Oxidative stress markers and disease severity among children with sickle cell anaemia

Background: Sickle cell anaemia has been associated with oxidative stress. Total Antioxidant Capacity (TAC), Total Oxidant Status (TOS) and Oxidative Stress Index (OSI) are cumulative markers of oxidative stress. Objective: To evaluate the serum levels of oxidative stress markers in children with sickle cell anaemia (SCA) and determine the relationship between these markers and disease severity. Method: One hundred and fifty-six children, comprising 78 with SCA, aged 1 - 15 years and 78 age- and sex-matched Haemoglobin AA controls were studied. Serum TOS, OSI, and TAC were determined using ELISA kits. The severity of the SCA was determined using clinical and laboratory parameters. Result: Children with SCA had lower mean serum TAC (0.83±0.31UAE) than controls (1.19±0.24UAE) with p< 0.001) but positive correlation with TOS (r = 0.3, p = 0.008) and OSI (r = 0.6, p < 0.001). Conclusion: Children with SCA had lower TAC but higher TOS and OSI than matched controls. Oxidative stress markers had a significant relationship with SCD severity

Evaluation of Hbr (MITE) markers for assessment of genetic relationships among maize ( Zea mays L.) inbred lines.

Recently, a new type of molecular marker has been developed that is based on the presence or absence of the miniature inverted repeat transposable element (MITE) family Heartbreaker ( Hbr) in the maize genome. These so-called Hbr markers have been shown to be stable, highly polymorphic, easily mapped, and evenly distributed throughout the maize genome. In this work, we used Hbr-derived markers for genetic characterization of a set of maize inbred lines belonging to Stiff Stalk (SS) and Non-Stiff Stalk (NSS) heterotic groups. In total, 111 markers were evaluated across 62 SS and NSS lines. Seventy six markers (68%) were shared between the two groups, and 25 of the common markers occurred at fairly low frequency (

Estimation of sampling variance of molecular marker data using the bootstrap procedure.

Knowledge of genetic relationships among genotypes is useful in a plant breeding program because it permits the organization of germplasm and provides for more efficient sampling. The genetic distance (GD) among genotypes can be estimated using random restriction fragment length polymorphisms (RFLPs) as molecular markers. Knowledge of the sampling variance associated with RFLP markers is needed to determine how many markers are required for a given level of precision in the estimate of GD. The sampling variance for GD among all pairs of 37 maize (Z. mays L.) inbred lines was estimated from 1202 RFLPs. The 1202 polymorphisms were generated from 251 enzyme-probe combinations (EPC). The sampling variance was used to determine how large a sample of RFLPs was required to provide a given level of precision. The coefficient of variation (CV) associated with GD has a nearly linear relationship between its expected standard deviation and mean. The magnitude of the decrease in the mean CV for GD with increasing numbers of bands was dependent upon the sampling unit; e.g., individual polymorphic bands vs EPC, and the degree of relatedness among the inbreds compared. The rate of reduction in mean CV with increasing sample size was the same regardless of the restriction enzyme used, BamHI, EcoRI or HindIII, when the bootstrap sampling units were individual polymorphic bands. In constrast, although the rate of reduction (slopes) was the same, the intercepts of the mean CVs were different when EPCs were used as the bootstrap sampling unit. This difference was due to the higher number of bands per EPC in BamHI (4.94) compared with EcoRI (4.83) and HindIII (4.63).

Comparing test cultivars using reliability functions of test-check differences from on-farm trials.

An approach to selection is proposed that is based on the probabilities that a test cultivar outperforms a check by more than an amount d in a future environment. The function that gives these probabilities for all possible values of d is called the reliability function. When d=0, the value of the reliability function is the probability that the test cultivar outperforms the check. The method is illustrated using data from on-farm maize (Zea mays L.) strip test trials grown cooperatively by Pioneer Hi-bred International and farmers. Results indicate that reliability functions are useful for evaluating how test cultivars perform relative to a check across a range of environments since the location, slope and shape of the reliability function may be used to describe a test cultivar's performance, similarity to the check and stability, and identify environments where the test cultivar has performance problems.

