February 20, 2002 UD, 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 mutations per nucleotide per year (Gaut et al 1996, PNAS 93, )

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... SNP Individuals Indel

Haploypes DNA Sequence A G - G T A A G - A G A G T A SNP Individuals Indel AG- GTA AG- AGA GTA Haplotypes

1.AG- 2.GTA 3.AG- 4.AGA 5.GTA 6.GTA 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) GGA C A Set of SNP polymorphisms: a SNP haplotype

Diploids vs Haploids Haploid cell Diploid 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 Diploid Heterozygous Example: sex cells

Problem of Phase Chr1 GA T C SNP1 SNP2 Observed: SNP1 G / T SNP2 A/C Possible Haplotypes: GA, TC or GC, TA

How to resolve the problem of phase ? GA T C SNP1 SNP2 T C SNP1 SNP2 GA Experimental solution

Computational solution GA T C GA T C A T GA T C A T GA T C A T GA GA T C A T GA Not all combinations occur. Need to observe several haplotypes in various combinations

Linkage Disequilibrium: “Non-random association of alleles ” Equilibrium Disequilibrium Locus 1 Locus 2 D’=0 6 6 Locus 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.

Redundant A-T AIC AIT C-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 recombination Size 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 GATATTCGTACGGAT GATGTTCGTACTGAT GATATTCGTACGGAT GATGTTCGTACTGAT SNP Individuals Genetic Map Phenotype Resistant Sensitive Resistant Sensitive

DNA Sequence SNP Haplotypes GATATTCGTACGGAT GATGTTCGTACTGAT GATATTCGTACGGAT GATGTTCGTACTGAT SNP Individuals with contrasting phenotypes Genetic Map Candidate gene Phenotype distribution

Allelic series and phenotypes (Hypothetical example) Haplotype information provides better resolution! 102

Haplotype diversity in maize elite germplasm Ada Ching, Dinakar Bhattramakki, Antoni Rafalski

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

Extent of linkage disequilibrium in maize elite breeding germplasm

18 genes individuals

No decline of LD within 500 bp was detected

LD at Adh1: ~120kb D’ = 1.0 R 2 = 0.85 Fisher P< ,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 ACACC G ATAT TAC G C A G A T G (After Lander et al. 2001) Low recombination Conserved haplotype Low recombination Conserved haplotype High recombination

A.R