The grain amaranths (Amaranthus sp.) and quinoa (Chenopodium quinoa Willd.) are important seed crops in South America. These crops have gained international attention in recent years for their nutritional quality and tolerance to abiotic stress. We report the identification and development of functional single nucleotide polymorphism (SNP) assays for both amaranth and quinoa. SNPs were identified using a genome reduction protocol and next generation sequencing. SNP assays are based on KASPar genotyping chemistry and were detected using the Fluidigm dynamic array platform. A diversity screen consisting of 41 amaranth accessions showed that the minor allele frequency (MAF) of the amaranth markers ranged from 0.05 to 0.5 with an average MAF of 0.27. A diversity screen of 113 quinoa accessions showed that the MAF of the quinoa markers ranged from 0.02 to 0.5 with an average MAF of 0.28. Linkage mapping in amaranth produced a linkage map consisting of 16 linkage groups, presumably corresponding to each of the 16 amaranth haploid chromosomes. This map spans 1288 cM with an average marker density of 3.1 cM per marker. Linkage mapping in quinoa resulted in a linkage map consisting of 29 linkage groups with 20 large linkage groups, spanning 1,404 cM with a marker density of 3.1 cM per SNP marker. The SNPs identified here represent important genomic tools needed for genetic dissection of agronomically important characteristics and advanced genetic analysis of agronomic traits in amaranth and quinoa. We also describe in detail the scalable and cost effective SNP genotyping method used in this research. This method is based on KBioscience's competitive allele specific PCR amplification of target sequences and endpoint fluorescence genotyping (KASPar) using a FRET capable plate reader or Fluidigm's dynamic array high throughput platform.

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The potential value of the Y chromosome for forensic applications has been recognized for some time with the current work dedicated to Short Tandem Repeat analysis and Single Nucleotide Polymorphism (SNP) discovery. This study examined the ability of two different SNP analysis methods to determine if they could be utilized in forensic applications and ultimately be developed into an established system for Y chromosome SNP analysis. This study examined two principle SNP analysis systems: single base extension and Pyrosequencing. Pyrosequencing was determined to be superior to single base extension, due to the wealth of information provided with sequencing and the flexibility of designing primers for analysis. Using Pyrosequencing, 50 Y chromosome loci were examined and the minimum loci required for maximum diversity for the development of a Y chromosome SNP analysis system were chosen. Thirteen loci were selected based on their ability to discriminate 60 different individuals from three different racial groups into 15 different haplogroups. The Y chromosome SNP analysis system developed utilized nested PCR for the amplification of all 13 loci. Then they were sequenced as groups, ranging from one to three loci, in a single reaction. The Y chromosome SNP analysis system developed here has the potential for forensic application since it has shown to be successful in the analysis of blood, buccal swabs, semen, and saliva, works with as little as 5 pg of starting DNA material, and will amplify only male DNA in the presence of male/female mixtures in which the female portion of the sample overwhelmed the male portion 30,000 to 1.

京都大学 / 新制・課程博士 / 博士(法学) / 甲第24865号 / 法博第298号 / 新制||法||180(附属図書館) / 京都大学大学院法学研究科法政理論専攻 / (主査)教授 曽我 謙悟, 教授 建林 正彦, 教授 待鳥 聡史 / 学位規則第4条第1項該当 / Doctor of Laws / Kyoto University / DGAM

The genus Mycobacterium contains many pathogenic bacteria that are known to cause serious diseases in humans. One of the most well-known of these bacteria is Mycobacterium tuberculosis, or Mtb, which is the causative agent of tuberculosis. It infects nearly one-third of the world’s population and kills 1.4 million people annually. Another important mycobacterial pathogen is Mycobacterium abscessus, or Mabs, which causes respiratory infections in cystic fibrosis patients. One of the biggest difficulties in combating these pathogens is the lack of effective diagnostics, as current strategies hold many pitfalls and can be unreliable. One common method used is sputum smear microscopy which involves acid fast staining of the bacteria present in a patient’s sputum. This method of detection fails to detect more than 50% of infections and is unable to differentiate between species of mycobacterium. This project introduces a novel method of mycobacterial diagnostics using binary deoxyribozymes (DNAzymes). Binary DNAzymes recognize bacteria-specific nucleic acid sequences and bind to them, forming a catalytic core which cleaves a substrate molecule. This cleavage separates a quencher molecule from a fluorophore, which results in a fluorescent output. This flexible assay platform has great potential for the detection of Mtb or Mabs. Our data shows the specificity of the DNAzymes allowing for a differential diagnosis of various species of Mycobacteria. It also shows the limit of detection of this technology and its additional utility in molecular typing of Mtb clinical isolates as well as drug resistance characterization. This multipurpose tool can contribute to disease management in multiple ways.

