Construction of a Microsatellite-Enriched Genomic Library of Physalis philadelphica

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Construction of a Microsatellite-Enriched Genomic
Library of Physalis philadelphica
Maria Chacon
March 19 2003
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Purpose

• Present progress on building a Physalis
  philadelphica genomic library with a high
  proportion of inserts containing microsatellite
  repeats. The protocol used was modified from the
  one developed at the Natural History Museum of
  the Smithsonian Institution

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Outline

1. Microsatellite definition and mutation process
2. Application of microsatellite markers
3. Advantages of microsatellites
4. Drawbacks of microsatellites
5. Protocol
6. Results
7. Conclusions and future work

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1. Microsatellite definition and mutation process
Also known as simple sequence repeats (SSR) or
short tandem repeat (STR). These terms are used
to describe tandem repeats of short sequence
motifs from mono to penta-nucleotides.
Classification Mono (A)11
AAAAAAAAAAA Di (AT)8
ATATATATATATATAT Tri (ATC)7
ATCATCATCATCATCATCATC Tetra (CTAG)6
CTAGCTAGCTAGCTAGCTAGCTAG Imperfect
microsatellite GTGTGTGTATGTGTGT Interrupted
microsatellite GTGTGTGTCCCGTGTGTGT Compound
microsatellite GTGTGTGTCTCTCTCTCTCT
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Genomic distribution of microsatellites
They are abundant in the eukaryotic genome and
are distributed throughout the genome The
genomic frequency of microsatellites is inversely
related to their repeat number, the higher number
of repeats the less frequent Microsatellites
not based on a unit of three are rare within
coding sequences as these can give rise to
frameshift if they mutate
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A microstellite mutation model
Microsatellite are exposed to a mutational
process called DNA (replication) slippage this
causes length instability of tandem repeats and
generates polymorphisms
(after Schlotterer and Harr, 2001)
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2. Applications of microsatellite markers
Several hundreds of microsatellites are present
in eukaryotic genomes and each locus is subjected
to DNA slippage they are therefore a huge
reservoir for polymorphic genetic markers

1. Population genetic studies of natural
   populations Hybridization, population history
   and phylogeography, divergence among populations,
   inbreeding, conservation genetics
2. Behavioral ecology male mating success
   determined by paternity testing, social
   organization of populations (identification of
   relatedness) and multiple paternity
3. Genetic mapping Microsatellites are distributed
   more or less evenly throughout the genome which
   makes them appropriate markers for mapping

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3. Advantages of microsatellites

• They probably exist in most of the species
• They are codominant markers
• They occur throughout most species genomes
• They can be isolated through the construction of
  a genomic library enriched for microsatellites or
  by the use of primers originally design for
  related species
• High heterozygosity level and high mutation rate
• Once isolated, microsatellites are amplified by
  PCR. Multiplex amplification of up to five loci
  is possible in a single PCR reaction which makes
  the scoring of multiple genotypes faster and
  cheaper

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4. Drawbacks of microsatellites

• Some organisms are very difficult to obtain
  microsatellite from Some plants, invertebrates
  such as Lepidopterans and birds
• Problems associated with PCR
• A. non-amplification of certain alleles due to
  substitutions, insertions or deletions within the
  priming sites generating null alleles
• B. Taq polymerase may generate slippage
  products or add an extra dNTP which cause single
  base shifts making typing difficult
• Problems associated with size or length homology
  alleles may converge on the same size via
  different types of events in or surrounding the
  repeat array. This has limited their use in
  resolving evolutionary relationships

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Size or length homoplasy

1. Addition or deletion of another type of repeat
   unit within the array
2. Nonrepeated sequences or a partial repeat within
   the array
3. Changes in the sequence flanking the array

Five SSR markers in Poplar. Tree Genetic
Engineering Research Cooperative
Six bovine SSR markers. Kovar et al. LI-COR
environmenral products
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5. Protocol

