Hamid Ashrafi

Overview: A broad understanding is lacking as to the importance of polyploidy and hybridization in woody angiosperm clades that share two particularly unusual properties: a propensity for producing unreduced gametes, and weak isolating barriers among species. These factors have been invoked alone or together to explain the origins of species in Vaccinium section Cyanococcus, the true blueberries. The Cyanococcus clade provides an ideal system for investigating the processes driving population- and species-level evolution, with outcomes that include a clear window into the generation of diversity via polyploidy and a stabilized taxonomy for the constituent species responsible for a billion-dollar agroindustry. The proposed research will marry genomic data with a detailed morphological taxonomic investigation to advance knowledge on phylogenetic theory, species diversity, polyploidy, and cryptic speciation in this ecologically and economically important group, and will result in an updated classification that will serve as the foundation for better understanding the evolutionary history of blueberries and increase clarity in breeding programs. Preliminary results from genomic data and careful examination of herbarium material simplify the approach by clearly defining hypotheses to be tested and serve to delimit subprojects well suited to student investigators. The proposed research will integrate data from museum specimens, field observations, a common garden, flow cytometry, and phylogenomics. Intellectual Merit: With diploids, tetraploids, and hexaploids resulting from both autopolyploidy and allopolyploidy, Cyanococcus is an ideal model for understanding polyploidy, hybridization, and cryptic speciation in flowering plants. In fact, these taxa were central to the development of the modern synthesis and have played an important role in furthering our understanding of the evolutionary consequences of polyploidization. Camp������������������s work on this polyploid complex stands as a monument within the body of knowledge developed in the 1930s and 1940s through the incorporation of cytogenetics, phytogeography, ecology, and morphology. In parallel, geneticists and plant breeders leveraged this natural system to develop one of the few woody fruit crops derived from North America. Although crop domestication has moved forward, the vast number of hypotheses generated to explain observations in the field, lab, and herbarium remain largely untested with genomic data. The proposed systematic study will test many of these hypotheses and provide a revised and updated classification of a widespread and economically important yet poorly understood plant group that will act as the foundation for understanding evolutionary history and provide increased clarity in breeding programs. The ubiquity and importance of Cyanococcus to heathlands and a range of acidophilic plant communities and as a food source for wildlife and humans underscores the broad interest of the group. The proposed research will provide important insights into the behavior of polyploids in phylogenetic analyses and will serve as a model for future studies attempting to disentangle polyploid species complexes. Broader Impacts: The proposed research will allow for numerous training opportunities and enhancement of resources including the addition of specimens to herbaria, annotations of existing collections, a germplasm collection, and genomic resources. The project will culminate in a synthesis of morphology, ploidy, and phylogenomic results that will inform professional and public sectors on how this biodiversity is best recognized, communicated, and understood. The multifaceted nature of the proposed research will provide an excellent training opportunity for undergraduates, graduate students, and a postdoc. Manos will mentor a postdoc in professional development and evolutionary plant biology. Together, they will mentor undergraduates in herbarium curation, plant taxonomy, wet lab work, and computational analysis. Fritsch will mentor a graduate student in these methods u

The North Carolina State University Blueberry Breeding Program has long supported and collaborated with NC blueberry growers to breed for blueberry cultivars that stand up to our challenging climate and meet the needs of high yield, quality, and machine harvest-ability. New machine-harvest trials coupled with on-farm grower trials will inform the release of new NC State blueberry cultivars, while new technologies such as machine learning and in vitro micropropagation will allow us to grow and test larger numbers of selections in our berry evaluations. In addition, our cooperative breeding and partnership with NC blueberry growers help develop new varieties faster.

In blueberry growing regions of North Carolina, almost 60% of blueberries are handpicked and sold fresh in local and national markets. A lower percentage (20%) of blueberries are machine harvested and sold in the fresh fruit market. The remaining 20% of the crop is being harvested by machines and sold in the processing market. Handpicking of small fruit crops, especially blueberries, requires a large labor force, which is increasingly hard to recruit and to manage. With increasing cultivation areas and competition between GA and NC to hire farm workers, and with overlapping seasons, farm operations have increasingly become challenging in NC in some years. The use of mechanical harvest is one of the alternative options for many fresh market blueberry growers where labor is scarce. The mechanical harvest approach requires an investment in research and development of methods to make blueberry plants more amenable to the mechanical approach. In addition to breeding for new cultivars that are machine harvestable, we are also focusing on breeding for early- and late-ripening southern highbush blueberries to enable blueberry producers to extend the harvest season beyond June in North Carolina. Cultivars that can escape the late spring frost by late flowering and eventually heat tolerant to start the ripening process beyond June in NC with hot and humid summers. Our objectives in this proposal are to phenotype the mapping populations that were developed during the first phase of this project (2016-2018), and to map QTLs for fruit quality-related traits including firmness, bruising index, shelf life, acidity and soluble solids, size, weight, and anthocyanin content.

