Search

CHAPTER L GENERAL INTRODUCTION 1 Overview I Dissertation organization 2 Literature Review 2 References 8 CHAPTER 2. ANALYSIS OF MUTANTS OF THREE GENES THAT INFLUENCE ROOT HAIR DEVELOPMENT IN ZEA MAYS (GRAMINEAE) SUGGEST THAT ROOT HAIRS ARE DISPENSABLE 16 Abstract 16 Introduction 17 Materials and Methods 18 Results 24 Discussion 29 Literature Cited 31 CHAPTER 3. MOLECULAR CLONING AND ANALYSIS OF THE rthS GENE THAT AFFECTS ROOT HAIR DEVELOPMENT IN MAIZE 44 Abstract 44 Introduction 45 Materials and Methods 46 Results 50 Discussion 54 References 55 CHAPTER 4. IDENTIFICATION AND ANALYSIS OF A CANDIDATE CLONE OF THE MAIZE rth I LOCUS REQUIRED FOR ROOT HAIR DEVELOPMENT 69 Abstract 69 Introduction 70 Materials and Methods 71 Results 75 Discussion 81 References 84 CHAPTERS. GENERAL SUMMARY 100 Conclusions 100 Recommendations for Future Research 102 References 102 ACKNOWLEDGEMENTS 106

Root hairs are tubular extensions of the epidermis. Root hairs of the monogenic recessive maize mutant roothairless 6 (rth6) are arrested after bulge formation during the transition to tip growth and display a rough cell surface. BSR-Seq in combination with Seq-walking and subsequent analyses of four independently generated mutant alleles established that rth6 encodes CSLD5 a plasma membrane localized 129 kD D-type cellulose synthase with eight transmembrane domains. Cellulose synthases are required for the biosynthesis of cellulose, the most abundant biopolymer of plant cell walls. Phylogenetic analyses revealed that RTH6 is part of a monocot specific clade of D-type cellulose synthases. D-type cellulose synthases are highly conserved in the plant kingdom with five gene family members in maize and homologs even among early land plants such as the moss Physcomitrella patens or the clubmoss Selaginella moellendorffii. Expression profiling demonstrated that rth6 transcripts are highly enriched in root hairs as compared to all other root tissues. Moreover, in addition to the strong knock down of rth6 expression in young primary roots of the mutant rth6, the gene is also significantly down-regulated in rth3 and rth5 mutants, while it is up-regulated in rth2 mutants, suggesting that these genes interact in cell wall biosynthesis.

Rates of Mu transposon insertions and excisions are both high in late somatic cells of maize. In contrast, although high rates of insertions are observed in germinal cells, germinal excisions are recovered only rarely. Plants doubly homozygous for deletion alleles of rad51A1 and rad51A2 do not encode functional RAD51 protein (RAD51−). Approximately 1% of the gametes from RAD51+ plants that carry the MuDR-insertion allele a1-m5216 include at least partial deletions of MuDR and the a1 gene. The structures of these deletions suggest they arise via the repair of MuDR-induced double-strand breaks via nonhomologous end joining. In RAD51− plants these germinal deletions are recovered at rates that are at least 40-fold higher. These rates are not substantially affected by the presence or absence of an a1-containing homolog. Together, these findings indicate that in RAD51+ germinal cells MuDR-induced double-strand breaks (DSBs) are efficiently repaired via RAD51-directed homologous recombination with the sister chromatid. This suggests that RAD51− plants may offer an efficient means to generate deletion alleles for functional genomic studies. Additionally, the high proportion of Mu-active, RAD51− plants that exhibit severe developmental defects suggest that RAD51 plays a critical role in the repair of MuDR-induced DSBs early in vegetative development.

