1,721,204 research outputs found
Genome-wide association studies to dissect the genetic architecture of yield-related traits in maize and the genetic basis of heterosis
Get PDFThis dissertation includes a general introduction (Chapter 1), three journal manuscripts (Chapters 2 to 4) and a section of general conclusions (Chapter 5). The paper in Chapter 2, which compares three statistical approaches for conducting GWAS and identifies the genetic architecture controlling for KRN trait, has been submitted for publication. I made major contributions include designing and performing the experiments, analyzing data and writing the manuscript under the guidance of Dr. Schnable. Dr. Nettleton and Dr. Dekkers provided technical support and conceptual advice for this work. The paper in Chapter 3, which investigates the modes of inheritance of trait-associated variants for seven yield-related traits and provides insight into heterosis, will soon be submitted for publication. I made major contributions to this experiment that include designing the experiments, supervising the data collection, data investigations, and writing the manuscript under the guidance of Dr. Schnable. Dr. Nettleton provided technical support and conceptual advice for this work. The paper in Chapter 4 reports a new method (termed XP-GWAS,) that uses pools of extreme-phenotype for conducting GWAS, will also be submitted for publication. My contributions to this paper include developing the concept, designing experiments, analyzing data and writing the manuscripts under the guidance of Dr. Schnable. The co-first author, Haiying Jiang assisted with data collection. Dr. Nettleton advised on the data analysis and wrote some custom R scripts for the data analysis.</p
Distinct genetic architectures for phenotype means and plasticities in Zea mays
Get PDFPhenotypic plasticity describes the phenotypic variation of a trait when a genotype is exposed to different environments. Understanding the genetic control of phenotypic plasticity in crops such as maize is of paramount importance for maintaining and increasing yields in a world experiencing climate change. Here, we report the results of genome-wide association analyses of multiple phenotypes and two measures of phenotypic plasticity in a maize nested association mapping (US-NAM) population grown in multiple environments and genotyped with ~2.5 million single-nucleotide polymorphisms. We show that across all traits the candidate genes for mean phenotype values and plasticity measures form structurally and functionally distinct groups. Such independent genetic control suggests that breeders will be able to select semi-independently for mean phenotype values and plasticity, thereby generating varieties with both high mean phenotype values and levels of plasticity that are appropriate for the target performance environments.This is a manuscript of an article published as Kusmec, Aaron, Srikant Srinivasan, Dan Nettleton, and Patrick S. Schnable. "Distinct genetic architectures for phenotype means and plasticities in Zea mays." Nature plants 3, no. 9 (2017): 715. doi: 10.1038/s41477-017-0007-7. Posted with permission.</p
Molecular characterization of CER2, an Arabidopsis gene involved in cuticular wax accumulation
No full textCuticular waxes are complex mixtures of very long chain fatty acids (VLCFAs) and their derivatives. The CER2 locus of Arabidopsis is involved in cuticular wax accumulation on stems, siliques, and hypocotyls. The CER2 gene was cloned via chromosome walking. This cloned sequence is able to complement the cer2 mutant phenotype. It is a single-copy sequence in the Arabidopsis genome and encodes a novel protein with a predicted mass of 47 kD. Anti-CER2 polyclonal antibodies detected a 47 kD polypeptide. Cell fractionation and immunoblot analyses demonstrated that the CER2 protein is not membrane-bound and localized in nuclei. These results suggest that CER2 might have a regulatory role in cuticular wax accumulation. The expression patterns of the CER2 gene were studied by in situ RNA hybridization and the analysis of Arabidopsis transgenic plants harboring an in-frame fusion of -1009/+234 of the CER2 gene to [beta]-glucuronidase (CER2-GUS). These analysis demonstrate that CER2 gene expression is developmentally regulated and organ- and tissue-specific. Consistence with the visible phenotype associated with cer2 mutants, the CER2 gene is highly expressed only on the epidermal cells of stems, siliques, and hypocotyls. In addition, CER2 expression was observed in guard cells, trichomes, petioles, sepals, petals, ovaries, pedicels, the tapetum layer of anthers, and pollen grains. CER2-GUS expression was not detected in roots or in the pavement cells of leaves. The observation of CER2 expression in anthers and pollen grains is in agreement with the fact that the CER2 gene is involved in pollen fertility. Light, drought, high osmotic pressure, heat or cold shock, and wounding were not be found to cause visible changes in CER2-GUS expression patterns. However, exogenous application of the cytokinin, BAP, induced ectopic expression of CER2-GUS in leaves, suggesting that CER2 gene expression might be mediated by endogenous cytokinins.</p
