Doubled haploidy (DH) is a plant breeding technique that is often utilized by plant breeders to minimize the time required to reach homozygosity in breeding lines. The first objective of this study was to compare two methods of generating DH lines in tobacco (Nicotiana tabacum L.). Inbred burley tobacco varieties TN 90LC and GR 149LC were used to produce both androgenic derived doubled haploids (ADDH) and maternally derived doubled haploids (MDDH). The relative agronomic performance of TN 90LC and GR 149 LC ADDH and MDDH lines was compared when used either as pure-line cultivars or when used for the production of the KT 204LC and TN 97LC hybrid cultivars, respectively. The ADDH method was more efficient than the MDDH method in generating large numbers of haploid plants. On average the ADDH TN 90LC population was statistically inferior to the inbred TN 90LC for several agronomic traits; this inferiority of the ADDH method was not observed in the GR 149LC populations. For both genotypes, the MDDH populations were comparable to the inbred parental genotypes. The ADDH method was inferior for TN 90LC, but several individual TN 90LC ADDH lines were equal or superior to the inbred source. The agronomic variability observed in both ADDH and MDDH lines was decreased when they were used to produce hybrid cultivars. Less variation was observed in the DH-derived hybrids KT 204LC and TN 97LC compared to the ADDH and MDDH TN90LC and GR149LC parental lines, respectively. The significant inferiority of ADDH TN 90 lines in comparison to inbred TN 90LC was not observed in the ADDH derived KT 204 population compared to KT 204LC. The second objective of this study was to compare DH Lines derived from an F1 breeding population versus DH lines derived from a segregating F2 population where plants used for DH were pre-screened for quantitatively inherited resistance to soil-borne diseases black shank (Phytophthora nicotianae) and/or Fusarium wilt (Fusarium oxysporum f. nicotianae). There was a clear difference in susceptibility to black shank between the F1 and F2 derived DH populations, both in terms of average disease incidence, and more importantly, in the percentage of individual lines displaying high disease resistance. For two different burley crosses, DH lines derived from the F1 generation were considerably more susceptible to black shank than DH lines derived from the F2 generation. No differences in the incidence of Fusarium wilt were observed between DH lines of F1 and F2 generations; this was likely due to low overall disease incidence. Although delaying the DH process in tobacco from the F1 to the F2 generation could add time to the development of homozygous breeding lines, the delay may be offset by having to screen fewer finished DH lines to identify superior lines.

Plant cell walls are complex structures that must not only constrain cellular turgor pressure but also allow for structural modification during the dynamic processes of cell division and anisotropic expansion. Cell walls are composed of highly glycosylated proteins and polysaccharides, including pectin, hemicellulose and cellulose. The primary cell wall polysaccharide is cellulose, a polymer composed of high molecular weight !- 1,4-glucan chains. Although cellulose is the most abundant biopolymer on Earth, there is still a lot to learn about its biosynthesis and regulation. This research began by applying a variety of analytical techniques in an attempt to understand differences in cell wall composition and cellulose structure within the plant body, between different plant species and as a result of acclimation by the plant to different environmental conditions. Next, a number of different Arabidopsis thaliana lines possessing mutations affecting cell wall biosynthesis were analyzed for changes in cellulose structure (crystallinity) and biomass saccharification efficiency. One of these mutants, isoxaben resistance1-2 (ixr1- 2), which contains a point mutation in the C-terminal transmembrane region (TMR) of cellulose synthase 3 (CESA3), exhibited a 34% lower biomass crystallinity index and a 151% improvement in saccharification efficiency relative to that of wild-type. The culmination of this research began with a chemical screen that identified the molecule quinoxyphen as a primary cell wall cellulose biosynthesis inhibitor. By forward genetics, a semi-dominant mutant showing strong resistance to quinoxyphen named aegeus was identified in A. thaliana and the resistance locus mapped to a point mutation in the TMR of CESA1. cesa1aegeus occurs in a similar location to that of cesa3ixr1-2, illustrating both subunit specificity and commonality of resistance locus. These drug resistant CESA TMR mutants are dwarfed and have aberrant cellulose deposition. High-resolution synchrotron X-ray diffraction and 13C solid-state nuclear magnetic resonance spectroscopy analysis of cellulose produced from cesa1aegeus, cesa3ixr1-2 and the double mutant shows a reduction in cellulose microfibril width and an increase in mobility of the interior glucan chains of the cellulose microfibril relative to wild-type. These data demonstrate the importance of the TMR region of CESA1 and CESA3 for the arrangement of glucan chains into a crystalline cellulose microfibril in primary cell walls.

