• The Hardy-Weinberg equilibrium states that both the allele frequencies and the genotype frequencies are constant from generation to generation in a large random-mating population when they are not influenced by the evolutionary processes.
• In other words, the Hardy-Weinberg Law states that under a limited set of conditions, it is possible to calculate the predicted frequencies of genotypes in a population if the frequency of the distinct alleles in a population is known.
• It was discovered in 1908 by the English mathematician G.H. Hardy and the German physician Wilhelm Weinberg
• The Hardy-Weinberg equilibrium can be disturbed by a number of processes, including mutations, non-random mating, genetic drift, natural selection, and gene flow.
• For example, mutations disturb the equilibrium of allele frequencies, by the introduction of new alleles into a population.
• Likewise, the Hardy-Weinberg equilibrium is disrupted by natural selection and nonrandom mating as they result in shifts in gene frequencies.
• This happens as certain alleles help or harm the survival chances of the organisms that carry them.
• Genetic drift, which occurs when allele frequencies grow higher or lower by chance and usually takes place in small populations, is another aspect that may disrupt this equilibrium.
• The Hardy-Weinberg equilibrium can also be altered by gene flow, which occurs when mating between two species transfers new alleles into a population.
• The Hardy-Weinberg equilibrium scarcely applies in nature, since all of these destructive forces usually occur in nature.
• Thus, the Hardy-Weinberg equilibrium explains an idealized state, and genetic variations in nature can be evaluated as changes from this equilibrium state.

Hardy-Weinberg equation and analysis:

• The Hardy-Weinberg Theorem concerns with Mendelian genetics in the frame of reference of populations of diploid, individuals reproducing sexually.
• On a set of conditions, this theorem states that:
  • In a group, allele frequencies will not alter from generation to generation.
  • If p and q are the allele frequencies in a population with two alleles B and b, at the locus, then p², 2pq, and q² are the predicted genotype frequencies.
• Once a population is in a Hardy-Weinberg equilibrium, this frequency distribution will not alter from generation to generation.
• For instance, if the frequency of allele B in the population is p and the frequency of allele b in the population is q, then the frequency of genotype BB= p², the frequency of genotype Bb= 2pq, and the frequency of genotype bb= q².
• If there are only two alleles at a locus, then by mathematical necessity, p+q equals one.
• The frequencies of the Hardy-Weinberg genotype, p² + 2pq + q², reflect the binomial expansion of (p + q)², and also amount to one.
• If the frequency of B allele is 0.7, then we know that the frequency of b allele is 0.3.
• If there are three alleles, B₁, B₂, and B₃, with frequencies p, q, and r, the equilibrium frequencies corresponding to the six possible genotypes (shown in parentheses) will be determined as follows:
• (p+q+r)²= p²(B₁B₁) +q² (B₂B₂) +r²(B₃B₃) + 2pq(B₁B₂) + 2pr(B₁B₃)+ 2qr(B₂B₃)
• The Hardy-Weinberg Theorem can be applicable to loci with more than two alleles, in which case the predicted frequencies of the genotype are given by the multinomial expansion for all k alleles segregating in the population: (p1 + p2 + p3 +…+ pk)².
• The results of the Hardy-Weinberg Theorem apply only when the population corresponds to the following assumptions:
• Natural selection does not function on the locus concerned (i.e. there are no consistent differences in likelihoods of survival or reproduction between genotypes).
• New alleles are not introduced into the population by either mutation (the origin of new alleles) or migration (the transport of individuals and their genes into or out of the population).
• Population size is infinite, which suggests that genetic drift does not cause random variations in allele frequencies from one generation to the next due to sampling error. All natural populations are finite and thus subject to drift, of course, but it is expected that the effects of drift to be more pronounced in small populations than in large ones.
• With regard to the locus in concern, individuals in the population mate randomly. Even if non-random mating does not alter allele frequencies from one generation to the next when the other assumptions hold, it can create deviations from predicted genotype frequencies, and it can set the stage for natural selection to trigger evolutionary change.
• If the genotype frequencies in a population deviate from the standards of Hardy-Weinberg, it takes only one generation of random mating to put them into equilibrium proportions, given that the above-mentioned assumptions hold that male and female allele frequencies are identical (or else that individuals are hermaphrodites) and that the locus is autosomal.
• It takes two generations of random mating to achieve the Hardy-Weinberg equilibrium if allele frequencies vary between the sexes.
• Since one sex has two copies of the gene and the other sex has only one, sex-linked loci take many generations to reach equilibrium.
• Given these cases, if we consider random mating in terms of the likelihood of generating each genotype through random union of gametes into zygotes, the predicted Hardy-Weinberg genotype frequencies can be easily derived.
• If each allele occurs in sperm and eggs at the same frequencies, and gametes combine at random to create zygotes, then the likelihood of any two alleles combining to form a specific genotype is equal to the product of the frequencies of the alleles.
• Since the heterozygous genotype (B egg and b sperm, or b egg and B sperm) is produced in two ways, we sum up the probabilities of those two forms of union to meet the predicted Hardy-Weinberg frequency of the heterozygous genotype (2pq).
• It is essential to note that the Hardy-Weinberg equilibrium is a neutral equilibrium, which implies that after a single generation of random mating, a population disrupted by its Hardy-Weinberg genotype frequencies will still achieve equilibrium (if it conforms to the other assumptions of the theorem), but if allele frequencies have changed, it will be a new equilibrium.
• This property differentiates a neutral equilibrium from a stable equilibrium, where a disturbed system returns to the same equilibrium state.
• It makes good sense that the Hardy-Weinberg equilibrium is unstable, since a change from the equilibrium genotype frequencies will usually be linked with a change in allele frequencies (p and q), which will in turn yield new values of p², 2pq and q².
• After that, a population that matches Hardy-Weinberg assumptions will persist at the new equilibrium until disturbed again.

A punnet square showing all the possibilities of genotypes at a diallelic Mendelian locus in a populations that meets the Hardy-Weinberg assumptions:

The Hardy-Weinberg Theorem’s Evolutionary Implications:

• The Hardy-Weinberg Theorem shows that in the absence of forces that change allele frequencies, Mendelian loci segregating for multiple alleles in diploid populations can maintain predictable levels of genetic variation.
• Plotting p², 2pq and q² as a function of allele frequencies is a common way of visualising these expectations.
• Two major implications of the Hardy-Weinberg theory are emphasized in this graphical presentation:
• The heterozygosity of the population (frequency of heterozygotes) is greatest when p = q = 0.5.
• Rare alleles are mostly found in heterozygotes, as they must be, provided that when q is near zero, q² is much smaller than 2pq and when p is near zero, p² is much smaller than 2pq.
• If we consider the ability for natural selection to affect the frequencies of new mutations, the second statement takes on specific significance.
• If a population conforms to all other Hardy-Weinberg assumptions, a beneficial allele in the population will inevitably be set by selection so that all individuals are homozygous for that allele.
• A uncommon, advantageous, dominant allele’s initial rise in frequency is higher than that of a rare, advantageous, recessive allele.
• This is because, as we have noticed, rare alleles are often present in heterozygotes, so that natural selection will not see a new recessive mutation until it reaches a sufficiently high frequency (perhaps through drift in a true, finite population) to begin to occur in homozygotes.
• However a new dominant mutation is noticeable to natural selection immediately because its effect on fitness is seen in heterozygotes.
• Even though Hardy(1908) showed that dominance alone does not alter allele frequencies at a locus, there can be a major impact on evolutionary trajectories of the dominance relationships between alleles.
• The processes that cause changes in allele frequencies are selection, mutation, migration, and genetic drift, and when one or more of these factors function, the population violates the assumptions of Hardy-Weinberg, and evolution happens.

Thus the Hardy-Weinberg Theorem is a null model for the discipline of population genetics and is central to evolutionary research

The post Hardy-Weinberg equilibrium appeared first on Online Biology Notes.

• A representation of the genetic differences that occur within a population is termed as genetic variation.
• The genetic variation of whole the species is usually termed as genetic diversity.
• The differences in genes or DNA segments are genetic variations and each variation of a gene is termed as an allele.
• For instance, there is a high amount of genetic variation in a population of several different alleles at a single chromosome locus.
• For natural selection, genetic variation is important, because natural selection can only increase or decrease the frequency of alleles that already exist in the population.
• Since conditions are unpredictable, genetically diverse populations may be able to adapt faster than those that do not involve genetic variation to evolving circumstances.
• All examples of genetic variations that can occur in a human population are the skin tone, hair color, dimples, freckles, and type of blood of an individual.
• The modified leaves of carnivorous plants and the development of flowers that mimic insects to attract plant pollinators are examples of genetic variation in plants.
• Albinism, cheetahs with markings, snakes flying, animals that play dead, and animals that resemble leaves are examples of genetic variation in animals.
• These variations allow the animals in their habitats to better adapt to conditions.

What are the causes of genetic variation?

