Genetics and Molecular Biology
Botany Major, Semester V
BT 502C
Unit I Genetics-I
1 Mendelian Genetics:
Principles of segregation and independent assortment,concept of Dominance, Incomplete dominance, Codominance, Multiple allele, Penetrance, Expressivity, Pleotropism, Phenocopy effect and Atavism.
2. Determining allelism of mutants
Complementation test, Definition of Cistron,Muton & Recon, Concept of pseudoallele, Fine structure of gene -- structure of phase rII locus.
3. Gene interactions with modified dihybrid ratios
12:3:1
9:7
9:4:3
9:6:1
13:3
15:1
Unit II Genetics -II
1. Cytoplasmic Inheritance:- Features, Plastid inheritance - leaf colour in Mirabilis jalapa, Mitochondrial inheritance -:Poky &Petite mutation, Maternal effect - shell coiling in snail.
2. Linkage :- Definition of complete linkage & incomplete linkage, coupling phase, repulsion phase, linked group,
Crossing over:- definition and cytological basis of crossing over ( Creigton & McClintock Experiment).
3. Recombination - Basic concept, Recombination frequency, Two point & Three point Test cross, Gene mapping from three point test cross data, coefficient of correlation , interference.
Unit-III Genetics-III
1. Chromosomal abberation:- Numerical changes:-( aneuploidy and euploidy), Polyploidy types.
Structural changes: Definition and types of deletion, Duplication, Inversion, Translocation
Meiotic behaviour of inversion and translocation heterozygote,
Position Effect.
2 Sex determination : Mechanism of sex determination in Human and Drosophila.
3. Sex linkage: Sex linked inheritance, Dosage compensation & Lyon's hypothesis, Sex limited and Sex influenced traits, problems on Sex linkage( Haemophilia & Colour blindness).
Unit IV Genetics IV
1 Population genetics :- Concept of Gene pool, Allele frequency and genotype frequency, Hardy- Weinberg law, Conditions for HW equilibrium, Numerical problems based on HW equation.
2. Factors affecting changes in gene frequency: Migration, Mutation,Selection and Genetic Drift:- definition and effects on gene frequency.
3.Quantitive inheritance - Characters of quantities traits, Heritability- Narrow sense and Broad sense Heritability ,
Polygenic inheritance: Regulation of kernel colour in Wheat
Cistron is a segment of DNA coding for a polypeptide .
(It is a segment of DNA containing the genetic information for the sequence of amino acids of a polypeptide chain i.e. the blueprint of a protein is actually stored in DNA as a cistron).
(2)Define intron and exon .
In Eukaryotes, the structural gene is monocistronic and coding sequences are interrupted by intervaining sequences.
Coding sequences are expressed as exons and intervaining sequences are expressed as introns in pre -mRNA/ hn RNA during transcription process .Introns are excised by spliceosome at processing time and only exons are appeared and joined to make a mature/processed mRNA.
The monocistronic structural genes are also known as split gene.
(3) Define transcription .
The process of copying/transfer of genetic information from template strand of DNA into mRNA maintaining "the principle of complimentarity" is known as transcription .
(4) Why both strands of DNA are not copied during transcription ?
There are two reasons :
(i) If two strands of a structural genes act as template then the two transcribed mRNA will be of different sequences , as a result of that transcribed proteins will differ from each other . Hence , a gene will be coding two different genes ,which contradicts the definition of a gene.
(ii) The transcribed mRNA molecules will be complementary to each other , hence, they will form ds RNA and translation pracess will prevented .
Unit I:
Chapter 1 Mendalian genetics
1.1.Mendel's first law/ Law of Segregation:
This law states that allele pairs separate during gamete formation, ensuring that each gamete received only one allele for a given trait.
Non - Blending: When two different alleles ( e.g., T & t) are brought together in an F1 hybrid (Tt) , they remain distinct entities and do not blend or contaminated one another.
Complete Seperation: During gamete formation, these paired alleles segregate purely and completely , meaning individual gametes carry only a single , unmixed allele from the pair.
Explanation with the help of Monohybrid cross
To understand this law, inheritance of single trait- plant height in pea plants is considered.
•Dominant allele (T) : Tall plant
•Recessive allele (t) : Dwarf plant
1. Parent Generation: (P) : A pure - breeding homozygous tall plant(TT) is crossed with a pure-breeding homozygous dwarf plant ( tt).
Or, when homozygous round seeded (RR) pea plant is crossed with a homozygous wrinkled seeded plant is crossed a hybrid (Rr) with round phenotypic offspring appeared in F1 generation which one selfing produce 3:1 phenotypic and 1:2:1 genotypic ratio in F2 generation.
2. First Generation (F1): All offsprings receive one allele from each parent , resulting them heterozygous (Tt) . Because T is dominant,all F1 plants appear tall.
3. Second Generation (F2): When F1 plants self-pollinated (Tt x tt) , both tall and dwarf plants appear in the next generation.