A new genetic map of maize, ISU–IBM Map4, that integrates 2029 existing markers with 1329 new indel polymorphism (IDP) markers has been developed using intermated recombinant inbred lines (IRILs) from the intermated B73 × Mo17 (IBM) population. The website http://magi.plantgenomics.iastate.edu provides access to IDP primer sequences, sequences from which IDP primers were designed, optimized marker-specific PCR conditions, and polymorphism data for all IDP markers. This new gene-based genetic map will facilitate a wide variety of genetic and genomic research projects, including map-based genome sequencing and gene cloning. The mosaic structures of the genomes of 91 IRILs, an important resource for identifying and mapping QTL and eQTL, were defined. Analyses of segregation data associated with markers genotyped in three B73/Mo17-derived mapping populations (F2, Syn5, and IBM) demonstrate that allele frequencies were significantly altered during the development of the IBM IRILs. The observations that two segregation distortion regions overlap with maize flowering-time QTL suggest that the altered allele frequencies were a consequence of inadvertent selection. Detection of two-locus gamete disequilibrium provides another means to extract functional genomic data from well-characterized plant RILs.

The roothairless1 (rth1) mutant is impaired in root hair elongation and exhibits other growth abnormalities. Unicellular root hairs elongate via localized tip growth, a process mediated by polar exocytosis of secretory vesicles. We report here the cloning of the rth1 gene that encodes a sec3 homolog. In yeast (Saccharomyces cerevisiae) and mammals, sec3 is a subunit of the exocyst complex, which tethers exocytotic vesicles prior to their fusion. The cloning of the rth1 gene associates the homologs of exocyst subunits to an exocytotic process in plant development and supports the hypothesis that exocyst-like proteins are involved in plant exocytosis. Proteomic analyses identified four proteins that accumulate to different levels in wild-type and rth1 primary roots. The preferential accumulation in the rth1 mutant proteome of a negative regulator of the cell cycle (a prohibitin) may at least partially explain the delayed development and flowering of the rth1 mutant.

Temperature gradient capillary electrophoresis (TGCE) can be used to distinguish heteroduplex from homoduplex DNA molecules and can thus be applied to the detection of various types of DNA polymorphisms. Unlike most single nucleotide polymorphism (SNP) detection technologies, TGCE can be used even in the absence of prior knowledge of the sequences of the underlying polymorphisms. TGCE is both sensitive and reliable in detecting SNPs, small InDel (insertion/deletion) polymorphisms (IDPs) and simple sequence repeats, and using this technique it is possible to detect a single SNP in amplicons of over 800 bp and 1-bp IDPs in amplicons of approximately 500 bp. Genotyping data obtained via TGCE are consistent with data obtained via gel-based detection technologies. For genetic mapping experiments, TGCE has a number of advantages over alternative heteroduplex-detection technologies such as celery endonuclease (CELI) and denaturing high-performance liquid chromatography (dHPLC). Multiplexing can increase TGCE's throughput to 12 markers on 94 recombinant inbreds per day. Given its ability to efficiently and reliably detect a variety of subtle DNA polymorphisms that occur at high frequency in genes, TGCE shows great promise for discovering polymorphisms and conducting genetic mapping and genotyping experiments.

Five ab initio programs (FGENESH, GeneMark.hmm, GENSCAN, GlimmerR and Grail) were evaluated for their accuracy in predicting maize genes. Two of these programs, GeneMark.hmm and GENSCAN had been trained for maize; FGENESH had been trained for monocots (including maize), and the others had been trained for rice or Arabidopsis. Initial evaluations were conducted using eight maize genes (gl8a, pdc2, pdc3, rf2c, rf2d, rf2e1, rth1, and rth3) of which the sequences were not released to the public prior to conducting this evaluation. The significant advantage of this data set for this evaluation is that these genes could not have been included in the training sets of the prediction programs. FGENESH yielded the most accurate and GeneMark.hmm the second most accurate predictions. The five programs were used in conjunction with RT-PCR to identify and establish the structures of two new genes in the a1-sh2 interval of the maize genome. FGENESH, GeneMark.hmm and GENSCAN were tested on a larger data set consisting of maize assembled genomic islands (MAGIs) that had been aligned to ESTs. FGENESH, GeneMark.hmm and GENSCAN correctly predicted gene models in 773, 625, and 371 MAGIs, respectively, out of the 1353 MAGIs that comprise data set 2.

