a look into the process of marker development matt robinson
Post on 19-Dec-2015
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TRANSCRIPT
![Page 1: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/1.jpg)
A Look into the Process of Marker Development
Matt Robinson
![Page 2: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/2.jpg)
Outline
• Background
• Current Research– Creating Degenerate Primers– Primer Testing
• Looking Ahead– Populations sequence variation
![Page 3: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/3.jpg)
Outline
• Background
• Current Research– Creating Degenerate Primers– Primer Testing
• Looking Ahead– Populations sequence variation
![Page 4: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/4.jpg)
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.
![Page 5: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/5.jpg)
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
![Page 6: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/6.jpg)
Goals
• With the same markers I am obtaining sequence data to explore:– Patterns of variability– Patterns of linkage disequilibrium– Geographic structure– History of domestication
![Page 7: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/7.jpg)
Outline
• Background
• Current Research– Creating Degenerate Primers– Primer Testing
• Looking Ahead– Populations sequence variation
![Page 8: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/8.jpg)
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
![Page 9: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/9.jpg)
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
![Page 10: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/10.jpg)
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
![Page 11: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/11.jpg)
Change to picture from Grandillo, et al.
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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
![Page 13: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/13.jpg)
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
![Page 14: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/14.jpg)
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).
![Page 15: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/15.jpg)
Outline
• Background
• Current Research– Creating Degenerate Primers– Primer Testing
• Looking Ahead– Populations sequence variation
![Page 16: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/16.jpg)
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
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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.
![Page 18: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/18.jpg)
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.
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Picture of cloning in E. coli
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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
![Page 21: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/21.jpg)
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
![Page 22: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/22.jpg)
Outline
• Background
• Current Research– Creating Degenerate Primers– Primer Testing
• Looking Ahead– Populations sequence variation
![Page 23: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/23.jpg)
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
![Page 24: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/24.jpg)
Questions
![Page 25: A Look into the Process of Marker Development Matt Robinson](https://reader031.vdocuments.us/reader031/viewer/2022032703/56649d2a5503460f949fedab/html5/thumbnails/25.jpg)
Thanks to…
• Todd, Maria, Jason, and the rest of my fellow lab members