**2.1 Karyotype trend**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

vice-versa [1–3].

and races [4–6].

**2. Concept of karyotype and its components**

somes in a chromosome complement [8, 9].

euchromatin and heterochromatin [10, 11].

Facultative heterochromatins are not permanently conserved or condensed and in unstable form i.e. easily changed from euchromatin to heterochromatin and

Heterochromatic regions could be easily recognized on chromosome structure in the form of chromomeres, chromocentres and knobs. Chromomeres are regular features of all prophase chromosomes but their number, size, distribution and arrangements are specific for a particular species at a particular stage of development. Chromocentres are the regions with varying size near the centromere in the proximal regions of chromosome arms. Some of the Chromocentres could be resolved into large number of strings of chromomeres which are much larger in size as compared to the chromomeres found in the distal region of the chromosmes arms during the mid-prophase. The relative distribution of the chromocentres on the chromosome structure, sometimes considered to be of significant evolutionary value. Knobs are considered to be a spherical bodies or regions with spherical in shape and sometimes diameter of these spherical bodies is equal in width to chromosome arm, but the size may vary i.e. less or more than the diameter of chromosome arm. For example, a very distinct such type of chromosome knob could be observed in maize (*Zea mays*) at pachytene stage of meiosis I. It could be considered as a valuable chromosome marker for distinguishing chromosome of related species

Karyotype may be defined as the study of chromosome morphology of a chromosome complement in the form of size, shape, position of primary constriction or centromere, secondary constriction, satellite, definite individuality of the somatic chromosomes and any other additional features. Karyotype highlights closely or distantly related species based on the similarity or dissimilarity of the karyotypes. For example, a group of species resemble each other in the number, size and form of their chromosomes. There may be 12 different types of karyotype categories depending on increasing asymmetry in chromosome complement [7]. The degree of asymmetry of chromosome complement depends on the four arm ratios (1 to 4) and the size of the smallest and largest chromosome and three different proportions of the metacentric chromosomes (ABC) of a given chromosome complement. Arm ratio 1 being the most symmetrical and 4 is the most asymmetrical. There are various quantitative karyotypic ratios to observe the karyotype variations and precise description of the karyotype such as relative length, centromeric index, total form percent, disprsin index, disparity index, coefficient of variation, volume of chromosomes, value of relative chromatin and so on. Asymmetric karyotype may be defined as the huge difference between the largest and smallest chromosome as well as less number of metacentric chromosomes in a chromosome complement. Similarly, symmetric karyotype may be defined as the small difference between the largest and smallest chromosome as well as more number of metacentric chromo-

The principle ways in which karyotypes differ from each other are (i) basic chromosome number, (ii) form and relative size (V➔J or L➔I) of different chromosomes of the same set, (iii) number and size of satellites (related to those positions of the chromosome which form nucleoli) and secondary constrictions (NOR region of chromoosmes), (iv) absolute size of the chromosomes, (v) distribution of material with different staining properties i.e.

**46**

Karyotypic trend may be defined as the evolutionary changes in the chromosome complement by increasing or decreasing its base chromosome number which showed a definite direction of movement or pattern of its movement either from polyploidy to diploid or vice versa. For example, *Luzula* species (Juncaceae), also called wood rush, a monocot with holocentric chromosomes showed huge variation in genome and pattern or direction of chromosome movement from diploid to polyploidy or vice versa through symploidy and agmatoploidy phenomenon. The phenomenon could be related to the ascending or descending dysploidy which is also known as pseudoaneuploidy where chromosomes rearrange themselves within or between the chromosomes to decrease or increase the chromosome number in the chromosome complement of a particular species. Simploidy is the phenomenon of fusion of chromosomes together to reduce the chromosome number while the agmatoploidy breaks the chromosomes (fission) to increase the chromosome number for a particular species (**Figure 1**). The trend of *Luzula* species are as follows, L. purpureo-splendens (2n = 2x = 6; chromosome length 6.66 μm), L.elegans (2n = 2x = 6; chromosome length 4.62 μm), *L. alpinopilosa* (2n = 2x = 12 ± 1; chromosome length 2.55 μm), L.nivea (2n = 2x = 12; chromosome length 1.70 μm), L. sylvetica (2n = 2x = 12; chromosome length 1.48 μm), *L. multiflora* (2n = 6x = 36; chromosome length 1.32 μm), and *L. sudetica* (2n = 8x = 48; chromosome length 0.52 μm). The trend could be explained to understand that species with chromosome number 12 has merged their chromosomes together through a process of symploidy to occur speciation of a new diploid with 2n = 6. This could be possible because the size of the chromosomes increasing in L elegans and L. purpurosplendans. Similarly, there is a possibility of agmatoploidy phenomenon has been occurred and the size the chromosomes decreased in *L. sudetica*. Moreover, it clearly


