• A mutation is a change in the amount, arrangement or structure of DNA of an organism. This produces a change in genotype which may be inherited by cells derived by mitosis or meiosis from the mutated cell. A mutation may results in the change in appearance of a characteristics in a population.
• Mutation occurring in a gamete are inherited in offspring, whereas those occurring in somatic cells can only be inherited by daughter cells produced by mitosis. Therefore, mutation can be germ-line and somatic.
• A mutation resulting from a change in the amount or arrangement of DNA or chromosome is known as chromosomal mutation or chromosomal aberration whereas mutation of DNA at a single locus is termed as gene mutation or point mutation
• Mutation involving either entire chromosome or large portions of chromosome are referred as chromosomal mutation. Such mutation in chromosome is caused by either by inheritance of extra fragment/whole chromosome or loss of fragment/whole chromosome or by rearrangement of genes.

Types of chromosomal mutation:

There are two categories of chromosomal mutation

1. Chromosomal aberration:
   • The chromosomal mutation that involves changes in gross morphology or architecture of chromosome known as chromosomal aberration.
   • This is the structural abnormality of chromosome.
   • The chromosome generally remains unchanged but during certain condition leading to mutation causes certain changes in structure of chromosome which may result in loss or gain of genes or rearrangement of position of genes.
   • Variation in chromosomal structure are due to- deletion, duplication, translocation and inversion
2. Numerical aberration of chromosome:
   • The chromosomal mutation which result from the change in number of chromosome known as numerical aberration of chromosome.
   • This is the phenomenon of ploidy

Chromosomal mutation-chromosomal aberration (structural mutation) and genomatic mutation (numerical aberration)

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• Genomatic mutation is a chromosomal mutation in which the numerical changes in chromosomes or variation in chromosomal numbers occurs, known as numerical chromosomal aberration. The variation in chromosome numbers is due to error in meiosis and mitosis. The measure of number of chromosome in a cell is called called ploidy.
• The minimum number of chromosome which function as a harmonious and integrated unit is called basic chromosomal number.
• The chromosome number present in gamete formed by diploid cell is called haploid (n). The chromosome occurs in pair in somatic cell called diploid (2n). For eg. In Human 23 pairs of chromosome is present, which is the basic chromosomal number.
• The chromosomal number is maintained from generation to generation in a species, however, certain mutation causes change in chromosomal number in somatic cell. This condition is called ploidy.
• The change in chromosome number involves- addition or deletion of individual chromosome or of a complete set of chromosome.

Types of ploidy:

Changes in chromosome number are of two basic types: changes in one or more chromosome number in a set, resulting in a condition called aneuploidy and changes in whole chromosome sets, resulting in a condition called aberrant euploidy.

1. Aneuploidy
2. Euploidy

Aneuploidy:

• It is the condition in which one or more chromosome is added or deleted from basic chromosomal number in a diploid cell.
• In aneuploidy, the chromosome number is not multiple of basic chromosomal number.
• The organism with such abnormal conditions are called aneuploids or heteroploids.

There are two class of aneuploidy

• Hyperploid: addition of one or more chromosome to a diploid set
• Hypoploid: deletion of one or more chromosome from a diploid set

Causes of aneuploidy

• i). Non-disjunction:
  • It is the condition in which one or more pairs of chromosome (bivalent chromosome) fails to separate during anaphase of meiosis-I.
  • Because of irregular distribution of chromosome at poles, one daughter cell receives one or more extra chromosome whereas other daughter cell lacks one or more chromosome and they form respective gametes.
  • When the gametes having extra chromosome fuse with normal haploid gametes, it result in hyperploids.
  • And when the gamete lacking one or more chromosome fuse with normal gamete, it result in hypoploids.
• ii). Non orientation of one or more bivalent at metaphase-I of meiosis-I
• iii). Loss of individual chromosome in meiosis or mitosis
• iv). Irregularities in segregation of chromosome during meiosis in polyploidy condition also results in aneuploidy
• v). Multipolar mitosis with irregular distribution of chromosome to daughter cell

Types of aneuploidy

i. Monosomics: 2n-1

• It is the result of loss of one copy of chromosome from a diploid complement set.
• It’s chromosome number is represented by 2n-1
• A diploid cell missing a single chromosome is monosomic.  And if a cell misses two nonhomologous chromosomes, it is called double monosomic.
• In most diploid organisms, loss of one chromosome copy from a pair is deleterious.
• In humans, monosomics condition in any autosomes are fatal. And also, monosomic in X-chromosome are fatal, however, few viable cases are present. Eg. Turner syndrome
• Example: Turner’s syndrome: (44+X)
  • It occurs when an abnormal egg (O) fuse with normal sperm (X). The individuals have 45 chromosome (44 autosome and one X).
  • The affected individual is sterile female with under-developed breasts, reduced ovaries, short stature, and often have a web of skin extending between the neck and shoulders lacks menstrual cycle and few male like characters

ii. Nullisomic: 2n-2

• It is the result of loss of a pair of chromosome from diploid set.
• In this case, a diploid organism lacks a pair of homologous chromosome.
• It’s chromosomal number is represented by 2n-2
• It is generally lethal in an organism.
• Example: In wheat, they can tolerate a nullisomic mutation

iii. Trisomic: 2n+1

• An organism containing one extra chromosome in addition to diploid set.
• In normal meiosis-I, chromosome pair of bivalent separates and goes to each of the daughter nuclei. But very rarely, one pair of chromosome fails to disjoin and finally it moves to one pole, so half of the daughter cell receive extra chromosome and other half of daughter cell lose one. Such that (n+1) and (n-1) gametes are formed.
• When n+1 gamete fuse with normal gamete, it gives trisomic organism.
• Example: Down syndrome; trisomy 21
  • It is due to an extra chromosome number 21.
  • The individual with Down syndrome have 47 chromosome.
  • The disorder is characterized by mental retardation, short body stature, swollen tongue, eyelid folds resembling Mongolian race.
• Example: Klinefelter’s syndrome:
  • It is characterized by 2n+1 (44+XXY) genotype.
  • It occurs when an abnormal egg (XX) fuse with normal sperm (Y)
  • The affected individual is sterile male and is characterized by unusually long body, obese and female like characteristics

iv. Tetrasomic: 2n+2

• An organism having one extra pair of chromosome in addition to its diploid set.
• It is represented as 2n+2
• Examples: tetrasomy 9p, tetrasomy 18p, tetrasomy 12p (Pallister-Killian syndrome), tetrasomy 22 (Cat eye syndrome)

Euploidy:

It is the condition of addition or loss of complete one set or more than one set of chromosome in diploid organism.