Quantitative analysis of two-dimensional protein profiles of inbred lines of maize (Zea mays L.).

Two-dimensional electrophoresis and fluorography of [35S]methionine labeled maize germinated embryo proteins were performed at Cold Spring Harbor Laboratory. Fluorographs of 63 gels representing 37 inbred lines were subsequently scanned and spot-detected at Protein and DNA Imageware Systems (Huntington Station, NY). The digitized images were then matched with the aid of PDQUEST-II computer software. Over 1500 different protein spots were included in the resulting dataset. The optical density data were normalized to parts per million, then transformed to their natural logarithms. Analyses of variance were performed on each spot in order to select for further study those spots with most of their variation partitioned among inbred lines rather than within inbred lines. Using this method of spot selection, over 100 protein spots were included in the set of spots which display significant differences among inbred lines of maize.

Similarities among a group of elite maize inbreds as measured by pedigree, F1 grain yield, grain yield, heterosis, and RFLPs.

Genetic distances were calculated among 37 inbred lines representing a wide range of related and unrelated elite Corn Belt germ plasm of maize (Zea Mays L.), using 257 probe restriction enzyme combinations. Genetic distances based on RFLP data were highly correlated with coefficients of parentage among pairs of lines. The RFLP-based distance had a higher correlation with single-cross grain yield performance and grain yield heterosis than any of the other measures of similarity we calculated using these same lines. The coefficients of determination (r (2)) from regressing the coefficient of parentage, grain yield, and grain yield heterosis on Nei's measure of genetic similarity based on RFLP data were 0.81, 0.87 and 0.77, respectively. A cluster diagram based upon the RFLP data grouped the lines into families consistent with the breeding history and heterotic response of these lines. We believe that measures of similarity calculated from RFLP data, coupled with pedigree knowledge and using molecular markers to locate quantitative trait loci (QTL), could allow maize breeders to predict combinations of lines that result in high-yielding, single-cross hybrids.

Associations among inbred lines of maize using electrophoretic, chromatographic, and pedigree data : 2. Multivariate and cluster analysis of data from Iowa stiff stalk synthetic derived lines.

Associations among 17 "Iowa Stiff Stalk Synthetic" derived inbred lines of maize (Zea mays L.) were determined using multivariate and cluster analysis. Objectives were to assess the level of unique characterization among lines afforded by reversed-phase high-performance liquid chromatography (RP-HPLC) of zeins and starch gel electrophoresis of isozymes and to compare associations among lines revealed by biochemical and pedigree data. Isozymic data for 33 loci provided unique discrimination among 88% of the lines; 2 closely related lines were indistinguishable. Seventy-one percent of the lines could be uniquely and unambiguously identified by RP-HPLC. Biochemical data showed associations between lines that would be expected on the basis of pedigree. Nevertheless, different associations were revealed by allozymic and chromatographic data. Although these data permitted a high degree of unique identification, additional markers, covering a larger proportion of the genome, are needed to more adequately monitor similarities among genes that respond to selection during plant breeding.

Variation in kafirin and alcohol-soluble glutelin chromatograms of sorghum inbred lines revealed by reversed-phase high-performance liquid chromatography.

Separations of kafirin and alcohol soluble glutelin proteins by reversed-phase high-performance liquid chromatography (RP-HPLC) from 7 inbreds and one hybrid of sorghum [Sorghum bicolor (L.) Moench] and one source of Johnsongrass [Sorghum halapense (L.) Pers.] were compared. Objectives were to assess the stability of protein profiles for seed sources produced at different locations and in different environments to examine the potential of RP-HPLC to provide genotypic profiles for sorghum. Analyses of variance data showed that levels of variation due to environments and locations were small; the majority of variation (93%) was among genotypes. Associations among inbreds revealed by multivariate and cluster analysis showed similarity with those that would be expected on the basis of pedigree. A chi-square analysis showed no deviation in the hybrid profile from the expected 2∶1 ratio of peaks from the female and male inbred parents, respectively. Improvements in the ability to correctly assign common peaks are necessary before associations among numerous sorghum genotypes can be reliably demonstrated by analysis of data from reversed-phase high-performance liquid chromatography (RP-HPLC).

