VTechWorks Repository :: Browsing by Author "Veilleux, Richard E."

Browsing by Author "Veilleux, Richard E."

Now showing 1 - 20 of 93

AFLP Marker Analysis Of Monoploid Potato
Varrieur, John Michael (Virginia Tech, 2002-05-17)
Potato haploids have been recent components in protoplast fusion research, strategies to combine wild and cultivated potato germplasm and the generation of economically valuable mutant phenotypes. Additionally, most major genetic mapping and QTL analyses in potato have utilized haploid germplasm to simplify linkage-mapping computations. The accuracy of genetic assumptions concerning the randomness and genetic purity of haploid genomes may directly affect the statistical validity of many results in current potato research. In the present study, AFLP analysis was conducted on two sibling S. phureja "BARD 1-3" monoploid populations derived by androgenesis in anther culture, and gynogenesis through the use of a haploid-inducing pollinator, S. phureja "IVP 101." Little indication of somaclonal variation and haploid-inducer gene introgression was found in the monoploid band data suggesting genomic stability. Segregation of marker alleles that were heterozygous in the parent was distorted from the expected 1:1 ratio in both populations, ranging from 35% in the gynogenic monoploids (GM) to 46% in the androgenic monoploids (AM). Genetic diversity appeared more random among the monoploid populations after skewed marker data was removed from phylogenetic analyses. Bilateral and unilateral marker skewness in the monoploid populations may respectively indicate common and unique segregation distorting loci (SDL) present in the AM and GM genomes. Representatives of both SDL types were located on a partial linkage map created using androgenic monoploid data.
Amplified fragment length polymorphism (AFLP) analysis of genetic variability in Phalaenopsis
Chang, Yeun-Kyung (Virginia Tech, 2008-07-23)
Amplified fragment length polymorphism (AFLP) markers allow a rapid assessment of the level of genetic variation that would be difficult to evaluate using a limited number of morphological markers. AFLP was used to assess the level of genetic variation among 16 different Phalaenopsis species and hybrids. Ten AFLP primer combinations were used for genetic analysis of these Phalaenopsis and 95% of polymorphism in 16 Phalaenopsis species and hybrids was detected. The genetic similarity among Phalaenopsis species and hybrids ranged from 0.298 to 0.774 based on Dice coefficient. The dendrogram derived by UPGMA analysis clustered into two main groups. A significant linear relationship (r² = 0.524, P < 0.0001) was observed between known pedigrees and AFLP-derived genetic similarity for 136 pairwise comparisons of Phalaenopsis species and hybrids. The results indicate that there is an abundance of genetic diversity among within Phalaenopsis and that AFLP can be used to distinguish morphologically similar genotypes. In a second study, the effect of gametophytic selection on genetic diversity in Phalaenopsis was examined by AFLP analysis. Sixteen F1 seedlings resulting from cross-pollination that occurred within high (30 ºC) and low (14 ºC) temperature incubators between two hybrid Phalaenopsis [P. (Taisoco Windian Ã Sogo Yukidian) by P. hybrid unknown], were subjected to genetic analysis by AFLP. A total of 651 fragments ranging in size from 100 to 350 bp were detected using six primer combinations, of which 387 (59.4%) were polymorphic. Seedlings derived from different temperature treatments exhibited 25.5% to 35.9% polymorphism. The genetic similarity among 16 F1 seedlings ranged from 0.825 to 0.946 based on the Dice coefficient. A dendrogram based on 387 polymorphic markers was derived by UPGMA analysis resulting in three major groups and one subgroup. The dendrogram analysis showed clear clustering in Phalaenopsis hybrids pollinated under different temperature treatments, suggesting that several loci may have been selected during the divergent temperature stress treatments during pollination and early pollen tube growth.
Analysis of anther-derived plants of Solanum phureja: variation in ploidy, photosynthetic efficiency and structure of the nuclear genome
Pehu, Eija (Virginia Polytechnic Institute and State University, 1986)
The ultimate goal of the· breeding scheme, of which the present study is a part, is to introduce exotic germplasm into existing cultivars of Solanum tuberosum through· 4x X 2x crosses using the South American diploid potato species Solanum phureja as the pollen parent. The first phase of this program includes the 'reconstruction' of a highly heterozygous diploid, pollen parent by .first : reducing the chromosome number of the S. phureja clones to the monoploid level and subsequently fusing genomes of two unrelated monoploid plants either by somatic hybridization or by cross-pollination between fertile doubled monoploids. Within this framework, the objectives of this research were to analyze variation among anther-derived plants of Solanum phureja regarding their: 1) ploidy level and morphology, 2) net photosynthesis and its biochemical components, and 3) nuclear genomic structure, particularly with regard ·to the amplification of rRNA genes as influenced by the anther-culture process. Based on the analysis of several morphological characters of the anther-derived plants by canonical discriminant function, four characters (anther length, number of chloroplasts/pair of guard cells, leaf width, corolla width) were selected for most effective assignment of plants to their ploidy groups by clustering procedures. Clustering of the anther-derived plants proved to be an efficient means of separating monoploids from higher ploidy levels. To assess the impact of the process of anther-culture on the physiology of the resulting plants and to evaluate the possibility of selecting anther-derived genotypes for further breeding efforts, monoploid, diploid and tetraploid anther-derived plants were studied regarding their net photosynthesis and its component characteristics. Leaf area, net photosynthesis and chlorophyll content increased with increasing ploidy' Among .the. monoploids I. Rubisco activity and concentration displayed a. significant genotypic effect; whereas in the diploid group variation among genotypes was significant for total protein content and maximum specific activity of ribulose bisphosphate carboxylase. Among the tetraploid genotypes, significant differenc.es were found with respect to net photosynthesis and specific leaf weight. Two exceptional genotypes were identified: a monoploid with an increase of 28% fcfr maximum activity of ribulose bisphosphate carboxylase and a tetraploid with an increase of 30% for net photosynthesis over the anther-donor plant. To evaluate DNA variation among the anther-derived plants, the nuclear genomes of anther-derived monoploid and diploid plant were studied by DNA reassociation kinetics. It was found that the nuclear genome of the monoploid has undergone differential replication resulting in an increase of sequences consisting of highly· repetitive DNA. Free solution RNA-DNA hybridization showed that the monoploid DNA contained 30% more rDNA sequences than the diploid. Southern blot analysis using rRNA as the probe revealed variation for copy number of certain restriction fragments and for restriction enzyme cleavage sites.