Types of euploidy:

1. Monoploidy or Haploidy
2. Diploidy
3. Polyploidy

Monoploidy or haploidy:

• The total amount of genetic material in a haploid cell or in prokaryotic organism is called genome.
• Monoploidy or haploidy involves loss of complete one set of chromosome from a diploid cell.
• Monoploid or haploid organism contains single genome (n) in their cell.
• They contains one member of each kind of chromosome.
• Haploid cell is formed during gametogenesis is diploid organism.
• Viruses and bacteria contains single genome and are haploid
• Majority of lower plants particularly thallophyta and bryophyte exists in monoploid form.
• In higher plants, haploidy develops as a result of parthenogenesis.
• In some animals, like honey bees and wasps, male drone are haploid.

Characteristics of haploids

• Haploid plants are usually weaker and smaller than diploid, but in pepper the haploid are as healthy as normal diploid plant
• Leaves of haploid plants are generally small and Plants have low viability.
• In monoploid male honey bees, during spermatogenesis the meiosis is bypassed by
  mitosis. As a result, their sperms are haploid and viable.

Diploidy:

• Normally all higher plants and animals occurs in diploid form.
• They contains two copy of each chromosome

Polyploidy:

• Polyploidy is a condition of addition of one or more complete set of chromosome in diploid cell.
• Polyploidy results due to failure of separation of chromosomal sets during mitosis or meiosis such that more than two chromosomal sets are present in a cell.
• Polyploidy is more common and sometimes  resulting in evolution  of  new  plant  species  with better  yield
• A number of agriculturally important crops, such as wheat, oats, cotton, potatoes, and sugar cane are polyploids.
• Generally, Organism possess two sets of chromosome (2n) called diploid.
• The organism with three sets of chromosome called triploid, 4 sets; tetrapolid, 5 sets; pentaploid and so on. These all are polyploids and the condition is known as polyploidy.

Artificial method of polyploidy:

i. Radiation:

• X rays, gamma rays induces rate of cell division in seeds, buds, flowers and also causes multiplication of chromosome number (somatic chromosome doubling)

ii. Injury:

• Injured part of plants forms callus. Callus growth is enhanced by a chemical Coumerine which also brings chromosomal doubling.
• Eg. Tetraploids tomato is developed from injured part

iii. Chemical treatment:

• Chemicals such as colchicine, 8-hydroxyquinolin, acetophenon, nitrous oxide, granosan, chloroform, choral hydrate, some alkaloids induce chromosome doubling.
• Colchicine is an alkaloid drug obtained from the corms of plants–Colchicum autmunale and C. luteum) and its aqueous solution is found to prevent the formation and organization of spindle fibres. So, when the cell is treated with colchicine, it prevent metaphasic plate formation of chromosomes and cell division do not proceed further. Colchicine even prevent cytokinesis. Thus, cell contains double chromosomes number in each treatment for diving cells.

Types of polyploidy

1. Autopolyploidy:
2. Allopolyploidy

Autopolyploidy:

• It is the condition in which an individual organism contains more than two sets of same genome (homologous chromosome).
• For examples: if an organism has two set of chromosome (homologous chromosome ie AA) then the autotriploid (an autopolyploidy condition) will have similar three chromosome AAA.
• Autopolyploidy condition is multiplication of same basic set of chromosome within same species.

Cause of Autoployploidy:

• Autopolyploidy arises due to the failure of disjunction of chromosome during anaphase resulting in duplication of genomes and or failure to separate cell during cytokinesis resulting in tetraploid cell.
  • The  failure  of  all  chromosomes  to  segregate  during  meiosis  gives  rise  to  a  diploid  gametes which  when  fertilized  by  a  haploid  gamete,  the  resulting  zygote  has  three  sets  of chromosomes.
  • Fertilization of an ovum by two sperms results in triploid zygote
  • Triploids can be generated experimentally by crossing diploids with tetraploids

Characteristics of autopolyploids:

• Autopolypolids are not very common and have of a little evolutionary consequences.
• Autopolyploids plants are more fertile, vigour and large size, resistant to disease. Eg. Banana, grapes, sugar beet, tomato, watermelon, marigold, corn, snapdragon etc
• Examples:
  • Autotriploid plants (3n); developed by fertilization of diploid (2n) and haploid (n) gametes
  • Autotetraploid (4n); developed by fertilization of two diploid gametes
• Autotetraploids  (4n)  are more common in nature than autotriploid becauses they have  an  even  number  of  chromosomes,  and  produces  genetically balanced  plants.
• Autotetraploids can be produced experimentally from diploid cells by Heat shock or cold shock treatment and also by colchicine treatment to somatic cell. If the  chromosome of the diploid cell  undergo  replication,  but  the  cell did not divide, it  results  in  the  doubling  of  the  chromosome  number.
• Colchicine prevents separation of chromosome during anaphase by interfering with spindle fiber formation. Upon removal of  colchicine the  cell can re-enter into  interphase  during which the  paired  sister  chromatids  separate  and  uncoil.  Now, the nucleus has four sets of chromosomes giving rise to a condition called autotetraploids (4n).

Significance of Autopolyploidy:

• Generally Autopolyploidy leads to increase in size, vigour and strength and often larger than their diploid counterparts. In some cases autopolyploids are smaller and weaker then diploid
• Pollen grains, stomatal guard cell and xylem parenchyma are larger in size in autoployploids than diploids
• Autopolyploids generally show reduced fertility due to high irregularities during meiosis which causes genotypic imbalance leading to physiological disturbances.
• Generally autopolyploids reproduce by vegetative propagation.
• The flower and fruits per plant in autopolyploids are usually less in number than diploids
• Autopolyploidy is much successful in species with low chromosome number and in cross pollinated species.
• Autopolyploidy is used in horticulture for ornamental plants like roses, dahlias and also in production of seedless plants. Examples; apples, pears, banana, grapes, orange etc

Allopolyploidy:

• Allopolyploidy is a condition developed by hybridization between two genetically distinct species followed by doubling of chromosomes.
• For examples; hybridization between species X with AA set of chromosomes and species Y with BB set of chromosomes results in hybrid species XY with AB set of chromosomes. On doubling the hybrid chromosomes set, resulting individuals have AABB set of chromosome, condition known as Allopolyploidy
• Generally the hybrid with AB set of chromosome are sterile but when the chromosomes is doubled (AABB), then resulting allopolyploids (amphidiploids) are fertile as they can produce gametes.

Examples of Alloploidy:

• George karpechenko (1927) performed a polyploidy experiment on Raphanus sativus (Raddish; 2n=18) and Brasssica oleracea (cabbage; 2n=18) by inter-generic crossing.
• The hybrid (Raphanobrassica) had 18 chromosome, 9 from raddish and 9 from cabbage but were sterile
• When the chromosome of sterile Raphanabrassica were doubled by artificial means (colchicine), a fertile Allopolyploids Raphanabrassica with 36 chromosome is produced.
• * Raphanabrassica has rooting system of cabbage and fruiting body of raddish.

