Hugo K. Dooner - US grants

Most of the maize genome (>90%) is made up of repetitive DNA, a large fraction of which is methylated. Genes comprise less than 3% of the genome and are found in contiguous stretches of less methylated DNA known as hypomethylated islands. Because of the repetitive DNA content in maize, sequencing the entire maize genome (2.5xl09 bp) is impractical. Some laboratories, mostly in industry, have adopted the strategy of large-scale sequencing of the RNA products transcribed from genes (cDNAs). An attractive alternative is to specifically sequence genes. One can take advantage of the tendency of the maize transposon Activator (Ac) to insert in hypomethylated DNA, the genomic component containing genes, to identify genes as sites into which Ac transposes (tac sites) and, then, to sequence the DNA adjacent to the transposon. An advantage of this approach is that, in addition to a sequence that can be compared to the existing database, it generates an insertion library. The collection of lines carrying Ac at many different locations in the maize genome will enable investigators to screen for subtle mutant phenotypes, particularly after obtaining information on where in the plant the genes are expressed. Many genes are expected to have minor effects and could be missed from a conventional transposon mutagenesis screen designed to identify gross changes in phenotype.

Taking advantage of the powerful endosperm genetics of maize, a simple and efficient Ac transposition assay based on the well-studied endosperm markers bz (bronze) and wx (waxy) has been developed. A collection of over 1200 independent Ac transposants has been generated and over one-third of these Ac sites have been mapped relative to the donor locus. In parallel, a panhandle PCR method, originally used in the human genome, has been adapted for the isolation of DNA adjacent to the insertion (tac sites). By sequencing tac sites, insertions have been identified, for example, in genes encoding a sulfur transporter, a MAPKK, and a sesquiterpene synthase.

This Ac mobilization scheme allows the isolation of tac sites that are either genetically linked or unlinked to the donor locus. However, because Ac has a strong tendency to transpose to closely linked sites, about one-half of the tac sites are linked to wx on chromosome 9. Clearly, it would be desirable to mobilize Ac from different starting sites in the genome. Toward that end, suitable maize lines that are readily transformable and regenerable are being developed. These lines will be transformed with a construct carrying an Ac element modified to facilitate the isolation of tac DNA. This construct should integrate at random sites in the genome, providing starting platforms for future Ac mobilization.

The expected outcomes of this proposal are: (a) The sequence of more than 1000 maize genes or gene fragments. (b) The elucidation of the function of a set of 50 genes based on the sequence of tac sites, the phenotype of Ac insertion mutations, and the pattern of gene expression. (c) The map location of those genes that correspond to unique sequences in the maize genome. (d) The development of transgenic maize lines that will facilitate the future isolation of genes (tac sites) from any location in the genome.

During the tenure of the previous award, molecular characterization of recombination events at and around bz has revealed the following: (1) Recombination within bz is very high, 100 times higher than in the average DNA segment in maize. (2) In the immediate proximal side of bz, consisting mostly of repetitive DNA, it is at least 50-fold lower. (3) Recombination within the bz gene is uniform and does not show polarity as it does in yeast. (4) Insertions and clustered single bp heterologies suppress recombination and have the effect of making recombination nonuniform across the gene. (5) Intragenic sequence polymorphisms affect resolution of the recombination intermediates, leading to an almost exclusive recovery of parentally marked IGRs. (6) Gene conversion, thought to be rarer in plants than in yeast, appears to occur frequently in nonpolymorphic heterozygotes. (7) Meiotic recombination exhibits high fidelity and is not mutagenic. The analysis of meiotic recombination in maize will be continued by combining the power of the genetics of the bz locus with that of modern molecular techniques. Specific aims are as follows:
(1) Examine the effect of single bp polymorphisms on the distribution of recombination junctions in the absence of insertion heterozygosity.
(2) Extend the analysis of recombination at bz to the 5' upstream region.
(3) Analyze gene conversion tracts in the presence and absence of heterologies.
(4) Test whether all "intergenic" recombination between bz and the closely proximal marker tac2094 occurs in genes.

Homologous meiotic recombination is essential to sexually reproducing organisms: it creates novel genotypes and ensures proper chromosome segregation at the first meiotic division. From a practical standpoint, it allows plant geneticists to construct genetic maps that have multiple applications, from marker-assisted selection of desirable genotypes to the map-based cloning of valuable genes. Yet in spite of its importance, our knowledge of the process in higher plants remains rudimentary. The bronze locus of maize, a uniquely advantageous system, will be used to elucidate the process of meiotic recombination in plants.

