Graduate Thesis Or Dissertation
 

The development and analysis of sequence-based DNA markers in sunflower for DNA fingerprinting and candidate gene analysis

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  • Molecular DNA markers have become widely used in all areas of genetic research. The objectives of this thesis were to develop polymorphic markers in sunflower and utilize the markers for genetic and candidate gene analyses. Amplified fragment length polymorphism (AFLP) markers were used to estimate genetic similarities and assess the genetic diversity among 24 public oilseed inbred lines of sunflower (Helianthus annuus L.). A total of 359 AFLP markers were scored by using six AFLP primer combinations. Genetic similarities ranged from 0.70 to 0.91, polymorphism rate ranged from 7 to 24%, and polymorphic information contents (PICs) ranged from 0.0 to 0.5. Principal coordinate and cluster analysis separated the lines into two groups, B-lines and R-lines, illustrating breeding history, basic heterotic pattern and the widespread practice of using each group to develop new lines. Δ9 stearoyl-ACP desaturase (SAD) and Δl2 oleate desaturase (OLD) cDNAs were cloned and sequenced. DNA fragment length polymorphism (DFLP), single strand conformational polymorphism (SSCP), and simple sequence repeat (SSR) markers were developed for the SAD6 and SAD17 genes among eight elite inbred lines. PICs for DFLP, SSCP, and SSR markers were 0.18, 0.37, and 0.30, respectively. Length variants were due to long monomeric repeats, insertions, and deletions in intron sequences, thereby producing polymorphic markers. OLD desaturates 18:1-PC (oleoyl phosphatidylcholine) to 18:2-PC, thereby converting oleic to linoleic acid. It is a likely candidate gene to be causing the high oleic phenotype in mutant sunflower. The expression of OLD7 in developing seeds was greatly reduced in mutant as opposed to wildtype backcross-derived lines. The restriction fragment length polymorphism (RFLP) patterns suggest that OLD7 is duplicated and rearranged in mutant lines. Utilizing sunflower SAD gene sequences and 27 inbred lines, intron fragment length polymorphism (IFLP) markers were developed for automated genotyping. These IFLP markers with ~470 to ~850 bp in length had a mean PIC score of 0.414, versus 0.336 for DFLP markers, and 0.582 for SSCP markers. One and two nucleotide length polymorphisms were reliably detected in PCR fragments up to ~150 and ~680 bp, respectively.
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