Significance of Allopolyploidy:

• Used in crop breeding
• Used as a bridge species in transfer of desired characters from one species to another. For eg. Modern wheat
• For production of new crop species; Raphanabrassica
• Played vital role in evolution of species: 1/3 rd of flowering plants are polyploids and most are allopolyploids

Numerical chromosomal aberration/ Chromosomal genomatic mutation

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• Sex determination is defined as the developmental decision that directs the bipotential gonad to develop as a testis or an ovary. In mammals, sex determination is genetically controlled depending on a developmental time and gene expression.
• Primarily sex determination is made at fertilization, but the embryo acquires its definite sex characteristics by a more complex mechanism called sex differentiation.
• Sex determination is not a single step method. But rather it involves various steps of sex differentiation.
• In human beings and other animals sex determination occurs in the following differentiation steps :

1. Genetic Sex:
2. Gonadal sex:

1. Genetic sex: chromosomal mechanism of sex determination

• Normal females have two X chromosomes and normal males have one X and one Y chromosome. Therefore, female is denoted as XX and male as XY.
• In Human as well as in other mammals, Y –chromosome controls sex.
• Conclusive evidence of this theory of Y-chromosome determining sex come from certain abnormal genetic syndromes such as Turner’s syndrome (XO Female; sterile female) and Klinefelter’s syndrome (XXY male; sterile male).
• In Turner’s syndrome, an individual is female despite having one X-chromosome and in Klinefelter’s syndrome, an individual is male despite having two X- chromosome.
• This evidence suggests that in human beings sex determination mechanism is Y- chromosome dependent like in plant (Melandrium) but not like Drosophila.
• There are genes on these sex chromosomes which determine femaleness or maleness.

Dosage Compensation:

• From the structure of X and Y chromosome, it was found that the X-chromosome carries much more genetic information than Y chromosome.
• Since females have two X- chromosome, they contain double dose of X-linked genes, whereas the male has only a single dose of these X-linked genes. This inequality in x-linked genes in female is tolerated by dose compensation mechanism.
• Dose compensation in human or mammal is achieved by inactivation of one X-chromosome in homogametic female (XX).
• In X-chromosome inactivation in mammals one X-chromosome gets characteristically condensed and inactivated. Such chromatin material is called facultative heterochromatin, since it becomes inactive in certain part of the life cycle and resumes activity before entering the germ line.
• The phenomenon of inactivation of X chromosome was confirmed by the observation of the Barr body.
• Barr and Bertram (1949) reported a deeply stained chromatin body (a chromocentre) in the nerve cells of female cat which was absent in the male. This chromatin body is called sex chromatin or Barr body after the name of its discoverer. Such Barr body has also been observed in most of the body cells (e.g., skin, oral epithelium and blood cells) of man and other mammals.
• Human females have the Barr body in the nuclei of their body cells in higher proportion than males and are, therefore, referred to as sex chromatin positive.
• The human males are sex chromatin negative.
• It has been demonstrated that the sex chromatin is derived only from one of the two X-chromosomes. The other X chromosome behaves like an autosome.
• Later, Lyon (1972) confirmed the existence of Barr body in normal females (XX), meta-females or super females (XXX) and in Klinefelter males (XXY).

2. Gonadal sex: transfer of Bipotential gonad into testis or ovary

• Until six weeks of foetal life, human embryo have identical undifferentiated gonads in male and female.
• Embryo before six weeks has two system of reproductive duct; wolffian duct and Mullerian duct.
• Wolffian duct: it develops into male reproductive tract
• Mullerian duct: it develops into female reproductive tract
• During this period of six weeks, the undifferentiated gonad have been invaded by the primary XX or XY cells.
• If gonad have been invaded by XY cells, the genes located in Y-chromosome called SRY gene causes the undifferentiated gonad to differentiate into a testis. Testis then begin producing two hormones; testosterone and anti-mullerian hormone.
• Anti-mullerian hormone (AMH) is mullerian inhibiting hormone which degenerates mullerian duct.
• Combination of events of these genes and other results in development of male phenotype.
• Testosterone and its other derivatives dihydro-testosterone induce formation of male sex organs and male reproductive duct.
• Similarly, if the gonad have been invaded by XX cells, the absence of SRY gene allows the gonad to become an ovary.
• Ovary begins producing estrogen hormone which induce development of uterus and cervix from mullerian duct.
• The development of gonad into a testis starts as soon as the gonocytes (primordial germ cells) from the yolk sac have finished their migration into the gonadal ridge.
• Gonocytes of the male (XY) migrate deeper into the gonadal blastema forming the medulla.
• Gonocytes of female gonocytes (XX) remain at the periphery of blastema, forming a thick cortical layer.
• Hence, the XX genotype develops into ovarian gonadal sex (female) and XY genotype develops into testicular gonadal sex (male)

Genes involved in sex differentiation in human:

#Role of Male sex-determining genes;

1. SRY gene:

• SRY gene of 35kpb on short arm of Y-chromosome known as sex determining region of Y (SRY) is the master regulator of sex determination.
• SRY is the segment of gene on Y –chromosome known as testis determining factor (TDF) in human. The TDF gene is the master switch that when turned on, activate on entire series of genes whose function is sex differentiation.
• In human or mammals, SRY encodes a transcriptional factor that activates testis formation after six weeks of foetal development.
• SRY gene activates other genes located in different chromosome and responsible for sex differentiation.

2. SOX9 gene:

• Sox9 is a member of the SOX transcription factor family which is located in autosome.
• SOX9 is induced by SRY gene.
• Immediately after expression of SRY gene, Sox9gene expresses in XY male gonad in lateral side and in sertoli cell precursors.
• Expression of SOX9 gene initiate differentiation of sertoli cell as well as production of testosterone and Anti-mullerian hormone (AHM) hormone. AHM causes degeneration of mullerian duct.
• Mutation in SOX9 gene results in loss of function and causes sex reversal from XY male to XY female as well as skeletal deformation.
• Therefore, SOX9 is responsible for sex determination as well as bone formation.
• Meanwhile, the duplication of SOX9gene in an SRY-negative female-to-male sex reversal patients demonstrated that SOX9 plays a crucial role in male sex determination and differentiation.
• Homozygous deletion of Sox9in developing mouse XY gonad leads to irregular sex cord formation, abnormal Sertoli cell differentiation, and reduced expression of male-specific makers

3. FGF9 gene:

• Fibroblast growth factor 9 (Fgf9) gene expression overlaps with SXO9 gene and results in early differentiation of sertoli cells.
• Loss of FGF9 gene results in male-to-female sex reversal.
• Therefore, FGF9 is also responsible for sex determination.

4. DMRT1 (Double sex and mab-3 related transcription factor 1) gene:

• Two copies of DMRT1 gene are present in human (Mammals) which are necessary for testis formation even in absence of SRY gene.
• DMRT1 gene are located in chromosome 9, so both sex (male and female) contains two copies of DMRT1 gene.