Peppers (Capsicum) are a valuable commodity throughout the world. They provide food, coloring additives, vitamins, and ornamental aesthetics. Peppers can be grown in many different areas of the world and their success is based on variety selection. Breeding cultivars for superior performance is critical to success. Recent consumer desire for less chemical use throughout the growing process has resulted in new cultivar developments.. The objective of this research was to investigate how cultivar and type affect the yield and weight of field grown peppers. This study provides valuable information for growers in determining which cultivars are suitable for production in Kentucky. The research was conducted at the Western Kentucky University Agriculture Research and Education Complex in Bowling Green, Kentucky in 2016. Plants were provided by Ball Horticultural and Pan American Seed, Elburn, Illinois. The experimental design was a random design, with a 12 plant experimental unit consisting of four three plant rows. Thirty-six cultivars were divided into groups based on the type of pepper produced. Harvest occurred approximately every 7 days and were grouped into 3 periods. Data were obtained on yield, weight, and mean weight. Individual group selection is determined by the producers and their goals for production. There were wide differences in the types of peppers, including size, shape, and color. When comparing all groups, two of the thirty six cultivars, Cultivar 23 (Tapered) and Cultivar 34 (Chili) performed best for both yield and mean weight.

New soybean lines have been developed with significantly higher oil, protein + oil and higher meal protein. These soybeans contain a VgD1 gene (highly active acyl-CoA:diacylglycerol acyltransferase, DGAT from Vernonia galamensis (VgDGAT1A) produces much higher oil synthesis and accumulation activity in soybean. Soybean with active DGAT from Vernonia galamensis (VgDGAT1A) has active TAG biosynthesis relative to other DGATs including from soybeans and Arabidopsis. DGATs catalyze the final step of TAG synthesis: DAG (diacylglycerol) + acyl-CoA → TAG + CoASH (Coenzyme A is notable for its role in the synthesis and oxidation of fatty acids, and the oxidation of pyruvate in the citric acid cycle). A thorough analysis of the major components in VgD1 lines, especially those of nutritional or anti-nutritional value including what else changed (decreased); and what remained at normal levels was conducted. A field study was conducted in Spindletop and Princeton KY, reviled no reduction in yield nor protein, and about 4 % (DW) more oil was obtained in Princeton and 2% (DW) in Spindeltop. No consistent reduction in the other seed composition.VgDGAT1A soybean lines indicated noticeably early maturation compared to the parental line. This is associated with higher expression of the flowering genes FT2 (FLOWERING LOCUS T2) and FT5 (FLOWERING LOCUS T5), for the high oil lines. A single recessive mutation in soybean (MIPS) myo-inositol 1-phosphate synthase, confers a seed phenotype of increase inorganic phosphate (Pi) crossed with high oil lines expressing a DGAT from Vernonia galamensis (VgDGAT1A) (VgD). The oil and protein were maintained compart to VgD. VgD X MIPS (VM), had 21.2, and 22 % oil in 2015, and 23.3 and 24.0 oil in 2016, and protein 46, 49 in 2015, and 37 and 39 % in 2016. Phosphate results suggesting the cross MV is still segregating for MIPS and more selection and planting are needed. Measurement of seed phosphate levels is an established technique for screening for low phytate mutants but to date, it has not been performed non-destructively from single soybean seeds. A protocol was developed greatly reducing the sample size thereby reducing the cost and time and saving a generation in the selection of low phytate mutant seeds based on the high Pi phenotype. Genotyping single seeds are useful in breeding and genetics while maintaining high germination rates. Nondestructive single-seed genomic DNA extraction protocols using 12 mg cotyledon tissue with a modified cetyl trimethyl ammonium bromide (CTAB) technique and a commercial seed DNA extraction kit using 1 mg cotyledon tissue were developed for dry soybean seeds and cross-verified with leaf DNA analysis.