• The main causes of genetic variation are:
  • Mutation
  • Gene flow
  • Sexual reproduction
  • Genetic drift
• However, there are other causes of genetic variation:
  • Random mating
  • Crossing over
  • Random fertilization
  • Non-random mating
  • Environmental variance

i. Mutation:

• In general, mutation is the primary source of genetic variation, which is the raw material for evolution through natural selection.
• A change in the DNA sequence is a mutation.
• Often these differences in gene sequences may be beneficial for an organism.
• By modifying genes and alleles in a population, mutations result in genetic variation.
• They may affect an individual gene or a whole chromosome.
• Though mutations alter the genotype of an organism (genetic makeup), they do not necessarily change the phenotype of an organism.
• They may affect an individual gene or a whole chromosome.
• Though mutations alter the genotype of an organism (genetic makeup), they do not necessarily change the phenotype of an organism.
• During replication, mutations occur in the DNA molecule, such that the daughter cells vary in sequence or DNA quantity from the parent cells.
• A mutation initially appears in a single cell of the body, but it is transferred on to all cells descended from the first.
• Mainly two types of mutation are discussed they are gene mutations and chromosomal mutations.

ii. Gene mutations:

• In the case where the nucleotide sequence is modified and is passed on to the offspring, gene mutation occurs.
• The alteration might be either insertion or deletion or substitution of one or a few nucleotides.
• The addition or deletion of nucleotides within the DNA sequence in the coded protein shifts the reading frame and hence results in highly altered sequence of amino acids.
• Substitutions of nucleotides, however, may or may not severely affect the biological function of the protein.
•  Those substitution that results in terminator codon are supposed to be harmful.
• Gene mutation take place spontaneously without being purposedly induced by humans.
• Ultraviolet light, X-rays, and other high-frequency electromagnetic radiation, as well as exposure to such mutagenic substances, such as mustard gas, can also trigger them.
• The effects of gene mutations can vary from negligible to lethal.
• Mutations that modify one or even many amino acids can have a minor or undetectable impact on the ability of the organism to live and replicate if the coded protein’s basic biological role is not hampered.
• But when an amino acid substitution influences an enzyme’s active site or changes a protein’s essential function in some other way, the effect can be severe.

iii. Chromosomal mutations:

• The mutation process produces several new genetic variants for each generation. The nucleus of each cell contains chromosomes that hold the inherited material, or DNA.
• Chromosomes occur in pairs and each of them is inherited from each parent.
• In the pair, the two members are termed as homologous chromosomes.
• As a rule, every cell of an organism and all people of the same species have the same number of chromosomes.
• However, the number, size, and organization of chromosomes differs between species.
• Alterations in the number, size, or organization of chromosomes within a species are referred as chromosomal mutations, chromosomal abnormalities, or chromosomal aberrations.
• By fusion of two chromosomes into one, by fission of one chromosome into two, or by addition or subtraction of one or more whole chromosomes or sets of chromosomes, changes in number can occur.
• Polyploidy is the condition in which one or more additional sets of chromosomes are acquired by an organism.
• Changes in the arrangement of chromosomes may occur by inversion when a chromosomal segment rotates 180 degrees in the same place; by duplication when a segment is added; by deletion when a segment is lost; or by translocation when a segment changes from one place to another.
• These are the mechanisms by which chromosomes evolve.
• The amount of DNA is not changed by inversions, translocations, fusions, and fissions.
• The significance of these mutations in evolution is that they alter the linkage relationships among genes.
• Genes that are closely related to each other are isolated and vice versa; this may impact their expression because genes are mostly sequentially transcribed, two or more at a time.

iv. Gene flow:

• It is also termed as gene migration.
• New genes are introduced into a population as organisms migrate into a new environment.
• The availability of new alleles in the gene pool makes it possible for new gene combinations.
•  The emigration, i.e., moving out of organisms from a population also alters the gene frequencies.
• The emigration results in the lack of genetic diversity.
• Immigration of new organisms into a population may favor organisms to adapt in changing environmental conditions.
• Sustained gene flow between two populations can also result to a combination of the two gene pools, decreasing the genetic variation between the two groups.
• Gene flow strongly plays role against speciation.
• It does so by the recombination of the gene pools of the groups, and hence, repairment of the developing differences in genetic variation that would have resulted to full speciation and formation of daughter species.
• For instance, if a species of grass grows on both sides of a road, pollen is likely to be migrated from one side to the other and vice versa.
• If this pollen is capable of fertilizing the plant where it ends up and producing viable offspring, then the pollen alleles have effectively connected the population to the other on one side of the road.

v. Sexual reproduction:

• Sexual reproduction enhances genetic variation by producing various gene combinations.
• The process by which gametes or sex cells are formed is known as meiosis.
• As alleles in the gametes are separated and haphazardly united upon fertilization, genetic variation occurs.
• During meiosis, the crossing over or swapping of homologous chromosomes also results in the genetic recombination of genes.

vi. Genetic drift:

• Genetic drift is the alteration of a population’s allele frequencies owing to occurrences of random chance, such as natural disasters.
• The converse of natural selection is genetic drift.
• The theory of natural selection maintains that certain individuals in a population have characteristics that allow more offspring to survive and produce, whereas other people have characteristics that are harmful and can cause them to die before reproducing.
• These selection pressures will alter the gene pool and the traits within the population over successive generations.
• A huge, strong male gorilla, for instance, will mate with more females than a small, weak male, so more of his genes will be passed on to the next generation.
• His descendants will continue to control the troops and also pass on their genes.
• Over time, in the gorilla population, the selection pressure would cause the allele frequencies to change towards big powerful males.
• Unlike natural selection, genetic drift explains the impact of chance on populations in the absence of positive or negative selection pressure.
• Allele frequencies within a population may change through random sampling, or the survival or reproduction of a random sample of individuals within a population.
• Instead of a male gorilla generating more offspring since he is stronger, he may be the only male accessible when a female is ready to mate.
• His genes are transferred on to future generation because of chance, not as he was the biggest or the strongest.
• Genetic drift is the transition of alleles within a population due to chance events that cause random samples of the population to produce offspring or not.
• Small populations are more prone to the powers of genetic drift.
• On the other hand, large populations are buffered against the consequences of chance.
• If one individual in a population of 10 happens to die at a young age before leaving any offspring to the next generation, all of their genes (1/10 of the gene pool of the population) will instantly be lost.
• The individual comprises only 1 percent of the total gene pool in a population of100; thus, genetic drift has much less effect on the genetic structure of the larger population.
• The Bottleneck effect:
  • Natural disasters, such as a natural catastrophe that destroys a large portion of the population at random, may also magnify genetic drift.
  • When only a few individuals survive, the bottleneck effect arises and reduces diversity in a population’s gene pool.
  • The survivors’ genetic structure becomes the whole population’s genetic structure, which could be very different from the pre-disaster population.
• The founder effect:
  • If any part of the population leaves to start a new population in a new location or if a population gets separated by a physical barrier of some sort, another scenario arises in which populations may experience a strong impact of genetic drift.
  • In this scenario, it is not likely that those individuals are representative of the total population, which leads to the founder effect.
  • When the genetic structure changes to match that of the founding fathers and mothers of the new population, the founder effect occurs.
  • The underlying effect is believed to have been a key factor in the genetic history of Dutch settlers in South Africa’s Afrikaner population, as evidenced by mutations that are prevalent in Afrikaners, but rare in most other populations.
  • This was likely due to the fact that these mutations were carried by a higher-than-normal proportion of the founding colonists.
  • As a result, Huntington’s disease (HD) and Fanconi anemia (FA), a genetic condition known to cause blood marrow and congenital defects, including cancer, have exceptionally high incidences in the population.

vii. Non-random mating:

• If individuals nonrandomly mate with the other individuals within a population, i.e., they select their mate, selections can drive evolution in a population.
• There are several explanations for the phenomenon of nonrandom mating.
• Simple mate choice or sexual selection is one reason; female peahens, for instance, may prefer peacocks with larger, brighter tails.
• Traits that contribute to more matches for an individual lead to more offspring and ultimately lead to a higher prevalence of that feature in the population through natural selection.
• An individual’s desire to mate with partners that are phenotypically similar to themselves is one common type of mate choice, called positive assortative mating.
• Physical location is another cause of nonrandom mating.
• This is particularly true in large populations distributed over large geographical distances where not all individuals would have equal access to each other.
• These may be miles apart across forests or over rugged terrain, while others may live nearby immediately.

viii. Environmental variance:

• Genes are not the only players involved in population variance determination.
• Other factors, such as the environment, also affect phenotypes.
• For instance, due to daily exposure to the sun, an environmental factor, a beachgoer is likely to have darker skin than a city dweller.
• For certain animals, some major features, such as gender, are determined by the environment.
• For instance, some turtles and other reptiles have temperature-dependent sex determination (TSD).
• TSD implies that individuals grow into males if their eggs are incubated over a certain temperature range, or females at a distinct temperature range.
• Geographic separation among populations can contribute to variation in the phenotypic variation between certain populations.
• In most populations, this geographical variation is seen and can be important.
• One sort of geographic variation, named a cline, could be seen as populations of a given species differ progressively across an ecological gradient.

Gene pool:

• The term gene pool in evolutionary science refers to the collection of all available genes that can be passed down in the population of a single species from parents to offspring.
• The more variation there is in the population, the greater the gene pool.
• The gene pool decides the phenotypes (visible features) are present at any given time in the population.