#### **Figure 1.**

*Karyotype trend in* Luzula *species by fission and fusion of the chromosomes.*

suggests the trend of chromosome size decrease from diploid species towards polyploidy species or vice-versa [12, 13].

## **2.2 Karyotype evolution and speciation**

Karyotype evolution may be defined as a phenomenon of change in chromosome number with time and space where fusion or fission and rearrangement may take place among chromosomes to decrease or increase its chromosome complement as well as to adapt themselves in available climatic conditions at that particular place and for their survival over a period of time (**Figure 2**). For example, triticeae genome with a basic chromosome number 7 had undergone 5 centric and 7 nested fusion to reach the present 5 chromosome structure karyotype in *Brachypodium distachyon*. The fusion in the genome of triticeae and *B. distachyon* involved different combinations of ancestral chromosomes and therefore they were independent of each other [14]. When triticeae genome was crossed with *B. distachyon* genome [triticeae, 2n = 2x = 14 (TT) × *B. distachyon*, 2n = 2x = 10 (BB) = hybrid, 2n = 2x = 12 (TB)], a hybrid of 2n = 2x = 12 chromosomes was considered to be the ancestor of present day *B. distachyon* (2n = 2x = 10). It was considered that approximately 7 nested fusion (large number of breaks in the chromosomes and then repositioning of the fragments) suggest descending dysploidy in the ancestor to reach the present *B. distachyon* (2n = 2x = 10). The evolution of eudicot and monocot lineages is driven by two counteracting processes i.e. whole genome duplication (WGD) and diplodization. It is inferred that allthe present grass genomes evolved from an intermediate ancestor with 12 chromosomes which itself arose from 5 or 7 chromosome ancestor through WGD and two reciprocal translocations (**Figure 3**). Although this particular rearrangements is common in grasses, it rarely occurs in eudicots in which end to end fusion are mostly responsible for reduction in chromosome number [15, 16].

**49**

**Figure 4.**

*Characteristics of various basic banding techniques.*

*Chromosome Banding and Mechanism of Chromosome Aberrations*

Similarly, evolutionary history of a karyotype is oftenly difficult to trace for older events and with time the accumulation of chromosome rearrangements remove the exact identity, number and order of the events occurred along the lineages leads to an extant karyotype. There are techniques to reconstruct the Extant karyotypes by extracting the information's from the extinct and its close relatives to get the hint for the direction of evolution of karyotype under paleogenomics [17].

This is a technique for the identification of chromosomes and its structural abnormalities in the chromosome complement. Chromosome identification depends on their morphological characteristics such as relative length, arm ratio, presence and absence of secondary constrictions on the chromosome arms. Therefore, it is an additional and useful tool for the identification of individual chromosome within the chromosome complement. Further, it could be used for identification of chromosome segments that predominantly consist of either GC or AT rich regions or constitutive heterochromatin. The technique which involves denaturation of DNA followed by slow renaturation permits identification of constitutive heterochromatin as it mainly consists of repetitive DNA. On banded chromosome, darkly stained or brightly fluorescent transverse bands (positive bands) alternate with the lightly stained or less fluorescent (negative bands). The bands are consistent, reproducible and are specific for each species and each pair of homologous chromosomes. Banding techniques also revealed the extensive genetic polymorphism manifested as inter-individual differences in the size and stain ability of certain chromosomal segments. Initially four basic types of banding techniques were recognized for the identification of Human chromosome complement (Q, C, G and R bands) and later on two additional major type of bands were developed (N and T bands) for complete identification of the chromosome complement (**Figure 4**). Now present bands and newly developed bands or molecular

