Cotton is a crop with a large global economic impact as well as a large, complex genome. Most industrial cotton production is from two tetraploid species (Gossypium hirsutum L. and Gossypium barbadense L.) which contain two subgenomes, specifically the AT and DT subgenomes. The DT subgenome is nearly half the size of the AT subgenome in tetraploid cotton and is closely related to an extant D-genome Gossypium species, G. raimondii Ulbr. Characterization of the structural variants present in diploid D-genome should provide greater insight into the evolution of the DT subgenome in the tetraploid cotton. Bionano (BNG) optical mapping uses patterns of fluorescent labels inserted at specific endonuclease sites to create physical maps of the genomes which can then be examined for structural variation. To develop optical maps in G. raimondii, we first developed a de novo PacBio long read sequence assembly of G. raimondii. This sequence assembly consisted of 2,379 contigs, an average contig length of 413 Kb and a contig N50 of 4.9 Mb. Using BNG technology, we developed two optical maps of the diploid D genome of G. raimondii. One was created using the Nt.BssSI endonuclease and one with the Nt.BspQI endonuclease. Using the BNG optical maps, the PacBio assembly was hybrid scaffolded into 100 scaffolds (+ 5 unscaffolded contigs) with an average scaffold length of 7.5 Mb and a scaffold N50 of 13.1 Mb. A comparison between the Nt. BssSI BNG optical map and the two sequence assemblies identified 3,195 structural variants. These were used to validate the accuracy of the reference sequence of G. raimondii and structural variants were used to create a new phylogeny of nine major cotton species.

Doctor of Philosophy / Genetics Interdepartmental Program / Jesse A. Poland / Wheat is perhaps the most important component in human diet introduced since the conception of modern agriculture, which provides about 20% of the daily protein and calorie intake to billions of people. Adaptable to wide range of climates, wheat is grown worldwide, lending it the potential to mitigate the imminent risk of food security for future population of 9.5 billion people. For developing improved crop varieties in the future, genetic diversity is a key factor in plant breeding. Constraints in wheat evolution and artificial selection practices have resulted in erosion of this ingredient in elite germplasm. However, wheat wild relatives, such as Ae. tauschii, D-genome donor of wheat, are a storehouse for unexploited genetic diversity that can be used for improving wheat for disease and insect resistance, yield, quality, and tolerance to abiotic stresses. More than 1700 genebanks around the world hold over 7 million accessions of these wild relatives. These genebanks are expensive to maintain, therefore, efficient curation is necessary. We developed and implemented a protocol to identify duplicate accessions using genomic tools. Implementing this approach with three genebanks, we identified over 50% duplicated accessions across genebanks. There are over a million Triticeae accessions held collectively, and it is likely as more number of genebanks are tested, there will be decreasing number of unique accessions. Selecting and utilizing the wild genetic diversity is no easy task. Historically, breeders and geneticists have chosen the accessions primarily based on associated phenotypic data. Unless focusing on a targeted trait, this practice is imperfect in capturing the genetic diversity with some other limitations, such as confounding phenotypic data with the testing environment. Utilizing next-generation sequencing methods, we selected a MiniCore consisting of only 40 accessions out of 574 capturing more than 95% of the allelic diversity. This MiniCore will facilitate the use of genetic diversity present in Ae. tauschii for wheat improvement including resistance to leaf rust, stem rust, Hessian fly, and tolerance to abiotic stresses. Hessian fly is an important insect pest of wheat worldwide. Out of 34 known resistance genes, only six have been mapped on the D sub-genome. With swift HF evolution, we need to rapidly map and deploy the resistance genes. Some of the undefeated HF resistance genes, such as H26 and H32, were introgressed from Ae. tauschii. In this study, we mapped three previously known genes, and a new gene from Ae. tauschii accession KU2147. Genes were mapped on chromosomes 6B, 3D, and 6D. Further, identification and cloning of resistance genes will enhance our understanding about its function and mode of action. In conclusion, wild wheat relatives are genetically diverse species, and utilizing the novel genetic diversity in Ae. tauschii will be fruitful for wheat improvement in the wake of climate change to ensure future food security to expected 2 billion newcomers by 2050.