To enhance gene discovery, expressed sequence tag (EST) projects often make use of cDNA libraries produced using diverse mixtures of mRNAs. As such, expression data are lost because the origins of the resulting ESTs cannot be determined. Alternatively, multiple libraries can be prepared, each from a more restricted source of mRNAs. Although this approach allows the origins of ESTs to be determined, it requires the production of multiple libraries. A hybrid approach is reported here. A cDNA library was prepared using 21 different pools of maize (Zea mays) mRNAs. DNA sequence „bar codes” were added during first-strand cDNA synthesis to uniquely identify the mRNA source pool from which individual cDNAs were derived. Using a decoding algorithm that included error correction, it was possible to identify the source mRNA pool of more than 97% of the ESTs. The frequency at which a bar code is represented in an EST contig should be proportional to the abundance of the corresponding mRNA in the source pool. Consistent with this, all ESTs derived from several genes (zein and adh1) that are known to be exclusively expressed in kernels or preferentially expressed under anaerobic conditions, respectively, were exclusively tagged with bar codes associated with mRNA pools prepared from kernel and anaerobically treated seedlings, respectively. Hence, by allowing for the retention of expression data, the bar coding of cDNA libraries can enhance the value of EST projects.

Cytoplasmic male sterility is a maternally transmitted inability to produce viable pollen. Male sterility occurs in Texas (T) cytoplasm maize as a consequence of the premature degeneration of the tapetal cell layer during microspore development. This sterility can be overcome by the combined action of two nuclear restorer genes, rf1 and rf2a. The rf2a gene encodes a mitochondrial aldehyde dehydrogenase (ALDH) that is capable of oxidizing a variety of aldehydes. Six additional ALDH genes were cloned from maize and Arabidopsis. In vivo complementation assays and in vitro enzyme analyses demonstrated that all six genes encode functional ALDHs. Some of these ALDHs are predicted to accumulate in the mitochondria, others in the cytosol. The intron/exon boundaries of these genes are highly conserved across maize and Arabidopsis and between mitochondrial and cytosolic ALDHs. Although animal, fungal, and plant genomes each encode both mitochondrial and cytosolic ALDHs, it appears that either the gene duplications that generated the mitochondrial and the cytosolic ALDHs occurred independently within each lineage or that homogenizing gene conversion-like events have occurred independently within each lineage. All studied plant genomes contain two confirmed or predicted mitochondrial ALDHs. It appears that these mitochondrial ALDH genes arose via independent duplications after the divergence of monocots and dicots or that independent gene conversion-like events have homogenized the mitochondrial ALDH genes in the monocot and dicot lineages. A computation approach was used to identify amino acid residues likely to be responsible for functional differences between mitochondrial and cytosolic ALDHs.

Root hairs are instrumental for nutrient uptake in monocot cereals. The maize (Zea mays L.) roothairless5 (rth5) mutant displays defects in root hair initiation and elongation manifested by a reduced density and length of root hairs. Map-based cloning revealed that the rth5 gene encodes a monocot-specific NADPH oxidase. RNA-Seq, in situ hybridization and qRT-PCR experiments demonstrated that the rth5 gene displays preferential expression in root hairs but also accumulates to low levels in other tissues. Immunolocalization detected RTH5 proteins in the epidermis of the elongation and differentiation zone of primary roots. Because superoxide and hydrogen peroxide levels are reduced in the tips of growing rth5 mutant root hairs as compared with wild-type, and Reactive oxygen species (ROS) is known to be involved in tip growth, we hypothesize that the RTH5 protein is responsible for establishing the high levels of ROS in the tips of growing root hairs required for elongation. Consistent with this hypothesis, a comparative RNA-Seq analysis of 6-day-old rth5 versus wild-type primary roots revealed significant over-representation of only two gene ontology (GO) classes related to the biological functions (i.e. oxidation/reduction and carbohydrate metabolism) among 893 differentially expressed genes (FDR