Choice of method for identifying germplasm with superior alleles : 1. Theoretical results.

Elite, adapted germplasm is not likely to contain all the favorable alleles available in a species. Three statistics were evaluated for screening populations for their ability to contribute favorable dominant alleles not available in an elite single cross: (1) a statistic proposed by Dudley (SD)=[(P x I1-I1)(I1 x I2-I2)-(P x I2-I2) (I1 x I2-I1)]/[2(I1-I2)]; (2) the upper bound minimum (P x I1-I1, P x I2-I2) ; and (3) the testcross to the single cross [TC(SC)]=P x (I1 x I2), where P is the population to be evaluated and I1 and I2 are homozygous parents of the elite single cross I1×I2. A superiority measure for a population was defined as the product of frequencies of favorable alleles and effects summed over loci where I1×I2 is homozygous unfavorable. Of the statistics considered, TC (SC) should have the highest genetic correlation with the superiority measure under the assumptions made, require the fewest testing resources and have the smallest standard error. Methods considered for screening inbreds were: (1) SDI proposed by Dudley=[(I1 x IW)+(I2 x IW)-I1-I2-IW-(I1 x I2)]/4 ; (2) TC(SC)=IW x (I1 xI2); and (3) UBND=minimum where Iw is the inbred to be evaluated. The superiority measure of an inbred Iw was defined as the relative number of loci where I1 and I2 are unfavorable and Iw is favorable. The genetic correlation with the superiority measure should be highest for SDI. The larger number of measurements used in calculation, the necessity of evaluating potentially unadapted inbreds and larger testing resources required for SDI suggest further research should be done to evaluate these statistics.

Choice of method for identifying germplasm with superior alleles : 2. Computer simulation results.

An accurate and efficient method of screening the many germplasm sources available for their ability to improve elite, adapted germplasm is needed. The superiority measure (SX) of a population (P) was defined as the product of the frequency and relative superiority of the alleles in P that are more favorable than the best in an elite, adapted reference single cross I1×I2. A computer simulation was done to determine the correlations between various screening methods and the SX. The genetic model used included multiple alleles, no linkage, two types of non-epistatic gene action (additive and complete dominance) and two types of epistatic gene action (complementary and duplicate). Genetic variances in the populations and a statistic proposed by Dudley (SD={[P x I1-I1] [I1 xI2-I2]- [P x I2-I2]-[P x I2-I2] [I1 x I2-I1]}/{2[I1-I2]{) were inconsistently correlated with the SX over all types of gene action on the basis of rank correlations. The testcross to the single cross (TC[SC]=P x [I1 x I2]) and the upper bound on the SX (UBND=minimum [P x I1-I1, P x I2-I2]) were both consistently highly genetically correlated with the SX. In the set of populations simulated, there were positive correlations between products of allelic frequencies and effects at different classes of loci. The UBND usually had a higher rank correlation coefficient with the SX than did the TC(SC). The differences between their correlation coefficients were often insignificant. Although the TC(SC) gives no indication as to which inbred the population is more closely related, its ease of use and expected lowers standard error compared with the UBND indicate that it would be an appropriate choice of screening method for identifying superior populations in the sense defined.

Comparisons of zein profiles from inbred, F1, and F 2 generations of maize as revealed by reversed-phase high-performance liquid chromatography.

