Phenotypic and genetic variation in an Apios americana breeding collection; and characterization of the HD-Zip gene family, involved in abiotic stress responses in Glycine max
This dissertation has two main objectives: (1) morphological and genetic characterization of a little-studied edible legume native to North America, Apios americana; and (2) characterization of the soybean (Glycine max) homeodomain leucine zipper (HD-Zip) transcription factor family (involved in abiotic stress responses), and identification of candidate genes for dehydration and salt stress. In these projects, next generation sequencing (NGS) is evaluated as a tool for rapidly characterizing genetic variation (in Apios) and fine-scale genetic responses to abiotic stress (in soybean).
Apios, commonly called “potato bean,” is a nitrogen-fixing legume that is adapted to diverse climatic conditions of central and eastern North America. It produces tubers (modified stem-tubers) that are rich in protein, have a long shelf life under refrigeration (>1 year), contain isoflavones, and have low levels of reducing sugars (potentially making the tubers useful for fried chips, for example). The plant was once a staple wild-collected food of Native American Indians, and is now a cultivated crop in Japan and South Korea. William J. Blackmon and Berthal D. Reynolds evaluated Apios as a new edible tuber crop in the US during the 1980s. Their breeding efforts during 1985-1994 lead to a collection of improved genotypes. As of 2010, 53 genotypes remained from Blackmon and Reynolds’ work. As part of this dissertation project, phenotypic evaluation of these 53 genotypes was performed for multiple years, in multiple environments (Iowa, Virginia and Pennsylvania), and in three growing conditions (field, 305-mm [12-in.] pots and 381-mm [15 in.] grow-bags). Twenty traits were recorded, including 10 aboveground and 10 belowground measurements. There was significant variation among the genotypes for all but two emergence traits. Several genotypes produced high yields - up to 1,515 g of tuber yield/plant. Transcriptome sequencing of multiple tissues from a single genotype generated both a high-quality de novo reference transcriptome assembly and an expression catalog. Re-sequencing of the leaf transcriptome from all the genotypes in the collection allowed identification of 58,154 high-quality SNPs and 39,609 gene expression markers (GEMs). Both SNPs and GEMs revealed population structure and pedigree relationships in the collection. Transcripts mapped to Phaseolus vulgaris (another legume in the Phaseoleae clade as Apios, with the same chromosome number and presumably similar genome structure) helped in building pseudo-Apios chromosomes. Linkage disequilibrium decay was investigated in the collection using putative genomic locations of the SNP markers, derived using the pseudo-Apios chromosomes. Association analysis conducted using SNPs and GEMs identified marker-trait associations for at least 11 traits. In summary, this study demonstrates accelerated and holistic (genomic and phenotypic) exploration of an underutilized crop.
The HD-Zip transcription factor family includes genes involved in abiotic stress. HD-Zip genes are well characterized in Arabidopsis thaliana, but not yet in soybean. As part of this dissertation project, HD-Zip genes were identified in the soybean genome using homology searches and Hidden Markov Model guided sequence alignments. Phylogeny reconstruction enabled placement of HD-Zip sequences into four previously described subfamilies. Syntenic paralog pairs were retained following polyploidy in Glycine ~13 Mya. RNA-Seq analysis identified 20 differentially expressed HD-Zip genes in the roots of soybean cv. ‘Williams 82’, at least at one of the three time points (1, 6, or 12 hr) under dehydration and salt stress. This indicates the role of HD-Zip genes in abiotic stress responses. Expression profiles generated for genes expressed in roots at 0, 1, 6 and 12 hr under dehydration and salt stress will serve as an important resource for soybean genomic studies, and will aid in understanding plant responses to dehydration and salt stresses.