February 20, 2002UD, Newark, DE

SNPs, Haplotypes, Alleles

Sequence differences

• Intra-specific differences: between individuals within species

• Inter-specific differences: between orthologous genes in different species

Intra-specific and inter-specific variation

• Mutations• radiation• chemicals• replication errors• transposable elements• somatic vs. germinal

• Mutation frequency• maize: 6.5 x 10-9 mutations per nucleotide per

year (Gaut et al 1996, PNAS 93, 1997-2001)

Frequency of SNPs

• Intra-specific diversity• Humans: 1 in 1000 nt = 3,000,000 ea• Maize: 1 in 60~120 nt = 35,000,000 ea• Soybean: 1 in 350 nt =3,000,000 ea• Melon: 1 in 700 nt = 1,400,000

• Inter-specific sequence difference - dependent on evolutionary distance• Humans - chimpanzees 1 in 100 nt

Types of sequence variation

• Single Nucleotide Polymorphisms (SNPs)

• Insertions / Deletions (Indels)

• Silent mutations vs. amino acid changing mutations

• Nonsense mutations

• Missense mutations

• Frameshifts

• Simple sequence repeats

SNPs and Indels

DNA Sequence

...GATATTCGTACGGATGT-TCCA...

...GATGTTCGTACTGATGTATCCA...

...GATATTCGTACGGATGT-TCCA...

...GATATTCGTACGGATGTATCCA...

...GATGTTCGTACTGATGTATCCA...

...GATGTTCGTACTGATGTATCCA...

SN

P

SN

P

Indi

vidu

als

1 2

3

4

5 6

Ind

el

Haploypes

DNA Sequence

A G - G T A A G - A G A G T A G T A

SN

P

SN

P

Indi

vidu

als

1 2

3

4

5 6

Ind

el

AG-GTAAG-AGAGTAGTA

Haplotypes

Haplotypes

1.AG-2.GTA3.AG-4.AGA5.GTA6.GTA

Haplotype Sequence Frequency

1 AG- 2/6

2 GTA 3/6

3 AGA 1/6

Haplotypes provide more information than individual SNPs

Haplotype list

Haplotype

• A set of closely linked genetic markers present on one chromosome which tend to be inherited together (not easily separable by recombination). Some haplotypes may be in linkage disequilibrium

(from Birgid Schlindwein's Hypermedia Glossary Of Genetic Terms)

G G A C A

Set of SNP polymorphisms: a SNP haplotype

Diploids vs Haploids

Haploid cellDiploid cell

Chr1 Chr2 Chr1 Chr2

Homo vs. Hetero

Chr1 Chr2 Chr1 Chr2

Homozygous Heterozygous

Haploid > Diploid

Haploid cell

Chr1 Chr2

Haploid cell

Chr1 Chr2 Chr1 Chr2

DiploidHeterozygousExample: sex cells

Problem of Phase

Chr1 G AT C

SNP1 SNP2

Observed: SNP1 G / T SNP2 A/CPossible Haplotypes: GA, TC or GC, TA

How to resolve the problem of phase ?

G AT C

SNP1 SNP2

T C

SNP1 SNP2G A

Experimental solution

Computational solution

G AT C

G AT C

A TG A

T CA T

G AT C

A TG A

G AT C

A TG A

Not all combinations occur. Need to observe several haplotypes in various combinations

Linkage Disequilibrium: “Non-random association of alleles”

Equilibrium

1 21 2

Disequilibrium

3 3

3 3

Locus 1

Loc

us 2

D’=0

6

6

Locus 2

Loc

us 2

D’=1

Linkage Disequilibrium

1 2

Disequilibrium

Single marker is enough to completely define haplotypes in this example. Second marker provides redundant information.In a general case, a subset of the markers will be sufficient to define major haplotypes

Linkage Disequilibrium

1 2

Disequilibrium

For example, marker in the 3’-UTR will be completely predicted by the marker at the 5’-end of the gene and vice versa, if LD extends across the gene.

A A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D TG A T A C A A I/D TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C T

Redundant

A-TAICAITC-T

Uniquely defined haplotypes:

Stearoyl-ACP desaturase

Linkage disequilibrium

• The extent of linkage disequilibrium (LD) in the germplasm is important for association mapping

• The LD in the population depends on population history

• LD is also expected to vary along the length of the genome: regions that recombine less will have more LD and vice versa.