5. FOXL2 (Forkhead box L2):

• Recent studies have shown that two genes, doublesex and mab-3 related transcription factor 1 (Dmrt1) and forkhead box L2 (Foxl2), play critical roles in maintaining sexual phenotype in mice

6. Steroidogenic factor 1 (SF1 gene):

• SF1 gene is present in both sex and is required to form bipotential gonad from genital ridge.
• SF1 gene encode splicing factor 1 which is involved in production of steroid during embryogenesis. SF1 factor is also known as adrenal 4 binding protein which regulates number of other genes.
• SF1 gene is more activated in XY male than in XX female.
• Interaction of SF1 with SOX9 gene and acts on leydig cell to produce testosterone and also interacts with sertoli cell to produce AMH resulting in wolffian duct formation.
• While interaction of SF1 with DAX1 inhibit AMH and testosterone, resulting in mullerian duct formation.

#Role of female sex determining gene:

• XX females lack Y chromosome and SRY gene.
• Ovary formation for many years was considered to be a “default” gonad development pathway resulting from the absence of SRY
• Number of gene are responsible for ovary development. Such as-β-catenin, follistatin (Fst), FOXL2, R-spondin (RSPO1), and WNT4, without which ovary development cannot take place.

1. DAX1 (Dosage sensitive sex reversal) gene:

• DAX1 is encoded by NROB1 gene located on short arm of X-chromosome.
• Function of DAX1 is antagonistic to SRY, which means it is a negative modulator which decrease SF1 expression.
• DAX1 down regulate Anti-mullerian hormone leading to development of mullerian duct and ovary.
• Mutation on this gene results in reversal of XX female to XX male.

2. WNT4 gene:

• WNT4 is an autosomal gene present in chromosome 1.
• WNT4 is antagonistic to TDF preventing testis formation and it is required for ovary formation.
• Interaction of DAX1 with SF1 expresses WNT4 gene which is required for ovary formation.
• Mutation in WNT4 gene of XX female causes no ovary development and reversal of XX female into XX male.

Sex determination in human and role of various genes

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Three point test cross in Drosophila:

• Wild-type Male Drosophila was crossed with female Drosophila homozygous for three recessive X-linked mutations—scute (sc) bristles, echinus (ec) eyes, and crossveinless (cv) wings to obtain F1 progeny.
• Wild Male Drosophila= (sc+, ec+, cv+)
• Mutated female Drosophila = (sc, ec, cv)
• Then F1 progeny were intercrossed to produce F2 flies, which are then classified and counted.
• The F1 males carried the three recessive mutations on their single X chromosome. Thus, this intercross was equivalent to a testcross with all three genes in the F1 females present in the homozygous form.
• The F2 progeny flies from the intercross comprised eight phenotypically distinct classes, two of them are parental and six recombinant.

Gene order:

• The parental classes were by far the most numerous (1158+1455=2613). The less numerous recombinant classes each represented a different kind of crossover chromosome.
• To figure out which crossovers were involved in producing each type of recombinant, we must first determine how the genes are ordered on the chromosome.
• There are three possible gene orders :

1. sc—ec—cv
2. ec—sc—cv
3. ec—cv—sc

• Four of the recombinant must have come from a single crossover in one of the two regions of the genes. The other two recombinant must have come from double crossing over—one exchange in each of the two regions. Because a double crossover switches the gene in the middle with respect to the genetic markers on either side of it, it is used for determining the gene order.
• Again, intuitively, double crossover occur much less frequently than a single crossover. Therefore, among the six recombinant classes, the two rare ones must represent the double crossover chromosomes
• From the given example, the double crossover must have occurred in class 7 (sc ec+cv) and class 8 (sc+ec cv+), each containing a single recombinant F2 progeny.
• Comparing these rare recombinant to parental class 1 (sc ec cv) and class 2 (sc+ec+Cv+), the echinus allele has been switched with respect to scute and crossveinless.
• Consequently, the echinus gene must be located between the other two.
• Therefore the correct gene order is sc–ec–cv.

Map distance:

• It is the distance between each pair of gene and it is obtained by estimating the average number of crossovers.
• Total map distance between these three genes is map distance between sc and ec plus map distance between ec and

i. Map distance between sc and ec:

• We can obtain the length of the region between sc and ec by identifying the recombinant classes that involved a crossover between these genes.
• There are four such classes: class 3 (sc ec+cv+), class 4 (sc+ec cv), class 7 (sc ec+cv), and class 8 (sc+ec cv+).
• Classes 3 and 4 involved a single crossover between sc and ec, and classes 7 and 8 involved two crossovers, one between sc and ec and the other between ec and
• We can therefore use the frequencies of these four classes to estimate the average number of crossovers between sc and ec:
• Average crossover between sc and ec =(163+130+1+1) /3248

=0.091 Morgan

=9.1 centiMorgan or Map unit

• Thus, in every 100 chromosomes coming from meiosis in the F1 females, 9.1 had a crossover between sc and ec.
• The distance between these genes is therefore 9.1 map units.

ii. Map distance between ec and cv:

• In a similar way, we can obtain the distance between ec and cv.
• Four recombinant classes involved a crossover in this region: class 5 (sc ec cv+), class 6 (sc+ec+cv), class 7 and class 8.
• The double recombinants are also included here because one of their two crossovers was between ec and cv.
• The average cross between ec and cv =(192+148+1+1)/3248

=0.105 morgan

= 10.5 centiMorgans or map unit

Total map distance:

• Combining the data for the two regions, the map is sc—9.1— ec—10.5— cv
• Thus map distances between sc and cv= 9.1 cM +10.5 cM =19.6 cM

Alternative way of calculating map distance:

• Directly calculating the average number of crossovers between these genes:
• Recombination frequency (RF)= Non–crossover + Single crossover + Double crossover

= (0)*(1158+1455)/3248 + 1 (163+130+192+148)/3248  + 2 (1+1)/3248

= 0 + 0.195 + 0.0006

= 0.196 Morgan

= 19.6 CentiMorgan

Inference and coefficient of coincidence:

• Inference is the phenomenon of inhibition of crossover of by another crossover nearby.
• For example, the crossover frequency between sc and ec in region I was (163 +130 +1+1)/3248 =0.091, and crossover frequency between ec and cv in region II was (192+148 +1 +1)/3248 =0.105.
• If we assume both crossover are independence of each other, the expected frequency of double crossovers in the interval between sc and cv would be 0.091 *0.105 = 0.0095.
• But actual observed frequency of double crossover is (1+1)/3248 = 0006
• Double crossovers between sc and cv were much less frequent than expected.
• The result suggest one crossover inhibited the occurrence of another nearby, a phenomenon called interference
• The extent of the interference is measured by the coefficient of coincidence (C).
• Coefficient of coincidence is the ratio of observed frequency to double cross to expected frequency to double cross.
• C= (observed frequency of double crossovers)/(expected frequency of double crossovers)

=0.0006/0.0095

C=0.063

Level of inference (1-C):

• Level of inference = 1-C
• =1-0.063
• =0.937
• Because in this example the coefficient of coincidence is close to zero, its lowest possible value, interference was very strong (I is close to 1).
• cases:
• if a coefficient of coincidence equal to 1; no interference between crossover at all which means the crossovers occurred independently of each other.
• If a coefficient of coincidence is equal to 0; very strong inference between crossover therefore double cross do not occur.
• ** map distance less than 20cM has very strong inference. Thus, double crossovers seldom occur in short chromosomal regions.
• The strength of interference is therefore a function of map distance

Three point test cross: gene order, map distance, inference and coefficient of coincidence and level of inference

The post Three point test cross: gene order, map distance, inference and coefficient of coincidence and level of inference appeared first on Online Biology Notes.

