Molecular markers

PCR based

A-priori sequence

knowledge

not required1courtesy of Carol Ritland

PCR markers a prior sequence knowledge

• RAPD

• AFLP

• SCARS

• CAPS

• AP-PCR

• RAMPO

2

RAPD

• Randomly Amplified Polymorphic DNA• Using a short primer (8-12 nucleotides)• No prior sequence knowledge is required• Require intact genome• Dominant marker• A major short fall = Lack of reproducibility• Welsh, J. and McCelland M. Nucleic Acid Res

1990 18:7213-7218• Williams et al. Nucleic Acid Res. 1990 18:6531-

65353

3 individualsOligos (8 to 12 nucleotides)

Oligos will anneal on both strandssearch for palidome sequences onboth strands

Will produce products when primers are close together to produce fragment sizes that can be visualized

4

PCR products formed for all individuals

5

PCR products for only two individuals

6

7

8

• RAPD marker has a major problem with dominance

• Previous example of individuals are shown as 2N

• In the next slide we will use chromatids (4 per individual) to demonstrate dominance

9

10

= sizeladder

RAPD Gels

11

Example of RAPD gel

12

Scoring RAPD gel

Sample name

(120bp) (130bp) (180bp) (220bp)

1101 1 1 0 1

1102 1 0 0 0

1103 0 1 1 1

1104 0 0 0 1

Sample name

Locus A (Allele type)

Locus B Locus C Locus D

1 2 2 1 2

2 1 2 1 2

3 2 1 2 1

4 1 2 1 1

13

A B C D E F G H I J

  Ladder (bp) A B C D E F G H I J1                      2                      3                      4                      5                      6                      7                      8                      9                      10                      

2000bp

 

800bp

600bp

 

300bp

 

100 bp

 

Score the following RAPD gel for these 10 samples (A-J).Indicate the loci you are scoring with an arrow on the right side of the image

14

•Some reproducibility problems, needs to use same lot for all chemicals eg. buffer, Taq, dNTP etc.•Same band on gel = same DNA fragment?•One band on gel = one DNA fragment?

(Allele homoplasy)•Selecting the band or lack of them to score

Issues to consider

15

•Anonymous markers - but can be converted to SCARs or CAPs•Dominant markers - homozygotes cannot be distinguished from heterozygotes•Fast, easy and cheap - commercial primer sets available•Scoring is subjective and individual dependent

More issues….

16

Applications

• Genetic Maps

• Fingerprinting isolates and cultivars

• Limited use today

17

•Amplified Fragment Length Polymorphism•Very sensitive•Good reproducibility but can be technically demanding•Combining RFLP and RAPD technique•Dominant marker•Generate fingerprint•Can use DNA and cDNA•Vos et al. Theor. Appl. Genet. 1995 23:4407-4414

AFLP

18

Courtesy of Ritland and Ritland

AFLP flowchart:

19

Courtesy of Ritland and Ritland

AFLP flowchart:

20

Genomic DNA

5’ - AATTC T - 3’ 3’ - G AAT - 5’

Ligate adapters + Ligase

5’ - CTCGTAGACTGCGTACCAATTC TTACTCAGGACTCAT - 3’ 3’ - CATCTGACGCATGGTTAAG AATGAGTCCTGAGTAGCAG - 5’

EcoRI adapter MseI adapter

Amplication(+n) AATGAGTCCTGAGTAGCAG

MseI primer

Preamplification

EcoRI and MseI

5’ - CTCGTAGACTGCGTACCAATTC TTACTCAGGACTCAT - 3’ 3’ - CATCTGACGCATGGTTAAG AATGAGTCCTGAGTAGCAG - 5’

(+1 to +3) AATGAGTCCTGAGTAGCAGMseI primer

5’ - CTCGTAGACTGCGTACCAATTC TTACTCAGGACTCAT - 3’ 3’ - CATCTGACGCATGGTTAAG AATGAGTCCTGAGTAGCAG - 5’

EcoRI primer GACTGCGTACCAATT (+1 to +3)

No selective bases= No PCR products

= label primer

EcoRI primer GACTGCGTACCAATT (+n)

Courtesy of Ritland and Ritland 2000Molecular Methods in Ecology

AFLP flowchart:

21

Complex

Simple

22

Linanthus Courtesy of Carol Goodwille

Samples 1 2 3 4 5 27 28

A

BC

D

E

23

Sample Locus A (bp)

Locus B Locus C Locus D Locus E

1 1 1 0 1 0

2 0 1 0 1 1

3 1 0 1 1 1

4 1 0 1? 1 1

5 1 1 0 1 0

Example of sampling from Linanthus data (see previous slide for image)

24

25

  Size (bp) A B C D E F G H I J K L M N O P

1                                  

2                                  

3                                  

4                                  

5                                  

6                                  

 

2550bp

 

2300bp

 

2004bp2000bp 

1750 bp

 

Scoring AFLP exercise: Samples = 16 (A-P) Indicate the loci you are scoring with an arrow on the right side of the image

Issues to consider• Discriminating homozygotes from heterozygotes

requires band quantification (possible gel scanner)• Bands are anonymous - interpretation of patterns

can be challenging• Same position equal only one band?• Dominant marker (difficult to test Hardy-Weinberg)• Can try to look for co-dominant bands• More expensive than RAPD markers• Much more repeatable than RAPD markers• Need to be technically consistent• Need clean intact genomes and min. amount (500ng)• May not be useful for population assignment pending on

population structure• Subjective scoring problem much like RAPD markers• Non independence bands are difficult to detect, problem

for phylogeny construction26

Applications for AFLP:• Physical mapping• Genome mapping• Population structure (clone detection)• Genetic diversity• DNA fingerprinting isolates or cultivars• Detection of somatic clone contaminations• Can convert segregating bands to co-dominant markers• Forensic sciences• QTL analysis• Locating possible genes related to complex traits (cDNA

template)

27

Clonality study• Wilson A.S.G., van der Kamp, B.J. and Ritland C. (2005) Can J. Bot 83:1126-1132

Maianthemum dilatatum

•Clone identification•Clone diversity•Spatial structure

28

Comparing RAPD to AFLP

• Kjolner, S., Sastad, S.M.,Taberlet, P. and Brochmann, C. (2004) Molecular Ecology 13:81-86

• 4 populations (13-15 individuals per population)

Saxifraga cernua 29

Comparing AFLP and RAPD

• Both markers produced similar results in estimating clone identity, clone relationships, gene diversity, linkage disequilibrium

• AFLP is superior in terms of efficiency but RAPD may still be used as a cheaper method

• Major caution with repeatability

30