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Chapter 3 -- Genetics 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 NH2-Val-His-Leu-Thr-Pro-Glu-Glu-COOH Sickle-Cell hemoglobin NH2-Val-His-Leu-Thr-Pro-Val-Glu-COOH Measuring Genetic Diversity 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”. 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: 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: There now exists several methods for directly or indirectly measuring DNA sequence variation. 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 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 1 - 5 bp is length -gtagacGTGTGTGTGTGTGTGTccatag catcagCACACACACACACACAggtatc Number of repeats is highly variable due to “slippage” during DNA replication. Genotyping with microsatellites Locus G10C BIBE1 BIBE15 BIBE16 DNA Sequencing Terms: Genome: The complete genetic material of a species or individual. All the DNA, all the loci, or all the chromosomes. Locus (loci): A segment of DNA (e.g., microsatellite) or an individual gene. Alleles: Different forms of the same locus that differ in DNA base sequence: A1, A2, A3, etc. Genotype: The combination of alleles present at a locus in an individual. Homozygote: An individual with two copies of the same allele at a locus -- A1A1 Heterozygote: An individual with two different alleles at a locus -- A1A2 Allele Frequency: Frequency of an allele in a population (often referred to a gene frequency). Example: If a population has 8 A1A1 individuals and 2 A1A2 individuals, then there are 18 copies of the A1 allele and 2 copies of the A2 allele. Thus, the A1 allele has a frequency of 18/20 = 0.9 and the A2 allele has a frequency of 2/20 = 0.1 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 0.95. 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 polymorphic (P): Number of polymorphic loci divided by the total number of loci sampled. Example: If you survey genetic variation at 10 loci and only 3 loci are polymorphic then, P = 3/10 = 0.3 Average Heterozygosity (H): Sum of the proportion of heterozygotes at all loci divided by the total number of loci sampled. 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 = (0 + 0.1 + 0.2 + 0.05 + 0)/5 = 0.07 Allelic Diversity (A): Average number of alleles per locus. Example: if the number of alleles at 6 loci are 1, 2, 3, 2, 1, 1 Then A = (1 + 2 + 3 + 2 + 1 + 1) = 1.67 Haplotype: Allelic composition for several loci on a chromosome, e.g., A1B3C2 This term is also used to refer to unique mtDNA sequences for a particular locus. Polymorphic sites within mtDNA haplotypes of 144 southwestern black bears Variable nucleotide positions Haplotype A B C D E 36 T . . C C 93 T . . C C 351 T . – . – 352 462 466 T A G – . . – . . – G A – G A BIBE N = 31 MM N = 29 B E A D BGWMA N=9 SDB N = 60 B SDC N=5 B A B SMM N=4 B 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-pi2) i=1 Where n is the number of gene copies in the sample, k is the number of haplotypes, and pi is frequency of the ith haplotype Example: population size = 50, 5 haplotypes. A B C D E n 46 1 1 1 1 pi 0.92 0.02 0.02 0.02 0.02 pi2 0.8464 0.0004 0.0004 0.0004 0.0004 0.848 h = 50/49(0.848) = 0.1551 n 10 10 10 10 10 pi 0.2 0.2 0.2 0.2 0.2 pi2 0.04 0.04 0.04 0.04 0.04 0.2 50/49(0.8) = 0.8163 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)( pipjdij) i=1 j<i Where pi is the frequency of haplotype i and pj is the frequency of haplotype j, and dij is an estimate of the number of mutations having occurred since the divergence of haplotypes i and j, k is the number of haplotypes. Example: 2 populations of size 30, each having 3 haplotypes. Population A A 10 B 10 C 10 Population B F 10 G 10 H 10 Haplotype diversity in each population = 0.6897 What is nucleotide diversity in each population? Sequenced 478 bp and obtained the following: Population 1 Haplotype A 10 A Haplotype B 10 B Haplotype C 10 C Nucleotide Diversity = 0.0019 Population 2 Haplotype D 10 D Haplotype E 10 E Haplotype F 10 F Nucleotide Diversity = 0.0115 A -1 1 B C -2 1 D -8 11 E F -5 -- SEQUENCED 478 BP A B C A --0.0021 0.0021 B 1 --0.0042 C 1 2 --- D E F D --0.0167 0.0230 E 8 --0.0105 F 11 5 --- Population 1 pi A vs. A 0.333 A vs. B 0.333 A vs. C 0.333 B vs. B 0.333 B vs. C 0.333 B vs. A 0.333 C vs. C 0.333 C vs. A 0.333 C vs. B 0.333 pj 0.333 0.333 0.333 0.333 0.333 0.333 0.333 0.333 0.333 dij 0 0.0021 0.0021 0 0.0042 0.0021 0 0.0021 0.0042 ij 0 0.00023 0.00023 0 0.00047 0.00023 0 0.00023 0.00047 0.00186 = (30/29) X 0.00186 = 0.00192 YOU SHOULD DO THE CALCULATIONS FOR POPULATION #2 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 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: PI = n3(2a22 - a4) - 2n2(a3 + 2a2) + n(9a2 + 2) - 6 (n - 1)(n - 2)(n - 3) n = sample size, ai = pji where pj = frequency of jth allele.