Holistic and growth stage-specific screening is needed for identifying tolerant genotypes and for formulating strategies to mitigate the negative effects of abiotic stresses on crops. The objectives of this study were to characterize the genetic variability of 100 rice lines for early-season vigor, growth and physiological plasticity, and drought and temperature tolerance. Five studies were conducted to accomplish these objectives. In study 1 and 2, 100 rice genotypes consisting of several cultivars and experimental breeding lines were characterized for early-season vigor using several shoot and root morphological, physiological, and yield related traits. In study 3, low- and high-temperature tolerance assessed on select rice cultivars/hybrids during early-season. In study 4, genotypic variability in response to drought stress tolerance using morpo-physiological traits including roots was assessed under pot-culture conditions in a mini-greenhouse conditions. In study 5, the 100 rice genotypes were used to identify and validate SNP markers, and genome-wide association study (GWAS) to generate genotypic and phenotypic data with the objective of identifying new genetic loci controlling drought stress traits. Significant variability was recorded among rice genotypes and treatments for many traits measured. Early-season cumulative vigor response indices (CVRI) developed by summing individual responses indices for each trait varied among the rice genotypes, 21.36 (RU1404196) to 36.17 (N-22). Based on means and standard deviation of the CVRI, rice genotypes were classified as low- (43) and moderately low- (33), high- (16), and very high-vigor (5) groups. Total low-temperature response index values ranged from 18.48 to 23.15 whereas total high-temperature responses index values ranged from 42.01 to 48.82. Antonio, CLXL 745, and Mermentau were identified as sensitive to cold- and heat, and XL 753 was highly cold and heat tolerant genotypes tested. A cumulative drought stress response index (CDSRI) values varied between 14.7 (CHENIERE) and 27.9 (RU1402174) among the genotypes tested. This preliminary analysis of GWA indicated that substantial phenotypic and genotypic diversity exists in the 100 rice genotypes, despite their narrow genetic pool. The stress tolerant and high vigor rice genotypes will be valuable for rice breeders for developing new genotypes best suited under growing environments prone to early-season drought and temperature.

<p>Complex disorders are caused by multiple genetic, environmental, and lifestyle factors, and their interactions. Most human diseases are complex, including many psychiatric, autoimmune, neurodegenerative, and cardiovascular disorders. Understanding their genetic background is an essential step toward developing effective preventive and therapeutic interventions for these disorders. In this dissertation, we present an overview of state-of-the-art methodology that is used to help elucidate the genetic basis of complex diseases and apply these methods to understand the genetic background of different complex disorders. First, we carried out a GWAS for myasthenia gravis (MG), a rare autoimmune disorder, and detected a novel risk locus, AGRN, which encodes a protein, involved in neuromuscular junction activation. Additionally, we observed significant genetic correlation between MG and ADs, and variants with pleiotropic effects. Second, we explored the genetic and phenotypic relationships among 11 different autoimmune disorders (ADs), using GWAS results o to calculate polygenic risk scores (PRS) and performing a PRS- phenome-wide association study (PheWAS) analysis with 3,281 phenotypes available in the UK Biobank. We observed associations of ADs PRS with phenotypes in multiple categories, including lifestyle, biomarkers, mental and physical health. We also explored the shared genetic components among the ADs, through genetic correlation and cross-disorder meta-analysis approaches, where we</p> <p>identified pleiotropic variants among the correlated ADs. Finally, we performed a meta-analysis GWAS of Tourette Syndrome (TS) followed by post-GWAS analyses including biological annotation of the results, and association tests of TS PRS with brain volumes. We detected a novel locus, NR2F1, associated with TS, supported by eQTL and Hi-C data. TS PRS was significantly associated with right and left thalamus volumes and right putamen volume. Overall, our work demonstrates the power of GWAS and related methods to help disentangle the genetic basis of complex disease and provides important insights into the genetic basis of the specific disorders that are the focus of our studies.</p>

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