Genetic basis of variation for root traits and response to heat stress in durum wheat

Durum wheat is the second most important wheat species worldwide and the most important crop in several Mediterranean countries including Italy. Durum wheat is primarily grown under rainfed conditions where episodes of drought and heat stress are major factors limiting grain yield. The research presented in this thesis aimed at the identification of traits and genes that underlie root system architecture (RSA) and tolerance to heat stress in durum wheat, in order to eventually contribute to the genetic improvement of this species. In the first two experiments we aimed at the identification of QTLs for root trait architecture at the seedling level by studying a bi-parental population of 176 recombinant inbred lines (from the cross Meridiano x Claudio) and a collection of 183 durum elite accessions. Forty-eight novel QTLs for RSA traits were identified in each of the two experiments, by means of linkage- and association mapping-based QTL analysis, respectively. Important QTLs controlling the angle of root growth in the seedling were identified. In a third experiment, we investigated the phenotypic variation of root anatomical traits by means of microscope-based analysis of root cross sections in 10 elite durum cultivars. The results showed the presence of sizeable genetic variation in aerenchyma-related traits, prompting for additional studies aimed at mapping the QTLs governing such variation and to test the role of aerenchyma in the adaptive response to abiotic stresses. In the fourth experiment, an association mapping experiment for cell membrane stability at the seedling stage (as a proxy trait for heat tolerance) was carried out by means of association mapping. A total of 34 QTLs (including five major ones), were detected. Our study provides information on QTLs for root architecture and heat tolerance which could potentially be considered in durum wheat breeding programs.

Excellent agreement between genetic and hydrogen breath tests for lactase deficiency and the role of extended symptom assessment

Clinical manifestations of lactase (LCT) deficiency include intestinal and extra-intestinal symptoms. Lactose hydrogen breath test (H2-BT) is considered the gold standard to evaluate LCT deficiency (LD). Recently, the single-nucleotide polymorphism C/T(-13910) has been associated with LD. The objectives of the present study were to evaluate the agreement between genetic testing of LCT C/T(-13910) and lactose H2-BT, and the diagnostic value of extended symptom assessment. Of the 201 patients included in the study, 194 (139 females; mean age 38, range 17-79 years, and 55 males, mean age 38, range 18-68 years) patients with clinical suspicion of LD underwent a 3-4 h H2-BT and genetic testing for LCT C/T(-13910). Patients rated five intestinal and four extra-intestinal symptoms during the H2-BT and then at home for the following 48 h. Declaring H2-BT as the gold standard, the CC(-13910) genotype had a sensitivity of 97% and a specificity of 95% with a of 0.9 in diagnosing LCT deficiency. Patients with LD had more intense intestinal symptoms 4 h following the lactose challenge included in the H2-BT. We found no difference in the intensity of extra-intestinal symptoms between patients with and without LD. Symptom assessment yielded differences for intestinal symptoms abdominal pain, bloating, borborygmi and diarrhoea between 120 min and 4 h after oral lactose challenge. Extra-intestinal symptoms (dizziness, headache and myalgia) and extension of symptom assessment up to 48 h did not consistently show different results. In conclusion, genetic testing has an excellent agreement with the standard lactose H2-BT, and it may replace breath testing for the diagnosis of LD. Extended symptom scores and assessment of extra-intestinal symptoms have limited diagnostic value in the evaluation of LD.

Coastal upwelling is linked to temporal genetic variability in the acorn barnacle Balanus glandula

Dispersal and recruitment are central processes that shape the geographic and temporal distributions of populations of marine organisms. However, significant variability in factors such as reproductive output, larval transport, survival, and settlement success can alter the genetic identity of recruits from year to year. We designed a temporal and spatial sampling protocol to test for genetic heterogeneity among adults and recruits from multiple time points along a similar to 400 km stretch of the Oregon (USA) coastline. In total, 2824 adult and recruiting Balanus glandula were sampled between 2001 and 2008 from 9 sites spanning the Oregon coast. Consistent with previous studies, we observed high mitochondrial DNA diversity at the cytochrome oxidase I locus (884 unique haplotypes) and little to no spatial genetic population structure among the 9 sites (Phi(ST) = 0.00026, p = 0.170). However, subtle but significant temporal shifts in genetic composition were observed among year classes (Phi(ST) = 0.00071, p = 0.035), and spatial Phi(ST) varied from year to year. These temporal shifts in genetic structure were correlated with yearly differences in the strength of coastal upwelling (p = 0.002), with greater population structure observed in years with weaker upwelling. Higher levels of barnacle settlement were also observed in years with weaker upwelling (p < 0.001). These data suggest the hypothesis that low upwelling intensity maintains more local larvae close to shore, thereby shaping the genetic structure and settlement rate of recruitment year classes.