How do gene pools change?

• Due to the movement of individuals into or out of a population, the gene pool can shift within a geographic region.
• When people with population-specific characteristics move abroad, the gene pool in that population shrinks and the characteristics are no longer available to be passed on to the offspring.
• In the other hand, as new people with new special features immigrate into the population, they expand the gene pool.
• A new form of diversity is added into the population when these new individuals interbreed with individuals already present.
• The size of the gene pool directly impacts the population’s evolutionary trajectory.
• The theory of evolution claims that natural selection works on a population to benefit the favorable features of that environment while weeding out the undesirable characteristics at the same time.
• The gene pool changes since natural selection operates on a population.
• Within the gene pool, beneficial adaptations become more frequent, and the less desirable characteristics become less prevalent or may even vanish entirely from the gene pool.
• Populations with larger gene pools are more likely than those with smaller gene pools to survive as the local climate changes.
• This is due to the fact that a broader range of traits are available to larger populations with wider diversity, which gives them an advantage as the environment changes and demands new adaptations.
• If there are few to no individuals with the genetic diversity needed to survive changes, a smaller and more homogeneous gene pool puts the population at risk of extinction.
• The more diverse the population, the more likely they are to survive significant changes in the environment.

Examples of gene pool in evolution:

• Individuals who are antibiotic-resistant are more likely to withstand any form of medical intervention in bacteria populations and live long enough to reproduce.
• The gene pool evolves over time (rather quickly in the case of rapidly reproducing organisms such as bacteria) to comprise only bacteria resistant to antibiotics.
• In this way, new strains of virulent bacteria are produced.
• A large number of plants that farmers classify as weeds are so tenacious because they have a diverse gene pool that helps them to adapt to a variety of environmental conditions.
• On the other hand, advanced hybrids also need very precise, even ideal conditions, because they have been bred to have a very small gene pool that prefers certain features, such as beautiful flowers or large fruits.
• It can be said that dandelions are superior to hybrid roses, on basis of size of gene pools.

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• Eukaryotic chromosomes are located within a separate cellular compartment termed as nucleus.
• The length of DNA must be compacted by a remarkable amount in order to fit it inside nucleus.
• The compacting of DNA is accomplished by the binding of the DNA to many different cellular proteins.
• The formation of a highly organized DNA-protein complex, termed as chromatin, which is a nucleoprotein complex completes the packing.
• Chromatin is a dynamic structure capable of changing its shape and composition during the life of a cell (cell cycle).
• Chromatin can be defined as highly condensed chromosomes at metaphase stage, and very diffuse structures in course of interphase.
  

Chromatin composition and packaging

Histones:

• Histones are most abundant proteins in chromatin.
• Histones are small and positively charged proteins and are of 5 major types: H1, H2A, H2B, H3 and H4.
• Histones are characterized by the presence of high percentage of basic amino acids arginine and lysine.
• These amino acids are positively charged that give the histones a net positive charge facilitating the binding of histones to the negatively charged DNA.
• Histone and DNA are present in equal amounts in chromatin.
• A heterogenous variety of non-histone chromosomal proteins also are found in eukaryotic chromosomes.
• There are times where variant histones, with different amino acid sequences, are integrated into chromatin in place of one of the major histone proteins.
• The amino acid sequences of histones H2A, H2B, H3 AND H4 are highly conserved, even between distantly related species.
• Evolutionary conservation of these amino acid sequences highly indicates that histones perform the same basic role in organizing the DNA in the chromosomes of all eukaryotes.
• Structural studies suggest that the histones classes do share a similar tertiary structure, showing that all histones are ultimately evolutionarily related.

Role of H1:

• The next level of condensation of chromatin is brought about by histone H1.
• H1, in contrast to the other histones is not the part of the core particle.
• H1 binds to 20-22 bp of DNA, where the DNA joins and leaves the octamer.
• H1 binds both to the linker DNA at one end of the nucleosome and to the middle of the DNA segment wrapped around core histones.
• H1 serves to restrict the DNA into place and functions as a clamp around the nucleosome octamer.
• The core particle and it’s associated H1 histone are altogether called as the chromatosome.

Nucleosomes:

• When the chromatin is isolated from the nucleus of a cell and observed under an electron microscope, it resembles beads on a string.
• The repeating core of protein and DNA produced by digestion with nuclease enzymes is the nucleosome.
• Nucleosome is the basic structural and fundamental unit of chromatin and is the simplest level of chromatin.
• Nucleosome is a core particle formed when the DNA is wrapped about 2 times around an octamer of eight histone proteins (2 copies each of H2A, H2B, H3, H4).
• The DNA in direct contact with the histone octamer is between 145 and 147 bp in length.
• This configuration compacts the DNA by six times.

Linker DNA:

• Each chromatosome encloses about 167 bp of DNA (147bp around nucleosome+20bp bound by H1).
• Chromatosomes are present at regular intervals along the DNA molecule and are apart from each other by linker DNA.
• The size of linker DNA varies among cell types, in most cells, linker DNA comprises of about 30-40 bp.

The 30nm chromatin fiber:

• The nucleosomes compact themselves into a structure about 30nm in diameter, now termed as the 30nm chromatin fiber.
• There are two possible models for the 30nm fiber.
• They are:
  • Solenoid model: In this model, a linear array of nucleosomes are coiled into a higher order left handed helix, entitled as solenoid, with around six nucleosomes per turn.
  • Helix model: In this model, nucleosomes are arranged in a zigzag ribbon that twists or supercoils.

Higher order structure of chromatin:

• The next higher level of chromatin structure is represented by a series of loops of 30nm fibers, each anchored at its base by proteins in the nuclear scaffold.
• On average, each loop encloses some 20-100kb of DNA and measures about 300nm in length.
• The 300nm loops are packed and folded to result a 250nm wide fiber.
• Tight helical coiling of the 250 nm, in turn, yields the structure that is visible in metaphase- individual chromatids approximately 700nm in width.

Overall, this packaging produces a chromosome that is about 10,000 times shorter and about 400 times thicker, than naked DNA.

Chromatin structure, composition and packaging

The post Chromatin structure, composition and packaging appeared first on Online Biology Notes.

• Molecular marker is identified as genetic marker.
• Molecular marker is a DNA or gene sequence within a recognized location on a chromosome which is used as identification tool.
• In the pool of unknown DNA or in a whole chromosome, these molecular markers helps in identification of particular sequence of DNA at particular location.
• Applications:
  • It plays a crucial role in gene mapping by identifying the position of linked genes in the chromosome which inherited together
  • It also detect any alteration in a sequence of DNA or any genetic oddity. It ascertains genes involved in genetic disorders.
  • It is used to determine different characters in a DNA sequence which is used to distinguish between individuals, populations or species.
• Different types of genetic polymorphism can be used as Genetic markers. On the basis of polymorphisms detected in the genetic makeup of individuals that may vary in the length of a DNA sequence or in the identity of nucleotides located at specific position in chromosome, some of the common genetic markers are- RFLP, SSLP, etc
• Genetic markers can be classified as PCR based and hybridization based.
  • PCR based genetic markers: RAPD, ISSR, EST-SSR, microsatellite, CAPS etc.
  • Hybridization based genetic markers: RFLP, VNTRs, in which targeted gene is digested with restriction enzymes and then hybridized with RFLP probe.

Quality for a good genetic marker:

• Genetic markers should be largely polymorphic in nature
• They should be selectively neutral
• Assay for detecting markers should be simple and rapid
• Genetic markers should occur frequently within genome
• The genetic marker (gene) should show codominant inheritance pattern.
• They should be highly reproducible
• They should not interact with other markers while using multiple markers at a same time

Types of genetic markers:

1. Random Amplified Polymorphic DNA (RAPD):

• RAPD was developed by Welsh and McClelland along with Williams in 1990.
• It is pronounced as ‘rapid’.
• It is based on PCR assay and it doesn’t need require any prior sequencing of DNA.
• This procedure uses short arbitrary primer of 8-12 bp that randomly amplifies the region of DNA.
• This primer serves as both forward and reverse primer.
• This reaction proceeds when a single primer anneals to the genomic DNA at two distinct sites on the complementary strand of DNA template.
• The amplification of segment of DNA depends on the positions complimentary to the primers’ sequence.
• The fragments obtained from RAPD are between 0.2 to 5.0kb and can be viewed by using agarose gel electrophoresis stained by ethidium bromide or with the help of polyacrylamide gel electrophoresis.
• If any mutation occurs in the primer binding region then no any PCR product will be produced, yielding a distinct pattern of amplified DNA segments on the gel.
• Application:
  • Distinct pattern of amplification is seen in different samples. This is why RAPD can be used for studying polymorphism.
  • RAPD is applicable for the mapping of genome, analyzing linkage, and individual specific genotyping.
  • RAPD markers are dominant in nature so it has restrictions for mapping purpose.
  • RAPD is strictly laboratory dependent so it requires sensitivity.
• Demerits:
  • It has demerits as poor reproducibility, yields faint products, problems occur in band scoring.