Mutations at the glossy1 (gl1) locus of maize (Zea mays L.) quantitatively and qualitatively affect the deposition of cuticular waxes on the surface of seedling leaves. The gl1 locus has been molecularly cloned by transposon tagging with the Mutator transposon system. The epi23 cDNA was isolated by subtractive hybridization as an epidermis-specific mRNA from Senecio odora (Kleinia odora). The deduced amino acid sequence of the GL1 and EPI23 proteins are very similar to each other and to two other plant proteins in which the sequences were deduced from their respective mRNAs. These are the Arabidopsis CER1 protein, which is involved in cuticular wax deposition on siliques, stems, and leaves of that plant, and the protein coded by the rice expressed sequence tag RICS2751A. All four proteins are predicted to be localized in a membrane via a common NH2-terminal domain, which consists of either five or seven membrane-spanning helices. The COOH-terminal portion of each of these proteins, although less conserved, is predicted to be a water-soluble, globular domain. These sequence similarities indicate that these plant orthologs may belong to a superfamily of membrane-bound receptors that have been extensively characterized from animals, including the HIV co-receptor fusin (also termed CXCR4).

Root hairs are specialized epidermal cells that are thought to play an important role in plant nutrition by facilitating the absorption of water and nutrients. Three maize mutants with abnormal root hair morphologies (rthl, rth2, and rth3) have been isolated from Mutator transposon stocks. All three root hair mutant phenotypes are controlled by single recessive alleles. The rthl mutant initiates normal-looking root hair primordia that fail to elongate. The normal-looking root hair primordia of the rth2 mutant elongate to only approximately one-fifth to one-fourth the length of wild type root hairs. Like rthl primordia, rth3 primordia undergo little elongation. However, unlike the relatively normal-looking rthl primordia, rth3 primordia are distinctly abnormal when viewed through a scanning electron microscope. The rthl mutant exhibits pleiotropic nutrient deficiencies, while the rth2 and rth3 mutants grow vigorously. This finding suggests that under some environmental conditions, root hairs are less important to plant growth than has been previously thought. The rthl, rth2, and rth3 genes have been mapped to chromosomes IL, 5L, and IS, respectively, via crosses with BA translocation stocks. The rth2 allele exhibits reduced transmission through the male gametophyte, but a normal rate of transmission through female gametophytes; rthl and rth3 are transmitted at normal rates. Root hairs are specialized root epidermal cells that are thought to play an important role in the absorption of water and nutrients from the rhizosphere by increasing the absorptive surface area of roots (Clarkson, 1985). The formation of root hairs occurs in two distinct stages: during "initiation," localized "bulges" (root hair primordia) form at the predetermined trichoblasts (hair-bearing epidermal cells); during "elongation," the "bulges" extend in a unipolar direction to form the tubular root hairs. This extension occurs via tip growth, a process common to root hairs and pollen tubes, and involves the addition of new membrane components at a cell's growing tip (Cormack, 1962; Newcomb and Bonnett, 1965; Sievers and

Summary The rth3 (roothairless 3) mutant is specifically affected in root hair elongation. We report here the cloning of the rth3 gene via a PCR-based strategy (amplification of insertion mutagenized sites) and demonstrate that it encodes a COBRA-like protein that displays all the structural features of a glycosylphosphatidylinositol anchor. Genes of the COBRA family are involved in various types of cell expansion and cell wall biosynthesis. The rth3 gene belongs to a monocot-specific clade of the COBRA gene family comprising two maize and two rice genes. While the rice (Oryza sativa) gene OsBC1L1 appears to be orthologous to rth3 based on sequence similarity (86% identity at the protein level) and maize/rice synteny, the maize (Zea mays L.) rth3-like gene does not appear to be a functional homolog of rth3 based on their distinct expression profiles. Massively parallel signature sequencing analysis detected rth3 expression in all analyzed tissues, but at relatively low levels, with the most abundant expression in primary roots where the root hair phenotype is manifested. In situ hybridization experiments confine rth3 expression to root hair-forming epidermal cells and lateral root primordia. Remarkably, in replicated field trials involving near-isogenic lines, the rth3 mutant conferred significant losses in grain yield.

