Date of Award

Winter 2023

Project Type

Dissertation

Program or Major

Genetics

Degree Name

Doctor of Philosophy

First Advisor

Thomas M Davis

Second Advisor

Anissa Poleatewich

Third Advisor

Marta Lima

Abstract

Quinoa (Chenopodium quinoa), an orphan crop native to South America, is tolerant of drought and saline soils and produces a highly nutritious grain. Given these qualities, the demand for this pseudocereal has been steadily increasing, and quinoa breeding programs can now be found throughout the world. I believe the introduction of quinoa-like cultivars developed specifically for Northern New England (NNE) with the region’s climate and disease pressures taken into consideration will benefit local farmers and consumers. Quinoa breeding efforts must consider its allopolyploid (2n = 4x = 36: AABB) genome composition as well as its reticulate ancestry, having descended via interspecific hybridization between diploid ancestors of genome composition AA and BB, respectively. The objective of this dissertation research was to utilize three species within the Chenopodium genus endemic to the NNE region, namely C. berlandieri (2n = 4x = 36: AABB), C. foggii (2n = 2x = 18: AA) and C. ficifolium (2n = 2x = 18: BB) as sources of germplasm and genetic data to improve quinoa yield, architecture, and tolerance to biotic and abiotic stresses. Through the analysis of quinoa × C. berlandieri hybrids and their progeny, and the development of C. ficifolium as a diploid model species, including genome assembly, this work investigated allelic variation in domestication-related traits and expanded genetic and germplasm resources to be used in molecular breeding programs.This research addresses three goals relating to quinoa breeding in NNE and establishing the foundational research required for de novo domestication of C. berlandieri; de novo domestication being a process by which undomesticated species are transformed to more closely resemble domesticated species via use of a gene editing system, while xiv retaining high-value traits such as stress tolerance from the undomesticated species (Zsögön et al., 2017). This process has been demonstrated to reduce the time required to produce an elite cultivar which may contain beneficial traits from two or more species or cultivars while eliminating the risk of beneficial traits segregating away in filial populations. Those goals are to: 1) determine which accessions from various species within the Chenopodium genus are best suited for breeding and ultimate cultivation in NNE; 2) establish efficient methods for making interspecific crosses and confirming hybridity; and 3) to develop a diploid model system representing quinoa which can be used to quickly establish marker-trait and gene-trait associations which themselves may be used to expedite quinoa breeding and define target genes for de novo domestication. The second chapter of this dissertation describes the foundational evaluation of local C. berlandieri germplasm and germplasm representing several other species including C. berlandieri, C. boscianum, and quinoa. These accessions were sourced from the USDA National Plant Germplasm Repository and from Brigham Young University collaborators Rick Jellen and Jeff Maughan and were used to determine which species and accessions perform well in the climate and soils of NNE. The data from field trials served to inform breeding directions by evaluating performance metrics of individual accessions such that those that are most able to thrive in NNE were selected for breeding. Data from two years of field trials indicate that the quinoa accession QQ065 and the C. berlandieri var. macrocalycium accession RB6 performed the best within their respective species for key traits which when found in combination would yield hybrid-derivative plants more fit than either parent for large scale agronomic deployment. xv Chapter III focuses on developing crossing methods to enable the production and validation of interspecific hybrids which may be themselves selfed or crossed to develop advanced lines suitable for usage by local farmers and growers, or they may be used in the creation of segregating progeny populations to establish marker-trait and gene-trait associations. This chapter explores methods used to try to produce interspecific hybrids, including hand emasculation and pollination under a dissecting microscope, and the use of the male-sterile quinoa accession, PI 510536. After putative hybrids were produced, several methods including PCR and sequence based approaches were evaluated to determine efficacy in establishing hybridity and parentage. It was found that typical crossing methods using two male-fertile parents resulted in zero confirmed hybrids, hence the shift to using male-sterile quinoa plants as the female parents, thus preventing selfing. The best validation method used involved whole genome resequencing which yielded enough high quality data to make parentage calls with relatively high certainty. The final chapter explores the development and use of C. ficifolium as a B genome diploid model system that can be used to inform breeding decisions in the tetraploid level species quinoa, C. berlandieri var. macrocalycium, and hybrids resulting from crosses involving those two species. The C. ficifolium genome was sequenced using the Pacific Biosciences HiFi platform and assembled using Hifiasm. This assembly was then corrected, annotated, and used to locate the causative region responsible for variations in height, flowering time, and number of branches within an F2 segregating population established by Madhav Subedi (Subedi, Neff and Davis, 2021). Within the single associated region lies the Flowering Locus T-Like (FTL) gene, which was previously implicated in the control of these traits by Madhav Subedi (Subedi, Neff and Davis, 2021), xvi thus reaffirming the prior results and demonstrating the usefulness of this species and reference genome for model system development. Much of the research presented within this dissertation serves as foundational knowledge aimed at the production of a quinoa-like crop option for NNE through traditional breeding of quinoa with C. berlandieri, or via de novo domestication. Further, this research serves to determine the possibility and practicality of de novo domesticating C. berlandieri through screening germplasm for beneficial traits, and research into crossing and hybrid identification methods which have yielded segregating populations that can be used in the isolation of genes and alleles of interest. Finally, the development of a high-quality annotated reference genome for the diploid model system C. ficifolium has been demonstrated to be useful in genome wide association studies furthering our ability to establish gene-trait associations. Currently, however, true de novo domestication is not feasible in Chenopodium. While a myriad of data exist both in germplasm and sequence, regeneration of C. berlandieri callus via organogenesis (study not presented within this dissertation) was wholly unsuccessful. Without a robust regeneration system to yield whole plants from modified callus tissue, de novo domestication within Chenopodium is virtually impossible at this time. Because of this, focus within this breeding program has turned towards more traditional methods utilizing male sterile plants within the accession PI 510536 to guarantee each seed produced from the male-sterile female parent is the product of an outcross. This process has been demonstrated successfully in chapter III. It appears the current path of least resistance towards developing a quinoa-like crop option is through the employment of male-sterile plants from within this accession in quinoa x quinoa crosses, and quinoa x C. berlandieri crosses, and the production of subsequent crosses by xvii locating male-sterile hybrids to be used as female parents in further crosses. While this process is less direct than de novo domestication and beneficial traits will likely segregate in hybrid populations, the use of, and further research within the CMS system, has the potential to yield elite hybrid-derivative quinoa-like cultivars possessing the biotic and abiotic stress tolerance of C. berlandieri and valuable domestication-related traits found within select quinoa accessions.

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