2. Restriction Fragment Length Polymorphism (RFLP):

• It was one of the first methods used for the analysis of DNA in various fields such as forensic science.
• It is a hybridization based technique.
• It was invented by Alec Jeffreys, an English scientist in 1984 during his research in genetic diseases.
• RFLP uses particular restriction endonuclease enzymes that cuts at its specific site yielding fragments of various lengths along with the fragment of interest.
• The length of the distinct fragments is determined by using blotting, now replaced with sequencing.
• RFLP markers are largely locus-specific and are co-dominant in nature due to the nature of restriction endonuclease used.
• Steps for RFLP are as follows:
  • DNA extraction is done from saliva, blood or other samples and is purified.
  • Restriction endonucleases digests the purified DNA resulting restriction fragments.
  • Now the restriction fragments are examined using gel electrophoresis.
  • The gel is now treated with luminescent dyes for the visibility of DNA bands.
• Applications:
  • RFLP was one of the first techniques applied for genetic fingerprinting/profiling.
  • It is used for identification of inherited diseases, carrier of that diseases, genetic mapping, and heterozygous detection.
  • The molecular basis of the RFLP is that any point mutations as such deletions, substitutions and insertions or alterations like duplications, inversions within the genome can eliminate or form new restriction sites. These alterations in genome can be detected by analyzing fragments of variable length, digested with restriction endonuclease enzyme
• Demerits:
  • requires relatively large DNA sample
  • laborious and tedious process
  • sensitivity and more precautions for contamination required

 3. Amplified Fragment Length Polymorphism (AFLP):

• Zabeau and Vos invented the AFLP technique in 1993.
• AFLP was originally developed by the KeyGene in 1990.
• It is a PCR based technique for fingerprinting. It includes both PCR and RFLP.
• The basis of AFLP is the amplification of selected fragments followed by restriction digestion of whole genomic DNA of specific organism.
• The steps for the AFLP are as follows:
• DNA extraction and its restriction digestion followed by ligation with the short adaptor sequences.
• Amplification of restricted fragments by PCR
• Analysis of results in gel electrophoresis or PAGE followed by autoradiography.
• Applications:
  • AFLP has its ability for rapid generation of marker fragments for any organism without prior sequencing of DNA is required.  
  • Also, it needs only small fragments of starting template DNA relative to RAPD and ISSR (inter-simple sequence repeats) and has much higher reproducibility.
  • AFLP is largely used for crop improvement programs, parentage and genomic interpretation of various crop species.
• Demerits:
  • AFLP require large DNA samples and require purification

4. Inter Simple Sequence Repeat (ISSR) markers:

• Inter simple sequence repeat (ISSR) technique is PCR based method.
• It was reported by Ztetikiewicz et al. in 1994.
• The ISSR markers are developed by PCR amplification of DNA segments between 2 similar microsatellites repeat regions by use of single primer consisting of microsatellite core regions.
• The primers can be usually 16-25 bp long, and unattached or attached at 3′ or 5′ end.
• Applications:
  • It is simple, rapid and economical like the RAPD technique and has higher reproducibility compared to RAPD because of longer primer length.
  • ISSR doesn’t require previous knowledge of genome for analysis and is highly polymorphic marker.
  • ISSR are used for identification of genetic diversity, phylogenetic analysis, to detect proximity of cultivars and to determine somaclonal variations in plants.
  • Due to the simple set up of ISSR, it is applicable for studying gene mapping, gene tagging, distinct strain identification, and parental recognition.
• Demerits:
  • ISSR has less reproducibility and non-homology of identical sized fragments due to multi locus feature.

5. Microsatellites or simple sequence length polymorphisms (SSLPs):

• Microsatellite was termed by Jeffery et al. in 1985.
• Microsatellites or simple sequence repeated (SSR) loci are PCR based markers which needs previous knowledge of gene sequence.
• In literature it is referred to as variable number of tandem repeats (VNTRs) or simple sequence length polymorphisms (SSLPs) or sequence tagged microsatellites (STMS).
• They are dispersed throughout the nuclear genomes in eukaryotes and to a few extent in prokaryotes.
• Microsatellite primers are short tandem repeats (STRs), or simple sequence repeats (SSRs), having 1-6 base pair long sequences repeated several times.
• Usually microsatellites are repeated less than 100 times.
• Microsatellites can be recognized by constructing a small-insert genomic library followed by screening of library and sequencing of positive clones.
• Microsatellites are used as markers for studying gene mapping, closeness among the species, and population genetics.
• The amplification of tandem arrays followed by visualization in gel helps to detect variation in DNA length.
• The main cause for the variation in DNA length is polymerase diminution during DNA replication, or slipped strand mispairing.
• Applications:
  • Microsatellite consists of co-dominance of alleles and requires low quantities of DNA templates.
  • It has high reproducibility and is economical in nature.
  • The screening of microsatellite variation can be automated.
• Demerits:
  • Assay is costly if sufficient primer sequences for the species of interest are not available.
  • Errors in genotype scoring occurs if alterations are seen in primer annealing sites.
  • chances of homoplasy ( some characters are present in more than one species but not present in their common ancestor because of convergence evolution)

6. Cleaved Amplified Polymorphic Sequence (CAPS):

• CAPS marker was described by Konieczny and Ausubel in 1993 for genetic mapping.
• CAPS is identical to RFLP and referred as PCR-RFLP.
• CAPS markers technique is based on the digestion of monomorphic PCR product for generation of polymorphism.
• It uses amplified DNA fragments which undergoes digestion by restriction endonuclease to display RFLP.
• The products of digestion are screened for their differential migration rate in course of electrophoresis by staining with ethidium bromide.
• CAPS primer can be developed on the basis of available information of sequence from genomic databank or cDNA sequences.
• Applications:
  • These markers are codominant in nature and are locus-specific.
  • It is used for gene mapping

 7. Expressed Sequence Tags (ESTs):

• ESTs are molecular markers synthesized by partial sequencing of random cDNA clones.
• Once cDNA is synthesized, then a few hundred nucleotides (500-800bp) from either end (3’ or 5’ end) is sequenced to create two different kinds of ESTs.
• Sequencing of the 5’ end of cDNA produces a 5′ EST which usually codes for a protein.
• These regions tend to be conserved across species and do not change much within a gene family.
• Sequencing of 3’ end of cDNA produces a 3′ EST which is non-coding, or untranslated regions (UTR) and it is less conserved among species.
• Therefore, ESTs are sub-sequence of cDNA which represents tag for the entire cDNA.
• Applications:
  • EST is used for the whole genome sequencing and studying gene of interest.
• In addition to it, it is used for cloning gene of interest and gene mapping.

8. Sequence Characterized Amplified Region (SCAR):

• SCAR is based on PCR-agarose gel electrophoresis.
• It uses longer primers of 15-30 nucleotides yielding high reproducibility.
• It needs prior sequence information for primer designing.
• As PCR is used, it needs only low quantities of template DNA.
• Applications:
  • It is simple, reliable and reproducible.
  • It is codominant in nature and locus-specific.
  • SCAR is applicable for studying gene mapping and largely used in plant selection studies.

9. Single nucleotide polymorphism (SNP):

• SNP was invented by Lander in 1996.
• SNP is formed when any alteration/mutation occurs in single nucleotide (A, T, C, or G).
• The point mutation as such substitutions, insertions or deletions in single nucleotide it represents SNP.
• SNPs are based on hybridization of detected DNA fragments with SNP chips (DNA probe arrays) and the SNP allele is named with respect to the hybridization results.
• Applications:
  • SNPs are widely used in biomedical research for comparing the case and control groups of disease.
  • It is also used in studying phylogenetics, genetic variation etc.
• Demerits:
  • The information obtained is low as compared to microsatellites and therefore large numbers of markers and complete genome sequencing is needed

References:

1. https://www.wur.nl/en/show/Random-Amplified-Polymorphic-DNA-RAPD.htm
2. https://www.ncbi.nlm.nih.gov/probe/docs/techrapd/
3. https://www.britannica.com/science/genetic-marker
4. https://www.news-medical.net/life-sciences/Restriction-Fragment-Length-Polymorphism-(RFLP)-Technique.aspx
5. https://www.ncbi.nlm.nih.gov/pmc/articles/PMC3513352/
6. https://www.wur.nl/en/show/Amplified-Fragment-Length-Polymorphism-AFLP.htm
7. https://www.researchgate.net/publication/257657433_Inter_Simple_Sequence_Repeat_ISSR_Polymorphism_and_Its_Application_in_Mulberry_Genome_Analysis
8. https://www.wur.nl/en/show/Sequence-Characterized-Amplified-Region-SCAR.htm
9. https://geneticeducation.co.in/different-type-of-genetic-markers/
10. https://www.researchgate.net/publication/307717534_Molecular_Markers_an_Introduction_and_Applications
11. https://link.springer.com/article/10.1023/A:1020691618797
12. https://www.ncbi.nlm.nih.gov/probe/docs/techcaps/

Molecular markers-types and applications

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• Induced mutations are induced by known factor- such as-physical (ionizing irradiation, ultraviolet light), chemical and biological mutagens (bacteria and viruses).

