Section 2 -- Genetics Diversity

Importance of Genetic DiversityImportance of Genetic Diversity -- Maintenanceof genetic diversity is a major focus of conservation biology and 1 of 3 global conservationissues of IUCN

Environmental change is a continuous process &genetic diversity is required for populations toevolve to adapt to such change.

Loss of genetic diversity is often associatedwith inbreeding and reduction in reproductivefitness.

Characterizing Genetic Diversity: Quantitative Variation

Quantitative (metric or polygenic) characters ofMost concern to conservation biology are thoseRelated to reproductive fitness such as:

Number of fertile offspring contributed by anIndividual that survive to reproductive age.

For endangered species, quantitative variation forreproductive fitness is involved in the majorgenetic concerns in conservation biology, namely:

Reduction in reproductive fitness due to inbreeding(inbreeding depressioninbreeding depression)

Loss of evolutionary potential due to small population sizes.

Impact of crossing between different populationson fitness, whether beneficial (heterosisheterosis) ordeleterious (outbreeding depressionoutbreeding depression).

Effects of translocating individuals from oneenvironment to another.

Correlations between molecular and quantitativemeasures of genetic diversity are low. Therefore,molecular measures of genetic variation provide,at best, only a very imprecise indication of evolutionary potential.

Quantitative characters typically have continuous,approximately normal distributions and includecharacters such as reproductive fitness, longevity,height, weight, disease resistance, etc.

It is not possible to directly infer genotype fromobserved phenotype for quantitative characters.

Individuals with the same genotype may have different phenotypic values and individuals withthe same phenotypic values may have different genotypes.

Underlying genetic basis to quantitative charactersis that they are affected by a number of loci,each possessing alleles that add to or detract fromthe magnitude of the character.

Loci affecting quantitative characters, individually,show usual Mendelian properties of segregationand linkage.

A major challenge in the study of quantitativegenetics is to determine how much of the observedvariation is due to genetics and how much is dueto environment.

One of the central concepts of quantitative geneticsis heritabilityheritability.

HeritabilityHeritability is the proportion of the totalphenotypic variance in a population due to geneticdifferences among individuals.

Algebraically, we can define the phenotypic valueOf an individual as the consequence of the allelesIt inherits together with environmental influencesAs:

P = G + EP = G + E

Where P = phenotype, G = Genotype, and E = Environment.

The genetic component can be partitioned fromthe environmental component as:

VVPP = V = VGG + V + VEE + 2Cov + 2CovGEGE

Where, CovGE is the covariance between geneticand environmental effects.

The covariance for this component is expectedto be 0 if conditions for different genotypesare equalized by randomly allocating individualsacross the range of environment, which is difficult to achieve in wild populations.

For example, in territorial species of birds andmammals, the genetically fittest parents mayobtain the best territories.

Offspring inheriting the best fitness genotypesalso inherit the best environments.

This results in a genotype X environmentcorrelation that increases phenotypic resemblanceamong relatives.

Differences in performance of genotypes in different environments is referred to asGenotype X Environment InteractionsGenotype X Environment Interactions.

These develop when populations adapt to particularenvironmental conditions, and survive andreproduce better in their native conditions thanin other environments.

Genotype X Environment InteractionsGenotype X Environment Interactions are of majorsignificance to the genetic management of endangered species as follows:

Reproductive fitness of translocated individualscannot be predicted if there are significantGenotype X environment interactions.

Success of reintroduced populations may be compromised by genetic adaptation to captivity.

For example, superior genotypess under captive conditions may perform relatively poorly when released to the wild.

Mixing of genetic material from fragment populations may generate genotypes that do notperform well under some, or all, conditions.

Knowledge of genotype X environment interactionscan strongly influence the choice of populationsfor return to the wild.

Genotype X Environment interactions must bedistinguished from the genotype X environmentcovariances and correlations.

Genotype X environment correlations occur whengenotypes are non-randomly distributed overenvironments.

By contrast, genotype X environment interactionsare detected by comparing all genotypes inseveral common garden environments; if theirrelative performances differ in the differentenvironments there is a genotype X environmentinteraction.

Likelihood of genotype X environment interactionincreases with the magnitude of both geneticand environmental differences.

Thus, it is more likely to be detected in specieswith wide geographic, ecological, or altitudinalranges.

Further, quantitative traits closely associated withreproductive fitness appear to be more prone togenotype X environment interactions than characters more peripheral to fitness.

Quantitative genetic variation has contributionsfrom the average effects of loci VA, from theirdominance deviations VD, and from interactions(epistatic) deviations among gene loci VI as:

VVGG = V = VAA + V + VDD + V + VII

These are referred to as additive genetic variance (VA), dominance variance (VD), andinteraction variance (VI).

Each of these has major conservation implicationsas follows:

VA and especially the ratio VA/VP (heritability)reflect the adaptive evolutionary potential of thepopulation for the character under study.

VD reflects susceptibility to inbreeding depression.

