Title page for ETD etd-09282012-161053


Type of Document Dissertation
Author Aulakh, Sukhwinder Singh
Author's Email Address ssaulakh@vt.edu
URN etd-09282012-161053
Title Characterization of activation tagged potato (Solanum tuberosum L.) mutants
Degree PhD
Department Horticulture
Advisory Committee
Advisor Name Title
Veilleux, Richard E. Committee Chair
Flinn, Barry S. Committee Co-Chair
Dickerman, Allan W. Committee Member
Mei, Chuansheng Committee Member
Nowak, Jerzy Committee Member
Keywords
  • GCN5
  • Sugar transporter
  • revertant
  • methylation
  • Solanaceae
  • BTB/POZ domain
  • RNA-seq
Date of Defense 2012-09-14
Availability unrestricted
Abstract
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.
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