Mechanism of induced mutation:

• Induced mutations occurs by at least three different mechanisms. They are-
  • By replacing nitrogenous base with base analogs
  • By base alteration-altering a base so that it specifically mispair with another base
  • By distortion of DNA molecule- damaging a base so that it no longer pair with another base

I. Incorporation of base analogs:

• Some chemical compounds are sufficiently similar to the normal nitrogenous bases of DNA known as base analogs and they can incorporated into DNA in place of normal bases.
• These analogs can base pair with other nitrogenous bases but they induce insertion of incorrect nucleotide during replication causing mutation.
• Uracil is halogenated in the carbon-5 position to give 5-bromouracil, 5-chlorouracil, and 5-iodouracil which can be incorporated into DNA in the place of thymine.
• 5-bromouracil (5-BU) bromine is formed by bromination at the carbon-5 position of uracil. The resulting structure of 5-bromouracil is similar to thymine  but thymine has CH3 group at C5 .
• 5-Bromouracil is most effective analog to thymine because size of bromine has same van der Waals radius as the methyl group in thymine.
• 5-bromouracil is highly mutagenic and it pairs with Adenine in normal condition. In 5-BU, the bromine atom is not in a position in which it can hydrogen-bond during base pairing, so the keto form of 5-BU pairs with adenine.
• However the frequency of tautomeric shift of 5-bromouracil is much higher than Thymine. It changes from keto form to either enol form or an ionized from amino form to keto form when protonated. Now, 5-bromouracil form hydrogen bond with Guanine instead of complementary base Adenine which result in base pair transition from T=A to C=G in subsequent replication cycle.
• Another commonly used base is 2-Aminopurine which is analog to adenine and pairs with thymine by two hydrogen bond. After a tautomeric shift due to protonation, it can form pair with cytosine.
• Therefore, 2-aminopurine can induce base pair transition from A=T to G=C in subsequent replication cycle.

II. Base alteration: alkylation, depurination, deamination and hydroxylation

• In this case, the mutagens do not incorporate with the DNA, however they can causes base alteration in a specific way.
• Some of the base alteration are- alkylation, depurination, deamination, hydroxylation etc.

Alkylation:

• Alkylating agents are most widely used mutagens which carry one, two or more alkyl groups in reactive form.
• Alkylating agents transfer their alkyl group (methyl or ethyl) group to nitrogenous bases of DNA and to phosphate group (alkylation).
• The most commonly used alkylating agents are- ethyl-methane sulfonate (EMS), Dimethyl sulphonate (DMS), Diethyl sulphonate (DES) etc , nitrosoguanidine (NG)
• Mechanism of alkylating agents:
  • Transfer alkyl group to phosphate group of DNA and produces unstable phosphate trimester which hydrolyses to give alkyl group. But some alkyl group may remain attached to phosphate which interfere with DNA replication cycle as well as cause breakage of sugar phosphate back bone.
  • Transfer alkyl group to 6-oxygen and 7-nitrogen of DNA bases. Among these alkylation of nitrogenous base, 7-ethylguanine is most common derivative. 7-ethylguanine is base analog to cytosine and can form base pair with thymine which ultimately cause base transition from G=C to AT.
  • Some difunctional or polyfunctional alkylating agents can crosslink with DNA strand and interfere DNA replication leading to chromosome breakage.

Depurination:

• Alkylation of purine base (Adenine and Guanine) give rise to unstable quaternary nitrogenous base which interfere with glycosidic bond between nitrogenous base and deoxyribose sugar.
• The loss of purine bases from DNA is termed as Depurination.
• Once the base is depurinated, it cannot specify complimentary base to the original purine during replication which result in incorporation of wrong base.
• Therefore, this causes base pair substitution.

Deamination:

• Some mutagens react with nitrogenous base containing amino group and remove it with another functional group.
• Nitrous acid (HNO2) a chemical which reacts with amino group containing nitrogenous bases (Adenine, cytosine and guanine) and replace it.
• Deamination of adenine by nitrous acid yields hypoxanthine (H) which can base pair with cytosine. This deamination at the position of adenine result in base transition from A:T to G:C in successive replication.
• Similarly, deamination of cytosine by HNO2 results in uracil. Now uracil can base pair with adenine. Therefore, change of base from cytosine to uracil results in base transition from G:C to A:U and then A:T in successive replication cycle.
• Other example of deamination: conversion of 5-methylcytosine to thymine.

Hydroxylation:

• The mutagen hydroxylamine (NH2OH) reacts with amino group of cytosine causing hydroxylation to yield hydroxylcytosine.
• Now the hydroxylcytosine can form base pair with adenine instead of guanine. This results in base pair transition.

III. Distortion of DNA molecule:

• Some of the mutagens directly reacts with DNA molecules causing distortion and modify the structure.

i. Intercalating agents:

• Certain florescent acridine dyes such as acridine orange, proflavin causes DNA mutation by insertion or deletion of nitrogenous bases.
• Acridine dyes are planar (flat) molecule that mimic nitrogenous bases and at low concentration it can inserts or intercalates between subsequent nitrogenous bases in DNA molecule.
• Insertion of the agent stretches the distance between adjacent base pair by 0.68nm which is twice the normal distance. This distortion result in single nucleotide deletion or insertion at this position during recombination.
• Intercalating agents result in insertion of bases:
  • Intercalation of acridine dye between two nitrogenous bases in template strand result in stretch of DNA molecule.
  • During DNA replication, a new base can be inserted in the newly synthesized strand opposite of acridine molecule.
  • Now during replication of newly synthesized strand, a complimentary base in added opposite to newly added base.
• Intercalating agents result in deletion of bases:
  • Insertion of acridine molecule in DNA strand may block the base in the template strand and does not allow to base pair.
  • During replication, one base is deficient in newly synthesized strand.

ii. DNA damage: by radiation

• Various kinds of radiations can induce mutation. Mutagenic radiations are two types- non-ionizing radiation (UV rays) and ionizing radiation (X-rays, gamma rays):

a. Mutation caused by UV rays: Pyrimidine dimer formation

• UV rays have a wavelength of 10-390nm.
• When UV rays falls on the genetic materials, it is absorbed and due to increase in energy level electrons are excited.
• The most effective wavelength of UV rays for inducing mutation is 260nm.
• UV rays are non-ionizing radiation and have a penetration power lower than X-rays.
• Exposure of DNA to UV rays causes nitrogenous bases to become highly reactively unstable free radicals.
• One of the consequences of UV rays exposure is photochemical fusion of two pyrimidine that are adjacent to each other on a same polynucleotide chain.
• Pyrimidine dimer formation is the primary effect of UV rays on DNA molecule.
• In case of two thymine, the fusion is called thymine dimer. UV rays causes adjacent thymine on the same DNA strand to bond together by covalent linkage between carbon 5 and 6 of adjacent thymine molecule forming cyclobutane ring.
• In case of thymine adjacent to a cytosine, the resulting fusion is a thymine-cytosine dimer in which the thymine is linked via its carbon atom 6 to the carbon atom 4 of cytosine.
• This dimer structure cannot fit into DNA helix and cause distortion of DNA molecule which result in failure of DNA replication and sometime leads to lethal effect.
• Pyrimidine dimer can also formed by cross linking of pyrimidine bases of adjacent DNA strand.
• There are three types of thymine dimers
• Thymine-thymine dimer= 50%
• Thymine-cytosine dimer= 40%
• Cytosine-cytosine dimer=10%

b. Mutation caused by Ionizing radiation (IR):

• The effects of different ionizing radiation are qualitatively the same.
• In direct effect, IR breaks the phosphate ester bond in DNA. The breakage may take place at one or more points. As a result of breakage, segment of DNA either get lost or rearranged during repair. Sometimes the effect is fatal.
• In indirect effect, the ionizing radiation ionize water producing free radicals which is extremely reactive.
• The free radicals react with DNA molecule to alter its structure.

Mechanism of induced mutation: base analogs, base alteration, intercalation, pyrimidine dimer formation

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Types of transposons in eukaryotes:

I. Class1: Retro-transposons:

• Examples: Ty elements in yeast, copia elements in Drosophila, LINEs and SINEs sequences in humans.