Chi-square analyses were performed on zein Chromatographic profiles of inbred lines, F1, F2, and reciprocal F1 seed for 10 hybrids of maize (Zea mays L.). The objective was to test the goodness of fit of observed profiles with those expected on the basis that the F1 and F2 generations represent a 2∶1 and 1∶1 addition of Female∶male parents of the F1, respectively. From 40 available comparisons, 39 showed no difference between the observed chromatograms and those that were expected on the basis of four models that were tested. The one exception was due to closely eluting peaks that were revealed as shoulders and not recorded as separate entities. chromatographic profiles of inbreds, F1, and bulk F2 seed sources can be accurately simulated. Even though the chromatographic profile of the F1 closely resembled that of the female parent, profiles of hybrids with common female but different male parents were distinguishable. The lack of novel peaks in both F1 and F2 generations compared with the inbred line thereby revealed no unpredictable interaction among zein loci. Zein protein data can be useful in registration, certification, and in the checking of hybrid pedigree especially when used in concert with isozymic data.

Associations among inbred lines of maize using electrophoretic, chromatographic, and pedigree data : 1. Multivariate and cluster analysis of data from 'Lancaster Sure Crop' derived lines.

Associations among 18 'Lancaster Sure Crop' derived inbred lines of maize (Zea mays L.) were determined using multivariate and cluster analysis. Objectives were to assess the degree of unique characterization among lines afforded by reversed-phase high-performance liquid chromatography (RP-HPLC) and starch gel electrophoresis of allozymes and to compare associations among lines revealed by biochemical and pedigree data. RP-HPLC revealed 11 different chromatograms that uniquely identified 79% of lines that differed by more than isogenic or near isogenic segments. Allozymic data for 21 loci provided unique discrimination among 93% of non-isogenic lines. Chromatographic and allozymic data together provided unique characterization of all non-isogenic lines. Cluster and multivariate analyses of biochemical data associated lines into three groups that would have been expected on the basis of pedigree breeding records. More detailed associations were dependent upon the data set employed. Multivariate and cluster analysis of chromatographic, electrophoretic, and pedigree data could be useful in revealing more detailed associations among elite germplasm than hitherto available, thus providing data pertinent to line and hybrid development, plant variety protection, and germplasm security.

Environmental effects on zein chromatograms of maize inbred lines revealed by reversed-phase high-performance liquid chromatography.

Alcohol soluble seed storage proteins (zeins and alcohol soluble glutelins) of maize (Zea mays L.) were separated by reversed-phase high-performance liquid chromatography (RP-HPLC). The objectives were to assess the reproducibility of chromatographic profiles using seed of inbred lines that had been produced in different locations and years. Reproducible differences between sources were seen but these were restricted to proteins that contributed 2% or less to an inbred profile. The majority of variation (93% for peak percent area; 99.8% for elution time) was between inbreds. RP-HPLC can therefore provide distinctive phenotypic profiles that are largely characteristic of genotype. Such qualitative and quantitative data will be valuable for studies of taxonomy, evolution, genetics, and germplasm identification.

The effect of parental genotype on initiation of embryogenic callus from elite maize (Zea mays L.) germplasm.

Embryogenic callus consisting of both Type 1, firm, compact, translucent, relatively slow growing callus and Type 2, highly friable, rapidly growing callus with well-formed somatic embryos, were observed in elite maize germplasm, notably B73 and hybrids with B73. Parental genotype is very important in the ability to identify and isolate embryogenic callus after 14 and 28 days in culture. A partial diallel analysis revealed that a large proportion of the genotypic variation was of the additive type although heterosis did positively increase culture response in most cases. A significant negative maternal effect for culture response was noted for inbred B73 from Reid-type germplasm while four lines sampled from Lancaster germplasm showed similar response whether used as male or female. Although significant media differences were observed in some genotypes, culture media did not preclude observation of Type 1 or Type 2 embryogenic cultures in this study after 14 and 28 days. Plants could be regenerated from all genotypes in this study after 14-days of culture, but not all genotypes were capable of sustained subculture and plant regeneration. Plant regeneration from Type 2 cultures of B73 and B73 hybrids has been obtained up to a year after initiation.