Genetic analysis of syndactyly in German Holstein cattle

Congenital syndactyly with a variable number of affected feet was observed in eight black and white German Holstein calves. Analysis of the pedigree data revealed that all affected individuals could be traced back to a single founder. The pedigree was consistent with monogenic autosomal recessive inheritance and variable expressivity. Bovine syndactyly or "mulefoot" has been previously shown to map on the telomeric end of bovine chromosome 15 and we performed PCR genotyping of microsatellite markers spanning 27 cM of this chromosomal region to test the new cases for genetic linkage with the phenotype. The haplotype segregation confirmed the suggested inheritance pattern of the mulefoot mutation in this family and markers RM004, BM848 and BMS820 showed significant linkage to the phenotype. The results confirmed the chromosomal location of the mulefoot gene in this pedigree. Furthermore the study demonstrated that although marker testing has been available for nearly a decade the use of mulefoot carriers in cattle breeding remains uncontrolled. The presented family provides a resource for positional cloning of the causative mutation.

Comparison of genetic gains per year for carcass traits among breeding programs in the Japanese Brown and the Japanese Black cattle

The breeding program for beef cattle in Japan has changed dramatically over 4 decades. Visual judging was done initially, but progeny testing in test stations began in 1968. In the 1980s, the genetic evaluation program using field records, so-called on-farm progeny testing, was first adopted in Oita, Hyogo, and Kumamoto prefectures. In this study, genetic trends for carcass traits in these 3 Wagyu populations were estimated, and genetic gains per year were compared among the 3 different beef cattle breeding programs. The field carcass records used were collected between 1988 and 2003. The traits analyzed were carcass weight, LM area, rib thickness, s.c. fat thickness, and beef marbling standard number. The average breeding values of reproducing dams born the same year were used to estimate the genetic trends for the carcass traits. For comparison of the 3 breeding programs, birth years of the dams were divided into 3 periods reflecting each program. Positive genetic trends for beef marbling standard number were clearly shown in all populations. The genetic gains per year for all carcass traits were significantly enhanced by adopting the on-farm progeny testing program. These results indicate that the on-farm progeny testing program with BLUP is a very powerful approach for genetic improvement of carcass traits in Japanese Wagyu beef cattle.

The common G-allele of interleukin-18 single-nucleotide polymorphism is a genetic risk factor for atopic asthma. The SAPALDIA Cohort Study

BACKGROUND: IL-18 is a pleiotrophic cytokine involved in both, T-helper type 1 (Th1) and Th2 differentiation. Recently genetic variants in the IL-18 gene have been associated with increased risk of atopy and asthma. OBJECTIVE: To examine the relationship of a genetic, haplotype-tagging promotor variant -137G/C in the IL-18 gene with atopic asthma in a large, well-characterized and population-based study of adults. METHODS: Prospective cohort study design was used to collect interview and biological measurement data at two examination time-points 11 years apart. Multivariate logistic regression analysis was used to assess the association of genotype with asthma and atopy. RESULTS: The G-allele of the IL-18 promotor variant (-137G/C) was associated with a markedly increased risk for the prevalence of physician-diagnosed asthma with concomitant skin reactivity to common allergens. Stratification of the asthma cases by skin reactivity to common allergens revealed an exclusive association of IL-18 -137 G-allele with an increased prevalence of atopic asthma (adjusted odds ratio (OR): 3.63; 95% confidence interval: (1.64-8.02) for GC or GG carriers vs. CC carriers), and no according association with asthma and concomitant negative skin reactivity (adjusted OR: 1.13; 0.66-1.94). The interaction between IL-18 -137G/C genotype and positive skin prick test was statistically significant (P=0.029). None of 74 incident asthma cases with atopy at baseline exhibited the CC genotype. CONCLUSION: Our results strongly suggest that this variant of the IL-18 gene is an important genetic determinant involved in the development of atopic asthma.