Recombination of ancestral haplotypes

Parents Progeny

Conserved ancestral haplotypes are reduced in size by recombinationSize of conserved segments depends on the history of the population and on

recombination frequency of the genome segment of interest

time

High LD Low LD

DNA Sequence

Sequence diversity

GATATTCGTACGGATGATGTTCGTACTGATGATATTCGTACGGATGATATTCGTACGGATGATGTTCGTACTGATGATGTTCGTACTGAT

SN

P

SN

P

Indi

vidu

als

1 2

3

4

5 6

Genetic Map

Phenotype

ResistantSensitiveResistantResistantSensitiveSensitive

DNA Sequence

SNP Haplotypes

GATATTCGTACGGATGATGTTCGTACTGATGATATTCGTACGGATGATATTCGTACGGATGATGTTCGTACTGATGATGTTCGTACTGAT

SN

P

SN

P

Indi

vidu

als

with

cont

rast

ing

phen

otyp

es

1 2

3

4

5 6

Genetic MapCandidate gene

Phenotypedistribution

Allelic series and phenotypes(Hypothetical example)

Haplotype Frequency Phenotypicmean

GATTGTA 0.35 96

GATTATA 0.55 89

GTTCATA 0.10 115

Haplotype information provides better resolution!

102

Haplotype diversity in maize elite germplasm

Ada Ching, Dinakar Bhattramakki, Antoni Rafalski

A A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TA A T A C G A TG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D CG A T A C A A I/D TG A T A C A A I/D TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C TG C A T T A C T

1

2

3

4

Genetic diversity in maize breeding germplasm

• This sample of 32 individuals provides an excellent representation of maize elite germplasm

• Conserved haplotypes over several hundered bp

• Small number of haplotypes (2-8)

• Lots of polymorphisms• SNP frequency: 1/61 bp

• Insertion / deletion frequency: 1/126 bp

Stearoyl-ACP desaturase

32 inbreds - 4 haplotypes

Two approaches to association mapping

• Candidate gene approach• Testing candidates for association with the trait

of interest

• Whole genome scan approach• Testing thousands of markers distributed along

the genome for association with trait• Suitable only for large linkage disequilibrium

situations

Consequences of LD

LD: High Low

Resolution Low High

Required number of markers Low High

Approach to associationmapping

Whole genomescan

Candidategene only

LD

Extent of linkage disequilibrium in maize elite breeding germplasm

Distance dependence of LD: abs(D')

0

0.2

0.4

0.6

0.8

1

1.2

0 100 200 300 400 500 600

Distance (bp)

LD

me

as

ure

: A

bs

(D')

18 genes32-35 individuals

Distance dependence of LD: R squared

0

0.2

0.4

0.6

0.8

1

1.2

0 100 200 300 400 500 600

Distance (bp)

LD

me

as

ure

: R

sq

ua

red

No decline of LD within 500 bp was detected

LD at Adh1: ~120kb

137522-137693T T T C T C G T T - G C C G G CT T T C T C G T T - G C C G G CG C C C C T A A A - G T C G G AG C C C C T A A A - A C C G G CT T T C T C G T T - G C C G G CT T T C T C G T T - A C C G G CT T T C T C G T T - G C C G G CG C C C C T A A A - G T C G G AT T T C T C G T T - A C C G G CG C C C C T A A A - G T C G G AT T T C T C G T T - G C C G G CT T T C T C G T T - G C C G G CT T T C T C G T T - G C C C T CG C C C C T A A A - G T C G G AT T T C T C G T T - A C C G G CG C C C C T A A A - G T C G G AG C C C C T A A A - G T C G G AT T T C T C G T T - A C C G G CT T T C T C G T T - A C C G G CT T T C T C G T T - G C C G G CG C C C C T A A A - G T C G G AG C C C C T A A A - G T C G G AT T T C T C A A A - A C C G G CA T T T C C G T T - G C A G G CT T T C T C G T T - G C C G G CT T T C T C G T T - A C C G G CT T T C T C G T T - G C C G G CT T T C T C G T T - G C A G G CT T T C T C G T T - A C C G G CT T T C T C G T T - G C C G G CT T T C T C G T T - A C C G G C

16523-16864

D’ = 1.0R2= 0.85Fisher P<0.001

)1()1(/ RRQQDr

21122211 D

121,171 bp

Mark Jung, Ada Ching

Conclusions

• Significant LD extending in some cases to >100 kb

• Whole genome scans may be possible

• More data is needed on LD in other regions of the genome and in other populations

• Haplotype association analysis adds power

• Work in progress (D. Bhattramakki)

Haplotype Organization of the Genome

A C A CC

G A T AT

T A C GC

A G A TG

(After Lander et al. 2001)

Low recombinationConserved haplotype

Low recombinationConserved haplotype

High recombination

A.R. 1-25-2001