F2 Generation Results:
•Phenotypic Ratio (Visual Appearance) :
Tall : Dwarf=3:1
•Genotypic Ratio( Genetic Makeup):
TT : Tt: tt = 1:2:1
•Allelles Remain Pure: Even though the F1 plants carry both T and t alleles together, the alleles do not mix, blend,or alter each other. They remain completely pure.
•Reason of Separation: The biological basis for this law occurs during Anaphase I of meiosis. Homologous chromosomes seperate into different gametes, taking the alleles located at corresponding positions on those chromosomes with them.As a result, a gamete will receive either T allele or the t allele, but never both.
1.2.Mendel's second law: The Law of Independent Assortment
The Law of Independent Assortment states that when two or more pairs of contrasting characters ( allelomorphic pairs) are combined in a hybrid, the segregation or separation of one pair is completely independent of the seperation of other pair of alleles during gamete formation.
Explanation:
(1) Gene location : Consider two genes located on two separate, non- homologous pairs of chromosomes.
(2) Allelic Distribution: Each gene pair consists of two alleles situated on homologous chromosome pairs.
(3) Meiotic Segregation: During Anaphase I of meiosis, homologous chromosome pairs separate independently of one another.
(4) Random Combination: The segregation of one pair of chromosomes does not influence the orientation or seperation of the other pair. Consequently, any allele of one gene is equally likely to combine with either allele of the second gene within a given gamete.
Fig: Mendel's dihybrid cross between two pea plants having yellow cotyledons with round seed and green seed with wrinkled cotyledons
Genetic Consequences:
Gamete formation in F1: A dihybrid organism (RrYy) produces four distinct types of gametes: RY, Ry, rY and ry in equal 1:1:1:1 ratios.
F2 Generation Ratios: The random fertilization among these four types of gametes yields 16 possible zygotic combinations.
Phenotypic ratio: 9:3:3:1
Genotypic ratio : 1:2:2:4:1:2:1:2:1
1.3.Concept of Dominance and recessiveness
Dominance occurs when one allele of a gene masks the phenotypic expression of another allele at the same locus. The allele that exerts the masking effect is dominant, while the allele whose effect is hidden is recessive.
In Mendel's monohybrid cross, crossing a homozygous tall (TT ) or heterozygous tall (Tt) pea plant with a homozygous dwarf (tt) plant yields offspring (F1 generation) that visually display only the dominant parents phenotype ( tall) .The unexpressed trait ( dwarf) remains hidden in this generation. However, when these F1 plants self-pollinate, the recessive trait reappears in the F2 generation, accounting for 25% ( a 3:1 phenotypic ratio) of the offspring.
Incomplete dominance
Incomplete dominance is a deviation from Mendelian Inheritance where two alleles of a gene mix together instead of one completely hiding the other. Instead showing one parent's trait , the heterozygous offspring ends up with a blended, middle - ground state.
The classical example of this is the formation of pink flowered offsprings ( Rr) in F1 generation when a cross is made in between homozygous red flower plant (RR) and another homozygous white flowered ( rr) plant of parent generation in Mirabilis jalapa or Four o'clock plant.
On selfing in F1 generation again deviation is observed . Both of phenotypic ratio and the genotypic ratio turned identical 1:2:1 instead of Mendelian Inheritance ratio of 3:1 and 1:2:1 respectively.
Here the intensity of colour formation in F1 generation (Rr) turned lower than the intensity of colour formation of RR parent. It resulted an intermediate between red and white i.e. pink.
Unit II : Genetics II
Chapter: : Maternal Effects and Cytoplasmic Inheritance
*.1.Maternal inheritance
The phenomenon of dextral and sinistral shell coiling in Lymnaea peregra was first discovered,analysed and published by A.E. Boycott and colleagues in 1923 and 1930. It is one of the best example of the maternal inheritance.
In Lymnaea peregra , the female gamete is physically larger than the male gamete and supplies the cytoplasm for the developing embryo. This cytoplasm contains factors originally transcribed from the female nuclear genes.
Maternal effect refers to a phenomenon where an offspring's phenotype is determined entirely by the nuclear genotype of the mother ,regardless of the offspring's own genotype or paternal influence.These traits are controlled by factors ( such as mRNAs or proteins) that are encoded by the nuclear genes in the mother and deposited into the cytoplasm of the egg.
Reciprocal cross of the above cross:
F2 generation (dd) : Although these individuals possess the homozygous recessive genotype (dd) , they exhibit a dextral ( right - handed) phenotype . This occurs because their mother ( the F1 generation with a Dd genotype) deposited maternal genes products-- specifically the functional dextral protein or m RNA into the egg cytoplasm during oogenesis. Consequently,the mother's phenotype ( Dd producing dextral offspring) dictates the physical trait of the F2 progeny regardless of their individual nuclear genotype.