• Gene mapping is the process of determining the genes and their location along the length of chromosome.
• T. D Morgan pave the foundation of gene map by identifying gene for white eye Drosophila on X-chromosome of mutant. Later his students able to locate other X-linked gene on X-chromosome.
• The procedure of gene mapping was developed by Alfred H Sturtevent. His procedure is based on the principle of linkage. The gene located on same chromosome inherits together known as linked gene. However, some gene on same chromosome could separate during meiosis and new combination of genes are formed. The phenomenon of recombination is due to crossover and chaismata formation during meiosis.
• Gene map is by counting the number of crossovers that occur during meiosis. However, because the actual crossover events cannot be seen, they cannot count them directly. So, recombination frequency is calculated to estimate the crossover.
• Chiasmata are counted through cytological analysis, whereas recombinant chromosomes are counted through genetic analysis.

Gene map distance:

• Gene map distance is the distance between points on a chromosome which can be estimated by counting the number of crossovers between them. Therefore, the distance between two points on the genetic map of a chromosome is the average number of crossovers between them. Genetic map distances are, in fact, based on such averages.
• Points that are far apart should have more crossovers between them than points that are close together. However, the number of crossovers must be understood in a statistical sense.
• In any particular cell, the chance that a crossover will occur between two points may be low, but in a large population of cells, this crossover will probably occur several times simply because there are so many independent opportunities for it. Thus, the quantity that we really need to measure is the average number of crossovers in a particular chromosome region.
• let us consider 100 oogonia undergoes gametogenesis (meiosis).
• In some cells, no crossovers will occur between sites A and B; in others, one, two, or more crossovers will occur between these loci.
• At the end of meiosis, there will be 100 gametes, each containing a chromosome with either zero, one, two, or more crossovers between A and B.
• We estimate the genetic map distance between these loci by calculating the average number of crossovers in this sample of chromosomes.
• The result from the data is 0.42.

Two point test cross in Drosophila:

• When wild-type Drosophila Females were mated to Males homozygous for two autosomal mutations—vestigial (vg) short wings, and black (b) body coloration. Ie. Female (vg+vg+ , b+b+) and male (vg vg, b b)
• All the F1 flies had long wings and gray bodies; thus, the wild-type alleles (vg+ and b+) are dominant.
• The F1 female progeny were then testcrossed to vestigial winged black body males (vg b), and the F2 progeny were obtained and there are classified on the basis of phenotypic characters and counted.
• There were four phenotypic classes, two abundant and two rare. The abundant classes had the same phenotypes as the original parents (vestigial wing black body and long wing and grey body), and the rare classes had recombinant phenotypes (ie. vestigial wings with grey body and long wings with black body).

• Number of F2 Progeny with vestigial wing and black body (vg vg, b b)= 405
• Number of F2 progeny with long wing and grey body (vg+ vg+, b+ b+)= 415
• Number of f2 progeny with vestigial wing and grey body (vg vg, b+ b)= 92
• Number of F2 progeny with long wing and black body (vg+ vg, b b)=88
• The genes for vestigial wings and black body are linked because the number of recombinants are much fewer than 50 percent of the total progeny counted in F2 generation. Therefore, these genes must be on the same chromosome.

Map distance:

• Map distance is the distance between genes.
• To determine the distance between the genes for vestigial wing and black body, we must estimate the average number of crossovers in the gametes of the doubly heterozygous F1 females (vg+vg, b+b)
• Average crossover is estimated by calculating the frequency of recombinant F2 progeny
• The average number of crossovers in the whole sample of progeny is therefore,

Frequency of recombination (RF) = (0) (415+405)/1000 + 1 (92+88)/1000

=0.82+0.18

=0.18 Morgan

=18 centimorgan or map unit

• This simple analysis indicates that, on average, 18 out of 100 chromosomes recovered from meiosis had a crossover between vg and b.
• Thus, vg and b are separated by 18 units
• 100 centiMorgans equal one Morgan (M). Therefore vg and b are 18 cM (or 0.18 M) apart.
• The map distance is equal to the frequency of recombination.

Gene mapping: two point test cross, map distance and frequency of recombination

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#Sex determination:

• Sex determination is the process by which the male and female gametes after fusion produce a zygote which develop the property of one sex or the other sex.
• Sex determination completes after fertilization

#Sex differentiation:

• It is the chain of developmental processes by which the genetically determined sex becomes translated into characteristic structure, functions and behavior pattern.
• Sex differentiation begins after fertilization and continues throughout embryonic development, childhood and adolescence.

Chromosomal mechanism of sex determination in animal

• Male and female differ from each other in respect to either number or morphology of sex chromosome (X and Y chromosomes). Autosomes do not differ between two sexes.

XX female-XY male type:

• In this type of sex determination, both sexes have got even number of sex chromosome; female have two X chromosome (XX) and male have one X and one Y chromosome (XY).
• Female are homogametic (XX) and after gametogenesis produces ova (female gamete) containing one X chromosome.
• Male are heterogametic and after gametogenesis produces two different types of sperm (male gamete); 50% sperm containing X chromosome and other 50% sperm containing Y chromosome.
• In fertilization; when X ova is fertilized by X sperm; it will produce XX female and when X ova is fertilized by Y sperm; it will produce XY male.
• This XX female and XY male mechanism of sex determination is common in Human as well as Drosophila and in plants.