Antioxidant responses of pea (Pisum sativum L.) protoplasts
Doulis, Andreas G. (Virginia Tech, 1994-01-15)
Freshly isolated protoplasts from pea leaves were used to investigate the responses of antioxidant enzymes to oxidative stress. Two cultivars, Progress (tolerant) and Nugget (sensitive), that have differing resistance with respect to oxidative stress at the whole plant level were used. Sulfite and the superoxide generating herbicide, paraquat, were used as the oxidants. Final sulfite concentrations during photosynthetic incubations ranged from 1.5 mM to 30.0 mM. During the polarographic estimation of photosynthesis, CO₂-dependent O₂ evolution did not decrease. At sulfite concentrations of 3.0 mM or less, light-dependent O₂ evolution increased and was probably due to a concomitant SO₂-dependent O₂ evolution. Photosynthesis determined as ¹⁴CO₂ fixation was not increased at these low concentrations of sulfite. Concentrations greater than 7 mM = sulfite inhibited photosynthetic ¹⁴CO₂ fixation. No difference in these responses was found between the two cultivars. At 0.1 µM paraquat, the relative resistance to oxidative stress was reversed compared to previous studies at the whole plant level. With the tolerant cultivar, activity of the plastid antioxidant enzyme, glutathione reductase, increased after a three-hour exposure. Changes in the steady state level of glutathione reductase protein, as judged by immunoblots, did not correlate with the observed changes in enzyme activity. No change in the de novo synthesis of glutathione reductase occurred over the same period as a consequence of paraquat application. A mechanism, unrelated to oxygen free radical scavenging, may contribute to the relative tolerance to low concentrations of paraquat. On the other hand, after an eight-hour exposure to 0.1 mM PQ in the presence of Gamborg’s basal salts, superoxide dismutase activity of Progress protoplasts was enhanced 288% above the preexposure levels while glutathione reductase activity decreased 70% and ascorbate peroxidase activity decreased 90%. The relationship of these changes to oxidative damage to the photosynthetic machinery remains to be assessed.
Apple Pollen Tube Growth Rates Are Regulated by Parentage and Environment
DeLong, Candace N.; Yoder, Keith S.; Combs, Leon; Veilleux, Richard E.; Peck, Gregory M. (American Society For Horticultural Science, 2016)
A greater understanding of apple (Malus domestica) pollen tube growth rates can improve crop load management in commercial orchards. Specifically, applications of caustic bloom-thinning chemicals need to occur when enough, but not too many, flowers have been fertilized to achieve crop load densities that balance yields with marketable fruit sizes. In this study, the pollen tube growth rates of five crabapple (Malus sp.) cultivars were measured in the styles of three maternal cultivars at 12, 18, 24, and 30 °C after 24 hours in a growth chamber. Pollen tube growth rates were greatest for ‘Selkirk’ and ‘Thunderchild’ at 12 °C, and greatest for ‘Indian Summer’, ‘Selkirk’, and ‘Thunderchild’ at 24 °C. Pollen tube growth increased with increasing temperatures until 24 °C. There were minimal pollen tube growth rate increases between 24 and 30 °C. Overall, ‘Snowdrift’ had the slowest pollen tube growth rate of the five evaluated crabapple genotypes. At 24 and 30 °C, ‘Indian Summer’ and ‘Thunderchild’ pollen tubes reached the base of the style most frequently, and ‘Snowdrift’ pollen tubes the least frequently. Pollen tube growth rate was also influenced by the maternal cultivar, with Golden Delicious having relatively faster pollen tube growth than Fuji at 24 and 30 °C. Interactions among paternal and maternal genotypes as well as temperature after pollination reveal complex biological and environmental relationships that can be used to develop more precise crop load management strategies for apple orchards.