The goals of this NSF Plant Genome project are to demonstrate that the transposon Ac (Activator) can be used as a gene identification and isolation tool, as well as a mutagen, in the complex maize genome, and to develop the necessary tools to facilitate that use. The first goal was met in the initial phase of this project where Ac was shown to insert exclusively in or close to genes and to be, therefore, an excellent gene-searching engine in the highly repetitive maize genome. A highly embryogenic bz wx inbred line was developed and transformed with Ac* and Ds* constructs that had been modified to facilitate the PCR isolation of the transposon-adjacent sequence. The main goal of the second phase of this project is to create a comprehensive set of transgenic lines that will serve as starting points for the production of future insertion libraries. This phase will occur in two well-defined stages. The first will be to demonstrate the germinal transposition of an engineered Ac* or Ds* element and the ready isolation of DNA adjacent to the transposon. The second will be to identify a method of maize transformation that will enable the production of a useful set of transposon lines for localized mutagenesis. Once the above two objectives have been met, the last stage of the project to produce a set of 124 transgenic lines with a uniquely marked element at evenly spaced locations in the genome will be initiated. In that set, most genes in the maize genome will be within 7 cM of a launching platform and will be, therefore, realistic targets in a localized transposon mutagenesis experiment. These lines will be deposited in the Maize Stock Center and will serve as starting materials for the generation of future insertion libraries by interested scientists.

Homologous meiotic recombination is an important process for sexually
reproducing organisms: it creates new genotypes by shuffling chromosomal
segments that otherwise would be inherited as a unit and promotes fertility by
ensuring that chromosomes segregate properly at meiosis. For close to a century,
geneticists have been using recombination as a tool to construct genetic maps,
which now find practical applications ranging from risk prediction for inherited
deleterious alleles to marker-assisted selection of desirable genotypes in
agriculture and map-based cloning of valuable genes in plants and animals. Yet,
in spite of its importance, our knowledge of the process of homologous meiotic
recombination in higher plants is rudimentary compared to other organisms, like
yeast. This project will increase our understanding of this basic biological
process in maize, a plant that is both an excellent model organism
for studies of recombination, and an economically important crop in American agriculture and industry.
The genetic system being used in the project is the bz locus, which affects seed
pigmentation and is uniquely advantageous for studies of recombination. This
project will continue the analysis of meiotic recombination in maize by combining the power of the genetics of the bz region with that of modern molecular tools. It is greatly influenced by the recent finding that the organization of genes and retrotransposons in the bz genomic region is polymorphic in different maize lines. There are five specific objectives of the project:
1. To analyze conversion tract lengths within the bz gene island in heterozygotes
lacking heterologies and extend the analysis to adjacent retrotransposon blocks.
2. To determine if the apparent conversion polarity at bz, detected with small
insertion mutations at the 5' and 3' ends of the gene, but not with more centrally
located point mutations, is due to the nature or the position of the mutations.
3. To test whether the presence of a highly methylated retrotransposon block
affects recombination in adjacent genes.
4. To determine if all recombination junctions in an interval made up of genes
and retrotransposons fall in genes.
5. To test if recombination between two sites in the genome separated by a
different number of genes in different lines is a function of the number of
common genes in the intervening segment.

Maize is probably the most diverse of all crop species. The most significant practical consequence of the huge genetic diversity within maize is the phenomenon of hybrid vigor (heterosis). Distantly related lines tend to produce highly vigorous F1 hybrids, which are planted across the entire Corn Belt. Yet, the molecular basis of hybrid vigor is not understood. However, analysis of allelic regions between inbreds has indicated that the same interval differs in sequence and gene content. Therefore, it has been hypothesized that hybrid vigor could be explained on the basis of different, yet complementing, gene sets in the two inbreds. To investigate the extent of occurrence of such plus-minus type changes, the genomic organization of two gene-dense genomic regions, bz and fie2-orp2, will be investigated in a group of 12 inbred lines, races, and relatives of maize. Twelve size-fractionated, NotI BAC libraries will be constructed and bz and fie2 clones will be isolated and sequenced from each library. Thus, >1 Mb of DNA sequence will be generated and deposited in central sequence repositories. The distribution of polymorphism will be examined with respect to differences among heterotic groups and to the potential role of inbreeding in genome rearrangement. BAC data will be analyzed with population genetic methods to contrast evolutionary histories between genes and examine the relationship between linkage disequilibrium and recombination.