Cultivated tomato plants are extremely susceptible to the two-spotted spider mite Tetranychus urticae Koch. Selection for pest resistance is usually a crucial step required to achieve successful genetic resistance transfer from wild into cultivated tomato genotypes. S. habrochaites LA2329, a wild relative of tomato, is highly resistant to arthropods. Its resistance has been attributed to the presence of a high density of type IV and type VI trichomes and abundant production of 7-epi-zingiberene, a sesquiterpene hydrocarbon. The interspecific backcross hybrids used in this research were derived from the cross between the wild relative tomato, S. habrochaites LA2329, and the cultivated tomato, S. lycopersicum ‘Zaofen 2’ (ZH2). This population has been directly selected for type IV trichome density and zingiberene. The arthropod resistance status of the backcross hybrids was unknown when this research was initiated. Thus, the main objective of the research was to verify the transfer of arthropod resistance from S. habrochaites to cultivated tomato. The effects of glandular trichome densities and leaf zingiberene contents on spider mite behavior and biology were also explored. Also, the chemical composition of the trichome secretions in the wild tomato donor is segregating for presence and abundance of sesquiterpenoids related to zingiberene. The bioactivity of these sesquiterpenoids was explored in this research. To evaluate the relative bioactivities of zingiberene alcohol and 7-epizingiberene, extracted from glandular trichomes of Solanum habrochaites accession LA2329, as well as alpha-zingiberene obtained from ginger oil, these were purified by silica gel chromatography and bioassayed with two-spotted spider mites, Tetranychus urticae Koch (Acari: Tetranychidae) using a bean leaf disc bioassay. Zingiberene alcohol was most efficacious and alpha-zingiberene, was least efficacious, while the efficacy of 7-epizingiberene was intermediate. Thus, tomato breeders should consider introgression of the genes responsible for the oxidation of 7-epizingiberene into zingiberene alcohol to potentially improve the spider mite resistance of cultivated tomato. Also, it is possible that this compound may be exploited as eco-biopesticide approach for integrated pest management against a broad spectrum of herbivorous pests. To verify transfer of arthropod resistance, a bioassay utilizing whole leaves was employed. Nine hybrids (BC3F3 and BC3F4) were chosen for this bioassay, based on variation of type IV trichome density and zingiberene concentration among the hybrids. The experiment also included three susceptible and three resistant control plants. Mite responses on some of the hybrids were similar to those on the resistant wild donor parent, S. habrochaites, as indicated by number of leaflet surfaces infested by mites, degree of mite webbing and feeding damage. Egg density on four backcross hybrids was similar to that on the S. habrochaites resistant controls. Based these results, we concluded that resistance had been successfully transferred from the wild accessions to the hybrids by deployment of backcrossing and indirect selection. There was a significant negative correlation of almost all mite behavioral and biological responses with Type IV trichome density and zingiberene content. This bioassay illuminated behavioral variations of mites associated with presence or absence of leaf compounds and glandular trichome densities. Also, the results support the idea that introgression of type IV trichomes and zingiberene has led to effective spider mite resistance. In another bioassay-based experiment to verify transfer of resistance, seven interspecific backcross hybrids (BC3F2), the resistant parent LA2329, and two susceptible cultivated tomato lines, the recurrent parent ZH2 and ‘Small Roma’, were used in thumbtack bioassays. Mite movement was measured by imaging bioassayed leaves at 15, 20, 30, 45, and 60 min intervals. In addition to confirming transfer of spider mite resistance, other objectives included determination of the relative contributions of type IV and VI trichome densities and leaf compounds to mite behavior over time intervals. Our findings confirmed the transfer of mite repellency from the wild resistant parent to advanced backcross hybrids. Several backcross hybrids performed similarly to the wild donor parent, displaying shorter distances traveled on the leaves after 15 and 30 min. The type IV and type VI trichome densities as well as zingiberene contents had a significant positive correlation with the number of spider mites remaining on tack. There was a significant negative correlation of type IV density and zingiberene concentration with the total distance travelled by mites for both the abaxial and adaxial surfaces across most time intervals. Stepwise multiple regression analysis showed that the type IV trichome density was the most critical factor, and zingiberene content was a secondary factor across over most time intervals. T. urticae remained longer on the thumbtack heads and traveled shorter distances on the leaf surface of the wild donor parent LA2329 and the interspecific hybrids compared to S. lycopersicum leaves. These results indicated that introgression of genetic resistance, especially repellence, against spider mite from the wild relative into cultivated tomato varieties has been successfully achieved. In conclusion, trichome type IV and/or zingiberene content has been successfully transferred from the wild relative into interspecific tomato hybrids, and the hybrids show significant adverse impact on spider mite behavior and/or biology in whole leaf and thumbtack bioassays. Type IV trichome density is the most crucial factor in mite deterrence while zingiberene seemed to be a second key factor across most of time durations for both surfaces. Collectively, several backcross hybrids had similar leaf characteristics to the S. habrochaites LA2329, also may be a potential source of resistance to other insect pests.