II. Class2: DNA transposons:

• Example: P element in Drosophila
• These are cut and paste transposons; similar to bacterial transposons

I. Class 1: Retro-transposons

• Retro-transposons are those transposable elements that utilize reverse transcriptase enzyme to convert RNA intermediate into DNA and then transpose into new position by itself. 
• They are of two types on the basis of Long terminal repeats (LTR)

1. LTR retro-transposons or retrovirus like element:

• All LTR retro-transposons have the same basic structure: a central coding region flanked by long terminal repeats (LTR) which are oriented in the same direction.
• The LTR are typically a few hundred nucleotide pairs long sequence having short inverted repeats at both ends like present in other types of transposons.
• Due to the characteristic LTR sequence which is similar to that of retrovirus, these transposons are known as LTR retrotransposons.
• The property of LTR retroposons is similar to retrovirus infection.
• LTR retrotransposons utilizes reverse transcriptase enzyme from retro virus that convert transposons RNA into DNA in the first stage and in in second stage the synthesized DNA is transposed into new location in the chromosome.
• Examples:  Ty elements of yeast, and copia elements of Drosophila 

Ty element in yeast

• Ty element is a LTR retro-transposons present in yeasts which is about 5.9 kbp long sequence having LTRs of about 340 base pairs long at both end.  
• Most yeast strains have about 35 copies of the Ty1 element in their genome.
• Ty1 elements have only two genes, TyA and TyB, which are homologous to the gag and pol genes of the retroviruses.
• Transposition of Ty1 element involves reverse transcriptase enzyme.
• After RNA is synthesized from Ty1 DNA, a reverse transcriptase encoded by the TyB gene uses it as a template to make double stranded DNA.
• Then the newly synthesized DNA is transported to the nucleus and inserted in targeted site in the genome, creating a new Ty1 element

2. Non-LTR retrotransposons (retroposons):

• The non-LTR retrotransposons are common retrotransposons present in eukaryotes.
• They are simply known as retroposons or non viral transposons
• Non-LTR retrotransposons lacks long terminal repeats (LTRs) at the terminal end. However, they have homogeneous sequence of A:T base pairs at one end which is derived from post transcriptional modification (poly A tail).
• These non-LTR retrotransposons are transposed via RNA intermediate which is reversed transcribed into double stranded DNA by reverse transcriptase enzyme encoded by the element itself and then transposed to the targeted site.
• Examples: Transposable element of human
  • LINEs (long interspersed nuclear elements)
  • SINEs (short interspersed nuclear elements)

Transposable element of human:

• At least 44 percent of human genome is repetitive which is derived from transposable elements, including retroviruslike elements (8%), retroposons (33 %), and several families of elements that transpose by a cut-and-paste mechanism (3 %)
• Class of human transposons:
• i.  LINEs
• ii. SINEs

Long Interspersed nuclear elements (LINEs):

• L1 retroposon is the principal human transposable element which belongs to long interspersed nuclear elements (LINEs) class.
• Two other LINEs sequences, L2 and L3 LINEs are also present in human genome, however both are transpositionally inactive.
• Complete L1 element is about 6 kb long sequence with an internal promoter that is recognized by RNA polymerase II, and two open reading frames (ORFs);
• ORF1 encodes a nucleic acid-binding protein,
• ORF2 encodes a protein with endonuclease and reverse transcriptase activities.
• The human genome contains about 3000 to 5000 copies of complete L1elements.

• Transposition of L1 element involves transcription of a complete L1 element into RNA in the first step and the reverse transcription of this RNA into DNA in next step.
• Both transcription and reverse transcription processes take place in the nucleus. However, before the L1 RNA is reverse transcribed, it travels to the cytoplasm where it is translated into polypeptides (nucleic acid binding proteins, endonuclease and reverse transcriptase).
• The polypeptides remain attached with RNA when it returns to the nucleus.
• The polypeptide encoded by ORF2 possesses an endonuclease activities that catalyzes cleavage of one strand of the DNA duplex at targeted site in a chromosome.
• The polyA tail of polypeptide juxtaposed with 5’end of cleaved DNA and the reverse transcriptase activity results into synthesis of single DNA strand.
• Utilizing 3’ end as a primer for DNA synthesis, the newly synthesized L1 DNA is subsequently made double-stranded. Therefore, new L1 element is placed in new location.

Short interspersed nuclear elements (SINEs):

• The short interspersed nuclear elements (SINES), are the second most abundant class of transposable elements in the human genome.
• SINEs are less than 400 base pairs long and have an internal promoter but do not encode proteins.
• Like all retroposons, SINEs do not have terminal repeats but instead they have a sequence of A:T base pairs at one end.
• Transposition of SINEs involves transcription of a SINEs DNA into RNA which is then reversed transcribed into DNA.
• However, SINEs do not have any open reading frame to encode any enzymes.  Therefore, it depends upon LINEs-type element for reverse transcriptase enzyme and other enzymes.
• Thus, the SINEs in depend on the LINEs to multiply and insert within the genome.
• Three families of SINEs are present in Human genome- Alu, MIR, and Ther2/MIR3 elements.
•  However, only the Alu elements is transpositionally active

II. Class 2: DNA Transposons

• Some mobile elements found in eukaryotes are DNA transposons and the mechanism of their transposition is similar to those of bacteria.
• The first transposable elements discovered by McClintock in maize are now known to be DNA transposons. However, the first DNA transposons to be molecularly characterized were the P elements in Drosophila.

P element in Drosophila:

• P element present in Drosophila resembles to Insertion sequence (IS element) of bacteria, which possesses a short inverted terminal repeats at both end and a single open reading frame which encode single protein (transposase).
• The P elements vary in size, ranging from 0.5 to 2.9 kb in length.
• P elements were discovered by Margaret Kidwell, who was studying hybrid dysgenesis

Dysgenesis in Drosophila:

• A phenomenon known as dysgenesis is present in Drosophila when laboratory reared female flies were crossed with wild male flies.
• In such crosses, the laboratory stocks are said to possess an M cytotype (cell type), and the natural stocks are said to possess a P cytotype. Laboratory flies do not have P element.
• In such cross of M (female) ×P (male), the F1 progeny show a range of surprising phenotypes characters in germ line including sterility, a high mutation rate, and a high frequency of chromosomal aberration and nondisjunction.
• These F1 hybrid progeny are biologically deficient and are known as dysgenic. The phenomenon is called dysgenesis
• However in reciprocal cross between P (female) × M (male), no dysgenesis is observed in F1 progeny.
• To explain the phenomenon of dysgenesis, the simple answer is that P-element transposition. In all adult P cytotype flies, they possess P-element which contains a transposase genes are silenced. However, the genes are activated in the F1generation resulting in dysgenesis when crossed M (female) with P (male)
• In the cross between M cytotype (female flies, no P elements) × P cytotype (male flies, P elements), each parent contribute respective gametes with respective cytotype. Male gamete provide P elements in the newly formed zygote which is in a silencing-free environment, since female gamete is M cytotype. The P elements derived from the male genome can now transpose throughout the zygote genome, causing a variety of damage as they insert into genes and all F1 progeny developed from such zygote are dysgenic.
• On the other hand, in reciprocal cross between P cytotype (female flies, P element) ×M cytotype (male flies, no P-element) no dysgenesis is observed. It is because, in this case female gamete provide P element in the newly formed zygote but the P-element is already silenced in female gamete due to presence of some component in cytoplasm of female gamate. Therefore Zygote developed from such gamete with already silenced P element prevent dysgenesis.

Transposable elements in eukaryotes

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1. Insertion sequences (IS elements)
2. Prokaryotic Transposons (Tn): Composite and non-composite transposons

1. Insertion sequences (IS element):

• IS elements are the simplest type of bacterial transposable sequences that can insert at different location of bacterial chromosome and plasmid through illegitimate recombination.
• They are typically short sequences and contains only one gene that encode the enzyme for transposition.
• IS elements were first identified as spontaneous insertion in certain lac operon mutations of E. coli which inactivate the gene and inhibit transcription and translation. The mutation of Lac operon gene was found to be unstable and molecular analysis reveals the presence of extra copies of DNA sequences near the lac gene. When the mutated E. coli undergoes reverse mutation, the extra DNA sequence is lost.
• A bacterial chromosome may contain several copies of a particular type of IS element. For example, 6 to 10 copies of IS1 are found in the E. coli chromosome.
• Plasmids may also contain IS elements

Characteristics of Insertion sequences (IS element):

• IS elements are compactly organized and containing about 1000 nucleotide pairs and contain only genes (open reading frame) which encode for enzyme for regulating transposition.
• Many distinct types of IS elements have been identified. The smallest IS element is IS1 which is 768 nucleotide pairs long.
• Each type of IS element contains inverted terminal repeats at both end and a transposon sequence in between those inverted repeats. Transposon is the only gene that code for transposition of IS element.
• The inverted terminal repeats is 9-40 base pair long and is the characteristics of most IS element
• IS element have the capacity to duplicate the inserted sequence at the site of insertion; known as target site duplication.

