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