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 3 rd 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. –1 st 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. 1 st Use this variation, along with fruit sizes to determine the PVE values of the Physalis QTLs. 2 nd Use this regions to get sequence var. from various genotypes

Questions

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