Belowground Fungal Community Change Associated with Ecosystem Development
Pineda Tuiran, Rosana P. (Virginia Tech, 2017)
Numerous studies have looked at biotic succession at the aboveground level; however, there are no studies describing fungal community change associated with long-term ecosystem development. To understand ecosystem development, the organisms responsible for shaping and driving these systems and their relationships with the vegetation and soil factors, it is critical to provide insight into aboveground and belowground linkages to ultimately include this new information into ecosystem theory. I hypothesized that fungal communities would change with pedogenesis, that these changes would correlate with vegetation community change, and that they should show change of composition and diversity as the seasons change. Chapter 1 discusses the main topics related to this dissertation. Chapter 2 includes a publication draft that describes a study of sand-dune soil samples from northern Michigan that were analyzed to pinpoint the structural change in the fungal community during the development of the ecosystem. The samples were analyzed by pyrosequencing the soil DNA, targeting the internal transcribed spacer region. Chapter 3 contains a coauthored published paper that describes plant invasion of fields in Virginia to determine how they impact soil bacterial and fungal communities. The bacterial and fungal communities that were invaded by 3 different plant species exhibited similar changes, regardless of plant species, suggesting that some functional traits of invasives may have similar impacts on belowground communities. Chapter 4 remarks the conclusions of this research.
Biosynthesis of Steroidal Glycoakaloids in Solanum chacoense Bitter
Mweetwa, Alice Mutiti (Virginia Tech, 2009-07-24)
Steroidal glycoalkaloids (SGAs) are secondary metabolites produced by approximately 350 species in the Solanaceae family. SGAs are reported to be important for pest resistance and flavor enhancement at low concentrations but are toxic to humans and other mammals at high concentrations. Studies on sterol / SGA biosynthesis have implicated squalene synthase as a key regulatory enzyme because it catalyzes an irreversible step from the mevalonic acid pathway. However, the regulatory mechanisms of squalene synthase are not yet known. A study was conducted to elucidate the distribution pattern of SGAs and to clone the squalene synthase gene in order to determine a relationship between SGAs and gene expression levels. Solanum chacoense, a wild potato species was used as a model plant from which tissues were harvested at specified developmental stages and analyzed for SGA content. The results from the SGA analysis suggest a qualitative and quantitative tissue- and age-dependent accumulation of SGAs. Regenerative tissues such as, axiliary shoots, flowers and floral buds had the highest levels of 88, 49 and 63 Âµmole/g DW, respectively. The roots, stems and tubers showed the lowest amounts of SGAs of 1 to 8, 5 to 15 and 7 to 15 Âµmole/g DW, respectively. Stolons and tubers accumulated higher amounts of α-chaconine (59 to 67%) than α-solanine (61 to 64%) at all developmental stages analyzed. On the other hand, in young expanding, fully expanded, and old senescing leaves where leptine and leptinines tend to dominate, α-solanine and α-chaconine together accounted for only 8 to 15%, 7 to 15%, and 8 to 45%, respectively. Plant organs that showed the highest biosynthetic activity for SGA production also had high levels of transcripts coding for genes of isoprenoid biosynthesis. The results from the cloning and characterization of squalene synthase suggest that the cloned cDNA fragment is a putative S. chacoense squalene synthase gene with an open reading frame / predicted protein precursor of 411 amino acids. The cloned cDNA has high similarity (68-100%) to known plant squalene synthase genes and contains six deduced peptide domains observed in other species. The 3â untranslated regions of floral buds, young leaves (early vegetative stage), and fully expanded leaves (anthesis) were different in length with, 249, 335, and 202 nucleotides, respectively. The Southern blot analysis suggests a single copy gene although the existence of a gene family cannot be ruled out.
Bulk segregant analysis for anther culture response and leptine content in backcross families of diploid potato
Boluarte, Tatiana (Virginia Tech, 1999-09-15)
Diploid potato populations between a primitive cultivated species, Solanum phureja, and a weedy species, S. chacoense, were used to examine the segregation of microsatellite markers and three traits in backcrosses. Two of the traits, anther culture competence and 2n pollen production, originated from S. phureja whereas the third, leptine production (a specific glycoalkaloid known to convey resistance to the Colorado potato beetle) originated from S. chacoense. Using CP2, a self-incompatible F₁ hybrid originating from a cross between S. chacoense clone 80-1 and S. phureja clone 1-3, three populations were developed: 1-3 x CP2 (PBCp), CP2 x 1-3 (PBCc), and CP2 x 80-1 (CBC). For the microsatellite study, four simple sequence repeat (SSR) primer pairs that amplified fragments within potato sequences found in the GenBank were used to look at segregation ratios in our backcross populations and to eliminate possible spurious genotypes bearing non-parental alleles in these populations. Seventeen spurious genotypes were discarded from PBCp; none was found in PBCc or CBC. Two SSR loci showed skewed segregation in PBCp (favoring transmissnion of the allele originally found in 80-1), PBCc showed normal segregation at all loci, and CBC showed distorted segregation at one locus (revealing a deficiency of homozygotes). In the study of anther culture, three components of ACR were investigated in a preliminary study: 1) embryos produced per anther (EPA), 2) embryo regeneration rate and 3) percentage of monoploids (2n=1x=12) among regenerants. CP2 was intermediate, 80-1 was low, and 1-3 was high for ACR. Only EPA was selected for further characterization in our populations. PBCp (78 genotypes) and CBC (57 genotypes), were characterized for anther culture response ACR/EPA in a series of studies. Nine high and ten low selections were identified in CBC, and ten high and ten low selections were identified in PBCp. EPA selections were used for bulk segregant analysis (BSA) using 214 RAPD primers. Two bands, one amplified by OPQ-10 and another by OPZ-4 were linked in coupling and in repulsion, respectively, to ACR in PBCp. One band amplified by OPW-14 primer was linked in coupling to ACR in CBC. One-way ANOVAs for data from remaining genotypes of the populations verified linkage of the markers to ACR/EPA. For 2n pollen production, a total of 77 PBCp genotypes was characterized; 80-1 produces low % 2n pollen, and 1-3 produces high % 2n pollen. Pollen samples were stained with propidium iodide and examined by flow cytometry. The frequency of 2n pollen varied continuously from 1.7 % to 40.6 % among the 41 genotypes that flowered sufficiently to allow three separate pollen collections. Variation due to the environment was observed where the frequency of 2n pollen appeared greater over a range of genotypes on single collection days. BSA could not be used due to limited population size and a low number of selections at the extremes of the distribution of phenotypes. The continuous variation for 2n pollen production suggests multigenic control of the trait. In the study of leptine content in reciprocal backcross populations, 87 genotypes within PBCp, and 42 genotypes within PBCc were characterized using gas chromatography of leaf samples. CP2 was intermediate, 1-3 had zero, and 80-1 was high for leptine content in the foliage. Leptines were present in low levels in 43 of 87 genotypes in PBCp, indicating simple genetic control. In PBCc, only 7 of 42 genotypes expressed leptines, generally at a higher level than in PBCp, indicating cytoplasmic inheritance. Ten high and ten nil selections within PBCp, and seven high and eight nil selections within PBCc were used for BSA using 214 RAPD primers. Three primers OPQ-2, OPT-16 and OPT-20 amplified bands segregating with high bulks in both populations. These markers were linked in coupling to leptine content in PBCp. Linkage was verified by ANOVAs for leptine content in the entire population.
