of SNPs in six cassava cultivars by analysis of 33 amplicons obtained from 3' EST and BAC end sequences. Overall, about 11 kb of DNA sequence was obtained for each cultivar. A total of 186 SNPs (136 and 50 from ESTs and BAC ends, respectively) were identified. Among these, 146 were intra-cultivar polymorphisms, while 80 were inter-cultivar polymorphisms. Thus the total frequency of SNPs was one per 62 bp. This information will help to develop new strategies for EST mapping as well as their association with phenotypic characteristics.

QTL mapping of grain quality traits from the interspecific cross Oryza sativa x O. glaberrima.

International rice export markets are increasing demands for rapid improvements in grain quality characteristics. The African rice Oryza glaberrima is a new potential source of genes that will enhance the eating, cooking, and milling properties of the rice grain. The objective of this research was to identify and characterize quantitative trait loci (QTLs) among 312 doubled haploid lines derived from the BC3F1 of an interspecific cross of O. sativa x O. glaberrima. Genetic material was planted in replicated plots and evaluated for ten grain quality traits in 2001 in Colombia. A linkage map was constructed with 100 polymorphic microsatellite markers using the mapdisto software program to adjust for segregation distortion. Transgressive segregation was observed for all traits. Interval and composite interval analyses identified 27 QTLs for nine characters located on 11/12 chromosomes. The chromosomal positions of QTLs for percentage amylose, alkali-spreading score, and percentage protein were in agreement with data reported by others, whereas QTL markers for percentage head rice, percentage milled rice, percentage protein, and percentage brown rice were different in our mapping population. Five major QTLs were found to be associated with improved percentage rice bran, percentage amylose, and alkali-spreading score. Seven QTLs for improved percentage rice bran, percentage milled rice, alkali-spreading score, percentage protein, and grain length/width ratio were derived from the O. glaberrima accession. Three new QTLs for percentage rice bran are reported here for the first time. Results from this study suggest that the African rice might be a valuable new source for introgression and improvement of several traits that affect quality traits demanded by the different rice export markets.

Isolation of Resistance Gene Candidates (RGCs) and characterization of an RGC cluster in cassava.

Plant disease resistance genes (R genes) show significant similarity amongst themselves in terms of both their DNA sequences and structural motifs present in their protein products. Oligonucleotide primers designed from NBS (Nucleotide Binding Site) domains encoded by several R-genes have been used to amplify NBS sequences from the genomic DNA of various plant species, which have been called Resistance Gene Analogues (RGAs) or Resistance Gene Candidates (RGCs). Using specific primers from the NBS and TIR (Toll/Interleukin-1 Receptor) regions, we identified twelve classes of RGCs in cassava (Manihot esculenta Crantz). Two classes were obtained from the PCR-amplification of the TIR domain. The other 10 classes correspond to the NBS sequences and were grouped into two subfamilies. Classes RCa1 to RCa5 are part of the first subfamily and were linked to a TIR domain in the N terminus. Classes RCa6 to RCa10 corresponded to non-TIR NBS-LRR encoding sequences. BAC library screening with the 12 RGC classes as probes allowed the identification of 42 BAC clones that were assembled into 10 contigs and 19 singletons. Members of the two TIR and non-TIR NBS-LRR subfamilies occurred together within individual BAC clones. The BAC screening and Southern hybridization analyses showed that all RGCs were single copy sequences except RCa6 that represented a large and diverse gene family. One BAC contained five NBS sequences and sequence analysis allowed the identification of two complete RGCs encoding two highly similar proteins. This BAC was located on linkage group J with three other RGC-containing BACs. At least one of these genes, RGC2, is expressed constitutively in cassava tissues.

Bacterial artificial chromosome (BAC) library resource for positional cloning of pest and disease resistance genes in cassava (Manihot esculenta Crantz).

Pest and disease problems are important constraints of cassava production and host plant resistance is the most efficient method of combating them. Breeding for host plant resistance is considerably slowed down by the crop's biological constraints of a long growth cycle, high levels of heterozygosity and a large genetic load. More efficient methods such as gene cloning and transgenesis are required to deploy resistance genes. To facilitate the cloning of resistance genes, bacterial artificial chromosome (BAC) library resources have been developed for cassava. Two libraries were constructed from the cassava clones, TMS 30001, resistant to the cassava mosaic disease (CMD) and the cassava bacterial blight (CBB), and MECU72, resistant to cassava white fly. The TMS30001 library has 55, 296 clones with an insert size range of 40-150 kb with an average of 80 kb, while the MECU72 library consists of 92 160 clones and an insert size range of 25-250 kb average of 93 kb. Based on a genome size of 772 Mb, the TMS30001 and MECU72 libraries have a 5 and 11.3 haploid genome equivalents and a 95 and 99 chance of finding any sequence, respectively. To demonstrate the potential of the libraries, the TMS30001 library was screened by southern hybridization using a cassava analog (CBB1) of the Xa21 gene from rice that maps to a region containing a QTL for resistance to CBB as probe. Five BAC clones that hybridized to CBB1 were isolated and a Hind III fingerprint revealed 2-3 copies of the gene in individual BAC clones. A larger scale analysis of resistance gene analogs (RGAs) in cassava has also been conducted in order to understand the number and organization of RGAs. To scan for gene and repeat DNA content in the libraries, end-sequencing was performed on 2,301 clones from the MECU72 library. A total of 1705 unique sequences were obtained with an average size of 715 bp. Database homology searches using BLAST revealed that 458 sequences had significant homology with known proteins and 321 with transposable elements. The use of the library in positional cloning of pest and disease resistance genes is discussed.

Inheritance of random amplified polymorphic DNA markers in cassava (Manihot esculenta Crantz).

The informativeness and inheritance of randomly amplified polymorphic DNA (RAPD) markers were investigated in an intraspecific F1 progeny derived from two heterozygous parents. The analysis confirmed the utility of RAPD markers for comparing candidate parents for the development of a molecular genetic map, and provided numerous markers for linkage analysis in a crop with a very limited history of classical or molecular genetic studies. Six potential parental lines (themselves F1 hybrid clones) showed between 1.82 and 0.62 segregating bands per primer in three hybrid families. Forty-three percent (309) of 722 primers produced polymorphic products in the most informative of these three crosses, revealing 328 single-dose (SD) markers segregating 1:1 for presence/absence in a progeny of 90 individuals. A second class of informative markers were those present in both parents but segregating in the progeny. Fifty-seven or 67% of the monomorphic but segregating markers exhibited the 3:1 ratio expected for SD dominant markers in a cross between heterozygotes. Linkage groups were constructed from the segregation of SD RAPD markers originating in the female (TMS 30572) and the male (CM2177-2) parent. Key words : RAPDs, molecular markers, genetic segregation, Manihot, single-dose markers.