Fusarium head blight (FHB) is a serious disease of wheat (Triticum aestivum) and other small grains; disease severity is affected by temperature and rainfall. This research comprised three studies: an artificially warmed experiment during 2016-2017, a morphology study and an FHB resistance screening study in 2015-2016, using approximately 250 wheat cultivars and breeding lines from programs in the eastern US. The location was the University of Kentucky Spindletop Research Farm in Lexington, KY. Higher levels of Fusarium damaged kernels and the toxin deoxynivalenol (DON) were observed in the warmed treatment compared to the control, and plant development was accelerated. In the FHB resistance screen, significant (p < 0.05) genotype differences for all traits were observed. A GWAS identified 16 SNPs associated with resistance and susceptibility, ranging from -2.14 to 4.01%. Three DON-associated SNPs reduced toxin levels by 3.2, 2.1, and 1.5 ppm. In the morphology study, negative correlations were observed among morphological and disease traits. Small effect SNPs were identified for all morphological traits, which might be useful in genomic selection; traits like spike length, spikelet number and inclination could be used in phenotyping. Response to warming indicates that existing resistance sources may be less effective in a warming climate.

Epigenetics is the study of heritable changes in phenotypes that are not the result of changes in DNA sequence. Examples of epigenetic affecters include methylation changes, chromatin modifications, transcription factors, and RNA-based changes. The molecular mechanisms behind epigenetic changes are not fully understood. Canalization is the buffering of gene expression against environmental changes over time, while habituation is semi-stable expression change over time due to selection. This work characterized the molecular changes associated with the kernel color changes of the R-sc:86-17pale allele at the maize red color1 (r1) locus to determine if the changes are epigenetic in nature. The research; 1) quantified the color differences between the progenitor and habituated sublines; 2) Determined that there are not sequence differences between the progenitor and habituated sublines at the 3` end of the Sc||nc1 gene that could account for changes in seed color; 3) and examined the cytosine methylation patterns at the 3` end of the Sc||nc1 gene of the habituated sublines and the progenitor to determine whether there are methylation differences that correspond with the kernel color changes. Quantification of the kernel colors of the R-sc:86-17pale selection sublines showed that there was a statistically significant difference in kernel color. The identical sequence of the R-sc:86 line and the R-sc:86-17pale Lightest and R-sc:86-17pale Darkest sublines at the 3` end of the Sc||nc1 gene is evidence that the kernel color change is not driven by differences in sequence within the r1 gene. The methylation data suggests that some methylation differences in the R-sc:86-17pale Lightest and R-sc:86-17pale Darkest sublines are present, and suggests that the molecular basis of the kernel color is epigenetic in nature.