Molecular cloning and characterization of gl8, a gene involved in maize cuticular wax biosynthesis
No full textThe gl8 locus of maize was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes. Sixty independently derived gl8 mutant alleles have been isolated from stocks that carried the Mutator transposon system. A DNA fragment that contains a Mu8 transposon and that co-segregates with one of these alleles, gl8-Mu3142, was identified and cloned. DNA flanking the Mu8 transposon was shown to represent the gl8 locus via allelic cross-referencing experiments. The gl8 probe reveals a 1.4-kb transcript present in wild-type seedling leaves, and in lesser amounts, in other organs and at other developmental stages. Sequence analyses reveal that the predicted GL8 protein exhibits significant sequence similarity to a class of enzymes that catalyze the reduction of ketone groups to hydroxyl groups. Polyclonal antibodies were raised against GL8 protein expressed in E. coli. Subcellular fractionation experiments indicate that the GL8 protein is associated with the endoplasmic reticulum. Furthermore, polyclonal antibodies raised against purified acyl-CoA elongase complex from leek can interact with the E. coli-expressed GL8 protein. In combination, these findings suggest that the GL8 protein is a component of the acyl-CoA elongase complex. This conclusion lends further support to the hypothesis that the GL8 protein functions as a reductase during the elongation of very long chain fatty acids required for the production of cuticular waxes.</p
Characterization of meiotic recombination in maize using the a1-sh2 interval as a model system
No full textMeiotic recombination across the ~130--140 kb a1-sh2 interval was characterized, aiming to answer the question "Why does recombination occurs non-randomly in the maize genome?" The a1-sh2 interval contains at least four genes (a1, yz1, x1 and sh2). Initially, the breakpoints associated with 101 recombinants isolated from a stock that carries maize A1 Sh2 and a1::rdt sh2 haplotypes were physically mapped relative to sequence polymorphisms. These breakpoints are concentrated in three recombination hot spots that are located in the proximal 10% of the a1-sh2 interval. Two hot spots are genic ( a1 and yz1) and one is apparently non-genic. The x1 gene is not a recombination hot spot. These results established that not all hot spots are genes and not all genes are hot spots. The retrotransposon fraction of the a1-sh2 interval is relatively inert recombinationally.;To test the roles cis-genetic modifiers play in regulating meiotic recombination, recombination events between the a1 and sh2 loci were isolated from three near-isogenic stocks that carry structurally distinct teosinte A1 Sh2 haplotypes (from Z. mays spp. mexicana Chalco, Z. mays spp. parviglumis and Z. luxurians ) and a common maize a1::rdt sh2 haplotype. Genetic distances across the a1-sh2 interval varied three fold. In each teosinte haplotype, over 85% of recombination events resolved in the proximal 10% of the a1-sh2 interval. Even so, significant differences were observed in the distributions of recombination breakpoints across subintervals among haplotypes. Each of the three previously detected recombination hot spots was detected in at least one of the three teosinte haplotypes and two of these hot spots were not detected in at least one teosinte haplotype. Moreover, novel hot spots were detected in two teosinte haplotypes. Due to the near-isogenic nature of the three stocks, the observed variation in the distribution of recombination events is the consequence of cis-modifications. Although generally negatively correlated with rates of recombination/Mb, frequencies of sequence polymorphisms do not fully account for the nonrandom distribution of recombination breakpoints. This study indicates that estimates of linkage disequilibrium must be interpreted with caution when considering whether a gene has been under selection.</p
Analysis of the maize (Zea mays L) genome using molecular, genetic and computational approaches