Specific deliverables are:
1. BAC libraries specific to the bz and fie2-orp2 regions from 12 accessions
2. 1 Mb sequence data deposited in GenBank and maizeGDB
3. Characterization of gene presence/absence and polymorphisms in the selected regions
4. Analysis of recombination rates across multiple accessions for the specific regions

Maize is the most important American crop today. Therefore, a thorough knowledge of the function of maize genes is highly desirable. That knowledge can be gained from maize functional genomics projects that use transposons to disrupt gene function. In the present maize functional genomics project, a genetic resource is being developed that will enable investigators to combine the power of the mutational approach to define gene function with that of DNA sequencing to define a gene's make-up. The resource consists of a comprehensive set of lines based on transposons of the Ac/Ds family, which because of their transposition pattern, will be useful to scientists interested in saturating specific regions of the maize genome with transposon insertions. A set of 370 randomly generated transgenic lines with active transposon 'launch pads' will be identified, from which we expect to identify 124 transgenic lines that are equally spaced across the genome. In this set most genes in the maize genome will be within 7 cM of a launcing platform and, therefore realistic targets in localized transposon mutagenesis experiments. The transposons have been engineered to greatly facilitate the isolation of DNA sequences adjacent to transposed elements. The simple genetic scheme to select for transpositions and the ease in isolation of the adjacent sequences should make this resource particularly user-friendly. The lines will be made freely available to the research community through the Maize Genetics Stock Center in Urbana, IL. They will be deposited approximately once a year.

Broader Impacts
The ability for researchers to do mutagenesis across the maize genome will enable gene discovery and empower the corn research community.

Plants possess a variety of mechanisms to protect themselves from attack by herbivores. One system that is induced upon attack by herbivorous insects is the production of terpenoids that can serve in either direct or indirect defense against the herbivore. It has previously been shown that the maize sesquiterpene cyclase gene stc1, located in 9S, is induced in corn seedlings being foraged by beet armyworm (BAW) larvae. The product of this gene is a volatile terpenoid that most likely serves to attract wasps that parasitize the BAW larvae. Preliminary work leading to this proposal has now shown that stc2, the ortholog of stc1 in 6L, is induced by a different insect pest, the southwestern corn borer (SWCB). The P.I.s propose a genetic, molecular, and biochemical dissection of the two orthologous maize genes involved in this novel type of inducible plant defense system, as follows:

1. Characterization of the induction of the stc2 gene by the SWCB and other related corn borers. These experiments will establish the pattern of stc2 induction in response to regurgitant from the SWCB and whether the plant response is specific to that insect species or not.

2. Determination of the complete structure of the stc2 gene by isolating a full-length stc2 cDNA from seedling sheaths that have been foraged by SWCB. This will enable identification of the N-terminus of the protein, confirmation of the predicted intron-exon structure of the transcript, and expression of the STC2 enzyme in a heterologous system for biochemical characterization of its activity.

3. Assessment of the possible role of stc2 as an insect resistance gene by isolating and sequencing the stc2 gene from the non-inducible, SWCB-susceptible Ki3 inbred line. This would identify the basis of the noninducibility of stc2 in Ki3 and provide further evidence that stc2 corresponds to a quantitative trait locus (QTL) for SWCB resistance that maps very close to the location of stc2 in 6L.

4. Determination of the nature of the terpenoid product catalyzed by the heterologously expressed STC1 and STC2 enzymes. These experiments will identify the nature of the terpenoids made by these enzymes and confirm whether the product of STC1 is a sesquiterpenoid, as suggested by the earlier biochemical genetics data.

5. Identification of the most likely subcellular localization of the STC proteins with constructs that fuse their putative chloroplast transit peptides to GFP. This will confirm whether the predicted N-terminal targeting sequence in each of the three sequenced stc1 alleles behaves as a chloroplast transit peptide in vivo.

The proposed activity will have clear broader impacts. The work combines genetics, molecular biology, and biochemistry to analyze the response of maize plants to insect pests. It will serve as excellent training ground for student and postgraduate researchers interested in applying biochemistry and molecular genetics to address practical problems in plant biology. Both the P.I and co-P.I. are members of minorities and their labs are heavily populated by under-represented groups. The project is relevant to long-term improvement in U.S. agriculture in that it addresses mechanisms by which maize defends itself from insect attack. The identification of genes involved in insect resistance will aid breeders in developing naturally resistant crop species. Understanding natural defense mechanisms in major crops will allow alternative insect control strategies and reduce our dependence on potentially toxic pesticides. Thus, the problem to be investigated is of scientific, economic and environmental significance.

