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Download Lab Meeting, Oct 16 2003
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A Look into the Process of Marker Development Matt Robinson Outline • Background • Current Research – Creating Degenerate Primers – Primer Testing • Looking Ahead – Populations sequence variation Outline • Background • Current Research – Creating Degenerate Primers – Primer Testing • Looking Ahead – Populations sequence variation Background • A Quantitative Trait Locus (QTL) is a region of the genome responsible for variation in a quantitative trait. • In tomato studies 28 QTLs have been identified as responsible for fruit weight variation between wt+ and domestic. • Similar studies have been done in eggplant and pepper. Several tomato fruit weight QTLs have homologs in these other species. Goals • Are these the same genes the ones that govern fruit size in Physalis? • To accomplish this I am isolating markers in Physalis homologous to markers close to the fruit weight QTL in tomato – Assumption: that the linkage between the marker and the gene in tomato is conserved in Physalis Goals • With the same markers I am obtaining sequence data to explore: – Patterns of variability – Patterns of linkage disequilibrium – Geographic structure – History of domestication Outline • Background • Current Research – Creating Degenerate Primers – Primer Testing • Looking Ahead – Populations sequence variation Why degenerate primers? • Degenerate primers for PCR – PCR uses two sequence specific primers, together with enzymes and other good stuff, to amplify a sequence of DNA. • Problem: we don’t know the Physalis sequence, we only know the tomato sequence • Solution: Degenerate primers (sets of primers with alternate possibilities at each base) allow for unknown sequence changes in Physalis Designing degenerate primers • Tomato sequence: CTC • Making 3rd codon position variable: CTN – CTA – CTT – CTC – CTG • Assuming conserved protein sequence, choosing residues that are the least degenerate Current Research: Creating Degenerate Primers • Chose 12 major fruit weight QTLs from a review of many wild x domesticated tomato crosses (Grandillo et al. 1999) • Used QTL with high values of percent phenotypic variance explained Change to picture from Grandillo, et al. Creating Degenerate Primers • Obtained sequence data of closely linked markers from SGN (Solanaceae Genome Network). • TBLASTX against DNA sequences at NCBI. – 1st against asterids (e.g. tobacco). – If no match was found then against all eudicotyledons (e.g. arabidopsis) • The alignments returned provide stretches of conserved protein sequences to make minimally degenerate primers Creating Degenerate Primers • A degenerate DNA sequence was made from the protein sequence of the stretch of alignment • Picture of the amino acids and their degenerate DNA sequences HERE Current Research: Creating Degenerate Primers • This degenerate sequences were scanned for possible primer regions which would allow for PCR of each of the QTL regions (using Primer3) • Candidate primer pairs were tested for melting temperature and other structural problems (internal repeats, reverse complementation). Outline • Background • Current Research – Creating Degenerate Primers – Primer Testing • Looking Ahead – Populations sequence variation Primer Testing • Primer pairs were tested at varying melting temperatures and enzyme mixtures. – This was to obtain a optimum reaction • Picture of gel of test conditions here Primer Testing • By comparing the length of the band in the gel of the PCR to the approximate length of the degenerate sequence which the primer pairs came from I am able to tell which lane contains a amplified product of a possible fruit weight QTL marker. Primer Testing • The bands which are approximately similar in size to the length of the original degenerate sequence are then cloned and sequenced to see if they share a homology to the QTL markers in tomato • The amplified PCR samples are inserted into a cloning Vector which is then inserted into E. coli. – Only one cloning vector will be inserted into the E. coli cells • The cells are then grown up overnight. Once the cells have grown individual colonies are picked and placed into a plate – These represent single colonies containing only one copy of the inserted PCR sample. Picture of cloning in E. coli Primer Testing • Next a sample is taking from each of the individual colonies in each well of the plate and placed in a PCR again to amplify the inserted sequence in the vector. – The product of this reaction is then run on a gel to find the correct band length for the clone • Picture of gel here Primer Testing • Once the correct band lengths are found in the clones I then sequence the clones – This gives me Physalis sequence data which I can compare to the tomato QTL markers at SGN – This also allows me to now create Physalis specific primer pairs Outline • Background • Current Research – Creating Degenerate Primers – Primer Testing • Looking Ahead – Populations sequence variation Looking ahead: Sequence Variation • sequences from the degenerate primers to create unique primers for physalis. • 1st Use this variation, along with fruit sizes to determine the PVE values of the Physalis QTLs. • 2nd Use this regions to get sequence var. from various genotypes Questions Thanks to… • Todd, Maria, Jason, and the rest of my fellow lab members