AFLPs (Amplified Restriction Fragment Polymorphism).
DNA markers based on sequencing:
SNPs (Single Nucleotide Polymorphism).
Timeline of development: 1970s-2000s.
Separation of Amplified Microsatellites by Gel Electrophoresis
Microsatellites are amplified using primers (Primer A and Primer B).
Example of three trees with different microsatellite genotypes:
Tree no. 1: Genotype 11 (ATATATAT TA TATATATA).
Tree no. 2: Genotype 12 (ATAT AT AT TA TA TA TATA).
Tree no. 3: Genotype 23 (AT AT AT AT AT TA TA TA TA TA TA TA TA).
Microsatellite genotypes are given as the number of bases, e.g., (166,170) (heterozygote) or (166,166) (homozygote).
Introduction to Parentage Analysis
Genetic fingerprinting is an important tool, e.g., in forensics (exercise "Blekingegadebanden revisited").
Knowledge of parentage is crucial in inheritance matters (exercise "Family affairs").
Applications beyond determining parentage:
Testing ecological or physiological hypotheses.
Variation in reproduction success.
Direct measures of gene flow.
Mating patterns.
Selection against self-fertilization (inbreeding), etc.
Introduction to Parentage Analysis II
Continuum of situations with increasing complexity:
Simple experimental set up – parentage analysis with all potential parents known.
More complicated – some parents known – some not.
No parents known.
Further complexity: e.g., data of varying quality.
Simple Experimental Set-up Example
Controlled crossings with pollen mix ("Pollen cloud" of pollen mix).
Seeds harvested and germinated.
Analysis with 2 SSR markers to decide fathers of the seeds.
Fractions indicating potential fathers:
1/6 = A. nord. Clone 19
1/6 = A. nord. Clone 46
1/6 = A. nord. Clone 81
3/6 = A. alba
Microsatellites
Microsatellites are located on chromosomes within the DNA.
The four bases: Guanine, Thymine, Cytosine, Adenine.
Different trees have microsatellites of different lengths.
Example:
Tree X Genotype 1,2
Tree Y Genotype 2,3
Determining Genotype
Tree's genotype in microsatellites is determined by measuring the length of the microsatellites, expressed in the number of base pairs.
Example:
Microsatellite NFF2 – genotype (111,115)
Microsatellite NFF3 – genotype (134,138)
Application of Microsatellites in Hybridization Experiment
One Mother (M1).
Microsatellite NFH15 genotype (103,105)
Microsatellite NFH3 genotype (118,132)
Four potential fathers (Far1 - Far4) with their respective allele combinations for NFH15 and NFH3.
Paternity Case: Offspring 1
Offspring 1 has specific allele combinations for NFH15 and NFH3.
Comparison with the mother and the four potential fathers to determine paternity.
Methods of Parentage Analysis
Uses incompatibilities between parents and offspring to reject particular parent-offspring hypotheses.
Most powerful with few candidate parents and highly polymorphic genetic markers.
Potential weaknesses of a strict exclusion approach that can contribute to false exclusions.
Exclusion - Weaknesses
Example: Mother and father with genotypes a/a and b/b respectively. Their offspring will have genotype a/b.
Genotyping Error: If the offspring's genotype is mistakenly scored as a/a, the actual father will be excluded from paternity.
Mutation: If the offspring's genotype has become a/a due to mutation, the actual father will be excluded from paternity.
Null Allele: If the father's genotype is actually b/null allele the offspring can mistakenly be scored as a/a, and the actual father will be excluded from paternity.
Null Alleles
A microsatellite null allele is any allele at a microsatellite locus that consistently fails to amplify to detected levels via PCR.
Potential causes:
Poor primer annealing due to e.g. point mutations or indels in one or both flanking primer sites.
Differential amplification of size-variant alleles.
PCR failure due to low template DNA quality or quantity.
Examples of SSR Null Alleles
Table of locus names, primer sequences, observed heterozygosity (HO), expected heterozygosity (HE), null allele frequency.
Example Data:
NFF2: HO = 0.669, HE = 0.906, Null allele Freq. = 0.149
NFF3: HO = 0.813, HE = 0.884, Null allele Freq. = 0.040
NFH15: HO = 0.731, HE = 0.822, Null allele Freq. = 0.059
NFH3: HO = 0.837, HE = 0.961, Null allele Freq. = 0.068
NFF7: HO = 0.455, HE = 0.950, Null allele Freq. = 0.352
Examples of Null Alleles Parent-Offspring Revelations
Shows examples of mother and offspring allele combinations for NFH3B and NFF2, highlighting potential null allele occurrences.
More Sophisticated Methods of Parentage Analysis
Likelihood techniques used to compensate for imperfect data (e.g., genotyping errors, low genetic variation, mutations, null alleles).
Assign progeny to non-excluded parents based on likelihood scores derived from their genotypes.
Likelihood evaluates hypotheses given certain data: Methods based on likelihood L(H∣D).
Likelihood
The likelihood of one hypothesis is evaluated relative to another → the likelihood ratio:
In parentage analysis, the data are genetic data from the offspring, the known parent (if one is known) and one or more candidate parents. For each candidate parent, the two alternative hypotheses are:
1. The candidate parent is the true parent
2. The candidate parent is not the true parent
The likelihood ratio is the likelihood that the candidate parent is the true parent divided by the likelihood that the candidate parent is not the true parent. P(D∣H</em>2)P(D∣H<em>1)=L(H</em>2∣D)L(H<em>1∣D)