Chapter 3 -- Genetics Diversity Importance of Genetic Diversity Importance of Genetic Diversity -- Maintenance of genetic diversity is a major focus of conservation biology. Environmental change is a continuous process & genetic diversity is required for populations to evolve to adapt to such change. Loss of genetic diversity is often associated with inbreeding and reduction in reproductive fitness.

IUCN recognizes the need to conserve genetic diversity as one of three global conservation priorities. Genes are sequences of nucleotides in a particular segment (locus) of a DNA molecule. Genetic diversity represents slightly different sequences.

DNA sequence variants may result in amino acid variation that may result in functional biochemical or morphological dissimilarities that cause differences in reproductive rate, survival, or behavior of individuals. Normal hemoglobin Glu NH 2 -Val-His-Leu-Thr-Pro-Glu-Glu-COOH Sickle-Cell hemoglobin Val NH 2 -Val-His-Leu-Thr-Pro-Val-Glu-COOH

Measuring Genetic Diversity Quantitative Characters Quantitative Characters: the most important form of genetic variation is that for reproductive fitness as this determines the ability to evolve. These traits and other measurable characters, such as height, weight, etc. are referred to as Quantitative Characters “Quantitative Characters”. Variation for quantitative characters is due to both genetic and environmental factors.

Therefore, methods are required to determine how much of this variation is due to heritable genetic differences among individuals and how much is due to the environment. While genetic variation for quantitative characters is the genetic diversity of most importance in conservation biology, it is the most difficult and time-consuming to measure.

Proteins Proteins: The first measures of genetic diversity using molecular methods were provided in 1966 using protein electrophoresis. This technique separates proteins according to their net charge and molecular weight.

Disadvantages of Protein Electrophoresis: Only about 30% of DNA substitutions result in charge changes so electrophoresis appreciably underestimates the full extent of genetic variation. Usually uses blood, liver, heart, or kidney in animals or leaves and root tips in plants therefore animals must be captured and many times killed.

DNA DNA: There now exists several methods for directly or indirectly measuring DNA sequence variation. Advantages Advantages: Sampling can often be done non-invasively Polymerase Chain Reaction (PCR) amplification allows the use of small quantities of sample.

Restriction Fragment Length Polymorphism (RFLP)

DNA Fingerprinting

Polymerase Chain ReactionPCR Polymerase Chain Reaction (PCR): Requires only extremely small quantities of sample to amplify a target sequence millions fold. Allows use of remote sampling (hair, skin biopsy, feathers, sperm, etc) and the use of degraded samples.

Randomly Amplified Polymorphic DNA (RAPD)

Microsatellite Repeats: Tandem repeats of short DNA fragments Typically bp is length -- GTGTGTGTGTGTGTGT gtagacGTGTGTGTGTGTGTGTccatag CACACACACACACACA catcagCACACACACACACACAggtatc Number of repeats is highly variable due to “slippage” during DNA replication.

Genotyping with microsatellites BIBE1 BIBE15 BIBE16 Locus G10C

DNA Sequencing

Terms: Genome Genome: The complete genetic material of a species or individual. All the DNA, all the loci, or all the chromosomes. Locusloci Locus (loci): A segment of DNA (e.g., microsatellite) or an individual gene. Alleles Alleles: Different forms of the same locus that differ in DNA base sequence: A 1, A 2, A 3, etc.

Genotype Genotype: The combination of alleles present at a locus in an individual. Homozygote Homozygote: An individual with two copies of the same allele at a locus -- A 1 A 1 Heterozygote Heterozygote: An individual with two different alleles at a locus -- A 1 A 2

Allele Frequency Allele Frequency: Frequency of an allele in a population (often referred to a gene frequency). Example Example: If a population has 8 A 1 A 1 individuals and 2 A 1 A 2 individuals, then there are 18 copies of the A 1 allele and 2 copies of the A 2 allele. Thus, the A 1 allele has a frequency of 18/20 = 0.9 and the A 2 allele has a frequency of 2/20 = 0.1

Polymorphic Polymorphic: Having genetic diversity. A locus in a population is polymorphic if it has more than one allele. Polymorphic loci are usually defined as having the most frequent allele at a frequency of less than 0.99 or less then Monomorphic Monomorphic: Lacking genetic diversity. A locus in a population is monomorphic if it has only one allele present in a population or if the frequency of the most common allele is greater than 0.99 or 0.95.