Characterization of activation tagged potato (Solanum tuberosum L.) mutants
Aulakh, Sukhwinder Singh (Virginia Tech, 2012-09-14)
Generation and characterization of activation tagged potato mutants could aid in functional genomic studies. Morphological and molecular studies were conducted to compare potato cv. Bintje, its two mutants, underperformer (up), and nikku generated using the activation tagging vector pSKI074, and nikku revertant plants. Mutant up exhibited a dwarf phenotype (plant height 42 cm vs. 73 cm in cv. Bintje), abundant axillary shoot growth (3.1 shoots/plant compared to 0.7 shoots/plant in cv. Bintje; in vitro plants), greater tuber yield, altered tuber traits and early senescence compared to wild-type Bintje under in vitro conditions. Under in vivo conditions, the dwarf and early senescence phenotypes of the mutant were consistent, but the tuber yield of up was less (250 g/plant compared to 610 g/plant in wild-type Bintje) and had fewer axillary shoots compared to wild-type (1.9 shoots/plant in up vs. 4.7 shoots/plant in Bintje). Mutant nikku plants exhibited an extremely dwarf phenotype (plant height 2 cm in nikku vs. 6 cm in Bintje), had small hyponastic leaves, were rootless, and infrequently produced small tubers when compared to cv. Bintje. The overall nikku phenotype was suggestive of a constitutive stress response, which was further supported by the higher expression levels of several stress-responsive genes in nikku. The nikku revertant plants exhibited near normal stem elongation, larger leaves and consistent rooting, and it was a case of partial reversion. Southern blot analyses indicated the presence of single T-DNA insertions on chromosome 10 in the up and on chromosome 12 in the nikku mutant. The reversion in the nikku plants was not associated with the loss of enhancer copies from the original nikku mutant. Reverse transcriptase PCR analyses indicated transcriptional activation/repression of several genes in the up and nikku mutants, suggesting pleiotropic effects. In revertant, the expression levels of several genes which were differentially regulated in the nikku mutant were similar to Bintje. The gene immediately flanking the right border of the T-DNA insertion, which encoded a novel BTB/POZ (Broad complex, Tramtrac, Bric a brac; also known as Pox virus and Zinc finger) domain-containing protein, was highly up-regulated in the up mutant. This protein domain plays an important role in several important developmental, transcriptional and regulatory pathways. The mRNA-seq analyses resulted in 1,632 genes that were differentially expressed between mutant up and Bintje and the total number of up-regulated genes (661) were less than the number of genes down-regulated (971 genes) in the up mutant. Further analyses indicated that a variety of biological processes including decreased cell division, cell cycle activity, and abiotic stress responses were modified in the up mutant. In the nikku mutant, two potato genes, encoding an Acyl-CoA N-acyltransferases (NAT) superfamily protein, and a predicted major facilitator superfamily protein (MFS) were identified and overexpression lines Bintje/35S::NAT1 and Bintje/35S::PMT1 were created for recapitulation of the nikku mutant phenotype. Methylated DNA-PCR between the nikku and the revertant indicated a change in methylation status of the 35S enhancers, suggesting that the nikku revertant phenotype may be associated with some epigenetic modification.
Characterization of delayed flowering in soybean in Virginia
Abeysiriwardena, D. S. de Z. (Virginia Tech, 1990-12-13)
Delayed flowering has the potential to overcome the problem of restricted vegetative growth, prior to flowering, that is often associated with double-cropped soybeans [Grycine max (L.) Merr.]. Objectives were to study delayed flowering in soybeans as influenced by date of planting, to estimate the lengths of the component vegetative periods in soybeans under short-day conditions, and to study the mode of inheritance of delayed flowering in soybeans. Date of planting experiments conducted in the field at two Virginia locations using 27 cultivars and breeding lines showed that genotypic differences exist for delayed flowering, especially between delayed and normal flowering isolines. Lengths of the juvenile and inductive periods were estimated for some selected early and late flowering genotypes. F85-84l7 had a longer juvenile period, and F85-1226 had both longer juvenile and inductive periods than their respective early flowering isolines and cultivar Essex. cultivar. The method of moving plants from inductive short-days to long-days, which has been used to estimate the length of inductive period, was adapted to estimate the length of the juvenile period as well. Delayed flowering in soybeans appeared to be controlled by two loci, each with two alleles, and delayed flowering appeared to be recessive. Anyone of the genes in the homozygous recessive state delayed flowering. F85-1226 may be segregating for both genes while F85-84l7 appeared to contain only one.