Two plaque-pure phage lambda clones designated as λhtX-l and λhtX-2 that hybridized to unfractionated bovine liver tRNA were isolated from a human X chromosome-specific library. The λDNAs were characterized by restriction mapping and Southern blot hybridization techniques. The human DNA segment in λhtX-l contains five or more presumptive tRNA genes and at least one Alu family member. The 19-kilobase human DNA insert in λhtX-2 contains two or more presumptive tRNA genes and at least three Alu family members. Another human genomic clone designated λhVKV7 hybridized to mammalian valine tRNA IAC. The clone was characterized by physical mapping and Southern blot hybridization techniques. The 18.5-kilobase human DNA fragment in λhVKV7 contains a cluster of three tRNA genes and at least nine Alu family members.

Master of Science / Department of Agronomy / Randall S. Currie / Mithila Jugulam / Italian ryegrass (Lolium perenne L. ssp. multiflorum (Lam.) Husnot), one of the problem weeds of the US, evolved resistance to multiple herbicides including glyphosate due to selection in Arkansas (AR). Glyphosate is a 5-enolpyruvylshikimate-3-phosphate synthase (EPSPS) inhibitor and amplification of EPSPS gene, the molecular target of glyphosate confers resistance to this herbicide in several weed species, including Italian ryegrass from AR. The objective of this study was to determine the expression of EPSPS gene and protein as well as distribution of EPSPS copies on the genome of glyphosate-resistant Italian ryegrass (ARR) using a known susceptible Italian ryegrass (ARS) from AR. EPSPS gene copies and expression of ARR and ARS were determined using quantitative PCR with appropriate endogenous controls. EPSPS protein expression was determined using Western blot analysis. Fluorescence in situ hybridization (FISH) was performed on somatic metaphase chromosomes to determine the location of EPSPS copies. Based on qPCR analysis, ARR plants showed a wide range of 12 to 118 EPSPS copies compared to a single copy in ARS. EPSPS gene expression correlated with the gene copy number in both ARR and ARS. Individuals with high EPSPS copies showed high protein expression in Western blot analysis. FISH analysis showed presence of brighter EPSPS signals, distributed randomly throughout the genome of ARR individuals compared to a faint signal in ARS plants. Random distribution of EPSPS copies was previously reported in glyphosate-resistant Palmer amaranth. Overall, the results of this study will help understand the origin and mechanism of EPSPS gene amplification in Italian ryegrass.

Wheat (genome AABBDD) is one of the essential crops, offering approximate 20% of human calorie consumption worldwide. Allopolyploidization of three diploid ancestors led to hexaploid wheat with narrowed genetic variation. Chromosome engineering is an applicable approach to restore the evolutionarily-omitted genetic diversity by homoeologous chromosomes recombination between wheat and its relatives. Two diploid relatives of wheat, Thinopyrum elongatum (genome EE) and Aegilops speltoides (genome SS), containing favorable genes, are used as gene resources for alien introgression and genome diversification in wheat. An advanced and effective experiment procedure was developed and applied for the production, recovery, detection, and characterization of homoeologous recombinants. Meanwhile, a novel recombinant chromosome recovery strategy was exploited with improved efficiency and accuracy. In this study, recombinants of wheat chromosomes 3B and 7B with their homoeologous chromosomes in Th. elongatum and Ae. speltoides (i.e. 3B-3E, 7B-7E, and 7B-7S) were produced and detected. Totolly, 81 3B-3E recombinants and four aberrations involving in distinct chromosomal regions were developed in three recombination cycles by fluorescent genomic in situ hybridization (FGISH). The secondary and tertiary recombination breakpoints occurred toward the proximal regions comparing to the primary recombination under this advanced recombination procedure. A novel recovery strategy was used to recover 7B-7E and 7B-7S homoeologous recombinants by chromosome-specific markers and FGISH verification. Marker-based pre-screening and subsequent FGISH verification identified 29 7B-7E and 61 7B- 7S recombinants, seven 7B-7E and four 7B-7S Robertsonian translocations, one 7E and five 7S telocentric chromosomes, and three 7S deletions. All the recombinants and aberrations were genotyped by high-throughput wheat 90K single nucleotide polymorphism (SNP) assay and the recombination breakpoints were physically mapped to wheat chromosome 3B or 7B according to their FGISH patterns, SNP results, and wheat reference genome sequence. Chromosome 3B was physically partitioned into 38 bins with 429 SNPs. Meanwhile, 44 distinct bins were resolved for chromosome 7B with 523 SNPs. A composite bin map was constructed for chromosomes 3B and 7B, respectively, with a comprehensive analysis of FGISH and SNPs results. In summary, this project provides a unique physical framework for further wheat genome studies and diversifies the wheat genome for germplasm development in wheat breeding.