Get PDFApproximately 80% of ~2,500 Mbp maize genome consists of highly repetitive sequences. Initial sequencing efforts have targeted the gene-rich regions of this genome. A new open reading frame (ORF) selecting vector (pORF-Rescue) was developed to efficiently distinguish between the coding and non-coding fractions of this complex genome. pORF-Rescue can enrich ORFs by ~15 fold and non-transposon coding sequences by ~3.6 fold. This vector can also be applied for rapid gene discovery from maize BAC DNA and to establish peptide/expression libraries;Methylation filtration (MF) and high-Cot (HC) sequencing strategies have also proven successful in maize gene-enrichment. To best utilize MF and HC genome survey sequences (GSSs), these GSS data were first examined by identifying types and frequencies of sequencing errors and then assembled into maize assembled genomic islands (MAGIs). Computational and biological quality assessments indicate that a very high percentage of the MAGIs accurately reflect the structure of the maize genome. In addition, we estimate that this assembly of the maize gene space has "tagged" >6,900 expressed genes that previously lacked evidence of transcription and that almost 350 of these expressed genes are "orphans"; i.e., they do not exhibit similarity to genes in other species;To construct a sequence-based maize genetic map using large-scale genic sequence data and intermated B73xMo17 recombinant inbred lines (IRILs) as mapping population will facilitate the map-based genome sequencing project, QTL and eQTL studies, and candidate gene cloning experiments. The observations that two segregation distortion regions overlap with maize flowering time QTLs suggest that the altered allele frequencies were a consequence of inadvertent selection during the development of the IBM IRILs. Detection of inter-chromosomal two-locus gamete disequilibrium suggests another means to discover potential functional relationships (e.g. epistasis) of different chromosome regions using well-characterized plant RILs.*;*This dissertation is a compound document (contains both a paper copy and a CD as part of the dissertation). The CD requires the following system requirements: Adobe Acrobat; Microsoft Office.</p
The effects of trans modifiers and tandem duplications on meiotic recombination in maize
Get PDFMeiotic recombination can be affected by factors that reside either within (i.e., cis-acting elements) or outside (i.e., trans -acting modifiers) the recombining interval. To assess how trans-acting genetic modifiers polymorphic within maize germplasm affect rates and patterns of meiotic recombination in the absence of polymorphic cis factors, meiotic recombination was characterized across a sequence-identical 140-kb multi-genic a1-sh2 interval in each of three genetic backgrounds in maize. Rates of recombination among the genetic backgrounds varied twofold. The distributions of recombination breakpoints across the a1-sh2 interval in each background established that trans -acting modifier(s) polymorphic among genetic backgrounds can increase or decrease rates of recombination in both genic and intergenic regions over relatively small genetic and physical intervals and can even convert a region to a recombination hotspot. In addition, at least some trans-acting modifiers do not globally affect recombination, but instead target specific regions of the genome. To study a potential trans-acting modifier, recombination at an a1 allele containing a non-autonomous Mu1 insertion was assayed in both the presence and absence of the corresponding autonomous transposon, MuDR. In the presence of MuDR the rate of crossing over increased by fourfold, thereby demonstrating that MuDR is a trans-acting modifier of meiotic recombination. Because MuDR catalyzes the excision of Mu1, this is the first in vivo evidence that DNA breaks stimulate meiotic recombination in plants. In addition to trans-acting modifiers, the effects of A1-b, a tandem gene duplication of the maize a1 locus and a cis-acting factor, on meiotic recombination were characterized. This study directly demonstrates that in this genotype the homolog is the preferred template for unequal recombination and that interchromatid recombination is regulated separately from interhomolog recombination (e.g., recombination breakpoints resolve differently between the two recombination templates). Regulation of unequal recombination appears to occur at many levels including the chromosome (i.e., sister chromatid vs. homolog recombination) and the components of the duplication (i.e., pairing configurations). Rates of unequal recombination at A1-b are similar to the rate of recombination between non-duplicated a1 alleles. Unequal recombination is therefore common and is likely to be responsible for the generation of genetic variability, even within inbred lines.</p