*DOI: http://dx.doi.org/10.5772/intechopen.96242*

*Schematic diagram to show the karyotype evolution in grasses.*

**3. Banding techniques**

**Figure 3.**

**Figure 2.** *Karyotype evolution in* Brachypodium *by fusion or descending dysploidy from the ancestor.*

*Chromosome Banding and Mechanism of Chromosome Aberrations DOI: http://dx.doi.org/10.5772/intechopen.96242*

#### **Figure 3.**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

polyploidy species or vice-versa [12, 13].

**2.2 Karyotype evolution and speciation**

suggests the trend of chromosome size decrease from diploid species towards

Karyotype evolution may be defined as a phenomenon of change in chromosome number with time and space where fusion or fission and rearrangement may take place among chromosomes to decrease or increase its chromosome complement as well as to adapt themselves in available climatic conditions at that particular place and for their survival over a period of time (**Figure 2**). For example, triticeae genome with a basic chromosome number 7 had undergone 5 centric and 7 nested fusion to reach the present 5 chromosome structure karyotype in *Brachypodium distachyon*. The fusion in the genome of triticeae and *B. distachyon* involved different combinations of ancestral chromosomes and therefore they were independent of each other [14]. When triticeae genome was crossed with *B. distachyon* genome [triticeae, 2n = 2x = 14 (TT) × *B. distachyon*, 2n = 2x = 10 (BB) = hybrid, 2n = 2x = 12 (TB)], a hybrid of 2n = 2x = 12 chromosomes was considered to be the ancestor of present day *B. distachyon* (2n = 2x = 10). It was considered that approximately 7 nested fusion (large number of breaks in the chromosomes and then repositioning of the fragments) suggest descending dysploidy in the ancestor to reach the present *B. distachyon* (2n = 2x = 10). The evolution of eudicot and monocot lineages is driven by two counteracting processes i.e. whole genome duplication (WGD) and diplodization. It is inferred that allthe present grass genomes evolved from an intermediate ancestor with 12 chromosomes which itself arose from 5 or 7 chromosome ancestor through WGD and two reciprocal translocations (**Figure 3**). Although this particular rearrangements is common in grasses, it rarely occurs in eudicots in which end to end fusion are mostly responsible for reduction in chromosome

**48**

**Figure 2.**

*Karyotype evolution in* Brachypodium *by fusion or descending dysploidy from the ancestor.*

number [15, 16].

*Schematic diagram to show the karyotype evolution in grasses.*

Similarly, evolutionary history of a karyotype is oftenly difficult to trace for older events and with time the accumulation of chromosome rearrangements remove the exact identity, number and order of the events occurred along the lineages leads to an extant karyotype. There are techniques to reconstruct the Extant karyotypes by extracting the information's from the extinct and its close relatives to get the hint for the direction of evolution of karyotype under paleogenomics [17].

### **3. Banding techniques**

This is a technique for the identification of chromosomes and its structural abnormalities in the chromosome complement. Chromosome identification depends on their morphological characteristics such as relative length, arm ratio, presence and absence of secondary constrictions on the chromosome arms. Therefore, it is an additional and useful tool for the identification of individual chromosome within the chromosome complement. Further, it could be used for identification of chromosome segments that predominantly consist of either GC or AT rich regions or constitutive heterochromatin. The technique which involves denaturation of DNA followed by slow renaturation permits identification of constitutive heterochromatin as it mainly consists of repetitive DNA. On banded chromosome, darkly stained or brightly fluorescent transverse bands (positive bands) alternate with the lightly stained or less fluorescent (negative bands). The bands are consistent, reproducible and are specific for each species and each pair of homologous chromosomes. Banding techniques also revealed the extensive genetic polymorphism manifested as inter-individual differences in the size and stain ability of certain chromosomal segments. Initially four basic types of banding techniques were recognized for the identification of Human chromosome complement (Q, C, G and R bands) and later on two additional major type of bands were developed (N and T bands) for complete identification of the chromosome complement (**Figure 4**). Now present bands and newly developed bands or molecular

