Construction of a Microsatellite-Enriched Genomic Library of Physalis philadelphica

Maria Chacon

March 19 2003

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

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

Classification:

Mono (A)11: AAAAAAAAAAADi (AT)8: ATATATATATATATATTri (ATC)7: ATCATCATCATCATCATCATCTetra (CTAG)6 CTAGCTAGCTAGCTAGCTAGCTAG

Imperfect microsatellite GTGTGTGTATGTGTGTInterrupted microsatellite GTGTGTGTCCCGTGTGTGTCompound microsatellite GTGTGTGTCTCTCTCTCTCT

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.

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

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)

2. Applications of microsatellite markers

I. Population genetic studies of natural populations: Hybridization, population history and phylogeography, divergence among populations, inbreeding, conservation genetics

II. Behavioral ecology: male mating success determined by paternity testing, social organization of populations (identification of relatedness) and multiple paternity

III. Genetic mapping: Microsatellites are distributed more or less evenly throughout the genome which makes them appropriate markers for mapping

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

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

4. Drawbacks of microsatellites

I. Some organisms are very difficult to obtain microsatellite from: Some plants, invertebrates such as Lepidopterans and birds

II. 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

III. 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

Size or length homoplasy

I. Addition or deletion of another type of repeat unit within the arrayII. Nonrepeated sequences or a partial repeat within the arrayIII. Changes in the sequence flanking the array

Five SSR markers in Poplar. Tree Genetic Engineering Research CooperativeSix bovine SSR markers. Kovar et al. LI-COR environmenral products

5. Protocol

I. Digestion of genomic DNA

II. Ligation of adapters

III. Enrichment steps with biotin-labeled SSR probes

IV. Removal of adapters

V. Cloning of enriched fragments

VI. PCR amplification of inserts

VII. Sequencing of inserts and design of primers

I. Digestion of genomic DNA

Genomic DNA is fragmented by digestion with restrictionEndonucleases. These are enzymes that cut the DNA atspecific recognition sequences

DNA was extracted from young leaves of Physalis philadelphicaand restricted with BamHI

5’-GTTCGATTGCGGATCCTCCTATTAGGATCCCGATCTGA-3’

3’-CAAGCTAACGCCTAGGAGGATAATCCTAGGGCTAGACT-5’

GTTCGATTGCGCAAGCTAACGCCTAG

GATCCTCCTATTAG GAGGATAATCCTAG

GATCCCGATCTGA GGCTAGACT

overhang overhang

overhang overhang

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

5’-GCGGTACCCGGGAAGCTTGG3’- CGCCATGGGCCCTTCGAACCCTAG

GATCCCAAGCTTCCCGGGTACCGC-3’ GGTTCGAAGGGCCCATGGCG-5

BamHI recognition sequences are restored at both ends of restriction fragments

III. Enrichment steps with biotin-labeled SSR probes

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net

Straptividine beads

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

The purpose of this step is to select the fragments containing microsatellite sequences

Biotin-labeled SSR probes

(AAC)10, (AAG)10, (AAT)10, (ACT)10, (AGT)10

(ATG)10, (ATC)10, (TTC)10, (TTA)10, (TTG)10

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

GATCCTCCAACAACAACAACAACTATTAG GAGGTTGTTGTTGTTGTTGATAATCCTAG

5’-GCGGTACCCGGGAAGCTTGG3’- CGCCATGGGCCCTTCGAACCCTAG

GATCCCAAGCTTCCCGGGTACCGC-3’ GGTTCGAAGGGCCCATGGCG-5

V. Cloning of insert DNA

Linear vectorBamHI restriction

Ligation of insert

Circular vector

pBluescript vector

Ampicillin lacZ’

MCS

Transformation

Transformation of XL1-blue strain of E. Coli

E. coli+plasmid without insert+f’ episome

E. coli+plasmid with insert+f’episome

Growing in selective medium

Ampicillin + Tetracycline + IPTG + X-GAL

E. coli+plasmid without insert, no f’ episome

lacZ

lacZgene

Ampr

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

Tetr

lacZ

Ampr

Tetr

lacZ

Ampr

White-blue color selection

FunctionallacZ gene

lac repressor

expression X-Gal degradesrepression inhibited by IPTG

lacZ

Ampr

lacZgeneAmpr

Non-functionallacZ gene

Non-expression X-Gal does not degrade

VI. PCR amplification of inserts

T7 primer T3 primer

VII. Sequencing of inserts

Partial sequence of an insert enriched with (TTG)10

6. Results

I. Enrichment was succesful for all microsatellite probes except for (ATG)10 and (TTC)10

II. 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

III. Insert size ranged from 300 bp up to 1500 bp. The most common sizes ranged from 300-700 bp

IV. 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

6. Results

Conclusions and future work

I. Physalis philadelphica contains AT-rich microsatellites as other plant species and this method have proved useful for isolating them

II. 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

III. Ninety six clones are going to be sequenced

IV. The aim is to isolate a minimum of 15-20 polymorphic loci