While NC growers are producing more blueberries than ever, competition from Florida, Georgia, and South/Central America is proving to be a fiscal challenge as they command some of the highest prices early in the year. The workhorses of NC early market are Star (1996) and Rebel (2008), which were released from Florida and Georgia, respectively, often underperform partially because of their unsuitability to NC climate. Newer varieties will pose the same risk as most have been released from the University of Florida, the University of Georgia and private companies such as Fall Creek Nursery (Lowell, OR), Oregon Blueberry Farms (Salem, OR), and Berry Blue LLC (Grand Junction, MI) and have been bred and tested in those climates. Growers spend time and resources trialing cultivars from these institutions to have them fail at crucial times because they lack North Carolina adaptations. An early, reliable, and productive replacement would be of immense value, especially if it is adapted for machine-harvesting. As our program reemerges into full production, we are confident that our team at NCSU can meet this goal.

The cultivar development time can be significantly shortened by tissue culture (TC) and multi-location -year tests if they can be done in the early stages of breeding. This requires a lot of land space and labor at NC State blueberry research farm at Castle Hayne, which impedes the implementation of such an experiment. Through collaborative work with stakeholders in the state, this problem should no longer be insurmountable

The overall goal of this project is to develop bioengineering pipelines for specific landscape crops with high economic value in order to provide the most immediate impacts for crop improvement. These systems will have broad utility and provide a foundation for developing improved crops with enhanced disease and insect resistance, non-invasiveness, unique phenotypes and greater commercial potential. Transgene-free end products will be realized by segregating out transgenes with selective breeding or with the use of DNA-free delivery systems.

Borlaug Fellowship Program for Turkey fellow to spend 12 weeks at NC State conducting research on apomixis at the gene level using a natural apomiet Boechera species and engineering apomixis in sexual crop plants using the model system of Arabidopsis thaliana.

Our long-term goal is to develop new ���������������crispy������������������ blackberry cultivars with enhanced firmness and superior postharvest performance that will be suitable for machine harvest. The primary objective of this proposal is to implement marker-assisted selection for texture in the UA fruit breeding program to reduce breeding cycle time and expedite the process of combining crispy texture with other desirable traits including primocane fruiting, thornlessness, sub-acid flavor, reduced internode length, large berry size, and high yield. Based on preliminary results in blackberry and studies in other Rosaceae fruit crops, we hypothesize that variation in fruit firmness in fresh-market blackberries is determined by mutations in a few gene(s) related to polysaccharide-modifying enzymes. We further hypothesize that crispy texture may be recessively inherited, with four copies of a non-functional allele at a gene coding for a polysaccharide-modifying enzyme, such as polygalactonurase, necessary to recover ���������������crispy������������������ texture. Allele dosage at this gene may contribute other quantitative variation in flesh firmness seen in the UA blackberry breeding program. We will leverage the new tools we have developed including two diploid reference genomes, a tetraploid blackberry resequencing panel, and a high throughput phenotyping protocol for red drupelet reversion to test these hypotheses and understand the underlying genetics controlling texture variation in cultivated blackberries.

Borlaug Fellowship Program for Tunisia fellow to spend 12 weeks at NC State conducting research on the conservation of Tunisian olive genetic resources and enhancing the quality of olive oil production by the use of molecular markers.

In tissue culture or response to wounds, plant cells produce an undifferentiated cell mass called callus. Different plant hormones can be used to differentiate the cells into various organs, such as shoots and roots. This technology is highly genotype-dependent and genetically controlled. Some genotypes are more amenable to regeneration than others. During the regeneration process, the new gene(s) can be introduced to the callus cells. These genes can be introduced from the same organism or have a foreign source. A genetically modified plant can be used to study the function of the genes by overexpression or gene knockout. However, more recently, a new technology named genome editing has been developed to repair the genes and make them functional. This technology has many advantages over introducing a new gene to the plants, which can have some pushback from society. Because first of all, we repair the gene(s) of the same organism, a process that naturally occurs in the cells by cell DNA repair mechanism, and second, there is no foreign DNA or The first step in genome editing of crop plants is to establish and identify the best method of transformation and regeneration. In woody plants with a prolonged traditional breeding process, genome editing is highly promising to create new genotypes with everything but lack one or two traits. For instance, a blueberry cultivar named New Hanover is highly productive with a unique aroma and flavor. Still, under certain environmental conditions, the fruit have a large picking scar and tear upon separation from the peduncle, making the crop unsuitable for packing and shipping. Furthermore, this technology in blueberry is poorly understood and studied. A few publications have reported the transformation and regeneration of one blueberry cultivar, Legacy; however, the method has not been replicable in our laboratories as the procedure has not been explained very well in the literature. There is a need to develop this technology in North Carolina for various reasons. Primarily, it enables us to perform functional genomics studies. In addition, it will help us to develop commercial cultivars faster and shorten the breeding process. Furthermore, we can test our methods in different genetic backgrounds and identify the impediments of transformation and regeneration in blueberry. Consequently, we can train next-generation breeders and make them ready for the current and future job market.