As an ancient segmental tetraploid, the maize (Zea mays L.) genome contains large numbers of paralogs that are expected to have diverged by a minimum of 10% over time. Nearly identical paralogs (NIPs) are defined as paralogous genes that exhibit ≥98% identity. Sequence analyses of the “gene space” of the maize inbred line B73 genome, coupled with wet lab validation, have revealed that, conservatively, at least ∼1% of maize genes have a NIP, a rate substantially higher than that in Arabidopsis. In most instances, both members of maize NIP pairs are expressed and are therefore at least potentially functional. Of evolutionary significance, members of many NIP families also exhibit differential expression. The finding that some families of maize NIPs are closely linked genetically while others are genetically unlinked is consistent with multiple modes of origin. NIPs provide a mechanism for the maize genome to circumvent the inherent limitation that diploid genomes can carry at most two “alleles” per “locus.” As such, NIPs may have played important roles during the evolution and domestication of maize and may contribute to the success of long-term selection experiments in this important crop species.

Temperature gradient capillary electropho- resis (TGCE) is a high-throughput method to detect segregating single nucleotide polymorphisms and InDel polymorphisms in genetic mapping populations. Exist- ing software that analyzes TGCE data was, however, designed for mutation analysis rather than genetic mapping. Genetic recombinant analysis and mapping assistant (GRAMA) is a new tool that automates TGCE data analysis for the purpose of genetic map- ping. Data from multiple TGCE runs are analyzed, integrated, and displayed in an intuitive visual format. GRAMA includes an algorithm to detect peaks in electropherograms and can automatically compare its peak calls with those produced by another software package. Consequently, GRAMA provides highly accurate results with a low false positive rate of 5.9% and an even lower false negative rate of 1.3%. Because of its accuracy and intuitive interface, GRAMA boosts user productivity more than twofold relative to previ- ous manual methods of scoring TGCE data. GRAMA is written in Java and is freely available at http:// www.complex.iastate.edu.

Recent sequencing efforts have targeted the gene-rich regions of the maize ( Zea mays L.) genome. We report the release of an improved assembly of maize assembled genomic islands (MAGIs). The 114,173 resulting contigs have been subjected to computational and physical quality assessments. Comparisons to the sequences of maize bacterial artificial chromosomes suggest that at least 97% (160 of 165) of MAGIs are correctly assembled. Because the rates at which junction-testing PCR primers for genomic survey sequences (90-92%) amplify genomic DNA are not significantly different from those of control primers (≈91%), we conclude that a very high percentage of genic MAGIs accurately reflect the structure of the maize genome. EST alignments, ab initio gene prediction, and sequence similarity searches of the MAGIs are available at the Iowa State University MAGI web site. This assembly contains 46,688 ab initio predicted genes. The expression of almost half (628 of 1,369) of a sample of the predicted genes that lack expression evidence was validated by RT-PCR. Our analyses suggest that the maize genome contains between ≈33,000 and ≈54,000 expressed genes. Approximately 5% (32 of 628) of the maize transcripts discovered do not have detectable paralogs among maize ESTs or detectable homologs from other species in the GenBank NR nucleotide/protein database. Analyses therefore suggest that this assembly of the maize genome contains approximately 350 previously uncharacterized expressed genes. We hypothesize that these “orphans” evolved quickly during maize evolution and/or domestication.

