Molecular methods in Behavioral Ecology and Forensics Clemens Küpper

Molecular methods in Behavioral Ecology and Forensics

Clemens Küpper

Outline

• Sex typing• DNA fingerprinting• Short tandem repeats / microsatellites- parentage- forensics- estimating relatedness

Sex typing

Sex typing

• in monomorphic species such as birds, reptiles and amphibians with genetic sex determination

• Birds: ZZ, ZW♂ ♀

• several sex typing markers available• CHD1 is found on Z and W, intron copies with

different length

Sex ratio and survival of Kentish plovers

Survival of sons is greater than daughters

Mating opportunities for deserting females are higher than for males

Polyandrous mating system

Male-biased population sex ratio

Székely et al. (2004) Behavioral Ecology

Kosztolanyi et al. (2011) J Evol Biol

Pitfalls of sex typing

• poor sample quality can lead to allelic dropout

• ZZ’ polymorphism in males in some species:

fragment pattern like females• Moorhens: ZZ’ male have low

survival

Lee et al. (2002) Evolution 56:2548-2553

DNA fingerprinting

• based on non-coding repetitive DNA (minisatellites)

• after DNA digestion and gel electrophoresis individual characteristic band pattern is visualized

• relatives share bands

Alex Jeffreys (1984)

DNA fingerprinting: Paternity tests

Red winged blackbirdWestneat (1991) Behavioral Ecology 4:49-60

DNA fingerprinting: Paternity tests

DunnockBurke et al. (1989) Nature 338:249-251

DNA fingerprinting: problems

Classic DNA fingerprinting is now rarely used:• laborious• large amounts of sample needed• band assignment by humans is subjective• fingerprints on different gels are hard to

compare

Microsatellites (or short tandem repeats)

• single locus co-dominant markers• alleles vary in repeat length• high mutation rate (10-3 - 10-5)• many microsats available• genetic profiles can be stored in data bases

2. DNA Polymerase stops during replication. Melting and wrong assembly of strands

ACCCCACACACACACACACATGGTACCTGTGGGGTGTGTGT GT

ACCCCACACACACACACACATGGTACCTGTGGGGTGTGTGTGTGTGTGTACCATGGAC

1. Microsatellite allele with 8 repeats

ACCCCACACACACACACACACATGGTACCTGTGGGGTGTGTGTGTGTGTGTGTACCATGGAC

3. Completion of synthesis and repair of strands results in two microsatellite alleles one with 8 and one with 9 repeats

ACCCCACACACACACACACATGGTACCTGTGGGGTGTGTGTGTGTGTGTACCATGGAC

Microsatellites: mutation process

Females increase heterozygosity in offspring through extra pair fertilizations

Foerster et al. (2003) Nature 425:714-717

Validation of Bateman’s principles in the rough-skinned newt

Jones et al. (2002) Proc R Lond B 269:2533-2539

Variance for mating (7x) and repr. success (19x) higher in than ♂♂ ♀

♀

Microsatellites in DNA profiling

• 13 highly polymorphic microsatellite markers with 5 to 20 alleles• Tetra- and pentanucleotide motifs• 1 in 113 000 000 0000 are expected to share identical genetic profiles• only needs one DNA molecule for profiling• FBI database (CODIS): 9 Mio entries in 2010, mainly offenders but also missing persons• 62k investigations aided in 2007

Problems with DNA profiling and data bases

• falsified DNA profiles (DNA spray, blood and saliva samples) • contamination (by sample takers / lab workers)• incomplete profiles e.g. if little sample is available•match can be higher (e.g. if you happen to have common alleles)• relatives share alleles, monozygotic twins have identical twins• security of DNA data bases

Calculating relatedness (r)

• r describes how much genetic information is shared between individuals • important for understanding of social evolution • occurrence of inbreeding and inbreeding avoidance • best estimate require pedigree but need long term studies and relatedness in first generation is unknown• dominant markers band sharing coefficient :

2Nab/(Na+Nb)

• co-dominant markers: Queller Goodnight’s r

Queller Goodnight’s r

x k lx

x k ly

PP

PPr

*

*

R = relatedness between individuals x and y where Px = frequency of allele l at locus k within individual xPy = frequency of same allele in individuals to which x is comparedP* = frequency of the allele in population at large (background allele frequency) rare alleles are more informative than common allelesnegative values possible : less related than pop average

Queller and Goodnight (1989) Evolution 43:258-275

Relatedness in haplodiploid species

Relatedness in hyaena packs

Van Horn et al. (2004) Mol Ecol 13:449-458

Problems with molecular r estimates

Csilléry et al. (2006) Genetics 173:2091-2101