Molecular characterization of wheat polyphenol oxidase (PPO).

It is well-established that the enzyme polyphenol oxidase (PPO) is involved in undesirable browning of noodles, chapattis, middle east flat breads and steamed breads. Methods for measuring PPO activity have been developed, and the variation of PPO activity among wheat ( Triticum aestivum L.) cultivars has been well documented. However, there is no report on the identification and characterization of a wheat PPO gene. PCR performed on wheat genomic DNA with oligonucleotide primers designed from conserved copper binding regions of other PPO genes resulted in amplification of a 444-bp DNA fragment. Sequence analysis identified the conserved amino acids of PPO genes indicating that the PCR product was part of the wheat PPO gene. Screening genomic and cDNA libraries using 444- and 760-bp DNA fragments as probes failed to identify a PPO gene based on conserved sequence, even though there were very strong hybridization signals for some isolates. Rapid amplification of cDNA ends (RACE) technique was used as an alternative to obtain the remaining DNA sequences in 5' and 3' directions based on the 444-bp partial wheat PPO gene sequence. With the use of ThermoScript Reverse Transcriptase (which functions at higher temperatures) and Advantage-GC cDNA kit, the complete DNA sequence in the 3' direction was obtained. A similar effort in the 5' direction resulted in amplification of a truncated 414-bp DNA sequence. Overall, 1,509-bp of putative wheat PPO DNA sequence was obtained. Alignment of deduced amino-acid sequences revealed similarity to the other PPO gene sequences, especially in the conserved copper binding regions. Southern-blot analysis performed with four different restriction enzymes revealed two to four DNA fragments, suggesting a limited number of PPO genes in wheat. Wheat genomic DNA restricted with HindIII and hybridized using a 760-bp wheat PPO probe revealed a clear distinction between wheat cultivars with high and low PPO activities. Northern-blot analysis indicated a transcript size of about 2.0-kb. PPO DNA fragment as well as RNA transcript was observed for the durum cultivar Renville which normally has very low PPO activity. Further study is needed to explain the relationship between PPO activity and the presence of PPO gene (s).

Development of a PCR marker for rapid identification of the Bt-10 gene for common bunt resistance in wheat.

In western Canada, the Bt-10 resistance gene in wheat (Triticum aestivum) is effective against all the known races of common bunt caused by Tilletia tritici and T laevis. The genotypes of 199 F2 plants, originated from a cross between BW553 containing Bt-10 and the susceptible spring wheat cultivar 'Neepawa,' were established in greenhouse and field inoculation studies. A ratio of 1:2:1 resistant : heterozygous : susceptible was observed for bunt reaction, indicating that Bt-10 was expressed in a partially dominant fashion. A polymorphic DNA fragment, amplified using RAPD, and previously shown to be linked to Bt-10 was sequenced and SCAR (sequence characterized amplified region) primers devised. However, SCAR primers failed to amplify the polymorphic fragment. Restriction of PCR products with DraI revealed a polymorphic fragment of 490 bp resulting from a single base pair difference between lines possessing Bt-10 and those lacking the gene. As per the base pair difference, FSD and RSA primers were designed to generate a 275-bp polymorphic DNA fragment. Both 275- and 490-bp polymorphic fragments were present in all of the 22 cultivars known to carry Bt-10, and absent in all 16 cultivars lacking Bt-10. A 3:1 ratio was observed for presence: absence of the 275-bp marker in the F2 population. Using Southern analysis, the 490-bp fragment was effective in differentiating homozygous resistant plants from those heterozygous for Bt-10, based on its presence and the hybridization signal strength. A 1:2:1 resistant : heterozygous : susceptible ratio was also observed for the molecular marker and corresponded to 88% of the phenotypes deduced from the original F2 population. The molecular marker was estimated to be between 1.1 cM and 6.5 cM away from the Bt-10 resistance gene, based on the segregation analysis. Segregation analyses of Bt-10 and the 275-bp marker, evaluated in three different Canada Prairie Spring (CPS) wheat populations, demonstrated a segregation ratio of 3:1 for the molecular marker in two of the populations. These results demonstrated that the PCR marker system using the FSD and RSA primer pair permitted a rapid and reliable identification of individual lines carrying the Bt-10 gene for resistance to common bunt.