**Figure 4.** *Characteristics of various basic banding techniques.*

bands are widely used in animals and plants for the identification of chromosome complement, chromosome aberrations as well as traces of phylogeny [18, 19].

#### **3.1 Banding pattern of Q, G, R and C bands on Human chromosome complement**

Chromosome band C and G clearly identifies the secondary constrictions of chromosome number 1, 9 and 16 sometimes slight or occasional staining were found for secondary constrictions of chromosome 9. C-band clearly stains and identifies peri-centromeric region on the chromosomes, while band Q slightly stains peri-centromeric region of chromosome 3. Both C and Q bands are equally important for staining the distal part of long arm of Y chromosome but for both the bands partial staining was recorded for satellites. Partial Q band staining was reported for chromosome 3, 13 and 21 while other chromosomes were recorded with intense staining. The C-band was found suitable to stain important regions and structures of the Human chromosome complement and widely used band. The G band is also known as golden band for the identification of the homologous pair within complement and could be considered a basic band before application of any sophisticated and molecular approach for further investigation [20].

#### **3.2 Code system for banding pattern**

There were 3 letter coding system for the banding procedure, for example, first letter codes for the type of banding to be done; second letter codes for the general technique to be used and third letter codes for the stain to be used. For instance, code QFQ indicates the Q-band to be done, fluorescence technique to be used and quinacrin mustard stain to be used during banding procedure. Similarly, other codes may be QFH, QFA, GTG, GTL, GAG, CBG, RFA, RHG, RBG, RBA, THG and THA depending on the bands, techniques and stains [21].

### **4. Chromosome bands**

#### **4.1 Q (quinacrine) band**

The band stains the chromosome with fluorochromequinacrine mustard or quinacrinedihydrochloride (atebrin), observed under fluorescence microscope, and shows a specific banding pattern [22]. The fluorescence intensity is determined by the distribution of DNA bases along the chromosomal DNA with which the dyes interacts. The AT-rich regions enhance the fluorescence while GC-rich regions quench the fluorescence. The brightly fluorescent Q bands show high degree of genetic polymorphism but the fluorescence of Q band is not permanent and fades rapidly, therefore, the banding must be observed on fresh preparation and selected metaphases photographed immediately for further analysis. The disadvantage of the technique is the application of an expensive fluorescent microscope.

Q banding could also be achieved by fluorochromes other than quinacrine or its derivatives e.g. daunomycin, hoechst33258, BrdUetc which enhances AT-rich regions and quenches GC-rich regions. Acridineornage stains AT-rich regions red and GC rich regions green.

#### **4.2 C (constitutive heterochromatin) banding**

C banding was developed as a by product of in situ hybridization experiments on the localization of the mouse satellite DNA [23]. Centromeric regions with

**51**

*Chromosome Banding and Mechanism of Chromosome Aberrations*

between maternal and foetal cells in amniotic fluid cell culture.

constitutive hterochromatin where satellite DNA was located stained more deeply with Geimsa than the rest of the chromosome [24]. The C banding technique is based on the denaturation and renaturation of DNA and the regions containing constitutive heterochromatin stain dark (C band) and could be visible near the centromere of each chromosome. The C bands are polymorphic in size which is believed to correspond to the content of the satellite DNA in those regions. C banding allows precise analysis of abnormalities in the centromeric regions and detection of isochromosomes. The C banding in combination with simultaneous T-banding in particular, extends to easy detection of dicentric rings [25]. Sometimes, C banding also permits to ascertain the parental origin of foetal chromosomes and distinguish