There are certain variation in chromosomal mechanism of sex determination in different organisms;

i. XX female –XO male type:

• In this type of sex determination, female possess normal pair of autosomes and normal sex chromosome (XX) while male possess normal pair of autosome and single X chromosome but no Y chromosome (XO).
• During gametogenesis female produces normal gametes containing a single X chromosomes
• During gametogenesis in male (spermatogenesis), two types of gametes are produced; one type containing X chromosome while other type lack sex chromosome.
• If the female gamete (ova) is fertilized by a sperm containing X chromosome, it will result in normal zygote containing XX chromosome and finally develops normal female (XX).
• If the female gamete (ova) is fertilized by sperm lacking sex chromosome (O), it will results XO zygote and finally develop XO male.
• This type of sex determination is common in grass hopper and some bugs.
• In human this is an example of genetic disorder called Turner’s syndrome in Human.

ii. ZW female-ZZ male:

• This mechanism is just reverse of XX female and XY male.
• In some organism such as Fowls, amphibian, reptiles, some fishes and butterflies, moths and in some plant species Frageria orientali., female are heterogametic (ZW) while male are homogametic (ZZ).
• Male chromosome is designed as ZZ while female chromosome is designed as ZW.
• In this example, female gamete determines the sex of individuals.
• During gametogenesis, male produce similar type of sperm containing one Z chromosome, while female produces two types of gametes; one type containing Z chromosome and other type containing W chromosome.
• When Z chromosome containing ova is fertilized by Z sperm, it will result in male offspring. Similarly when W chromosome containing ova is fertilized by Z sperm, it will result in female offspring.
• ZW-ZZ mechanism of sex determination is similar as XX-XY mechanism except it is reverse.

Sex determination in plants:

• Mechanism of sex determination in plants varies from genus to genus and species to species.
• Angiosperm are flowering plants. Some plants are dioecious or unisexual (male and female occurring individually) and most are monoecious or bisexual (male and female organ in same plant body or in same flower).
• Monoecious plants are of two types:
  • Monoecious plants with unisexual male and female flowers in same plant body (sporophyte). Examples;
  • Monoecious pant with male and female organs in dame flower. Such monoecious pant are known as hermaphrodite.
• In monoecious plants, there is no special sex chromosome however formation of ova and pollen is accomplished by histological differentiation.
• In dioecious plants, different cases of sex determination have been found:
• Mechanism of sex determination in plants are explained by genic theory, environmental theory and chromosomal theory.

Examples: chromosomal mechanism of sex determination in plants

• Genetic information of sex is located on chromosome. In animals, sex chromosomes are responsible for sex differentiation but in plant, it could be autosomes or sex chromosomes.
• In some selected plant species sex chromosomes have been identified and the expression of sex chromosomes seems to be biologically justified.

i. Normal XX female-XY male type:

• This is a very common mechanism of sex determination in most plants.
• For eg. In a bryophyte Sphaerocarpus,
• Sphaerocarpus is a haploid plant with 7 autosomes and a sex chromosome. In male gametophyte has (7A+Y) and female has (7A+X) chromosome.
• Fusion of male gamete and female gamete give a zygote (14A + X+Y) chromosomes.
• Zygote then develops into sporophyte and under goes meiosis producing haploid gametes of two types; one containing 7A+Y and other gamete type containing 7A+X chromosome.
• 7A+Y develops into male gametophyte and 7A+X develops into female gametophyte.

ii. XY male and XX female: Melandrium album

• It is a model plant for genetic study for sex determination of plants.
• It has total chromosome of 12 pairs, among them 11 autosomes and 1 sex chromosome. A+XX; female and A+XY; male
• X and Y sex chromosome of Melandrium is similar to that of human or Drosophila but mechanism of sex determination is different from usual XX-XY system.
• Y-chromosome play crucial role in sex determination in M. album. Y chromosome contains two main genes, they are;
  • Male determining gene
  • Female suppressing gene
• Both genes are necessary for production of fertile anther in male plant.
• Y-chromosome is longer than X –chromosome and during meiosis (spermatogenesis) in male plant, X and Y chromosome form heteromorphic bivalent and these chromosome divides into five segments. These segments control the development of sex of plant.
• Chromosome Y have 4 segments (I, II, III and IV) and chromosome X has two segments (IV and V). Segment IV is common in both X and Y chromosome.

• Segment I: Contains gene that suppress femaleness and inhibit development of female structure in flower
• Segment II: contains gene that initiate anther development
• Segment III: also initiate anther development
• Segment IV: homologous region of both X and Y chromosome, helps in homologous pairing during meiosis.
• Segment V: It is present in X Chromosome and contains gene for femaleness in absence of Y chromosome
• Female plant is homogametic (XX), it only produce single type of gamete containing X-chromosome. Male pant is heterogametic (XY) and it produce two types of gametes; one with Y-chromosome and other with X-chromosome.
• When X chromosome containing male gamete fuse with female gamete, it will develop into female plant and produces female flowers.
• When Y chromosome containing male gamete fuse with female gamete, it will develop into male pant and produces male flower.
• If segment I is deleted from male plant then it will produce bisexual flower ( because segment V of X chromosome expresses femaleness in absence of segment I of Y chromosome and segment II and III expresses anther formation, so bisexual flower with both male and female organs develops)
• Melandrium album occurs in diploid (2n), triploid (3n) and tetraploid (4n) forms. In each form presence of Y- chromosome irrespective of number of X-chromosome and autosome produces male plant.

Chromosomal mechanism of sex determination

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• The mechanism of sex determination are similar in both plants and animals.

I. Classical Theories of sex determination:

1. Metabolic theory of sex determination:

• This theory was developed by Riddle
• According to metabolic theory, sex is conditioned by metabolism of cells.
• The high rate of oxidation, large quantity of water content and less protein leads to maleness while reverse in there metabolic activity leads to femaleness.

2. Quantitative theory of sex determination:

• This theory is developed by Goldschmidt
• According to quantitative theory, the enzyme andrase is responsible for maleness and gynase for femaleness.
• The balance between these two enzymes produces different sexes and intersexes in varying degree.

II. Modern theories of sex determination:

1. Chromosomal theory of sex determination:

• In sexual reproduction in higher organism, male and female gametes are produced and fuse together to form zygote. These gametes are specialized cell with haploid number of chromosome. The chromosomes are of two types-autosomes and sex chromosomes.
  • Autosomes: it determines the phenotypic body chcaracters. These chromosomes (22 pairs) in diploid organism do not differs among male and female. Their number and morphology are conserved in male and female.
  • Sex chromosome: it determines sex.
• Herman Henking (1891) observed darkly stained chromatin element and named it as X body which was later found to be sex chromosome and named as X-chromosome.
• Miss Stevens (1905) observed Drosophila melanogaster and found four pair of chromosomes. In male flies, one pair of chromosome was peculiar, one of them resembles with the X-chromosome of female while other is unequal in size. Later Wilson (1909) term this unusual chromosome as Y-chromosome. So, Drosophila could be described as XX female and XY male.
• McClung (1902) suggests the chromosome was responsible for sex determination
• In human, there are 23 pairs of chromosomes
  • Female= (22 pairs of AA chromosomes + XX)
  • Male = (22 pairs of AA chromosome + XY)
• X and Y chromosomes differ from each other. In human Y chromosome is shorter than X- chromosome while In Drosophila Y chromosome is larger than X-chromosome.