A DNA marker for the Bt-10 common bunt resistance gene in wheat.

The Bt-10 bunt gene confers resistance to most races of the common bunt fungi, Tilletia tritici and T. laevis. The RAPD technique, employing a total of 965 decamer primers, was used to identify polymorphic markers between resistant (BW553) and susceptible ('Neepawa") near-isogenic lines. Primer 196 (5' CTC CTC CCC C 3') produced a 590 base pair (bp) reproducible fragment only in the resistant near-isogenic line. The 590-bp DNA fragment was present in all the 22 wheat cultivars known to carry the Bt-10 resistance gene and also in 15 resistant F2 lines obtained from a cross between the resistant parent, BW553, and the susceptible parent, 'Neepawa'. The 590-bp fragment was absent in 16 susceptible cultivars tested and in 15 susceptible F2 lines obtained from the cross described above. These results suggest a close linkage between the presence of the 590-bp fragment and the Bt-10 resistance gene. Primer 372 (5' CCC ACT GAC G 3') amplified a 1.0-kilobase (kb) fragment that was present only in the susceptible near-isogenic line. This 1.0-kb fragment was present in 13 of the 16 susceptible cultivars and in 13 of the 15 susceptible F2 lines. However, the primer also amplified the 1.0-kb fragment in some resistant cultivars and resistant F2 lines, suggesting a looser linkage between the occurrence of the fragment and the susceptible allele.

Genetic diversity of potato determined by random amplified polymorphic DNA analysis.

The RAPD procedure was used to establish genetic diversity of 28 potato genotypes including siblings and genotypes with no immediate relationship. In addition amplified DNA from three parents and Solanum chacoense were compared with that from six progeny to determine the genetic relationships. Amplification of genomic DNA from the 28 genotypes using PCR and 12 decamer primers yielded 158 amplified DNA fragments, ranging in size from 490 to 3200 bp. A total of 128 unique RAPD fragments were observed among the 28 potato genotypes. Similarity measures and principal coordinate analysis generally reflected the expected trends in relationships of the full and half-sib potato genotypes. However there were important exceptions to this general trend and it appears that related varieties can be as genetically different as varieties with no immediate relationship. The data suggest that RAPD analysis used in conjunction with pedigree information can provide a superior measure of genetic divergence than analysis based solely on pedigree information.

Goitre in Ethiopia.

A stratified goitre survey was conducted on 35,635 schoolchildren and 19,158 household members in all Regions of Ethiopia except Eritrea and Tigrai. The gross goitre prevalence (mean of male and female values) among schoolchildren and household members was 30.6 and 18.7% respectively, while that of visible goitre was 1.6 and 3.2% respectively. Prevalence was higher in females (27.3% in household members and 36.1% in schoolchildren) than in males (10.1% in household members and 25.1% in schoolchildren) and increased with age more in females than in males. The prevalence rates at higher altitudes were higher than those at lower altitudes in both schoolchildren and household members. Using an epidemiological model the consequences of iodine deficiency, including cretinism and maternal wastage, have been estimated.

RAPD DNA fingerprints and terpenoids: clues to past migrations of Juniperus in Arabia and east Africa.

Random amplified polymorphic DNAs (RAPDs) and leaf volatile terpenoids were used to compare junipers from Abha, Saudi Arabia with J. excelsa from Greece and J. procera from Addis Ababa, Ethiopia. Both the RAPDs and terpenoids clearly identified the Abha juniper as J. procera. The migration and evolution of J. excelsa or pre-J. excelsa junipers southward from Asia Minor into Africa is discussed. A computer program, PCO3D, is now available for 3-D ordination of RAPDs data. In addition, this research supports the recognition of both J. excelsa and J. procera as separate species.

The effects of plant polysaccharides and buffer additives on PCR.