1. Digestion of genomic DNA
2. Ligation of adapters
3. Enrichment steps with biotin-labeled SSR probes
4. Removal of adapters
5. Cloning of enriched fragments
6. PCR amplification of inserts
7. Sequencing of inserts and design of primers

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1. Digestion of genomic DNA

Genomic DNA is fragmented by digestion with
restriction Endonucleases. These are enzymes
that cut the DNA at specific recognition
sequences DNA was extracted from young leaves of
Physalis philadelphica and restricted with BamHI
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II. Ligation of adapters
Adapters are short DNA fragments of known
sequence that may or not contain at the 3 end an
overhang for a specific restriction enzyme.
Adapters are linked to both ends of each
fragment generated by restriction
digestion Adapters help manipulate the digested
fragments of unknown sequence
GATCCTCCTATTAG GAGGATAATCCTAG
BamHI recognition sequences are restored at both
ends of restriction fragments
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III. Enrichment steps with biotin-labeled SSR
probes
The purpose of this step is to select the
fragments containing microsatellite sequences
Adapter-ligated fragments are hybridized with
biotin-labeled SSR probes
Fragments that do not hybridize with probes are
washed away by attaching biotin to straptividine
beads and a magnet
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IV. Removal of adapters
Adapters are removed by digesting with BamHI
enzyme. The BamHI overhangs are restored at both
ends of the fragments. These overhangs are going
to complement overhangs of the vector for cloning
5-GCGGTACCCGGGAAGCTTGG 3- CGCCATGGGCCCTTCGAACCCT
AG
GATCCCAAGCTTCCCGGGTACCGC-3
GGTTCGAAGGGCCCATGGCG-5
GATCCTCCAACAACAACAACAACTATTAG
GAGGTTGTTGTTGTTGTTGATAATCCTAG
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V. Cloning of insert DNA
Linear vector
lacZ
Ampicillin
MCS
pBluescript vector
Ligation of insert
Circular vector
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Transformation of XL1-blue strain of E. Coli
pBluescript vector carries a partial copy of the
lacZ gene and F episome also carries a defective
lacZ gene which complement each other to produce
an active B-galactosidase gene The active gene
gives a blue color. The inactive gene gives a
white color
Ampr
lacZ
Tetr
lacZ
E. coliplasmid without insertf episome
lacZgene
Ampr
Tetr
lacZ
E. coliplasmid with insertfepisome
Ampicillin Tetracycline IPTG X-GAL
Ampr
E. coliplasmid without insert, no f episome
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White-blue color selection
Ampr
lacZ
expression
Functional lacZ gene
X-Gal degrades
repression inhibited by IPTG
lac repressor
lacZgene
X-Gal does not degrade
Non-expression
Ampr
Non-functional lacZ gene
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VI. PCR amplification of inserts
T7 primer
T3 primer
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VII. Sequencing of inserts
Partial sequence of an insert enriched with
(TTG)10
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6. Results

• Enrichment was succesful for all microsatellite
  probes except for (ATG)10 and (TTC)10
• Several clones with insert were obtained for the
  successful enrichment reactions
• (TTG)10 250 clones (AGT)10 126 clones
• (TTA)10 90 clones (AAC)10 50 clones
• (ACT)10 50 clones (AAT)10 130 clones
• (AAG)10(ATC)10 72 clones
• Total 768 clones

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6. Results

• Insert size ranged from 300 bp up to 1500 bp. The
  most common sizes ranged from 300-700 bp
• 33 clones 10 enriched for (TTG)10, 14 for
  (AGT)10, 5 for (TTA)10 and 3 for (AAC)10 were
  sent for sequencing. One third did not contain
  SSR including all those that were enriched for
  TTA

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Conclusions and future work

1. Physalis philadelphica contains AT-rich
   microsatellites as other plant species and this
   method have proved useful for isolating them
2. Microsatellite sequences can be isolated by doing
   one or two steps of enrichment without need for
   further screening such as hybridization of clones
   with SSR-probes
3. Ninety six clones are going to be sequenced
4. The aim is to isolate a minimum of 15-20
   polymorphic loci