6 Indirect Genetic Tests
ANIM 2503 Animal Breeding and Molecular Genetics
Instructor and Institution
Instructor: Dr. Nicholas Clark
Institution: University of Queensland, Gatton Campus, School of Veterinary Science
Contact: n.clark@uq.edu.au
Learning Objectives
Distinguish between direct and indirect genetic tests.
Define and describe markers used in indirect tests:
SNPs (Single Nucleotide Polymorphisms): Variants at a single nucleotide.
Microsatellites: Repetitive short sequence units.
Explain genetic heterogeneity, phenocopies, and multifactorial traits concerning genetic test interpretation.
Direct vs. Indirect Genetic Tests
Direct Genetic Tests
Analyze specific mutations in the gene of interest.
Example: Test for mutation in gene B.
Indirect Genetic Tests
Depend on genetic linkage—test genetic markers linked to the disease gene, not the causative mutation.
Example: If gene B is linked to gene A, testing marker A can help predict the presence of mutation in gene B.
Linkage and Recombination
Recombination breaks linkage between genes on the same chromosome, impacting test accuracy.
The recombination rate affects how closely markers are linked to the target mutations, influencing the predictive power of indirect tests.
The closer two genes are, the higher the chance they will recombine together, thus impacting the accuracy of the genetic tests used.
Types of Genetic Markers for Indirect Tests
SNPs (Single Nucleotide Polymorphisms)
Variants present in >1% of the population.
Typically have two alleles (e.g., C or T).
Microsatellites
Repetitive regions with short repeat units (e.g., CT repeated 25 times).
Found throughout the genome, often in intergenic regions.
SNP Genotyping Methods
Detecting SNPs:
Sequencing
Restriction enzyme digestion of PCR products
Real-time PCR with allele-specific probes
Conventional PCR with allele-specific primers
Allele-Specific Primers:
Use separate primers for each allele to determine which alleles are present.
Primer Extension Methodologies:
Include dye-labeled ddNTPs in capillary electrophoresis for detection.
Mass spectrometry assesses the mass of different dNTPs.
Case Study: Canine Diabetes Mellitus
Similar clinical symptoms as human diabetes: weight loss, polyuria, persistent hyperglycemia.
SNP discovery identified 15 SNPs linked with the CTLA4 gene, which is involved in immune response.
Investigated whether these SNPs were associated with an increased risk of diabetes in dogs.
Findings on CTLA4 Variants
Variants linked to diabetes risk based on allele frequency in different breeds:
Labrador: SNP9 variant associated with increased risk.
Border Terrier: SNP12 G variant associated with increased risk.
Samoyed: SNP9 T variant associated with decreased risk.
Genetic Testing in Beef Cattle
DNA Testing for Dehorning
New Poll Gene Marker Test:
Cost-effective ($25) and nearly 100% accurate.
Revolutionizes breeding practices to eliminate horned animals without traditional dehorning methods.
Time Reduction:
Breeding can be achieved in 8 years instead of 39 years.
Characteristics of Genetic Markers
Microsatellites
High polymorphism and mutation rate.
Characteristics:
Amplification by PCR using primers matching flanking sequences.
Length differentiation through electrophoresis.
Genetic Tests: Considerations
Assess genetic heterogeneity, phenocopies, and multifactorial traits when interpreting results:
Genetic Heterogeneity: Different mutations can produce the same phenotype.
Phenocopy: Non-genetic causes replicate genetic disease symptoms.
Multifactorial Traits: Combination of multiple genetic mutations, with or without environmental effects, influencing the phenotype.
Important Considerations in Genetic Testing
Identify correct causative gene?
Are there alternative genetic variants presenting the same phenotype?
Non-genetic causes for similar phenotypes?
Evaluate recombination rates for accuracy of indirect tests.
Understand the sensitivity and specificity of the tests.
Identify whether the trait is multifactorial or polygenic.