Characterization of Polymorphic Microsatellites in Strawberry and Their Transferability to Other Genera in the Rosaceae Family
Arora, Vishal (Virginia Tech, 2006-02-09)
We investigated the transferability of 20 Fragaria vesca microsatellite primer pairs to 13 Fragaria vesca accessions, six Fragaria species and ten commercially important species in Rosaceae. Genetic diversity studies were carried among 16 diploid Fragaria accessions using these polymorphic microsatellites. The average number of alleles amplified for a polymorphic locus was 4.7 with maximum being 8.0 and minimum being 3.0. Observed heterozygosity ranged from 0.00 to 0.84 with an average of 0.28. Expected heterozygosity ranged from 0.33 to 0.91 with an average of 0.76. Power of discrimination varied from 0.43 to 0.92 with an average of 0.78. Transferability of microsatellites to F. orientalis (4x) and F. Ã ananassa (8x) was high, i.e., 18 (90%) primers produced amplicons. Cross species amplification within Rosaceae using these primers showed limited transference. Four microsatellites showed amplification for different species in Rosaceae. Products generated by UDF-003 and UDF-018 primers were sequenced. Sequencing results for UDF-018 showed that three species, i.e., Pyrus calleryana, Prunus persica and Rubus idaeus contained the expected microsatellite whereas another four, i.e., Cotoneaster salicifolius, Rosa rugosa, Amelanchier arborea and Potentilla fruticosa had conserved regions resulting in generation of amplicons. For UDF 003, Spirea xbumalda and Prunus persica did not contain a microsatellite although there was some sequence similarity with Fragaria. Size homoplasy, i.e., alleles of identical size with different numbers of repeats within the SSR was observed among Fragaria and Rosaceae species for primer UDF-018, suggesting a need for caution when interpreting SSR variation from band migration in the absence of DNA sequences.
Characterization of T-DNA integration sites within a population of insertional mutants of the diploid strawberry Fragaria vesca L.
Ruiz-Rojas, Juan Jairo (Virginia Tech, 2010-11-01)
Cultivated strawberry (Fragaria × ananassa) is an octoploid (2n=8x=56) species that belongs to the Rosaceae family and the high ploidy level makes genetic and molecular studies difficult. However, its commercial success because of its unique flavor and nutritious qualities has increased interest in the development of genomic resources. Fragaria vesca L. is a diploid (2n=2x=14) species with a small genome size (206 Mbp), short reproductive cycle, and facile vegetative and seed propagation that make it an attractive model for genomic studies. The availability of an efficient transformation methodology for Fragaria vesca has facilitated the use of a T-DNA mutagenesis system to develop a collection of several hundred insertional T-DNA mutants at Virginia Tech, using either of two commercially available vectors, pCAMBIA 1302 and 1304. In this study, we have used expression of the green fluorescent protein (GFP) as a tool to identify homozygous mutant lines. Three different approaches were conducted, first we identified 11 homozygous lines by PCR, then another 55 homozygous lines by absence of segregation of GFP expression in T2 seedlings, and finally we attempted to distinguish homozygous from hemizygous lines by relative GFP expression measured using a commercially available GFP meter. The latter methodology was unsuccessful due to uncontrolled variability in the readings. Continuing the characterization of our mutant population, we used thermal asymmetric interlaced PCR (TAIL-PCR) to obtain the nucleotide sequence of the genomic DNA regions that flank the T-DNA insertion sites in independent transgenic strawberry lines. Primers were designed that would amplify the derived strawberry flanking sequences in the two parents of an interspecific mapping population between the two diploid species, F. vesca x F. bucharica. The amplified products were sequenced and examined for the occurrence of SNPs (single nucleotide polymorphisms). The same primers were then used on the F2 mapping population. Segregation of SNP markers with previously mapped genetic markers allowed us to position 74 SNP markers, and hence their corresponding insertional mutants, on a well-populated genetic linkage map for the diploid strawberry. Finally, we analyzed the insertion site from more than 190 mutants looking at both the right and left borders of the T-DNA where microsimilarities of a few base pairs between ends of T-DNA and genomic DNA were observed, indicating that T-DNA integration had not occurred randomly in strawberry. We have also characterized the insertion sites through gene annotation found in the strawberry genome database.