The banding could also be recognised as the modification of C banding procedure [26]. The technique permits the accurate identification of each pair of the chromosomal complement as well as recognition of the specific chromosomal rearrangements within complement. The preparations are permanent after staining with giemsa. A number of modifications for G bands have been developed and proposed such as pre-treatment with trypsin, urea, enzymes and salts, even though original ASG method (G like bands) as well as trypsin method often slightly modi-

G bands correspond exactly to chromomeres of meiotic chromosomes but the mechanism leading to the visualization of the basic chromosome pattern is still unclear. The process is believed to be associated with denaturation and distributin of non-histone proteins and rearrangements of chromatin fibres from G negative to

R banding patterns are based on the thermal treatment of chromosomes and in general the reverse of the Q and G bands developed and proposed by Dutrillaux and Lejeune [27]. The ends of the R banded chromosomes are almost or always found positive and the centromeric regions are easily distinguished. This permits the observation of minor abnormalities in the terminal regions of chromosomes and the precise determination of chromosomal lengths. The technique is performed on a fixed chromosomal preparation and is based on heat denaturation of chromosomal DNA. R bands (GC rich regions) are more sensitive to DNA denaturation than Q and G bands (AT-rich regions). Giemsa stained R bands can be observed under phase contrast microscope while acridine orange stained R bands

T bands are, in fact, the segments of the R bands that are most resistant to the heat treatment and the transition patterns between the R and T bands could be obtained by gradual treatments. Therefore, it may be regarded as the modifications of the R banding technique [28]. The clear marking of telomeric regions of chromosome with T banding enables the detailed analyses of the structural rearrangements at the ends of chromosomes. It also allows the detection of human chromosome 22 and its involvement in translocation. The usefulness of this method is for the detection of dicentric rings that were undetectable by other procedures. T bands can be

*DOI: http://dx.doi.org/10.5772/intechopen.96242*

**4.3 G (Giemsa) banding**

fied and most widely used.

**4.4 R (Reverse) banding**

require fluorescence microscope.

observed either after giemsa or acridine orange staining.

**4.5 T (Telomeric) banding**

G positive bands.

*Chromosome Banding and Mechanism of Chromosome Aberrations DOI: http://dx.doi.org/10.5772/intechopen.96242*

constitutive hterochromatin where satellite DNA was located stained more deeply with Geimsa than the rest of the chromosome [24]. The C banding technique is based on the denaturation and renaturation of DNA and the regions containing constitutive heterochromatin stain dark (C band) and could be visible near the centromere of each chromosome. The C bands are polymorphic in size which is believed to correspond to the content of the satellite DNA in those regions. C banding allows precise analysis of abnormalities in the centromeric regions and detection of isochromosomes. The C banding in combination with simultaneous T-banding in particular, extends to easy detection of dicentric rings [25]. Sometimes, C banding also permits to ascertain the parental origin of foetal chromosomes and distinguish between maternal and foetal cells in amniotic fluid cell culture.

#### **4.3 G (Giemsa) banding**

*Cytogenetics - Classical and Molecular Strategies for Analysing Heredity Material*

and molecular approach for further investigation [20].

THA depending on the bands, techniques and stains [21].

**3.2 Code system for banding pattern**

**4. Chromosome bands**

**4.1 Q (quinacrine) band**

and GC rich regions green.

**4.2 C (constitutive heterochromatin) banding**

bands are widely used in animals and plants for the identification of chromosome complement, chromosome aberrations as well as traces of phylogeny [18, 19].

