Candace Haigler

This project will focus on improving population level breeding tools and understanding genetic information in crop systems, particularly cotton. Bioinformatic analysis will use next-generation sequencing for identification of novel QTL and candidate gene loci and to understand population structures of breeding populations. Validation of novel QTL and candidate gene loci will be initiated through lab activities.

This is a proposal in response to a new FOA including calls for proposed renewal of existing Energy Frontier Research Centers. At NC State, we work in a multi-disciplinary collaboration between the College of Agriculture and Life Sciences and the College of Engineering to understand how cellulose is made by plants. Cellulose within plant cell walls is a major renewable resource with importance to many biomaterials and biomass feedstocks. We will combine advanced microscopy, genetics, and computational modeling to uncover the mechanisms regulating the formation of cellulose microfibrils and their biophysical properties.

The ever-increasing amount of large scale genomic and genetic information from agriculturally important organisms makes it essential to analyze the data insightfully in order to identify genes that confer advantages in the context of agricultural production systems and product quality. This project combines the skills of a research biologist and a bioinformatician in the computational data-mining process and subsequent data analysis and interpretation. The results of the project are expected to benefit cotton, among other crop plant or animal species of importance to the agricultural industry and consumers.

The objective of this project is to utilize whole genome sequencing and other sequencing techniques for cotton in order to perform bioinformatics analysis and identification of Quantitative Trait Loci for traits of interest. The project aims to develop tools for breeders and geneticists that are applicable to agricultural improvement of cotton. Via bioinformatic analysis results, important loci and breeders tools are expected to be additional objectives of this project.

This proposal is a revised renewal proposal for the Center for Lignocellulose Structure and Formation. Bridging three Colleges at NC State (CALS, COE, CNR) and two disciplines, the research in the renewal phase will be directed toward understanding and manipulating the structure of plant cell walls. The work will include plant genetics, biotechnology, and computational modeling of protein structure. The research has relevance to the improvement of cellulosic renewable biomaterials and biomass feedstocks, such as those used in biofuels production.

In this non-assistance cooperative agreement between the U.S. Dept. of Agriculture-Agricultural Research Service and North Carolina State University, we will perform bioinformatics analysis of deep sequencing data to identify Quantitative Trait Loci and relevant genes controlling traits of interest and importance for cotton improvement. We will use available software and improve software for comparative analysis of sequence variation in multiple genotypes. We will synergize large scale sequence data collection (with the assistance of collaborators), bioinformatics approaches, and biological insights to provide additional useful tools to cotton breeders.

In this non-assistance cooperative agreement between the U.S. Dept. of Agriculture-Agricultural Research Service and North Carolina State University, we will perform bioinformatics analysis of deep sequencing data to identify Quantitative Trait Loci and relevant genes controlling traits of interest and importance for cotton improvement. We will use available software and improve software for comparative analysis of sequence variation in multiple genotypes. We will synergize large scale sequence data collection (with the assistance of collaborators), bioinformatics approaches, and biological insights to provide additional useful tools to cotton breeders.

The goals of this project are to identify cotton genes and regulatory sequences that are important to cotton fiber cell development and cellulose biosynthesis and to develop genomic resources for sequencing and resequencing the genomes of cultivated allotetraploid species that account for >95% of world cotton production. Cotton fiber is our most important natural fiber and a major economic driver of the international economy. These experiments will lead to the eventual improvement of fiber length, strength, and dyeability, particularly in Gossypium hirsutum that is grown most extensively in the USA and around the world. Findings about the control of cellulose synthesis in cotton fiber are also relevant to the improvement of diverse fiber crops such as wood and biomass crops used for biofuel production. Molecular tools will be developed to aid the resequencing of tetraploid cotton genomes in a companion proposal that will be submitted to DOE Joint Genome Institute. A cotton genome sequencing initiative will be coordinated through national and international participants with the ultimate goal of generating the complete genome sequence for commercial tetraploid Gossypium hirsutum. An outreach program will be conducted to provide unique training and educational opportunities for undergraduate students and high school teachers in predominately underrepresented groups.

There are several Agrobacterium-mediated transformation protocols that have been developed for cotton. All these protocols require an extensive space in various controlled environments, personnel time, and protocol repetition in order to produce a sufficient number of fertile lines for robust evaluation of improved plant and fiber traits. Such protocols utilize embryogenic callus as explants for transformation and require an additional four months for initiation of embryogenic cultures before each transformation experiment. This approach is not feasible for high throughput cotton transformation studies. Our proposed project seeks to reduce these research and development barriers by providing a more time- and space-efficient protocol for regeneration of transformed cotton. This improved protocol has been already tested in the NCSU Plant Transformation Lab (PTL). In addition to improvements of our proposed system, embryogenic callus could be maintained for up to 12 months to provide a constant supply of plant material for transformation experiments. However, it is important to note that the long term in vitro cultures are known to reduce regeneration capability and increase sterility in regenerated plants. Therefore, we propose to test the regeneration capability of embryogenic callus cultures after maintaining them for 10-12 months followed by determining fertility/sterility ratio of regenerated plants. Since cotton is a very valuable crop many research groups are working on cotton plant and fiber improvements through molecular biology (functional genomics, proteomics), biotechnology, and breeding. One problem faced by cotton breeders and other researchers is the comparatively long time required for plant growth from seed to flower. One strategy that has been proven successful in other plants is to reduce the time required for flowering by over expressing the FLOWERING LOCUS T (FT) gene. Flowering time is affected by both temperature and day length. Although the leaves sense both environmental signals, the responses necessary for flowering occur at the apex of the plant, suggesting that the floral stimulus must travel a long distance within the plant. Florigen, the migrating signal, is encoded by the FT gene, a major floral activator that was first described in Arabidopsis. Under a long-day photoperiod, FT transcripts are produced in the vascular tissue of leaves and the FT protein migrates to the shoot apical meristem where it interacts with a transcription factor FLOWERING LOCUS D (FD) that is expressed only in the shoot apex. The interaction of FT and FD results in the induction of several floral genes. Over-expression of the FT gene in Arabidopsis and ornamental gentian plants has been shown to induce early flowering. It was also reported that over-expression of FT orthologs isolated from poplar, citrus, rice, and tomato could induce early flowering as well. Therefore, if the FT gene from Arabidopsis is over-expressed in cotton it could lead to earlier flowering and a shorter life cycle. Because of the exclusive regeneration/transformation capability of Coker 312 this genotype will be used to produce transgenic plants that can over-express the FT gene. Transgenic FT-expressing plants then can be used as donor plants for FT protein production. Since the cotton grafting technique has been reported already, any cotton genotype could be grafted onto the transgenic FT-expressing Coker 312 plants. The FT protein produced in the transgenic part of the plant can then migrate to the untransformed shoot apex to induce early flowering. The major advantage of this strategy is that the genetic makeup of grafted cotton will remain intact, but the life cycle could be temporarily shortened to facilitate breeding. Presently Coker 312 remains the only cotton genotype with relatively high regeneration/transformation capability that is publicly available. Unfortunately Coker 312 is not a valuable elite germplasm for cotton breeders any more. It is very desirable to identify new public cotton elite lines with high regeneration

This project will characterize an atypical form of cotton fiber already available and develop methods to generate cotton fibers with additional variations in morphology or other characteristics. The research will also use or explore methods that allow characterization of cotton fiber characteristics on ultra-small samples produced by laboratory methods.