Prorportion of loci polymorphicP Prorportion of loci polymorphic (P): Number of polymorphic loci divided by the total number of loci sampled. Example Example: If you survey genetic variation at 10 loci and only 3 loci are polymorphic then, P = 3/10 = 0.3

Average HeterozygosityH Average Heterozygosity (H): Sum of the proportion of heterozygotes at all loci divided by the total number of loci sampled. Example Example: If the proportions of individuals heterozygous at five loci in a population are: 0, 0.1, 0.2, 0.05, and 0, then H = ( )/5 = 0.07

Allelic DiversityA Allelic Diversity (A): Average number of alleles per locus. Example Example: if the number of alleles at 6 loci are 1, 2, 3, 2, 1, 1 Then A = ( ) = 1.67

Haplotype Haplotype: Allelic composition for several loci on a chromosome, e.g., A 1 B 3 C 2 This term is also used to refer to unique mtDNA sequences for a particular locus.

Variable nucleotide positions Polymorphic sites within mtDNA haplotypes of 144 southwestern black bears Haplotype ATT T T A G B... –.. C.. – –.. DCC. – G A ECC – – G A

SDC N = 5 MM N = 29 BIBE N = 31 BGWMA N = 9 SDB N = 60 SMM N = 4

Haplotype Diversityh Haplotype Diversity (h): this is also known as Gene Diversity and is equivalent to expected heterozygosity for diploid data. It is defined as the probability that two randomly chosen haplotypes are different in the sample. k h = (n/n-1)(1-  p i 2 ) i=1 n Where n is the number of gene copies in the sample, kp i k is the number of haplotypes, and p i is frequency of the i th haplotype

Example: population size = 50, 5 haplotypes. np i p i 2 np i p i 2 np i p i A B C D E /49(0.8) = h = 50/49(0.848) = /49(0.8) =

Nucleotide diversity  Nucleotide diversity (  ): also known as average gene diversity over L loci and is the probability that two randomly chosen homologous nucleotides are different. This is equivalent to gene diversity at the nucleotide level.

k  = (n/n-1)(   p i p j d ij ) i=1 j<i p i p j Where p i is the frequency of haplotype i and p j is d ij the frequency of haplotype j, and d ij is an estimate of the number of mutations having occurred since the divergence of haplotypes i and k j, k is the number of haplotypes.

Example: 2 populations of size 30, each having 3 haplotypes. Population APopulation B A10F10 B10G10 C10H10 Haplotype diversity in each population = What is nucleotide diversity in each population?

Sequenced 478 bp and obtained the following: Population 1ABC Haplotype A10A-- Haplotype B10B1-- Haplotype C10C121 Nucleotide Diversity = Population 2DEF Haplotype D10D-- Haplotype E10E8-- Haplotype F10F115-- Nucleotide Diversity =

ABC A---11 B C DEF D E F SEQUENCED 478 BP

Population 1 p i p j d ij  ij A vs. A A vs. B A vs. C B vs. B B vs. C B vs. A C vs. C C vs. A C vs. B  = (30/29) X =

YOU SHOULD DO THE CALCULATIONS FOR POPULATION #2

Probability of IdentityPI Probability of Identity (PI): Probability of randomly pulling two individuals from a population and them having the exact same genotype at all loci examined.

Probability of IdentityPI Probability of Identity (PI): Probability of randomly pulling two individuals from a population and them having the exact same genotype at all loci examined. The unbiased estimate of PI over multiple loci is: n 3 (2a a 4 ) - 2n 2 (a 3 + 2a 2 ) + n(9a 2 + 2) - 6 (n - 1)(n - 2)(n - 3)  PI =  n = sample size, a i =  p j i where p j = frequency of j th allele.