Characterization of the F Locus Responsible for Floral Anthocyanin Production in Potato
Laimbeer, F. Parker E.; Bargmann, Bastiaan O. R.; Holt, Sarah H.; Pratt, Trenton; Peterson, Brenda A.; Doulis, Andreas G.; Buell, C. Robin; Veilleux, Richard E. (Genetics Society of America, 2020-08-27)
Anthocyanins are pigmented secondary metabolites produced via the flavonoid biosynthetic pathway and play important roles in plant stress responses, pollinator attraction, and consumer preference. Using RNA-sequencing analysis of a cross between diploid potato (Solanum tuberosum L.) lines segregating for flower color, we identified a homolog of the ANTHOCYANIN 2 (AN2) gene family that encodes a MYB transcription factor, herein termed StFlAN2, as the regulator of anthocyanin production in potato corollas. Transgenic introduction of StFlAN2 in white-flowered homozygous doubled-monoploid plants resulted in a recovery of purple flowers. RNA-sequencing revealed the specific anthocyanin biosynthetic genes activated by StFlAN2 as well as expression differences in genes within pathways involved in fruit ripening, senescence, and primary metabolism. Closer examination of the locus using genomic sequence analysis revealed a duplication in the StFlAN2 locus closely associated with gene expression that is likely attributable to nearby genetic elements. Taken together, this research provides insight into the regulation of anthocyanin biosynthesis in potato while also highlighting how the dynamic nature of the StFlAN2 locus may affect expression.
Characterization of the soybean genome in regions surrounding two loci for resistance to soybean mosaic virus
Hayes, Alec J. (Virginia Tech, 2003-08-01)
Soybean mosaic virus (SMV), has been the cause of numerous and often devastating disease epidemics, causing reduction in both the quality and quantity of soybeans worldwide. Two important genes for resistance to SMV are Rsv1 and Rsv4. Alleles at the Rsv1 locus have been shown to control resistance to all but the most virulent strain of SMV. This locus has been mapped previously to the soybean F linkage group. Rsv4 is an SMV resistance locus independent of Rsv1 and confers resistance to all strains of SMV. This locus has not been mapped previously. The purpose of this study is to investigate the two genomic regions that contain these vitally important resistance genes. A population of 281 F2 individuals that had previously been genotyped for reaction to SMV was evaluated in a mapping study which combined bulk segregant analysis with Amplified Fragment Length Polymorphism (AFLP). A Rsv4-linked marker, R4-1, was identified that mapped to soybean linkage group D1b using a reference mapping population. More than 40 markers were mapped in the Rsv4 segregating population including eleven markers surrounding Rsv4. This will provide the necessary framework for the fine mapping of this important genetic locus. Previous work has located Rsv1 to a genomic region containing several important resistance genes including Rps3, Rpg1, and Rpv. An RFLP probe, NBS5, whose sequence closely resembles that of several cloned plant disease resistance genes has been mapped to this chromosomal region. The efficacy of using this sequence to identify potential disease resistance genes was assessed by screening a cDNA library to uncover a candidate disease resistance gene which corresponds to this NBS5 sequence. Two related sequence classes were identified that correspond to NBS5. Interestingly, one class corresponds to a full length gene closely resembling other previously cloned disease resistance genes offering evidence that this NBS5-derived clone is a candidate disease resistance gene. A new marker technique was developed by combining the speed and efficiency of AFLP with DNA sequence information from cloned disease resistance genes. Using this strategy, three new markers tightly linked to Rsv1 were identified. One of these markers, which maps 0.6 cM away from Rsv1, has motifs consistent with other cloned disease resistance genes, providing evidence that this approach is an efficient method for targeting genomic regions where disease resistance genes are located.
Characterization of two genes, trehalose-6-phosphate synthase/phosphatase and nucleotide binding protein, shown to be differentially regulated in roots of Cypripedium parviflorum var. pubescens grown with a mycorrhizal fungus Thanatephorus pennatus
Watkinson, Jonathan I. (Virginia Tech, 2002-04-23)
The analysis of gene changes associated with formation of the mycorrhizal symbiosis between orchid and fungi could have broad implications for plant pathogen interactions. Fungi associated with North American terrestrial orchids were once included in the pathogenic genus Rhizoctonia. This suggests that orchids are able to overcome or utilize normally pathogenic pathways to establish symbioses. A differential display technique was employed to analyze gene changes in orchid in response to a fungus. Samples of RNA from roots of Cypripedium parviflorum var. pubescens (CyPP) grown in the presence or absence of a mycorrhizal fungus; Thanatephorus pennatus, were analyzed using AFLP differential display. Forty-four fragments were selected out of 5000 as being differentially expressed, but only 15 sequences were obtained. Most showed homology to ribosomal genes. Two represented genes believed to be regulated by the mycorrhizal interaction: trehalose-6-phosphate synthase/phosphatase (Tps), which showed down-regulation and nucleotide binding protein (NuBP), which showed up-regulation. The Tps partial clone identifies 2100 bp at the 3' end of the gene and encodes a protein of 667 amino acids. The NuBP gene is approximately 1200bp in length and encodes a protein of 352 amino acids. The Tps gene exists in multiple copies with high expression in roots and low expression in rhizomes and leaves. The NuBP gene exists as a single copy and has a low level of expression in rhizomes and leaves. Expression of Tps is induced by sucrose, but reduced by trehalose. Cultivation of CyPP with non-mycorrhizal fungi did not affect expression of Tps or NuBP. Trehalose induced NuBP expression whereas sucrose did not. A second species of mycorrhizal fungi induced expression of NuBP but reduced expression of Tps. Analysis of Tps expression in Arabidopsis was done using promoter:GUS fusions. The Tps promoter:GUS plants revealed that Tps expression is constitutive in roots. Regulation of Tps driven GUS is expressed throughout seedlings. GUS was not detected in leaves of older plants but was detected in anthers and stigmatic surfaces of flowers. Expression of GUS driven by Tps showed a strong wound response and was present in the junction between siliques and pedicels.