A survey of the inhibitory effects of various plant polysaccharides on PCR amplification of a 974-bp section of rbcL in spinach revealed that most of the polysaccharides tested (arabinogalactan, carrageenan, dextran, gum guar, gum karaya, gum locust bean, inulin, mannan, pectin, starch and xylan) were not inhibitory. In contrast, two of the acidic polysaccharides (dextran sulfate and gum ghatti) were inhibitory. The addition of 0.5% Tween 20 reversed the inhibitory effects of gum ghatti (polysaccharide:DNA ratio of 500:1). The inhibitory effect of dextran sulfate (50:1) could be reversed by the addition of Tween 20 (0.25% or 0.5%), DMSO (5%) or polyethylene glycol 400 (5%), but none of these three additives were effective at a 100:1 ratio of dextran sulfate/DNA.

Potential taxonomic use of random amplified polymorphic DNA (RAPD): a case study in Brassica.

The potential use of RAPDs for taxonomic studies were investigated using Brassica, Sinapis and Raphanus taxa. Principal coordinate analysis of 284 RAPD bands revealed the classical U triangle relationship between diploid and amphidiploid Brassica taxa. Raphanus sativus and S. alba were distinct from the Brassica taxa. It appears that at least ten primers with approximately 100 total bands are needed to adequately portray these relationships. Cultivars of cabbage and cauliflower were separated by RAPDs. Analysis of RAPDs from individual plants of B. carinata cv. dodola resulted in 69 RAPDs, with 91.7% monomorphic and 8.3% polymorphic bands. RAPDs appear to be useful for taxonomic studies at levels ranging from populations to species and perhaps genera.

Xerophthalmia in Ethiopia: a nationwide ophthalmological, biochemical and anthropometric survey.

A total of 6636 children, aged from 6 months to 6 years and selected throughout the country using a multi-staged stratified sample design, were examined for signs of xerophthalmia. The concentrations of retinol and of beta-carotene were measured in 742 children, including those with xerophthalmia and every twentieth of the remaining children. Anthropometric measurements were made on 2909 of the children. Bitot's spots were seen in 1.0% of all children, with a higher prevalence in the pastoral (1.6%) and cropping (1.1%) agro-ecological zones than in the zones characterized by cash crops (0.4%) and 'ensete' (false banana, Ensete ventricosum) (0.0%). One case of corneal xerosis and 2 cases of corneal scar were also seen. Serum retinol levels were in the 'deficient' range (less than 0.35 mumol l-1) in 16% and 'low' (0.35-0.69 mumol l-1) in 44% of children. Serum retinol and clinical signs did not show any correlation with occupation and education of head of household, household size or anthropometric measurements. More stunting than wasting was observed, with peak prevalence of these signs of malnutrition being observed in the second year of life.

Seasonal energy deficiency in Ethiopian rural women.

The purpose of the study was to establish the existence of seasonal exposure to energy deficiency in rural areas of developing countries and to investigate the sequence of appearance and the nature of energy-sparing mechanisms utilized under real life conditions. The body weight of a group of 226 rural Ethiopian women was measured repeatedly over a one year period, at 45 day intervals. On a sub-group of 22 non-pregnant women total energy intake, TEI, total energy expenditure, TEE, and basal metabolic rate, BMR were also measured by the precise weighing method and by indirect calorimetry (Kofranyi-Michaelis respirometer or Douglas bag) and activity diaries. Body weight was found to have a moderate but statistically significant seasonal trend, with an overall loss of 1.6 kg. Women with higher BMI had larger seasonal swings of their body weight. Seasonal fluctuations were also found for TEI (maximum difference 420 kcal/d, not significant), and for BMR (maximum difference 200 kcal/day, P less than 0.000). TEE (mean yearly value of 1909 kcal/d, 42 kcal/kg) was very stable over the year and did not show any seasonal fluctuation. The present findings suggest that, under the study circumstances, seasonal exposure to fluctuation in food availability caused a moderate weight loss which was sufficient to induce metabolic adaptations, but not to cause any detectable change in physical activity.

Micropropagation of Phytolacca dodecandra through shoot-tip and nodal cultures.

A procedure for micropropagation of endod (Phytolacca dodecandra) is described. BA at 0.44 µM produced 3.1 new shoots per expiant in six weeks using shoot tips. Nodal expiants, however, produced up to 4.7 shoots per explant on medium with 0.44 µM BA and 0.27 µM GA,. IBA at 0.49 µM induced 90% rooting with minimal callus. Plantlets were successfully transferred to the greenhouse and some staminate clones produced flowers after six months.