VI influences the effects of outbreeding, whetherbeneficial or deleterious.

Therefore, VVPP = V = VGG + V + VEE = 2Cov = 2CovGEGE

More specifically,

VVPP = V = VAA + V + VDD + V + VII + V + VEE + 2Cov + 2CovGEGE

Evolutionary Potential and HeritabilityEvolutionary Potential and Heritability

Conservation genetics is concerned with the evolution of quantitative traits and how their abilityto adapt is affected by reduced population size,fragmentation, and changes in the environment.

Immediate evolutionary potential of a population isdetermined by the heritabilityheritability which is definedas the proportion of total phenotypic variationdue to additive genetic variation or hh22 = V = VAA/V/VPP..

Heritabilities range from 0 to 1.

Heritabilities of 0 are found in highly inbredpopulations with no genetic variation.

Heritabilities of 1 are expected for characters withno environmental variance in an outbred populationif all genetic variance is additive.

Heritabilities are specific to particular populationsliving under specific environmental conditions.

Heritability and VA are fundamentally measures ofhow well quantitative traits are transmitted fromone generation to the next.

Unfortunately, very few heritability estimatesexist for endangered species and there clearly isneed for many more estimates of heritability inthreatened and endangered species.

Measuring Genetic DiversityMeasuring Genetic Diversity

Quantitative CharactersQuantitative Characters: the most importantform of genetic variation is that for reproductivefitness as this determines the ability to evolve.These traits and other measurable characters, such as height, weight, etc. are referred to as“Quantitative CharactersQuantitative Characters”.

Variation for quantitative characters is due toboth genetic and environmental factors.

Therefore, methods are required to determinehow much of this variation is due to heritablegenetic differences among individuals and how much is due to the environment.

While genetic variation for quantitative charactersis the genetic diversity of most importance inconservation biology, it is the most difficult andtime-consuming to measure.

ProteinsProteins: The first measures of genetic diversityusing molecular methods were provided in 1966using protein electrophoresis.

This technique separates proteins according to their net charge and molecular weight.

DNA sequence variants may result in amino acidvariation that may result in functional biochemicalor morphological dissimilarities that cause differences in reproductive rate, survival, or behavior of individuals.

Normal hemoglobinNH2-Val-His-Leu-Thr-Pro-GluGlu-Glu-COOH

Sickle-Cell hemoglobinNH2-Val-His-Leu-Thr-Pro-ValVal-Glu-COOH

Disadvantages of Protein Electrophoresis:Disadvantages of Protein Electrophoresis:

Only about 30% of DNA substitutions result in charge changes so electrophoresis appreciablyunderestimates the full extent of geneticvariation.

Usually uses blood, liver, heart, or kidney in animals or leaves and root tips in plants thereforeanimals must be captured and many times killed.

DNADNA: There now exists several methods fordirectly or indirectly measuring DNA sequencevariation.

AdvantagesAdvantages:Sampling can often be done non-invasivelyPolymerase Chain Reaction (PCR) amplification

allows the use of small quantities of sample.

Restriction Fragment Length PolymorphismRestriction Fragment Length Polymorphism(RFLP)(RFLP)

DNA FingerprintingDNA Fingerprinting

Polymerase Chain ReactionPolymerase Chain Reaction (PCRPCR): Requires onlyextremely small quantities of sample to amplifya target sequence millions fold.

Allows use of remote sampling (hair, skin biopsy,feathers, sperm, etc) and the use of degradedsamples.

Randomly Amplified Polymorphic DNA (RAPD)Randomly Amplified Polymorphic DNA (RAPD)

Microsatellite Repeats:Microsatellite Repeats:

Tandem repeats of short DNA fragments

Typically 1 - 5 bp is length --

gtagacGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTGTccatagcatcagCACACACACACACACACACACACACACACACAggtatc

Number of repeats is highly variable dueto “slippage” during DNA replication.

Genotyping with microsatellites

BIBE1

BIBE15

BIBE16

Locus G10C

DNA Sequencing

Terms:

GenomeGenome: The complete genetic material of aspecies or individual. All the DNA, all theloci, or all the chromosomes.

LocusLocus (lociloci): A segment of DNA (e.g., microsatellite)or an individual gene.

AllelesAlleles: Different forms of the same locus thatdiffer in DNA base sequence: A1, A2, A3, etc.

GenotypeGenotype: The combination of alleles present at alocus in an individual.

HomozygoteHomozygote: An individual with two copies of thesame allele at a locus -- A1A1

HeterozygoteHeterozygote: An individual with two differentalleles at a locus -- A1A2

Allele FrequencyAllele Frequency: Frequency of an allele in apopulation (often referred to a gene frequency).

ExampleExample: If a population has 8 A1A1 individuals and 2 A1A2 individuals, then there are 18 copiesof the A1 allele and 2 copies of the A2 allele.

Thus, the A1 allele has a frequency of 18/20 = 0.9and the A2 allele has a frequency of 2/20 = 0.1

PolymorphicPolymorphic: Having genetic diversity. A locus ina population is polymorphic if it has more thanone 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.