2. Genic balance theory of sex determination:

• B Bridge (1925) put forward the genic balance theory of sex determination in which he stated that the sex of an individual is determined by a balance between the genes for maleness and those for femaleness present in an individual.
• While studying Drosophila, it was found that Y chromosome is mostly heterochromatin and play no significant role in sex determination, the gene for maleness is present in autosomes while the gene for femaleness is present in X-chromosome.
• Therefore all the individuals carry the gene for both sex-male and female. But it is actually the ratio between X-chromosome and the autosomes which governs the development of male or female sex. The ratio is known as sex index ratio.
• Sex index ratio (X/A) = (X chromosome number/ Number of autosome sets)
  • If the sex index ratio (X/A) is 0.5 or less, it gives male sex
  • If the sex index ratio (X/A) is 1 or more than 1 then it gives female sex
  • If the sex index ration (X/A) is between 0.5 and 1 then it gives intersex

• This table shows sex expression in Drosophila according to sex index ratio.
• Genic balance theory explain the sex determination in Drosophila on the basis of sex index ratio, where Y-chromosome does not play role in sex determination.
• However this genic balance theory of sex determination does not hold true for human or mammals and also in those individuals where Y chromosomes plays certain role in sex determination.

3. Haplo-diplo mechanism of sex determination:

• In haplo-diplo mechanism of sex determination, females developed from fertilized egg and are diploid whereas male developed from unfertilized egg by the process called parthenogenesis and are haploid.
• This method of sex determination is very common in Insects species such as bees, ants, wasps.
• In a study conducted by Whitings (1945) on wasp species (Habrobracon), it was found that all females were diploid whereas most of the males were haploid and developed from unfertilized egg and very few males were diploid.
• Diploid male were poorly viable.
• Similar type of result can be observed in honey bees, where male drone developed by parthenogenesis process.

4. Single gene sex determination:

• In microorganisms such as Chlamydomonas, Neurospora and Saccharomyces species, the sex is determined by a single gene. These microorganism have two separate mating type (+/-strains) which are determined by a pair of allele (gene). Sexual reproduction takes place when gametes from two opposite strain fused together. Gametes from same strain do not fuse in such organisms.
• Similarly in some plants and animals, sex is determined by a single gene. For examples, Male and female mosquito differ from each other in a single gene.
• An example of single gene sex determination is observed in maize. Maize is a monoecious plant with both male and female sex organs in same plant.  If gene for both bassen cob and gene for tassel ear are in recessive form, then the monoecious plant is converted into dioecious plant (female).

5. Cytoplasmic theory of sex determination:

• In bacteria eg E. coli, extra chromosomal DNA called sex factor (F-plasmid) is responsible for sexuality.
• The bacteria containing F-plasmid is known as donor (male) and denoted as F+ cell while the bacteria lacking F-plasmid is known as recipient (female) and denoted as F-cell.
• During bacteria conjugation, donor cell transfer its F-factor into recipient cell and finally F- cell also become F+ cell.
• F- factor may integrate with DNA of donor cell and become Hfr cell (high frequency recombination) and can transfer its genetic material to recipient cell
• However, cytoplasmic factor is also responsible for sexuality is some microorganisms is postulated by this theory.

6. Nutritional theory of sex determination:

• It was proposed by Sharp (1934)
• According to this theory, sex determination is not only genetic but also depends upon the nourishment of gametes.
• For examples; In Equisetum (puzzlegrass), when its spore is grown under good growth condition, it develops into female whereas when grown under unfavorable condition, it develops into male.
• Similarly, in a marine worm (Dinophilus), size of egg determines the nature of sex. The small egg develops into male whereas large egg develops into female.

7. Environmental theory of sex determination:

• According to this theory, environmental factor plays direct or indirect role in determining sex of an organism.
• Every zygote contains all the responsible gene required for development of sex and expression of particular sex (male or female) is determined by internal or external environmental condition.
• In certain environmental condition, particular sex gene might get inhibited such that other sex gene get activated giving one type of sex and similarly in other environmental condition the process might get revered giving other type of sex.
• For example, in reptile like turtles, high temperature (30-35) induce the expression of female and low temperature (23-28) induce male sex.
• Also in plants like Equisetum (puzzlegrass), spore grown under optimum condition develops into female gametophyte and when grown under unfavorable condition develops into male.
• Other examples: In cucumber and melon, day lights length, temperature etc also differentiate sex.

8. Hormonal control theory of sex determination:

• In certain animals, hormone plays an important role in sex determination (differentiation).
• For examples- A marine worm
• The worm Bonellia is morphologically distinct. Male worm is sessile, lives inside uterus of female worm and smaller in size than female worm which is about 2 inch long.
• After fertilization of egg, larvae are releases in water. All larvae are cytogenetically identical.
• Those larvae, which comes in contact with mature female get attached to their proboscis and develop into male. These male then migrate to female reproductive organs and live a parasite. But those larvae which do not comes in contact with mature female develops into female worm.
• It has been found that the proboscis of mature female worm secrete hormone like substance which inhibits the larval gene determining female sex and allowing male gene to express.
• Other example; freemartin or hormonal intersex cattle
• If a cattle develops dizygotic twins of opposite sex, there is more chance of development of sterile female calf (freemartin) and a normal male calf.
• Freemartin develops only when foetal membrane of two embryos fused together such that cross circulation between two embryos occurs.
• It was found that during cross circulation, some of the hormone released by male embryo cross the placenta of female embryo and it inhibits the secondary sexual characters in female embryo and at the same time induce male characters. Such that the female embryo after birth become more like male but it is sexually female.
• Freemartin is also known as hormonal intersex.

Theories of sex determination in organisms

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• The word ‘karyotype’ is derived from Greek word “kernel” which means content of nucleus.
• The term Karyotype is referred to a group of characteristics that allow the identification of particular chromosome pair on the basis of general morphology of a set of chromosome at metaphase of a somatic cell of an individual.
• Karyotype concept was derived by S. Navashin based on the observation that the number of chromosomes and morphology of each chromosome pair is normally constant and characteristics for a species.
• Karyotype concept is a phenotypic appearance of a chromosome and its homologous pair which helps to differentiate types of chromosome based on their morphology in an individual species.
• Karyotyping is a method of arrangement of pair of homologous chromosome of a cell in decreasing series of their size.
• Karyotype study is very important to identify abnormalities in chromosomal structure and number.
• Karyotype study also helps to determine origin and evolutionary relationship among different texa.

Types of karyotype on the basis of symmetry:

• Developed by G. Levitzky (1931)
• Two types

1. Symmetrical karyotype:

• In this all the chromosome of a set are approximately of same size and have median or sub-median centromere.
• Homogenous karyotype
• It is regarded as primitive karyotype

2. Asymmetrical karyotype:

• In this all the chromosome exhibit good degree of variation in chromosomal size and also there is variation in position of centromere.
• Position of centromere changes from median or sub-median to sub-terminal or terminal.
• It is regarded as specialized or advanced karyotype.