Effects of genetic and environmental factors on chronic lower airway disease in horses

BACKGROUND: Environment and genetics influence the manifestation of recurrent airway obstruction (RAO), but the associations of specific factors with mild, moderate, and severe clinical signs are unknown. HYPOTHESIS: We hypothesized that sire, feed, bedding, time outdoors, sex, and age are associated with clinical manifestations of mild, moderate, and severe lower airway disease. ANIMALS: Direct offspring of 2 RAO-affected Warmblood stallions (F1S1, n = 172; F1S2, n = 135); maternal half-siblings of F1S1 (mHSS1, n = 66); and an age-matched, randomly chosen control group (CG, n = 33). METHODS: A standardized questionnaire was used to assess potential risk factors and to establish a horse owner assessed respiratory signs index (HOARSI 1-4, from healthy to severe) according to clinical signs of lower airway disease. RESULTS: More F1S1 and F1S2 horses showed moderate to severe clinical signs (HOARSI 3 and HOARSI 4 combined, 29.6 and 27.3%, respectively) compared with CG and mHSS1 horses (9.1 and 6.2%, respectively; contingency table overall test, P < .001). Sire, hay feeding, and age (in decreasing order of strength) were associated with more severe clinical signs (higher HOARSI), more frequent coughing, and nasal discharge. CONCLUSIONS AND CLINICAL RELEVANCE: There is a genetic predisposition and lesser but also marked effects of hay feeding and age on the manifestation of moderate to severe clinical signs, most markedly on coughing frequency. In contrast, mild clinical signs were not associated with sire or hay feeding in our populations.

Genetic and pharmacologic manipulation of oxidative stress after neonatal hypoxia-ischemia

Oxidative stress is a critical component of the injury response to hypoxia-ischemia (HI) in the neonatal brain, and this response is unique and at times paradoxical to that seen in the mature brain. Previously, we showed that copper-zinc superoxide-dismutase (SOD1) over-expression is not beneficial to the neonatal mouse brain with HI injury, unlike the adult brain with ischemic injury. However, glutathione peroxidase 1 (GPx1) over-expression is protective to the neonatal mouse brain with HI injury. To further test the hypothesis that an adequate supply of GPx is critical to protection from HI injury, we crossed SOD1 over-expressing mice (hSOD-tg) with GPx1 over-expressing mice (hGPx-tg). Resulting litters contained wild-type (wt), hGPx-tg, hSOD-tg and hybrid hGPx-tg/hSOD-tg pups, which were subjected to HI at P7. Confirming previous results, the hGPx-tg mice had reduced injury compared to both Wt and hSOD-tg littermates. Neonatal mice over-expressing both GPx1 and SOD1 also had less injury compared to wt or hSOD-tg alone. A result of oxidative stress after neonatal HI is a decrease in the concentration of reduced (i.e. antioxidant-active) glutathione (GSH). In this study, we tested the effect of systemic administration of alpha-lipoic acid on levels of GSH in the cortex after HI. Although GSH levels were restored by 24h after HI, injury was not reduced compared to vehicle-treated mice. We also tested two other pharmacological approaches to reducing oxidative stress in hSOD-tg and wild-type littermates. Both the specific inhibitor of neuronal nitric oxide synthase, 7-nitroindazole (7NI), and the spin-trapping agent alpha-phenyl-tert-butyl-nitrone (PBN) did not reduce HI injury, however. Taken together, these results imply that H2O2 is a critical component of neonatal HI injury, and GPx1 plays an important role in the defense against this H2O2 and is thereby neuroprotective.

