Date of Graduation

Summer 2019

Degree

Master of Science in Biology

Department

Biology

Committee Chair

Laszlo Kovacs

Keywords

REN1, ‘Kishmish Vatkana’, plant disease resistance, Oxford Nanopore Technology, gene discovery, defense genes, haplotype

Subject Categories

Bioinformatics | Biology | Biotechnology | Genetics | Molecular Genetics | Other Genetics and Genomics | Plant Pathology

Abstract

The REN1 region of the grapevine ‘Kishmish Vatkana’ was mapped as the locus that confers resistance to the economically important disease, grape powdery mildew. The purpose of this work was to extend the nucleotide sequence information of this region. By sequencing a heretofore unknown bacterial artificial chromosome clone, the sequence information of this region was extended by 46,890 nucleotides. Sequencing was performed using the third-generation sequencing method, named Oxford Nanopore Technology (ONT). In order to improve the accuracy of the sequence data, a modified ONT library preparation method was developed. ONT sequencing of a library prepared with the modified protocol generated a single 69,750 nucleotide-long contig of the entire BAC insert. Comparative analysis of the Sanger, Illumina, and ONT sequence data of the same stretch of DNA has revealed, however, that ONT sequence quality still lagged behind that of the former two. Several gene-finding programs were able to identify in the BAC insert three new genes encoding a TCP-type transcription factor, a cinnamoyl-CoA reductase enzyme and CC-NBS-LRR-type resistance protein. Orthologs of all three of these proteins have been implicated in plant disease resistance. The newly acquired sequence information also made it possible to determine that the BAC insert represented the resistance haplotype of the ‘Kishmish vatkana’ REN1 region. Nonetheless, evidence that any of the three defense-related alleles discovered here contribute to the powdery mildew resistance phenotype will require more experimental work.

Copyright

© Dani Joseph

Open Access

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