Transcript Lab Meeting, Oct 16 2003

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