Domesticated species are vital to global food security and have also been foundational to the formulation and advancement of evolutionary theory. My dissertation employs emerging molecular genomic tools to provide an evolutionary context for crop improvement. I begin by providing a contemporary perspective on two components of domestication biology that have long been used to improve crop production: wild relatives of crop species and grafted rootstocks. First, I propose a method to systematically introgress crop wild relative diversity into crop breeding programs. Then, I explore rootstocks, the lesser-known half of the perennial crop equation, documenting prevalence and diversity, cataloging rootstock traits under selection, and discussing recent advances in rootstock biology. Both crop wild relatives and rootstocks remain largely underutilized resources and hold great promise for agricultural innovation. While humans have domesticated thousands of plant species, research has largely focused on annual crops, to the exclusion of perennials. To improve our understanding of how tree species respond to domestication, I examine the evolution and domestication of one of the world’s most important perennial tropical fruit crops, the mango, Mangifera indica, and its wild and semi-domesticated relatives. I generated a dataset suitable for studying Mangifera across evolutionary time using double digest restriction site associated DNA sequencing (ddRADseq). I present a multilocus phylogeny that informs the classification of Mangifera and reveals, for the first time, the evolutionary relationships of wild, semi-domesticated, and domesticated species in the genus. Narrowing my focus to the intraspecific level, I examine how the introduction of M. indica into regions of the world impacted its genetic diversity. My results show M. indica maintained high levels of genetic diversity during its introduction into the Americas. However, the novel diversity I detect in Southeast Asian mango cultivars suggests that M. indica has a more complex domestication history than previously assumed. I also find evidence that M. indica hybridized with multiple congeners following its introduction into Southeast Asia, forming two hybrid lineages that may be maintained by clonal polyembryonic reproduction. Collectively, my research provides a comprehensive framework for understanding the evolution and domestication of a tropical tree crop of global economic importance.

Isolated populations have potential to become new species that should have less genetic variation than their ancestors. Small populations are more likely to lose genetic variation, which is, thus, expected to be greater in ancestors. Aliciella caespitosa and A. tenuis, two endemic species, may be derived from small populations of A. subnuda, a widespread species. Chloroplast DNA sequences were used to test this hypothesis. Allozyme data were used to compare genetic variation and numbers of alleles. Chloroplast data do not support the proposed relationships between A. subnuda and the other two species. Allozyme data were not more variable in A. subnuda. The data suggest that A. tenuis is derived from A. caespitosa, although the former did not show lower allozyme diversity. I detected fewer alleles in A. tenuis. These data suggest that the original population of A. tenuis was not small enough to lose genetic variation relative to its progenitor.

Switchgrass (Panicum virgatum), a summer perennial grass native to North America, is currently being explored for its potential use in the production of biofuels. With these interests, genetic manipulation of switchgrass to produce plants that are easier to digest, have an increased resistance to diseases and stresses, and maintain viability longer in the field are required. Therefore, it was necessary to develop a reliable and efficient tissue culture system for the transformation of switchgrass. Current switchgrass tissue culture requires months for regeneration of transformants with relatively poor transformation efficiencies and are limited to derivatives of a single variety, Alamo. We have developed a tissue culture system, utilizing a novel media, LP9, which has demonstrated decreased time to the production of whole transgenic plants and with an increased efficiency. LP9 is not an MSO-based tissue culture system. It is comprised of both N6 macroelements and B5 microelements with the auxin, 2,4-D and does not include any cytokinin. After just 1 month on LP9 media, callus can be selected and used for Agrobacterium tumefaciens-mediated transformation or particle bombardment, and plants can be regenerated within 3 weeks of callus initiation. Our system is unique to previously explored MSO-based systems in that it is optimized for the production of type II callus, which has been shown to produce higher transformation efficiencies in other monocots. We have increased the transformation efficiency of switchgrass from to up to 4% to 34% efficiency by selecting for this type of callus.