Parameters used in karyotype preparation:

• Number of chromosome in the set
• Relative length of chromosome
• Arm ratio (ratio of length of long arm to length of short arm from the position of centromere)Position of secondary constriction
• Position and size of nucleolus and satellite
• Staining reaction and differentiation of bands of euchromatin and heterochromatin in the chromosome

Human karyotype:                                              

• Diploid human cells contain 46 chromosomes—44 autosomes and two sex chromosomes, which are XX in females and XY in males.
• At mitotic metaphase, each of the 46 chromosomes consists of two identical sister chromatids. When stained appropriately, each of the duplicated chromosomes can be recognized by its size, shape, and banding pattern.
• Chromosome 1: is the largest autosome
• Chromosome 21: is the smallest is number 21.
•  X chromosome (sex- chromosome):  is intermediate in size
• Y- chromosome (sex chromosome): is about the same size as chromosome 22

Idiogram:

• Idiogram is the diagrammatic representation of karyotype showing all the morphological feature of the chromosomes grouped on the basis of position of centromere and ordered in a series of decreasing size.
• Chromosome banding and painting technique are used to identify each chromosome. These technique are also used to distinguish each arm of a chromosome and to investigate specific regions within them.
• In some chromosome, a centromere which divides each chromosome into long and short arms. The short arm is denoted by the letter p and the long arm by the letter q.
• For example, the short arm of chromosome 5 is denoted simply by writing “5p.” Thus, in the short arm of chromosome 5, we have region 5p11, which is closest to the centromere, followed by regions 5p12, 5p13, 5p14, and 5p15, which is farthest from the centromere.
• Within each region, individual bands are denoted by numbers following a decimal point; for example, 13.1, 13.2, and 13.3 refer to the three bands that make up region 5p13. The pattern of bands within the chromosome is called an idiogram

Karyotype and Idiogram

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• Study of chromosome number and structure by staining the dividing cells with certain dyes and then examining them under microscope for cytogenetic analysis is called chromosome banding.
• Most cytological analysis is performed on dividing cells (metaphase of mitosis). Therefore, meristematic cell of root or shoot tip of plant and embryo cell of animals are used. However, the development of cell-culturing techniques has made it possible to study chromosomes in other types of cells. For example, human white blood cells can be collected from peripheral blood, separated from the non-dividing red blood cells, and put into culture. The white cells are then stimulated to divide by chemical treatment, and midway through division a sample of the cells is prepared for cytological analysis.
• The dividing cell on treatment when treated with chemical disables spindle fibre formation, such mitotically arrested cell are then immersed in a hypotonic solution that causes cell to take up water. On preparation for microscopic examination such cells are squashed on a microscopic slide, such that the chromosomes are spread out in an uncluttered fashion which can be observed by staining
• The stains such as Feulgen’s reagent or aceto-carmine stains the chromosome uniformly making difficult to distinguish one chromosome form other. Now a days, cytogeneticists use dyes that stain chromosomes differentially along their lengths.
• The staining of chromosome is known as banding technique because stains give rise to pattern of bands along the length of chromosome.

Types of chromosome banding technique:

1. Q-banding:

• Q banding used quinacrine stain (quinacrine dihydrochloride or quinacrine mustard) and it is the simplest and the first chromosomal banding method. Quinacrine stained chromosomes show a characteristic pattern of bright bands on a darker background. Because quinacrine is a DNA intercalating agent and a fluorescent compound, the bands appear only when the chromosomes are exposed to ultraviolet (UV) light.
• Ultraviolet light causes the quinacrine molecules to shine, therefore parts of the chromosome intercalated with quinacrine shine brightly, whereas other parts remain dark.
• This bright-dark banding pattern is highly reproducible and is also specific for each chromosome. Thus with quinacrine banding is used to identify particular chromosomes in a cell, and also to determine if a chromosome is structurally abnormal.
• Quinacrine is an intercalating agent and it intercalate between the base pairs of the DNA helix.  Quinacrine has more affinity for DNA sequence containing AT sequence. Therefore fluorescence of quinacrine is enhanced along AT rich sequence and appear bright than GC rich sequence.
• * quinacrine is carcinogenic.

2. G-banding:

• G-banding is the most frequent used technique for chromosome staining in cytogenetics.
• Giemsa staining is an excellent nonfluorescent staining techniques.
• Giemsa also creates a reproducible pattern of bands on each chromosome. It is still not clear why chromosomes show bands when they are stained with quinacrine or Giemsa
• Bright field microscope is used for visualization
• In G banding technique, before using Giemsa stain, there is always a pretreatment step.
• Usually proteolytic enzyme trypsin is used for pretreatment. Therefore the process is also known as GTG banding (G-banding by trypsin with Giemsa).
• There is an alternative to Giemsa stain and it is Wright stain.
• G-banding also produce same banding pattern as Q banding along the length of chromosome. Geimsa stain has more affinity for DNA sequence rich in AT content hence stained dark while sequence rich in GC content stain light.

3. C-banding:

• C-banding is called Centromeric heterochromatin staining
• In this technique, before using Giemsa stain the cell is pretreated with alkali. Theefore this technique is also known as CBG-staining (B-banding by base with Giemsa).
• Centromeric heterochromatin and distal part of Y chromosome containing highly repetitive DNA sequence (satellite DNA) is used to stain by C-binding technique.
• After staining, banding is viewed by bright-field microscope.

4. R-banding:

• R-banding is called Reverse chromosome banding.
• With this banding technique the band pattern produced in chromosome is reversed to the band produced by G-banding and Q-banding. ie. The dark band (AT rich region) observed in G-banding technique appears light in R-banding and vice versa.
• R-banding technique also uses Giemsa stain but before staining with Giemsa the slide is heated at 88°C in a buffer solution. Heating causes denaturation of DNA.
• Denaturation of chromosome at AT rich region occurs at faster rate resulting in loss of DNA from these regions but not from GC rich region.  The GC rich region is then stained by Giemsa stain which appears stained (R band)
• G-banding is usually preferred over R-banding. However R-banding can be used for chromosome identification.
• In some cases, R-banding is a useful complement to G-banding because some small light G band can be more easily detected when they are stained by R-banding.
• R-banding is also useful for visualization of telomere sequence at the ends of chromosomes. Telomeres stained dark with R-banding while light with G-banding.

Chromosome painting:

• It is the most advanced technique. With this technique, colorful chromosome images are created by treating chromosome spreads with fluorescently labeled DNA fragments that have been isolated and characterized in the laboratory.
• At first the DNA fragment isolated from desired gene is labelled with florescent dye and is applied to chromosomal spread on a slide. The florescent labelled DNA fragment will bind to chromosomal DNA that is complementary to it in sequence. This binding results in visual color at the position of complementary sequence.

Chromosome banding and painting

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