Common oat (Avena) has held a significant place within the global crop community for centuries; although its cultivation has decreased over the past century, its nutritional benefits have recently garnered increased interest for human consumption. No published reference sequences are available for any of the three oat subgenomes. Here we report a quality sequence assembly, annotation and hybrid optical map of the A-genome diploid Avena atlantica Baum and Fedak. The assembly is composed of a total of 3,417 contigs with an N50 of 11.86 Mb and an estimated completeness of 97.6%. This genome sequence will be a valuable research tool within the oat community.

Common oat (Avena) has held a significant place within the global crop community for centuries; although its cultivation has decreased over the past century, its nutritional benefits have recently garnered increased interest for human consumption. No published reference sequences are available for any of the three oat subgenomes. Here we report a quality sequence assembly, annotation and hybrid optical map of the A-genome diploid Avena atlantica Baum and Fedak. The assembly is composed of a total of 3,417 contigs with an N50 of 11.86 Mb and an estimated completeness of 97.6%. This genome sequence will be a valuable research tool within the oat community.

Agronomic outcomes are the product of a plant's genotype and its environment. Genomic technologies allow farmers and researchers new avenues to explore the genetic component of agriculture. These technologies can also enhance understanding of environmental effects. With a growing world population, a wide variety of tools will be necessary to increase the agronomic productivity. Here I use massively parallel, deep sequencing of RNA (RNA-Seq) to measure changes in cotton gene expression levels in response to a change in the plant's surroundings caused by conservation tillage. Conservation tillage is an environmentally friendly, agricultural practice characterized by little or no inversion of the soil prior to planting. In addition to changes in cotton gene expression and biological pathway activity, I assay the transcriptional activity of microbial symbiotes living in and around the cotton roots. I found a large degree of similarity between cotton individuals in different treatments. However, under conventional disk tillage I did find significantly greater activity of cotton phosphatase and sulfate transport genes, as well as greater abundance of the microbes Candidatus Burkholderia brachynathoides and Arthrobacter species L77. This study also includes the use of high-throughput physical mapping of DNA to examine the genomic structure of a wild cotton species, Gossypium raimondii, which is closely related to the economically significant crop species Gossypium hirsutum. This technology characterizes genomic regions by assembling large input DNA molecules labeled at restriction enzyme recognition sites. I created an efficient algorithm and generated 812 whole genome assemblies from two datasets. The best of these assemblies allowed us to detect 3,806 potential misassemblies in the current release of the G. raimondii genome sequence assembly.

Leaf rust caused by Puccinia hordei is an important disease of barley (Hordeum vulgare) in many regions of the world. Yield losses up to 62% have been reported in susceptible cultivars. The Rph5 gene confers resistance to the most prevalent races (8 and 30) of barley leaf rust in the United States. Therefore, the molecular mapping of Rph5 is of great interest. Genetic studies were performed by analysis of 93 and 91 F2 plants derived from the crosses 'Bowman' (rph5) x 'Magnif 102' (Rph5) and 'Moore' (rph5) x Virginia 92-42-46 (Rph5), respectively. Linkage analysis positioned the Rph5 locus to the extreme telomeric region of the short arm of barley chromosome 3H at 0.2 cM proximal to RFLP marker VT1 and 0.5 cM distal from RFLP marker C970 in the Bowman x Magnif 102 population. Synteny between rice chromosome 1 and barley chromosome 3 was employed to saturate the region within the sub-centimorgan region around Rph5 using sequence-tagged site (STS) markers that were developed based on barley expressed sequence tags (ESTs) syntenic to the phage (P1)-derived artificial chromosome (PAC) clones comprising distal region of the rice chromosome 1S. Five rice PAC clones were used as queries to blastn 370,258 barley ESTs. Ninety four non-redundant EST sequences were identified from the EST database and used as templates to design 174 pairs of primer combinations. As a result, 10 EST-based STS markers were incorporated into the 'Bowman' x 'Magnif 102' high-resolution map of the Rph5 region. More importantly, six markers, including five EST-derived STS sequences, co-segregate with Rph5. Genes, represented by these markers, are putative candidates for Rph5. Results of this study demonstrate the usefulness of rice genomic resources for efficient deployment of barley EST resources for marker saturation of targeted barley genomic region. / Ph. D.

No description available.