Homologous meiotic recombination is an important process because it creates new genotypes by shuffling chromosomal segments that otherwise would be inherited as blocks and promotes fertility by ensuring that chromosomes segregate properly. Recombination allows the construction of genetic maps, which are used in risk prediction for inherited disorders, in marker-assisted selection of desirable genotypes in agriculture, and in map-based cloning of valuable genes in plants. This project will increase our understanding of homologous meiotic recombination in maize, which is an excellent model organism for studies of recombination and has economic importance to American agriculture and industry. Much of the work is based on the discovery in the PI's lab of an unprecedented level of haplotype variability in maize. The project aims to study how recombination is affected by it. The project will continue to analyze meiotic recombination in maize by combining the power of the genetics of the bz region with that of modern molecular tools.

The project is relevant to U.S. agriculture in that it studies meiotic recombination, the phenomenon underlying most plant breeding. The project will be staffed by members of under-represented minorities and will serve as excellent training ground for undergraduate students interested in learning modern genotyping techniques. The P.I. himself is actively involved in collaborations with scientists from developing countries.

Intellectual merit. Homologous meiotic recombination is an important process because it creates new genotypes by shuffling chromosomal segments that otherwise would be inherited as blocks and promotes fertility by ensuring that chromosomes segregate properly. Maize is the species with the most diverse genome structure known. In many chromosomal segments, different inbred lines share just the gene sequences, but none of the surrounding repetitive DNA. The latter consists of transposons, retrotransposons making up more than half of the genome. This high level of structural variation affects recombination and, possibly, gene expression. This project aims to analyze those effects.

The genetic system used is the bz locus, which has unique advantages for studies of recombination. Previous work showed that genome structural variation affects the frequency and distribution of recombination events in maize in multiple ways. Although recombination is limited to genes, its distribution is highly nonuniform: some genes are hotspots and others are coldspots. Genes in Helitron transposons fail to recombine. Whether a sequence recombines or not depends on its C-methylation status and transposons are highly methylated. The presence of a retrotransposon block in only one homolog, a common situation in hybrids, inhibits recombination in adjacent genes. Recombination not associated with crossing over, called gene conversion, shows a strong polarity: bz mutations at both ends of the gene convert (i.e., recombine) more frequently than central ones. This project will continue to exploit the power of genetic analysis in the bz region. It will elucidate the pattern of recombination within a plant gene and will use recombination as a tool in the genetic analysis of gene expression differences. Its specific objectives are to: 1. Test whether genomic structural variation (+/- intergenic retrotransposons) impacts the frequency and polarity of conversion in an adjacent gene. 2. Determine if the stretch of DNA transferred from one homolog to another (conversion tract) at the bz 5 high conversion end extends into the adjacent gene. 3. Investigate whether conversion tracts are generally shorter when the recombining DNAs differ at only two sites than when they differ at many, as in the heterozygotes commonly studied. 4. Measure recombination and crossing-over interference in heterozygotes between identical chromosomes, i.e., an inbred, and contrast with values obtained from standard maize F1 heterozygotes, i.e., hybrids. 5. Define all genes in the sh-bz interval of two well studied lines and use recombinants to dissect the contribution of genomic structural variation to allelic expression differences.

Broader impacts. This project will train a graduate student in plant genetics and provide summer employment and training for students, including those from a predominantly undergraduate institution with which the Principal Investigator collaborates. It will incorporate several members of underrepresented groups and will help the Principal Investigator to maintain ongoing collaborations with investigators in developing countries. The project is relevant to the long-term improvement of U.S. agriculture in that it studies recombination, a cornerstone of genetics and the basis of most plant and animal breeding, and it utilizes maize, an excellent model organism and a crop of economic importance. Much of the proposed work is based on an earlier discovery in the Principal Investigator's lab of an unprecedented level of maize genome structure variation. The project aims to study how recombination and gene expression are affected by it.