Comparative Analysis of Regions with Distorted Segregation in Three Diploid Populations of Potato
Manrique-Carpintero, Norma C.; Coombs, Joseph J.; Veilleux, Richard E.; Buell, C. Robin; Douches, David S. (2016-08-09)
Genes associated with gametic and zygotic selection could underlie segregation distortion, observed as alterations of expected Mendelian genotypic frequencies in mapping populations. We studied highly dense genetic maps based on single nucleotide polymorphisms to elucidate the genetic nature of distorted segregation in potato. Three intra- and interspecific diploid segregating populations were used. DRH and D84 are crosses between the sequenced doubled monoploid DM 1-3 516 R44 Solanum tuberosum Group Phureja and either RH89-039-16 S. tuberosum or 84SD22, a S. tuberosum × S. chacoense hybrid. MSX902 is an interspecific cross between 84SD22 and Ber83 S. berthaultii × 2 × species mosaic. At the 0.05 significance level, 21%, 57%, and 51% of the total markers mapped in DRH, D84, and MSX902 exhibited distorted segregation, respectively. Segregation distortion regions for DRH were located on chromosomes 9 and 12; for D84 on chromosomes 2, 3, 4, 6, 7, and 8; and on chromosomes 1, 2, 7, 9, and 12 for MSX902. In general, each population had unique segregation distortion regions and directions of distortion. Interspecific crosses showed greater levels of distorted segregation and lower recombination rates as determined from the male parents. The different genomic regions where the segregation distortion regions occurred in the three populations likely reflect unique genetic combinations producing distorted segregation.
A Comprehensive Analysis of Rust Disease Resistance in the Bioenergy Plant Switchgrass (Panicum virgatum L.)
Frazier, Taylor Price (Virginia Tech, 2016-01-14)
Switchgrass is a C4 perennial grass that is currently being developed for use as a second generation lignocellulosic biofuel crop. For switchgrass to be fully utilized as a bioenergy crop, large-scale plantings of elite switchgrass germplasm, possibly in monoculture, are likely to occur. This practice may increase the selection pressure on plant pathogens, such as switchgrass rust, which could result in devastating disease epidemics. The identification and deployment of quantitative trait loci (QTLs) and major plant disease resistance genes (R) in switchgrass breeding programs could offer broad spectrum and durable disease resistance in commercial switchgrass cultivars. 'Alamo', a lowland cultivar, is generally resistant to switchgrass rust whereas 'Dacotah', an upland cultivar, is highly susceptible. I hypothesized that major R genes and/or QTLs were contributing to the differences in disease phenotypes of these two cultivars. In this dissertation, bioinformatics and molecular biology approaches were employed to dissect the genetic mechanisms underlying switchgrass rust disease resistance. Novel pseudo-F2 mapping populations were created from a cross derived from 'Alamo' and 'Dacotah'. RNA-sequencing of the pseudo-F2 progenies of 'Alamo' x 'Dacotah' was used to construct a genetic linkage map and to identify potential QTLs correlating with disease resistance. In addition, a homology-based computational method was used to identify 1,011 potential NB-LRR R genes in the switchgrass genome (v 1.1). These potential R genes were characterized for polymorphism and expression differences between 'Alamo' and 'Dacotah'. Moreover, I found that some NB-LRR genes are developmentally regulated in switchgrass. One of the major objectives of switchgrass breeding programs is to develop cultivars with improved feedstock quality; however, changes in the components of the plant cell wall may affect disease resistance. I hypothesized that genetically modified switchgrass plants with altered cell wall components will respond differently than the wild-type to switchgrass rust. Transgenic switchgrass plants overexpressing AtSHN3, a transcription factor with known functions in epicuticular wax accumulation and cell wall deposition, were created. I found that AtSHN3-overexpressing transgenic switchgrass lines were more susceptible than wild-type plants in their response to switchgrass rust. Overall, the results of this dissertation provide a platform for elucidating the molecular mechanisms underlying resistance of switchgrass to switchgrass rust. These findings will help breeders create switchgrass cultivars with improved disease resistance, and will ultimately allow switchgrass to be used for sustainable biomass production.
A Computer Vision Tool For Use in Horticultural Research
Thoreson, Marcus Alexander (Virginia Tech, 2017-02-13)
With growing concerns about global food supply and environmental impacts of modern agriculture, we are seeing an increased demand for more horticultural research. While research into plant genetics has seen an increased throughput from recent technological advancements, plant phenotypic research throughput has lagged behind. Improvements in open-source image processing software and image capture hardware have created an opportunity for the development of more competitively-priced, faster data-acquisition tools. These tools could be used to collect measurements of plants' phenotype on a much larger scale without sacrificing data quality. This paper demonstrates the feasibility of creating such a tool. The resulting design utilized stereo vision and image processes in the OpenCV project to measure a representative collection of observable plant traits like leaflet length or plant height. After the stereo camera was assembled and calibrated, visual and stereo images of potato plant canopies and tubers(potatoes) were collected. By processing the visual data, the meaningful regions of the image (the canopy, the leaflets, and the tubers) were identified. The same regions in the stereo images were used to determine plant physical geometry, from which the desired plant measurements were extracted. Using this approach, the tool had an average accuracy of 0.15 inches with respect to distance measurements. Additionally, the tool detected vegetation, tubers, and leaves with average Dice indices of 0.98, 0.84, and 0.75 respectively. To compare the tool's utility to that of traditional implements, a study was conducted on a population of 27 potato plants belonging to 9 separate genotypes. Both newly developed and traditional measurement techniques were used to collect measurements of a variety of the plants' characteristics. A multiple linear regression of the plant characteristics on the plants' genetic data showed that the measurements collected by hand were generally better correlated with genetic characteristics than those collected using the developed tool; the average adjusted coefficient of determination for hand-measurements was 0.77, while that of the tool-measurements was 0.66. Though the aggregation of this platform's results is unsatisfactory, this work has demonstrated that such an alternative to traditional data-collection tools is certainly attainable.