This presents results on training both finite state classifiers and interpolated Markov models as classifiers for polymerase chain reaction primers. The goal of the study is to find techniques to decrease the number of primers that fail to amplify correctly within a large genomics project. Standard primer design packages already select primers in a manner consistent with current knowledge of the biophysics of DNA. The classifiers trained in this effort are used to capture lab and organism specific features of primer data and are used to postprocess the output of standard primer design packages. The finite state classifiers in this study are trained with a novel evolutionary algorithm that uses an incremental fitness reward system and multipopulation hybridization. This hybridization is akin to population seeding, not the more usual hybridization of evolutionary computation with other techniques. The interpolated Markov model is a form of Markov model that adapts to data rich and data sparse portions of the training set by using a variable order in its modeling. The interpolated Markov models exhibited slightly superior performance and trains with far higher speed. The finite state classifiers provide a substantially different classification, however, and require less training data.

We present preliminary results on training finite state machines (FSMs) as good/bad classifiers for polymerase chain reaction (PCR) primers. Novel features of the work presented include hybridization of multiple populations of FSMs and an incremental fitness function. The system presented here is a post-production add-on to a standard primer picking program intended to compensate for organism and lab specific factors.

Cytoplasmic male sterility is a maternally transmitted inability to produce viable pollen. Male sterility occurs in Texas (T) cytoplasm maize as a consequence of the premature degeneration of the tapetal cell layer during microspore development. This sterility can be overcome by the combined action of two nuclear restorer genes, rf1 and rf2a. The rf2a gene encodes a mitochondrial aldehyde dehydrogenase (ALDH) that is capable of oxidizing a variety of aldehydes. Six additional ALDH genes were cloned from maize and Arabidopsis. In vivo complementation assays and in vitro enzyme analyses demonstrated that all six genes encode functional ALDHs. Some of these ALDHs are predicted to accumulate in the mitochondria, others in the cytosol. The intron/exon boundaries of these genes are highly conserved across maize and Arabidopsis and between mitochondrial and cytosolic ALDHs. Although animal, fungal, and plant genomes each encode both mitochondrial and cytosolic ALDHs, it appears that either the gene duplications that generated the mitochondrial and the cytosolic ALDHs occurred independently within each lineage or that homogenizing gene conversion-like events have occurred independently within each lineage. All studied plant genomes contain two confirmed or predicted mitochondrial ALDHs. It appears that these mitochondrial ALDH genes arose via independent duplications after the divergence of monocots and dicots or that independent gene conversion-like events have homogenized the mitochondrial ALDH genes in the monocot and dicot lineages. A computation approach was used to identify amino acid residues likely to be responsible for functional differences between mitochondrial and cytosolic ALDHs.

The gl8 locus of maize (Zea mays L.) was previously defined by a mutation that reduces the amount and alters the composition of seedling cuticular waxes. Sixty independently derived gl8 mutant alleles were 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 via allelic cross-referencing experiments to represent the gl8 locus. The gl8 probe revealed a 1.4-kb transcript present in wild-type seedling leaves and, in lesser amounts, in other organs and at other developmental stages. The amino acid sequence deduced from an apparently full-length gl8 cDNA exhibits highly significant sequence similarity to a group of enzymes from plants, eubacteria, and mammals that catalyzes the reduction of ketones. This finding suggests that the GL8 protein probably functions as a reductase during fatty acid elongation in the cuticular wax biosynthetic pathway.

The surfaces of the aerial parts of plants are coated with a layer of lipids collectively known as the cuticular waxes (1,2). This first, outer barrier to the environment is thought to play an important, but as yet undefined role in the survival of the organism. The cuticular waxes are a complex mixture of very long chain (20 – 36 carbons) aliphatic compounds, including fatty acids, alcohols, esters, aldehydes, ketones, and alkanes, which are biosynthetically derived from palmitic acid (2). We are taking a molecular genetic approach to clone genes involved in the biosynthesis and/or deposition of these compounds. In maize, mutants have defined 17 loci that affect the appearance of seedling cuticular waxes (the glossy loci) (3). In Arabidopsis, a total of 21 loci that affect cuticular wax accumulation on the stems and siliques have been defined genetically (the cer loci) (4).