Molecular markers

PCR based

Requiring sequence

knowledge

1courtesy of Carol Ritland

PCR markers prior sequence knowledge

• Microsatellites (SSR/STR/ STMS)

• SSCP

• ISSR

• T-RFLP

2

Microsatellite (SSR/STR/STMS)

• Also known as Short Sequence Repeat/Simple Tandem Repeats/Sequence-Tagged Microsatellite Sites

• Repeats are 1 to 10 nucleotides bp long• Mutation rate is higher than base rate (1X104 vs 1X108)• Related to VNTR (minisatellites)• PCR based• Require extensive labour prior to finding useable

markers• Can be expensive to find these markers• Co-dominant• Litt, M. and Lutty, J. A. Am. J. Hum. Genet. 1989

44:397-4013

Allelic Variation at a Microsatellite Locus

GCCATGACACACACAGTAACGT

GCCATGACACACACACACACACACAGTAACGT

Allele “A”Allele “B”

4

Mechanisms of mutation(slipped-strand mispairing)

Step 1

Step 2

Step 3

Step 4

ca ca ca ca

gt gt gt gt gt gt gt

ca ca ca ca ca ca

ca ca ca ca

gt gt gt gt gt gt gt

gt gt gt gt gt gt gt

5

•Construction of DNA library•Restriction Enzyme digestion•Ligation to plasmid •Screening for various repeats (Southern blot)•Sequencing positive clones•Primers design to flank microsatellites•Testing Primers for polymorphism (need segregating families preferably with known parents)•Focal vs Non focal species

Development of SSR

6

Step by Step….

Restriction Digestion

Alu I AGCTHae III GGCCRsa I GTAC

Gel electrophoresis

Isolate fragments

200 to 500 bpVector for cloning

Ligase

Cloning and screen for positive clones 7

Step by Step cont’d

Screen for repeats

CA or CAT or CATA

CA positive cloneUsing CACACA(25)probe

1X106 clones

Sequence all positive clones usually 96 at a time

Primer design from clones that show repeats

8

SSR primer design

•50% contain repeats < 10 bp•20% contain repeats starting at one end of the sequence•20% to 30% contain repeats that may be usable•Watch for complex repeats eg. Compound = GCGGCCATATAT(16)GCGATGATATAT(16)GCGAAIrregular = GCGGCCATATATCCATATAT(16)CCATATGCGComplex = GCGGCCATATATCCATAT(12)GCTGCT(10)GCG•Ideally design primers 18 to 24 nucleotides•Aim for PCR product sizes that are > 100bp to 400bp

9

Primer testing

• Require ideally >3 populations for testing • Ideally 6 individuals randomly sample per

population• 20% yield zero or poor amplicons• 24% yield multiple or uninterruptible bands• 18% monomorphic bands• 38% usable microsatellite marker• Squirrell et al. 2003 Mol. Ecol. 12:1339-1348

10

WRC paternity analysis A. Miscampbell

= size ladder

SSR gel

11

= male parental type= female parental type

Issues with Microsatellites (SSR)•Highly variable and somatically stable marker•Specific primers designed for target species (18-25 nt)•Highly reproducible and yet evolve quickly (mutation rate is higher than normal rates)•A co-dominant marker with high heritability•Excellent for paternity/pedigree analysis•Could be difficult to use between species (focal vs non focal species)•Null alleles (lacking one of the allelic band for some heterozyote individuals within a population) test with family; excessive homozygotes, under estimate of diversity•Stutter bands (due to Taq incomplete amplification)•Subjectiveness when scoring (be consistent)

12

Scoring microsatellites

• Require known ladder to run with samples

• Resolve 2 or more bases differences using polyacrylamide gel

• Use base size to score allelic differences

Sample Locus A Locus A’ Locus B Locus B’

Cat A 202 204 353 355

Cat B 200 206 353 357

Cat C 202 206 351 355

13

14

Score SSR gel:Samples = 21

  1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21

A1                            A2B1                            B2C!                            C2D1                            D2

204 bp200 bp 

 

175 bp

 

  

145 bp  

120 bp

 

 

Allelic variation and statistical analyses

•Matala, A.P., Gray, A.K., Heifetz, J. and Gharrett, A.J. (2004) Envior. Biololy of Fishes 69:201-210•Population structure of Shortkaker Rockfish

15

Example of allelic variation in microsatellites

Microsatellite variation and genetic relationship among Rajasthani sheep: Relevance for conservation

R Arora and S Bhatia

16