Map based candidate gene cloning and functional analysis of genes involved in VLCFAs synthesis
Get PDFVLCFAs are biosynthesized by an endoplasmic reticulum associated enzyme system, referred to as fatty acid elongase. Fatty acid elongase generates VLCFAs by elongating 18C fatty acids. The enzymatic machinery of fatty acid elongation is believed to be composed of 3-ketoacyl-CoA synthase, 3-ketoacyl-CoA reductase, 3-hydroxyacyl-CoA dehydratase, and enoyl-CoA reductase. However, the insoluble nature of the fatty acid elongase system has hampered efforts to purify the system and to identify the genes that encode the component proteins. In the current study a candidate gene cloning approach was used to demonstrate that the previously defined gl26 gene of maize encodes a predicted enoyl-CoA reductase (ECR). Based on the phenotypes of existing and newly isolated gl26 mutants, the GL26 protein is required for the normal accumulation of cuticular waxes (CWs). The specific alterations in the composition of the CWs and the transcripts of gl26 gene that accumulate on gl26 mutants are consistent with the GL26 protein being a ECR. Because some of the gl26 mutants exhibit conditional lethality, we conclude that ECR activity is essential in maize. Using the similar methods, two candidates of 3-hydroxyacyl-CoA dehydratase (HCD) were identified in maize.</p
Characterization of three root hair mutants in maize
No full textRoot hairs are thought to play an important role in the uptake of water and nutrients from the environment. Several root hair defective mutants were isolated from Mutator-tagged stocks and EMS-mutagenized lines of maize. These mutants all condition either non-elongated root-hair initials (rth1, rth3-1, rth3-2, rth3-3, rth4, rth5, rth6) or shorter-than-normal root hairs (rth2). The morphology of rth1, rth2, and rth3 root hairs were analyzed via SEM. The rth1, rth2, and rth3 genes map to chromosomes 1L, 5L, and 1S, respectively. The rth3 gene was cloned using Mu-tagged allele. The partial cDNA sequence of this gene suggests that it may be a cell wall protein. In addition, an rth1 candidate gene was cloned. This gene contains, 25 exons and encodes a protein with similarity to yeast proteins involved in protein trafficking.</p
Functional characterization of the rad51 genes in Zea mays and their roles in Mu transposition
Get PDFIn Saccharomyces cerevisiae, Rad51p plays a central role in homologous recombination and DNA repair. Double mutants of the two Zea mays L. rad51 homologs are viable, but male sterile and have ~22% of normal seed set. Light microscopic analyses of male meiosis in these plants reveal: many chromosomes are unpaired at diakinesis and over 33% of quartets carry cells that either lack a nucleolus or have two nucleoli, indicating that non-disjunction occurs at both meiotic divisions. FISH analysis shows that 70% pachytene cells have paired 5S rDNA loci. Thus, maize RAD51 is required for efficient chromosome pairing and proper chromosome segregation in meiosis. FISH data also indicate that RAD51 is not essential for chromosome pairing. Consistent with that, surviving female gametes produced by double mutants are euploid and exhibit near-normal rates of meiotic crossovers. These results differ from those of Arabidopsis in which a rad51 mutant exhibits completely disrupted chromosome pairing during meiosis. Although maize rad51 double mutants develop well under normal condition, RAD51 function is critical for the repair of radiation-induced double-stranded breaks (DSBs) during early vegetative development;In late somatic cells rates of Mu insertion and excision are both high. In contrast, although high rates of insertion are observed in germinal cells, germinal excisions are recovered rarely. In RAD51- plants (i.e., rad51a, rad51b double mutants) rates of germinal derivatives from al-m5216 are 40-fold higher than controls. Most of the germinal derivatives involve deletions of the MuDR insertion and the al gene. This suggests that in wild-type germinal cells MuDR excisions are efficiently repaired via RAD51-directed homologous recombination with the sister chromatid, which replaces the excised MuDR. Two experiments suggest that RAD51 is also required for repairing Mu-induced DSBs during early vegetative development. First, a high proportion of Mu-active RAD51- mutants exhibit severe developmental defects. Second, ear sectors of germinal derivatives were recovered at a higher rate from RAD51- mutants than from controls. The higher rate at which partial deletions of the a1 locus were recovered indicates that the rad51 double mutant stock offers an attractive means to generate knock-out alleles for functional genomic studies.</p
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