**3.1 Banding pattern of Q, G, R and C bands on Human chromosome complement**

Chromosome band C and G clearly identifies the secondary constrictions of chromosome number 1, 9 and 16 sometimes slight or occasional staining were found for secondary constrictions of chromosome 9. C-band clearly stains and identifies peri-centromeric region on the chromosomes, while band Q slightly stains peri-centromeric region of chromosome 3. Both C and Q bands are equally important for staining the distal part of long arm of Y chromosome but for both the bands partial staining was recorded for satellites. Partial Q band staining was reported for chromosome 3, 13 and 21 while other chromosomes were recorded with intense staining. The C-band was found suitable to stain important regions and structures of the Human chromosome complement and widely used band. The G band is also known as golden band for the identification of the homologous pair within complement and could be considered a basic band before application of any sophisticated

There were 3 letter coding system for the banding procedure, for example, first letter codes for the type of banding to be done; second letter codes for the general technique to be used and third letter codes for the stain to be used. For instance, code QFQ indicates the Q-band to be done, fluorescence technique to be used and quinacrin mustard stain to be used during banding procedure. Similarly, other codes may be QFH, QFA, GTG, GTL, GAG, CBG, RFA, RHG, RBG, RBA, THG and

The band stains the chromosome with fluorochromequinacrine mustard or quinacrinedihydrochloride (atebrin), observed under fluorescence microscope, and shows a specific banding pattern [22]. The fluorescence intensity is determined by the distribution of DNA bases along the chromosomal DNA with which the dyes interacts. The AT-rich regions enhance the fluorescence while GC-rich regions quench the fluorescence. The brightly fluorescent Q bands show high degree of genetic polymorphism but the fluorescence of Q band is not permanent and fades rapidly, therefore, the banding must be observed on fresh preparation and selected metaphases photographed immediately for further analysis. The disadvantage of

Q banding could also be achieved by fluorochromes other than quinacrine or its derivatives e.g. daunomycin, hoechst33258, BrdUetc which enhances AT-rich regions and quenches GC-rich regions. Acridineornage stains AT-rich regions red

C banding was developed as a by product of in situ hybridization experiments on the localization of the mouse satellite DNA [23]. Centromeric regions with

the technique is the application of an expensive fluorescent microscope.

**50**

The banding could also be recognised as the modification of C banding procedure [26]. The technique permits the accurate identification of each pair of the chromosomal complement as well as recognition of the specific chromosomal rearrangements within complement. The preparations are permanent after staining with giemsa. A number of modifications for G bands have been developed and proposed such as pre-treatment with trypsin, urea, enzymes and salts, even though original ASG method (G like bands) as well as trypsin method often slightly modified and most widely used.

G bands correspond exactly to chromomeres of meiotic chromosomes but the mechanism leading to the visualization of the basic chromosome pattern is still unclear. The process is believed to be associated with denaturation and distributin of non-histone proteins and rearrangements of chromatin fibres from G negative to G positive bands.

#### **4.4 R (Reverse) banding**

R banding patterns are based on the thermal treatment of chromosomes and in general the reverse of the Q and G bands developed and proposed by Dutrillaux and Lejeune [27]. The ends of the R banded chromosomes are almost or always found positive and the centromeric regions are easily distinguished. This permits the observation of minor abnormalities in the terminal regions of chromosomes and the precise determination of chromosomal lengths. The technique is performed on a fixed chromosomal preparation and is based on heat denaturation of chromosomal DNA. R bands (GC rich regions) are more sensitive to DNA denaturation than Q and G bands (AT-rich regions). Giemsa stained R bands can be observed under phase contrast microscope while acridine orange stained R bands require fluorescence microscope.

#### **4.5 T (Telomeric) banding**

T bands are, in fact, the segments of the R bands that are most resistant to the heat treatment and the transition patterns between the R and T bands could be obtained by gradual treatments. Therefore, it may be regarded as the modifications of the R banding technique [28]. The clear marking of telomeric regions of chromosome with T banding enables the detailed analyses of the structural rearrangements at the ends of chromosomes. It also allows the detection of human chromosome 22 and its involvement in translocation. The usefulness of this method is for the detection of dicentric rings that were undetectable by other procedures. T bands can be observed either after giemsa or acridine orange staining.