A thiazide test for the diagnosis of renal tubular hypokalemic disorders

Although the diagnosis of Gitelman syndrome (GS) and Bartter syndrome (BS) is now feasible by genetic analysis, implementation of genetic testing for these disorders is still hampered by several difficulties, including large gene dimensions, lack of hot-spot mutations, heavy workup time, and costs. This study evaluated in a cohort of patients with genetically proven GS or BS diagnostic sensibility and specificity of a diuretic test with oral hydrochlorothiazide (HCT test). Forty-one patients with GS (22 adults, aged 25 to 57; 19 children-adolescents, aged 7 to 17) and seven patients with BS (five type I, two type III) were studied; three patients with "pseudo-BS" from surreptitious diuretic intake (two patients) or vomiting (one patient) were also included. HCT test consisted of the administration of 50 mg of HCT orally (1 mg/kg in children-adolescents) and measurement of the maximal diuretic-induced increase over basal in the subsequent 3 h of chloride fractional clearance. All but three patients with GS but no patients with BS and pseudo-BS showed blunted (<2.3%) response to HCT; patients with BS and the two patients with pseudo-BS from diuretic intake had increased response to HCT. No overlap existed between patients with GS and both patients with BS and pseudo-BS. The response to HCT test is blunted in patients with GS but not in patients with BS or nongenetic hypokalemia. In patients with the highly selected phenotype of normotensive hypokalemic alkalosis, abnormal HCT test allows prediction with a very high sensitivity and specificity of the Gitelman genotype and may avoid genotyping.

Genetic maps of the proximal half of chromosome arm 2L of Drosophila melanogaster

Drosophila mutants have played an important role in elucidating the physiologic function of genes. Large-scale projects have succeeded in producing mutations in a large proportion of Drosophila genes. Many mutant fly lines have also been produced through the efforts of individual laboratories over the past century. In an effort to make some of these mutants more useful to the research community, we systematically mapped a large number of mutations affecting genes in the proximal half of chromosome arm 2L to more precisely defined regions, defined by deficiency intervals, and, when possible, by individual complementation groups. To further analyze regions 36 and 39-40, we produced 11 new deficiencies with gamma irradiation, and we constructed 6 new deficiencies in region 30-33, using the DrosDel system. trans-heterozygous combinations of deficiencies revealed 5 additional functions, essential for viability or fertility.

Mental health and quality of life after genetic testing for Huntington disease: a long-term effect study in Germany

Predictive genetic testing for Huntington disease (HD) might cause severe short-term psychological reactions in patients with poor mental health. Very few studies exist on the long-term effects of genetic HD testing. The aim of this study was to assess mental health and quality of life in persons who were tested for HD mutation, to compare mental health depending on the result of the genetic test (non-carriers, gene carriers, and patients with HD) and to identify predictors of mental health and quality of life via linear regression. The data were collected by self-report questionnaires. In total, 121 individuals participated in this study: 52 were non-carriers, 54 were gene carriers, and 15 were gene carriers suffering from HD. Non-carriers and gene carriers showed better mental health and quality of life than HD-patients but did not differ from each other. In non-carriers four variables predicted increased depression and low mental quality of life: low perceived social support, no intimate relationship, female sex and younger age. For gene carriers three predictors were found: low perceived social support, the expectation of an unfavorable genetic test result before the testing procedure and being childless. To prevent detrimental effects of HD testing on mental health and mental quality of life, specific attention should be paid to persons with limited social networks during genetic counseling. Assessment of expectations related to the test result and mental health prior to a genetic testing procedure may help to identify gene carriers at risk of poor coping after an unfavorable test result.

Control design and genetic algorithm optimization for electrostatic MEMS

This dissertation discusses structural-electrostatic modeling techniques, genetic algorithm based optimization and control design for electrostatic micro devices. First, an alternative modeling technique, the interpolated force model, for electrostatic micro devices is discussed. The method provides improved computational efficiency relative to a benchmark model, as well as improved accuracy for irregular electrode configurations relative to a common approximate model, the parallel plate approximation model. For the configuration most similar to two parallel plates, expected to be the best case scenario for the approximate model, both the parallel plate approximation model and the interpolated force model maintained less than 2.2% error in static deflection compared to the benchmark model. For the configuration expected to be the worst case scenario for the parallel plate approximation model, the interpolated force model maintained less than 2.9% error in static deflection while the parallel plate approximation model is incapable of handling the configuration. Second, genetic algorithm based optimization is shown to improve the design of an electrostatic micro sensor. The design space is enlarged from published design spaces to include the configuration of both sensing and actuation electrodes, material distribution, actuation voltage and other geometric dimensions. For a small population, the design was improved by approximately a factor of 6 over 15 generations to a fitness value of 3.2 fF. For a larger population seeded with the best configurations of the previous optimization, the design was improved by another 7% in 5 generations to a fitness value of 3.0 fF. Third, a learning control algorithm is presented that reduces the closing time of a radiofrequency microelectromechanical systems switch by minimizing bounce while maintaining robustness to fabrication variability. Electrostatic actuation of the plate causes pull-in with high impact velocities, which are difficult to control due to parameter variations from part to part. A single degree-of-freedom model was utilized to design a learning control algorithm that shapes the actuation voltage based on the open/closed state of the switch. Experiments on 3 test switches show that after 5-10 iterations, the learning algorithm lands the switch with an impact velocity not exceeding 0.2 m/s, eliminating bounce.

