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Genetic studies of candidate genes in the glycoalkaloid biosynthetic pathway of potatoManrique Carpintero, Norma Constanza 24 January 2013 (has links)
Potato (Solanum tuberosum L) is an outcrossing, highly heterozygous cultivated in which the elucidation of the genetic basis of quantitative traits, is more complex than in self-pollinated crops. Both a candidate gene approach and a whole genome SNP genotyping analysis were used to assess allelic variation and to identify loci associated with biosynthesis and accumulation of steroidal glycoalkaloids (SGAs). SGAs are secondary metabolites produced in Solanum species as defense against insects and pathogens. Fragments of genomic DNA coding for regions of five SGA biosynthetic candidate genes were amplified, cloned and sequenced [3-hydroxy-3-methylglutaryl coenzyme A reductase 1 and 2 (HMG1, HMG2); 2,3-squalene epoxidase (SQE); solanidine galactosyltransferase (SGT1); and solanidine glucosyltransferase (SGT2)]. A germplasm panel of six wild potato species [Solanum chacoense (chc 80-1), S. commersonii subsp. commersonii, S. demissum, S. sparsipilum, S. spegazzinii, and S. stoloniferum] and a cultivated clone S. tuberosum Group Phureja (phu DH) was used in an allelic variation analysis. A segregating interspecific F2 population phu DH �" chc 80-1 was screened to assess association with SGAs. Sequence diversity analysis showed a tendency of purifying selection and increased frequency of rare alleles in most of the candidate genes. Genes of primary metabolism (HMG1, HMG2 and SQE) had stronger selection constraints than those in secondary metabolism (SGT1 and SGT2). Sequence polymorphism in HMG2, SQE, SGT1 and SGT2 separated either the phu DH clone which produced no SGAs, or chc 80-1, the greatest SGA accumulator, from other accessions in the panel. Segregation analysis of the F2 population revealed that allelic sequences of HMG2 and SGT2 derived from chc 80-1 were significantly associated with the greatest SGA accumulation. In the whole genome analysis, SNP genotyping and cluster analysis based on putative association with SGA accumulation in the germplasm panel, allowed identification of eight informative SNPs that can be used in future studies. In the segregating F2 population, loci located on five pseudochromosomes were associated with SGA synthesis. Loci on pseudochromosomes 1 and 6 explained segregation ratios of synthesis for α-solanine and α-chaconine, the most common SGAs in most potato species. In addition, loci on seven pseudochromosomes were associated with accumulation. New candidate genes, putatively affecting synthesis and accumulation of SGAs, were identified in adjacent genomic regions of significant SNPs. This research demonstrates how the newly available genome sequence of potato and associated biotechnological tools accelerates the identification of genetic factors underling complex traits in a species with a difficult breeding structure. / Ph. D.
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Relationships among antioxidants, phenolics, and specific gravity in potato cultivars, and evaluation of wild potato species for antioxidants, glycoalkaloids, and anti-cancer activity on human prostate and colon cancer cells in vitro.Nzaramba, Magnifique Ndambe 15 May 2009 (has links)
Understanding the influence of environment and correlation/relationships among
traits is necessary in selection for crop quality improvement. Therefore, correlations
among antioxidant activity (AOA), total phenolics (TP), phenolic composition, and
specific gravity (SPG) in four potato (Solanum tuberosum, L.) cultivars (Atlantic, Red
La Soda, Russet Norkotah, and Yukon Gold) grown in nine states (California, Idaho,
Michigan, Minnesota, New Jersey, North Carolina, Oregon, Texas, and Wisconsin) for
three years, and in 15 advanced selections grown in Texas were investigated. Cultivars
within and between locations were significantly different in AOA, TP, and SPG.
Significant effects of cultivar, year, location and their interactions on AOA, TP, and SPG
were observed. There were significant positive correlations among the four cultivars
between AOA and TP, and negative correlations between AOA and SPG, and between
TP and SPG. However, correlations between AOA and SPG, and between TP and SPG,
in the advanced selections were not significant.
Some tuber-bearing wild potato species were higher in AOA and TP than the
commercial cultivars; therefore, they could be used as parental material in breeding for high AOA and TP. However, use of wild species that might be higher in total
glycoalkaloids (TGA) than cultivars could result in progenies with high TGA if the traits
are positively correlated. To elucidate the relationships among AOA, TP and TGA,
accessions of Solanum jamesii and S. microdontum from the US Potato Genebank were
screened for these traits and their correlations determined. Also, anti-proliferative and
cytotoxic effects of 15 S. jamesii tuber extracts (5 and 10 μg/ml) on human prostate
(LNCaP) and colon (HT-29) cancer cells was determined in vitro.
Alpha-solanine and α-chaconine were found in both species, while tomatine and
dehydrotomatine were quantified in some S. microdontum accessions. Both species were
higher in all above traits than the Atlantic, Red La Soda, and Yukon Gold cultivars.
More than 90% of S. jamesii accessions had TGA levels < 20 mg/100g fresh weight,
while only two accessions of S. microdontum, P1 500041 and PI 473171, exhibited TGA
< 20 mg/100g. Neither AOA nor TP was significantly correlated with TGA in both
species. Also, individual phenolics were not correlated with TGA. Solanum jamesii
accessions significantly reduced proliferation of HT-29 (5 and 10μg/ml) and LNCaP
(10μg/ml) cells and were not cytotoxic compared to the control (DMSO). Therefore,
since AOA and TP were not found to be correlated with TGA, using wild accessions in
breeding for increased health promoting compounds would not necessarily increase
glycoalkaloids in newly developed potato cultivars.
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Biosynthesis of Steroidal Glycoakaloids in Solanum chacoense BitterMweetwa, Alice Mutiti 02 September 2009 (has links)
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 µmole/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 µmole/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. / Ph. D.
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