A Look into the Process of Marker Development

1. A Look into the Process of Marker Development Matt Robinson

2. Outline • Background • Current Research • Creating Degenerate Primers • Primer Testing • Looking Ahead • Populations sequence variation

3. Outline • Background • Current Research • Creating Degenerate Primers • Primer Testing • Looking Ahead • Populations sequence variation

4. 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.

5. 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

6. Goals • With the same markers I am obtaining sequence data to explore: • Patterns of variability • Patterns of linkage disequilibrium • Geographic structure • History of domestication

7. Outline • Background • Current Research • Creating Degenerate Primers • Primer Testing • Looking Ahead • Populations sequence variation

8. 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

9. 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

10. 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

11. Change to picture from Grandillo, et al.

12. 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

13. 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

14. 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).

15. Outline • Background • Current Research • Creating Degenerate Primers • Primer Testing • Looking Ahead • Populations sequence variation

16. 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

17. 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.

18. 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.

19. Picture of cloning in E. coli

20. 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

21. 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

22. Outline • Background • Current Research • Creating Degenerate Primers • Primer Testing • Looking Ahead • Populations sequence variation

23. 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

24. Questions

25. Thanks to… • Todd, Maria, Jason, and the rest of my fellow lab members