Construction of Reference Chromosome-Scale Pseudomolecules for Potato: Integrating the Potato Genome with Genetic and Physical Maps
Sharma, Sanjeev Kumar; Bolser, Daniel; de Boer, Jan; Sonderkaer, Mads; Amoros, Walter; Carboni, Martin Federico; D'Ambrosio, Juan Martin; de la Cruz, German; Di Genova, Alex; Douches, David S.; Eguiluz, Maria; Guo, Xiao; Guzman, Frank; Hackett, Christine A.; Hamilton, John P.; Li, Guangcun; Li, Ying; Lozano, Roberto; Maass, Alejandro; Marshall, David; Martinez, Diana; McLean, Karen; Mejia, Nilo; Milne, Linda; Munive, Susan; Nagy, Istvan; Ponce, Olga; Ramirez, Manuel; Simon, Reinhard; Thomson, Susan J.; Torres, Yerisf; Waugh, Robbie; Zhang, Zhonghua; Huang, Sanwen; Visser, Richard G. F.; Bachem, Christian W. B.; Sagredo, Boris; Feingold, Sergio E.; Orjeda, Gisella; Veilleux, Richard E.; Bonierbale, Merideth; Jacobs, Jeanne M. E.; Milbourne, Dan; Martin, David Michael Alan; Bryan, Glenn J. (Genetics Society of America, 2013-11)
The genome of potato, a major global food crop, was recently sequenced. The work presented here details the integration of the potato reference genome (DM) with a new sequence-tagged site marker-based linkage map and other physical and genetic maps of potato and the closely related species tomato. Primary anchoring of the DM genome assembly was accomplished by the use of a diploid segregating population, which was genotyped with several types of molecular genetic markers to construct a new similar to 936 cM linkage map comprising 2469 marker loci. In silico anchoring approaches used genetic and physical maps from the diploid potato genotype RH89-039-16 (RH) and tomato. This combined approach has allowed 951 superscaffolds to be ordered into pseudomolecules corresponding to the 12 potato chromosomes. These pseudomolecules represent 674 Mb (similar to 93%) of the 723 Mb genome assembly and 37,482 (similar to 96%) of the 39,031 predicted genes. The superscaffold order and orientation within the pseudomolecules are closely collinear with independently constructed high density linkage maps. Comparisons between marker distribution and physical location reveal regions of greater and lesser recombination, as well as regions exhibiting significant segregation distortion. The work presented here has led to a greatly improved ordering of the potato reference genome superscaffolds into chromosomal pseudomolecules.
Controlling Growth in Echinacea Hybrids
Grossman, Mara Celeste (Virginia Tech, 2017-05-02)
New hybrid Echinacea cultivars, based on crosses of Echinacea purpurea (L.) Moench with several other Echinacea species, have generated interest and excitement in the marketplace due to novel flower colors and forms. However, these cultivars vary significantly in their growth habits and requirements from the species. We examined factors in the production of Echinacea hybrid cultivars to provide guidance to growers. Foliar sprays 600 mg·L⁻¹ benzyladenine (BA) increased numbers of branches between 19% and 83% in Echinacea cultivars while 400 mg·L⁻¹ dikegulac sodium or 500 mg·L⁻¹ ethephon did not improve branching. Of several height control PGRs applied to E. ‘Marmalade,’ only plants treated with two applications of 5000 mg·L⁻¹ daminozide were shorter (24%) compared to untreated controls although flowering was also reduced by 70%. Echinacea ‘Harvest Moon’ plants were shorter in response to all of the PGRs applied, with the best results seen in plants treated with foliar sprays of uniconazole (one application of 30 mg·L⁻¹ or two applications of 15 mg·L⁻¹ ), two applications of 5000 mg·L⁻¹ daminozide, or 4 mg·L⁻¹ paclobutrazol applied once as a drench. Supplying N at 150 mg·L -1 during the growing season provided Echinacea cultivars adequate nutrition and maximized numbers of branches and flowers and shoot dry weight. In overwintering, fertilization treatments that resulted in low substrate electrical conductivity going into dormancy, 5.0 kg·m controlled release fertilizer 15N-3.9P-10K or 150 mg·L⁻¹ N using 15N-2.2P-12.5K applied using constant liquid feed, resulted in the highest survival rates of Echinacea cultivars. As a monitoring tool, SPAD measurements were not successful in predicting tissue N levels in Echinacea hybrids. Twenty-one hybrid cultivars acquired as stage 3 tissue culture plantlets were grown under one of three photoperiods (10-hour, 16-hour, or 24-hour) for 10 weeks before being transplanted to larger containers and grown under natural daylength until flowering. Providing Echinacea hybrid cultivars with a 16-hour photoperiod during liner production resulted in plants which flowered soonest without negative effects on growth. The need for height control PGRs varied by cultivar; however, overall height control PGRs controlled flower stalk height and increased market rating.

Browsing by Author "Veilleux, Richard E."

Results Per Page

Sort Options