Transposition of insertion sequence in bacteria:

• IS element contains single open reading frame (ORF) which encodes for the enzyme transposase, catalyzing its own transposition.
• The enzyme transposase is like restriction endonuclease which binds to terminal inverted repeats (IR) of IS element which is the restriction site. Then the enzyme cut and excise IS elements from chromosome or plasmid.
• The excised IS element is mobile in nature and moves along the length of chromosome to recognizes the target site for insertion on same or different chromosome or plasmid.
• Once recognizing the target site, it generate staggered cleavage (cut the single strand of DNA) generating sticky and itself get inserted.
• As IS element get inserted, the proofreading mechanism of DNA results in duplication of the DNA sequence at the target site of the insertion such that one copy of target DNA is located on each side of IS element.
• Thus IS elements helps in target site duplication

2. Prokaryotic Transposons (Tn):

• Prokaryotic Transposons are similar to IS element but they are larger and also contains other genes (mostly antibiotic resistance gene) in addition to gene that encode transposase.
• Transposons are several thousand base pairs long and contains inverted terminal repeats.
• There are two types of prokaryotic transposons- composite and non-composite transposons.
• The composite transposons and Tn3-like elements are more complex than IS elements, containing some genes that encode products unrelated to the transposition process.

i. Composite transposons:

• Composite transposon are created when two IS elements insert near each other and the region between the two IS elements can then be transposed when the elements act jointly.
• For example: Tn10 is a composite transposons of 9.3kbp which contains 1.4 kbp terminal inverted repeats and in between them is gene for transposase and gene for antibiotic resistance.

ii. Non-composite transposons:

• The non-composite transposons is a sequence of DNA containing gene for trasnposase and multiple other gene in between terminal inverted repeats.
• Unlike composite transposons, it does not contains IS elements at each end but instead it contains simple inverted repeats 0f 38-40 nucleotide pairs at each end.
• For example; Tn3 is a non-composite transposons of 5kbp which contains three gene for beta-lactamase (bla), transposase (tnpA) and resolvase (tnpB).
• The beta lactamase provide resistance to the antibiotic ampicillin, and the other two enzymes play important roles in transposition and recombination.

Role of bacterial transposons in antibiotic resistance:

• As many bacterial transposons also carry genes for antibiotic resistance apart from gene for transposase and by the nature of transposable element, the genes move or translocate from one DNA to another and from chromosome to plasmid and vice versa. This results in genetic flux of antibiotic resistance genes in bacterial population.
• When the transposons undergoes recombination with plasmid vector within a bacterial cell then it can be transformed horizontally or vertically to other bacteria, spreading the drug resistance gene in bacterial population. This creates multidrug resistance pathogenic bacteria such that diseases become difficult to control.

Transposable elements in Prokaryotes (Bacteria)

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Characteristics of transposable elements:

• Transposable elements are specific sequence of DNA that codes for enzymes for their transposition from one position to another position in the chromosome.
• Such mobile transposable elements is commonly known as transposons or jumping genes and the phenomenon is known as transposition.
• Transposable elements undergoes both replication and recombination process. However, they lacks the site for origin of replication. So, they have to depend upon host DNA or plasmid for their replication.
• All transposable elements are commonly known as transposons or mobile gene or jumping genes that changes their location and insert into different location in a chromosome or into other chromosome.
• Transposable elements contributes to the repetitive sequences in the genome of the organisms. Human genome contains 50% repetitive sequences.
• Both prokaryotic and eukaryotic organisms such as; bacteria, fungi, plants, animals as well as humans have transposable elements in their genome and they have their significant structure and functional value.
• The transposable elements are broadly classified as- Insertion sequences (IS) and transposons.
• Insertion sequences are the simplest type of transposable element with short sequences of about 1000 base pairs, present in Bacteria.
• Transposons have the longer sequences of more than several thousand base pairs.

Mechanism of transposition:

• There are different type of transposable elements depending upon structure and mechanism. On this basis, there are three different mechanism of transposition (Replicative, conservative and Retro-transposition). Most prokaryotes and eukaryotes employed one of two-either replicative or conservative mechanism of transposition whereas retro-transposition is only employed by eukaryotes.
• Three types:
• Replicative transposition
• Conservative or non-replicative transposition
•  Retro-transposition

I. Replicative transposition or copy paste transposition:

• Replicative transposons are those transposons which at first replicates itself and then insert one copy of it into new position. Therefore, during replicative transposition, the transposable elements is replicated and one copy is inserted into new position while one copy remains at original position.
• This transposition is catalyzed by an enzyme transposase which is encoded by the transposable element itself.
• It is also known as copy and paste transposition.
• Replicative transposition is present only in prokaryotic organisms.
• Example: Non-composite transposons (Tn3) is a replicative transposons that undergoes transposition in two stage process.  In the first stage, two plasmid- (one containing Tn3 transposons; donor plasmid) and the other recipient plasmid undergoes fusion catalyzed by transposase enzymes giving rise to a structure called cointegrate. During the formation cointegrate, Tn3 is replicated, and one copy is inserted at each point where the two plasmids have fused. In the second stage of transposition, the tnpR-encoded enzyme resolvase which mediates a site-specific recombination between the two Tn3 copies at the resolution site, and when it is completed, cointegrate is resolved into its two constituent plasmids, each with a copy of Tn3.

II. Conservative or non-replicative: cut and paste transposition

• The conservative transposons are those which changes position by excision from one position and then insertion into another position within a chromosomes.  Therefore, during conservative transposition, the transposable element is physically cut from its original position and paste into another position in a chromosome.
• It is also known as cut and paste transposition
• This types of transposition is catalyzed by an enzyme called transposase which is coded by such transposable element itself.
• Conservative transposition is present in both prokaryotic and eukaryotic organisms

III. Retro-transposons:

• The retro-transposons are those transposons, whose RNA is reversed transcribed by reverse transcriptase enzyme into DNA and the synthesized DNA is inserted into new position in a chromosome.
• The mechanism of such transposition is known as retro-transposition
• Some of these retro-transposons are related to retro viruses and utilized their reverse transcriptase enzyme for transposition, such transposable elements are known as retroposons.
• Retro-transposons are only present in Eukaryotic organisms.

Transposable elements: characteristics and mechanisms of transposition

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• All the situations and examples discussed with inheritance of character according to Mendel’s law are inheritance of genes situated on different chromosomes.
• Cytological study have reveals that human possess 46 chromosomes in all somatic cells (Vegetative cells). Since human possess thousands of characters such as blood group, eye color, insulin production etc, it follows that each chromosome must carry a large number of genes.
• Genes situated on same chromosome are said to be linked. All genes on a single chromosome form a linkage group and usually pass into same gamete and are inherited together.
• According to Mendel’s law of independent assortment, the genes situated in different chromosomes assort and appear independently in next generation either appear together or apart.
• But if the genes are situated in the same chromosome and are fairly close to each other, they tend to be inherited together. This type of inheritance of linked genes in the same chromosome is known as Linkage.
• As a result of linkage, genes belonging to the same linkage group usually do not show independent assortment. Since these genes do not conform to Mendel’s principle of independent assortment they fail to produce the expected 9:3:3:1 ratio in a breeding situation involving the inheritance of two pairs of contrasting characters (Dihybrid cross). In linkage, a variety of ratios are produced.

In most breeding experiments involving linkage produces approximately equal numbers of parental phenotypes and a significant smaller number of recombinant phenotypes (new combination of characters) in equal numbers.

Therefore, two genes are said to be linked when phenotypes with recombinant characters occur less frequently than the parental characters.

• The theory of chromosomal inheritance of linked gene was proposed by T. H Morgan in 1911 as experiments shows linked genes in a same chromosome tend to remain in their original combinations.
• Before Morgan, W. Sutton and T. Boveri 1902), Sutton (1903) and Bateson and Punnett (1906) had put forward some hypothesis about the phenomenon of linkage.

Coupling and Repulsion hypothesis of linkage: by Bateson and Punnett

• Bateson and Punnett in 1906 described a cross in sweat pea (Latharus odoratus), where they find gene pairs do not assort independently as expected.
• While they were working on sweet pea, they noticed two variety of plant-plant with Blue flowers (BB) with long pollen grains (LL) and red flower (bb) with round pollen grain (ll).
• When the sweat pea variety having blue flower and long pollen grain (BBLL) were crossed with those of another variety having red flower and round pollen (bbll).
• In F1 generation, all the offspring having blue flower and long pollen (BbLl) were observed as expected. Because, blue color is dominant over red and long pollen is dominant over round.
• When these F1 offspring (BbLl) were test crossed with red flower and round pollen variety (bbll), they obtained (BbLl), (Bbll), (bbLl), (bbll) in the ration 7:1:1:7 instead of 1:1:1:1.
• This indicates that the dominant characters (Blue color and long pollen) tends to remain together. Similarly, the recessive characters (red color and round pollen) tends to remain together. This deviation or phenomenon is explained by Coupling and Repulsion hypothesis by Bateson and Punnett.
• Bateson and punnett conclude that- the pairs of genes from homozygous parents try to remains together in a cross due to which parental characters is more frequent. This is due to coupling. Two dominant gene for Blue flower and long pollen (BL) are linked in one plant while in other plant two recessive gene for red flower and round pollen (bl) are linked. Such linkage is called coupling.
• In another experiment, when a cross was made between sweet pea variety having blue flower and round pollen (BBll) with another variety having red flower and long pollen (bbLL)
• In F1 generation, all offspring with blue flower and long pollen were obtained (BbLl).
• When these F1 offspring (BbLl) were test crossed with red flower and round pollen variety (bbll), they obtained (BbLl), (Bbll), (bbLl), (bbll) in the ratio 1:7:7:1 instead of 1:1:1:1.
• It was observed in both experimental cases that plants with parental characters are obtained in excess number than the plants with recombinant characters. Also when dominant alleles are from different parents (heterozygous ) the frequency of recombination is more because of repulsion of gene (repulsion).
• In First experiment, BL are linked and such linkage is known as Coupling and in second experiment bL are linked and such linkage is known as Repulsion.
• The coupling and repulsion hypothesis is now discarded.