MonomorphicMonomorphic: Lacking genetic diversity. A locusin a population is monomorphic if it has only one allele present in a population or if thefrequency of the most common allele is greater than 0.99 or 0.95.

Prorportion of loci polymorphicProrportion of loci polymorphic (PP): Number ofpolymorphic loci divided by the total numberof loci sampled.

ExampleExample: If you survey genetic variation at 10loci and only 3 loci are polymorphic then,

P = 3/10 = 0.3

Average HeterozygosityAverage Heterozygosity (HH): Sum of the proportion of heterozygotes at all locidivided by the total number of loci sampled.

ExampleExample: If the proportions of individualsheterozygous 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 DiversityAllelic Diversity (AA): Average number of allelesper locus.

ExampleExample: 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

HaplotypeHaplotype: Allelic composition for several locion a chromosome, e.g., A1B3C2

This term is also used to refer to unique mtDNAsequences for a particular locus.

Variable nucleotide positions

Polymorphic sites within mtDNA haplotypes of 144 southwestern black bears

Haplotype 36 93 351 352 462 466A T T T T A GB . . . – . .C . . – – . .D C C . – G AE C C – – G A

E

D

B

SDCN = 5

MMN = 29

B

A

B

B

A

B

BIBEN = 31

BGWMAN = 9

SDBN = 60

SMMN = 4

Haplotype DiversityHaplotype Diversity (hh): this is also known asGene Diversity and is equivalent to expectedheterozygosity for diploid data.

It is defined as the probability that tworandomly chosen haplotypes are differentin the sample.

k

h = (n/n-1)(1-pi2)

i=1

Where nn is the number of gene copies in the sample,kk is the number of haplotypes, and ppii is frequency of the ith haplotype

Example: population size = 50, 5 haplotypes.

n pi pi2 nn ppii ppii

22

A 46 0.92 0.8464 1010 0.20.2 0.040.04B 1 0.02 0.0004 1010 0.20.2 0.040.04C 1 0.02 0.0004 1010 0.20.2 0.040.04D 1 0.02 0.0004 1010 0.20.2 0.040.04E 1 0.02 0.0004 1010 0.20.2 0.040.04

0.848 0.20.2

h = 50/49(0.848) = 0.1551 50/49(0.8) = 0.816350/49(0.8) = 0.8163

Nucleotide diversityNucleotide diversity (): also known as averagegene diversity over L loci and is the probability that two randomly chosen homologous nucleotides are different.

This is equivalent to gene diversity at the nucleotidelevel.

k= (n/n-1)( pipjdij) i=1 j<i

Where ppii is the frequency of haplotype i and ppjj isthe frequency of haplotype j, and ddijij is an estimate of the number of mutations having occurred since the divergence of haplotypes i andj, kk is the number of haplotypes.

Example: 2 populations of size 30, each having3 haplotypes.

Population APopulation A Population BPopulation BA 10 F 10B 10 G 10C 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 A B CHaplotype A 10 A --Haplotype B 10 B 1 --Haplotype C 10 C 1 2 1Nucleotide Diversity = 0.0019Nucleotide Diversity = 0.0019Population 2 D E FHaplotype D 10 D --Haplotype E 10 E 8 --Haplotype F 10 F 11 5 --Nucleotide Diversity = 0.0115Nucleotide Diversity = 0.0115

A B CA --- 1 1B 0.0021 --- 2C 0.0021 0.0042 ---

D E FD --- 8 11E 0.0167 --- 5F 0.0230 0.0105 ---

SEQUENCED 478 BP

Population 1pi pj dij ij

A vs. A 0.333 0.333 0 0A vs. B 0.333 0.333 0.0021 0.00023A vs. C 0.333 0.333 0.0021 0.00023B vs. B 0.333 0.333 0 0B vs. C 0.333 0.333 0.0042 0.00047B vs. A 0.333 0.333 0.0021 0.00023C vs. C 0.333 0.333 0 0C vs. A 0.333 0.333 0.0021 0.00023C vs. B 0.333 0.333 0.0042 0.00047

0.001860.00186 = (30/29) X 0.00186 = 0.00192= (30/29) X 0.00186 = 0.00192

YOU SHOULD DO THE YOU SHOULD DO THE CALCULATIONS FORCALCULATIONS FOR

POPULATION #2POPULATION #2

Probability of IdentityProbability of Identity (PIPI): Probability of randomly pulling two individuals from a populationand them having the exact same genotype at allloci examined.

The unbiased estimate of PI over multiple loci is:

nn33(2a(2a2222 - a - a44) - 2n) - 2n22(a(a33 + 2a + 2a22) + n(9a) + n(9a22 + 2) - 6 + 2) - 6

(n - 1)(n - 2)(n - 3)(n - 1)(n - 2)(n - 3)PI =

n = sample size, ai = pji where

pj = frequency of jth allele.