Genetic association studies under the population stratification, family pedigree and application to genome-wide association studies

This dissertation has three separate parts: the first part deals with the general pedigree association testing incorporating continuous covariates; the second part deals with the association tests under population stratification using the conditional likelihood tests; the third part deals with the genome-wide association studies based on the real rheumatoid arthritis (RA) disease data sets from Genetic Analysis Workshop 16 (GAW16) problem 1. Many statistical tests are developed to test the linkage and association using either case-control status or phenotype covariates for family data structure, separately. Those univariate analyses might not use all the information coming from the family members in practical studies. On the other hand, the human complex disease do not have a clear inheritance pattern, there might exist the gene interactions or act independently. In part I, the new proposed approach MPDT is focused on how to use both the case control information as well as the phenotype covariates. This approach can be applied to detect multiple marker effects. Based on the two existing popular statistics in family studies for case-control and quantitative traits respectively, the new approach could be used in the simple family structure data set as well as general pedigree structure. The combined statistics are calculated using the two statistics; A permutation procedure is applied for assessing the p-value with adjustment from the Bonferroni for the multiple markers. We use simulation studies to evaluate the type I error rates and the powers of the proposed approach. Our results show that the combined test using both case-control information and phenotype covariates not only has the correct type I error rates but also is more powerful than the other existing methods. For multiple marker interactions, our proposed method is also very powerful. Selective genotyping is an economical strategy in detecting and mapping quantitative trait loci in the genetic dissection of complex disease. When the samples arise from different ethnic groups or an admixture population, all the existing selective genotyping methods may result in spurious association due to different ancestry distributions. The problem can be more serious when the sample size is large, a general requirement to obtain sufficient power to detect modest genetic effects for most complex traits. In part II, I describe a useful strategy in selective genotyping while population stratification is present. Our procedure used a principal component based approach to eliminate any effect of population stratification. The paper evaluates the performance of our procedure using both simulated data from an early study data sets and also the HapMap data sets in a variety of population admixture models generated from empirical data. There are one binary trait and two continuous traits in the rheumatoid arthritis dataset of Problem 1 in the Genetic Analysis Workshop 16 (GAW16): RA status, AntiCCP and IgM. To allow multiple traits, we suggest a set of SNP-level F statistics by the concept of multiple-correlation to measure the genetic association between multiple trait values and SNP-specific genotypic scores and obtain their null distributions. Hereby, we perform 6 genome-wide association analyses using the novel one- and two-stage approaches which are based on single, double and triple traits. Incorporating all these 6 analyses, we successfully validate the SNPs which have been identified to be responsible for rheumatoid arthritis in the literature and detect more disease susceptibility SNPs for follow-up studies in the future. Except for chromosome 13 and 18, each of the others is found to harbour susceptible genetic regions for rheumatoid arthritis or related diseases, i.e., lupus erythematosus. This topic is discussed in part III.

Statistical methods for multi-marker testing in genetic association studies

As the development of genotyping and next-generation sequencing technologies, multi-marker testing in genome-wide association study and rare variant association study became active research areas in statistical genetics. This dissertation contains three methodologies for association study by exploring different genetic data features and demonstrates how to use those methods to test genetic association hypothesis. The methods can be categorized into in three scenarios: 1) multi-marker testing for strong Linkage Disequilibrium regions, 2) multi-marker testing for family-based association studies, 3) multi-marker testing for rare variant association study. I also discussed the advantage of using these methods and demonstrated its power by simulation studies and applications to real genetic data.