Chromosomal theory of linkage: by T. H Morgan

• Bateson and Punnett failed to explain the exact reasons of coupling and repulsion
• Later, T.H. Morgan who found coupling and repulsion hypothesis incomplete, while performing experiments with Drosophila, in 1910. Therefore, he proposed that the two genes are found in coupling phase because they are present on same chromosome and similarly on repulsion phase because they are preset on two different homologous chromosomes. There genes are then called linked genes and the phenomenon of inheritance of such linked genes is called linkage by Morgan.
• And the term coupling and repulsion were replaced by the terms, cis and trans by (Haldane, 1942).
• Morgan stated the linked genes have the tendency to remain together in original combination because they are located on same chromosome. And the strength of linkage depends upon the distance between the linked genes in the chromosome.
• The concept of linkage by Morgan establish the foundation of Cytogenetics and develop the theory of linear arrangement of genes in the chromosomes and helps to construct genetic map of the chromosome.
• According to Chromosomal theory of linkage:
• Chromosome contains genes and Genes lie in a linear order in a chromosome and distance between them is variable.
  • Each gene has a definite locus in a chromosome. The genes which are close to each other, shows the phenomenon of linkage
  • The linked genes cannot be separated during gametogenesis (inheritance process),  they inherited together
  • Tendency of genes to remain linked is due to their presence on same chromosome
  • The distance between the linked genes determines the strength of linkage. The closer the distance stronger is the linkage strength.
  • The linkage is not due to any relation between two genes but is simply because they happens to be located in the same chromosome

Linkage group:

• Genes situated on a chromosome are linked and all the genes on a single chromosome forms a linkage group. The gene located in different chromosome are unlinked genes.
• Usually linkage group as a whole passes into gamete during gametogenesis and are inherited together.
• The number of linkage group is equal to total number of chromosome in a cell. But actual number is restricted to haploid number of chromosome
• Human has 23 pairs of chromosomes and 23 linkage groups.
  • Male: 22 autosome + X-chromosome + Y chromosome= 24 linkage group
•  Drosophila has 4 pairs of chromosomes and 4 linkage groups.
• Maize (Zea mays) has 10 pairs of chromosomes and 10 linkage groups.

 Types of Linkage:

There are two types of linkage; complete linkage and incomplete linkage

Complete Linkage:

• When genes are very closely associated to each other, they tends to remains together and have no chance of separation from each other during inheritance. These genes are called linked genes and the phenomenon is known as complete linkage.
• In complete linkage the parental combination of characters appear together for two or more generations in a continuous and regular fashion.

Examples:

• Red eyed Normal winged Female Drosophila (RRWW) crossed with purple eyed vestigial wing male Drosophila (rrww).
• In F1 progeny, all the Drosophila obtained were heterozygous with red eyed normal winged (RrWw)
• When F1 males (RrWw) were test crossed to homozygous recessive female (rrww), only two types of F2 progeny individuals were produced (one type with red eyed normal winged (RrWw) and other type with purple eyed vestigial winged (rrww) in ratio 1:1 ).
• Recombinant type are absent. Frequency of recombination is Zero
• This is due to complete linkage between gene for eye color and wings. The F1 males produced only two types of gametes ie (RW) and (rw) instead of all possible four types of gametes.
• In most of the organism, crossing over takes places during gametogenesis. But in male Drosophila and in female Silkworm, the frequesncy of crossing over is either very area or not at all.
• Because of this, linked genes remains together during inheritance, showing complete linkage phenomenon.
• However, in practice, total linkage is rare.
• When F1 males were crossed with F1 females, the ratio would be 3:1 of parental characters. However, in practice 3:1 ratio never achieve, this is because total linkage is rare.

Incomplete Linkage

• The linked genes which are located at farther distance in a chromosome, do not always remains together and separate during gametogenesis are said to be incompletely linked gene and the phenomenon is known as Incomplete linkage.
• These linked genes separates because of crossing over between the homologous non-sister chromatids during pachytene stage of Prophase-I of meiosis.
• The incomplete linkage have been reported in various organisms including female Drosophila.

Examples:

• When dominant sweat pea homozygous for blue flower and long pollen (BBLL) cross with double recessive homozygous for red flower and round pollen (bbll).
• In F1 generation all the progeny plants obtained hybrid plant (heterozygous) for blue flower and long pollen (BbLl).
• When F1 hybrids weretest crossed with doule recessive parent (bbll),  all four types of progeny (BbLl, BbLl, bbLl and bbll) were obtained in F2 generation in the ratio 7:1:1:7 instead of 1:1:1:1.
• The parental phenotype ie blue flower-long pollen (BbLl) and red flower-round pollen (bbll) accounts for 87.4% whereas the recombinant phenotype ie. blue flower-round pollen (Bbll) and red flower-long pollen (bbLl) accounts for only 12.6%.
• This experiment shows that, the genes for color of flower and shape of pollen are linked genes and located at a distant position in a chromosome. During gametogenesis these linked genes tends to remains together, however being distant and due to crossing over they separated.

Genetic Linkage-Introduction, types, and chromosomal theory of linkage

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• Meiosis division occurs during gametogenesis in higher organism (eukaryotic cells).  The events of meiosis of a cell includes duplication of chromosomes which is followed by one reductional division and other equational division resulting into four haploid cells, each containing half the number of chromosomes.
• Meiosis consists of two rounds of chromosome segregation following a single replication.
• The phases of meiosis is divided into two; meiosis-I and meiosis-II.
• Before the onset of meiotic division, the chromosomes of the pre-gametic cell are replicated to yield two pairs of sister chromatids after which they enter a long meiotic prophase interval.
• This prophase-I is divided into five distinct stages which are termed as (a) leptotene, (b) zygotene, (c) pachytene, (d) diplotene, and (e) diakinesis
• In leptotene stage, the chromosomes condense along their axial elements.
• During the zygotene stage, the parental homologous chromosomes pair with the help of a structure known as the Synaptonemal complex (SC). These Synaptonemal complex have a tripartite structure, comprising a central element bounded by two lateral elements.
• The pachytene is the longest stage of the meiotic prophase, during which crossing over and genetic recombination takes place between the paired homologous chromosomes.
• In diplotene and diakinesis stages, nuclear membrane disappears and spindle fibres formations begins.
• Prophase I: chromosomes condense, undergo duplication and crossing over, and centrioles move to the poles
• Metaphase I: microtubules connect from the centrioles to the centromeres, and the paired chromosomes line up along the equator of the cell
• Anaphase I: paired chromosomes separate and are pulled to alternate poles
• Telophase I: chromosomes decondense, nuclear membrane and nucleolus forms, centrioles move back together
•  Prophase II: chromosomes condense and centrioles move to the poles
• Metaphase II: microtubules connect from the centrioles to the centromeres, and the paired chromatids line up along the equator of the cell
• Anaphase II: paired chromatids separate and are pulled to alternate poles
• Telophase II: chromosomes decondense, nuclear membrane and nucleolus forms, centrioles move back together.
• With the separation of the parental chromosome pairs in the subsequent stages, the first meiotic division results in the reduction of chromosomes by half, which is followed by another division, analogous to mitosis to produce haploid gametes.
• During meiosis, replicated homologous chromosomes pair and engage in recombination and then segregate to opposite poles. Sister chromatids are separated at meiosis II, and four recombined haploid gametes are formed.

Cytological basis of segregation of alleles:

• The cytological basis of segregation of alleles can be explained if we take the example of an individual which possesses two allelomorphs ‘A’ and ‘a’ on two different homologous chromosomes i.e., A on one chromosome and a on the second chromosome.
• In the prophase stage the two chromosomes will pair and duplicate
• In meiosis-I, allele Aand allele a from homologouschromosome will segregate or separate from each other.
• In the meiosis-II, the two chromatids of A chromosomes and those of a chromosome separate. Thus it becomes clear that both alleles of heterozygote separate during meiosis (gametogenesis) to produce gametes with only one chromosome with either dominant allele or recessive allele.
• No gamete will have both the genes and chromosomes of an allelic pair.

Historically, there have been three general approaches for the study of meiosis,

1. Morphological and cytological approaches
2. Genetic approaches:
3. Biochemical approaches: Characterization of the biochemical processes of meiosis.

1. Morphological and cytological approach:

• In this approach, morphological and cytological investigation of dividing cell was carried out and from these studies it was clear that chromosome behaves in certain ways during meiosis.
• From the behavior study of chromosome during prophase stage, it was found that chromosome comes together forming homologous pair which is brought together by a protein called synaptonemal complex.
• So, it is now very clear that these protein structures are an integral part of meiosis all living organisms.

2. Genetic approach:

• In genetic approach, from the study of several mutants such as yeast, Neurospora etc, it was found that several genes are responsible for meiosis process.

3. Biochemical approach:

• In this approach of characterizing biochemical processes in cell during meiosis, it was found that several enzymes and protein are responsible for chromosome pairing as well as DNA replication and other events.
• This approaches helps to characterized the role of enzymes and protein in DNA synthesis, chromosome pairing and in crossing over (recombination).

Physical behaviour of chromosomes during meiosis

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