PI: Hugo K. Dooner (Rutgers University)
CoPIs: Chunguang Du (Montclair State University) and Randall A. Kerstetter (Rutgers University)

Transposable element (TE) insertions cause mutations that help researchers to elucidate gene function. Collections of insertions that have been sequence-indexed (i.e., in which the host DNA adjacent to the insertion is known) are valuable resources in organisms with a sequenced genome and have been identified by the maize community as essential to fully exploit the maize genome sequence just generated. Recently, the feasibility of combining high-throughput DNA sequencing with efficient multi-dimensional pooling strategies to rapidly and simultaneously sequence and index hundreds of new insertions has been demonstrated. This project will develop and use a general method for generating, sequencing, and indexing Ac/Ds transposable element insertions in maize that is rapid, accurate, and cost-effective by taking advantage of next-generation sequencing technology. Specifically, the project will: (1) sequence-index a collection of existing lines that contain a unique transposed Ac element; (2) complete a set of 120 roughly equidistant transgenic Ds elements that serve as launching platforms and carry easily scored markers that will allow simple visual selection of element transposition from any region of the genome and, thus, enable researchers to generate regional gene knock-out collections, (3) sequence-index 10,000 Ds element insertion sites from model platforms using a novel method that should be applicable to any collection of insertions produced in a common genetic background; and (4) develop a web-searchable database of insertion site sequences cross-referenced to lines that will be freely available from the Maize Genetics Stock Center (http://maizecoop.cropsci.uiuc.edu). All relevant information from this project will be accessible from MaizeGDB (http://www.maizegdb.org).

This project addresses a critical need in that it will deliver a sequence-indexed reverse genetics resource, considered essential for researchers to fully exploit the maize genome sequence. It will integrate next-generation sequencing technology at Rutgers with bioinformatic sequence analysis at Montclair State University, a predominantly undergraduate institution in NJ. Students at MSU will assist with the assembly, annotation, and mapping of short reads as putative Ac-adjacent sequences and with the design of primers for validation. The project will also provide research training opportunities for MSU informatics and molecular biology students, many of whom are from underrepresented groups. Students at both Rutgers and MSU will also participate in the project as summer interns in the molecular biology lab and maize genetics nursery.

PI: Hugo K. Dooner (Rutgers University)

CoPI: Charles Du (Montclair State University)

The availability of a mutant line in which a single gene has been disrupted gives biologists a powerful tool in understanding the action of that gene. Thus, sequence-indexed collections of single insertions are critical resources for elucidating gene function in organisms with sequenced genomes and are deemed essential by the community to fully exploit the maize genome sequence. It has recently become feasible to combine high-throughput sequencing with multi-dimensional pooling strategies to sequence and index hundreds of new insertions at a time. This work will complete the production of a reverse genetics resource based on the transposon Ds that will enable the community to generate and build up a single-gene knockout resource for maize. Specifically, the work will: (1) characterize the transposition frequency of 78 single Ds-GFP (or Ds*) launching platforms generated by Agrobacterium transformation during the previous grant period and map them to the genome. Highly active platforms will be identified and deposited in the Stock Center to complement the 82 already mapped. These platforms will allow visual selection of transpositions from many regions of the genome and, thus, enable researchers to create regional gene knock-out collections; (2) generate collections of 480 Ds* transpositions each from 24 Ds* launching platforms located on all 10 chromosomes which, together with the 9 collections currently at hand, will serve as the foundation for a Ds*-based maize single-gene knockout resource; and (3) sequence-index these collections by high throughput MiSeq Illumina sequencing of 3-D DNA pools. To this end, a specific software package (InsertionMapper) was developed to extract Ds* transposon junctions from the large amount of sequencing data and map them to the maize genome.

This project will integrate high throughput sequencing at Rutgers with bioinformatic sequence analysis at Montclair State University, a predominantly undergraduate institution in NJ. MSU students will annotate all Ds*-adjacent sequences generated by the NextGen sequencer. The project will provide informatics and molecular biology students at that institution with the opportunity to participate directly in maize research and fulfill their independent research requirement for graduation. Students at both Rutgers and MSU will work in the project as summer interns in the molecular biology lab and maize genetics nursery of the PI. The PI and coPI are members of underrepresented groups and have a record of fruitful prior collaborations. They have collaborated in previous NSF PGRP-funded projects which led to the development of a bioinformatics tool and the publication of three joint papers. About a third of the Genomics students participating in the project at MSU are members of underrepresented minorities. MSU has added the research opportunity from this project to its outreach campaign to attract and retain minority high school graduates. This project is relevant to U.S. agriculture in that it deals with maize, the most important American crop today. It addresses a critical need in that it will deliver a sequence-indexed reverse genetics resource, considered essential for researchers to fully exploit the maize genome sequence. All data will be made available through a web-searchable database of insertion site sequences (http://www.acdsinsertions.org/) that are cross-referenced to ~10000 seed stocks available from the Maize Genetics Cooperation Stock Center (http://maizecoop.cropsci.uiuc.edu). All relevant information from this project will be accessible through http://www.acdsinsertions.org/ and long-term through MaizeGDB (http://www.maizegdb.org).