F3 generation (dd) : These dd offsprings develop a sinistral (Left -handed) phenotype. Their mothers belong to the F2 generation ( dd genotype) and, despite displaying a dextral phenotype themselves due to their maternal inheritance,their underlying genotype is homozygous recessive (dd). Therefore, these F2 mothers produce eggs lacking the dextral factors, resulting in the expression of the maternal genotype( dd) as a sinistral phenotype in the F3 generation.
Key Principles: The phenotype of an individual for this trait is entirely determined by the genotype of the mother , delayed by one generation.
*.2.Organellar inheritance/Plastid inheritance in Mirabilis jalapa
Plastid inheritance was first discovered by C. Correns in Mirabilis jalapa in 1908.There are three types of branches in this plant:-
(i)Complete green branches and leaves having chloroplasts,
(ii)White branches and leaves without chloroplasts and contains leucoplasts,and
(iii)Variegated branches with both kinds of plastids.
The following reciprocal crosses proved that phenotype of offsprings depend on the type of eggs , not on the nature of pollens.
Cross I : A cross between eggs from variegated plant/ branch ( female) and pollens from green plant/ branch ( male) :
Results:
(i) If the egg cytoplasm contains chloroplasts only then all F1 appear as green,
( ii) If the egg cytoplasm contains both of chloroplasts and leucoplasts then F1 all offspring appear as variegated., and
( iii) When the eggs have leucoplasts only then all offsprings appear as white. These white seedlings die early due to the lack of chloroplast and consequently lack of the photosynthesis process.
Cross II : It is reciprocal to the previous one. Eggs from green plant/ branches and pollens from variegated plant/ branches:
Results:
Only one kind of F1 offsprings appear which are green.
Conclusion: Since the results of the reciprocal crosses are phenotypically completely different from each other , it is proved that it is a case of maternal line and leaf variation is inherited non-Mendelianly through the cytoplasmic DNA or plastidDNA.
The following experiment also proves that plastid inheritance in Mirabilis jalapa is depend on the nature of female branchs :
In such cases the phenotype of F1 offssprings depend upon phenotype of branch on which flowers are pollinated.
*.3.Mitochondrial Inheritance
'Petite in Yeast
The petite phenotype is primarily caused by mutations in the mitochondrial genome , loss of mitochondria, deletions in mtDNA , or host cell genome mutation( neutral petite).
Genetic basis of petite: Vegetative petite which completely lack rho factor are called neutral petite. A vegetative petite having a defective rho factors is called suppressive petite.This rho is a cytoplasmic factor. The neutral petites are not transmitted and lack mtDNA, while suppressive petites are transmitted to a fraction of vegetative diploid progeny and contain mtDNA , that is often grossly altered in base composition with respect to wild mtDNA.
Experiments /Crosses:
A cross between a neutral petite yeast ( with defective mtDNA) and a wild type yeast:
Inference: Upon sporulation and tetrad analysis it is found that all four spores of an ascus are wild-type and there is no segregation of petite trait into 2:2 ratio, completely violating Mendel's first law .
Another cross between Suppressive petite and wild type:
Result: Formation of suppressive petite yeast in F1 generation.
Inference: In this cross resulted F1 generation are petite, because the mutated or deleted mtDNA actively overrides or ' swamps out' the wild type mitochondrial genome.Since, all four spores are petite or in irregular ratios in a single ascus, the mutation is extra nuclear .
Physiological basis of petite:
Inner chamber of mitochondria is the site of TCA cycle because of the presence of respiratory enzymes. In petite yeast there is lack of Cyt a, Cyt a3, Cyt b and a number of other changes in mitochondrial respiratory enzymes, or mitochondria with incomplete developement , hence petites have a defective aerobic respiratory mechanism. As a result of that petites fail to grow on carbon source like sucrose and only produce smaller colonies when grown on sugars like glucose.
2. Linkage
Definition of Linkage: Linkage is the tendency of genes or DNA segments located close to one another on the same chromosome to be inherited together during meiosis.
Classification of Linkages: Linkages are broadly classified into two primary categories based on the presence or absence of recombinant ( non- parental) combinations in subsequent generations :-
(i) Complete Linkage: Complete linkage occurs when two or more linked genes are inherited together with zero recombination, appearing exclusively in their parental combinations across successive generations.
* Molecular basis: The genes exhibiting complete linkage are situated extremely close to each other on the same chromosome.
* Crossing over: Because of there physical proximity, chiasma formation and subsequent crossing over donot occur between their loci during meiosis.
* Progeny output: Generates 100% parental phenotypes and strictly zero non- parental ( recombinant) progeny.
Classical example : Linkage observed in male Drosophila.
Income Linkage: Incomplete linkage is characterized by the production of both parental combinations and a measurable portion of non- parental (recombinant) combinations.
*Molecular basis: The genes are located at a distance from one another along the chromosome.
* Crossing over: During Prophase I of meiosis, , the physical breakage and exchange of chromosomal segments (crossing over) occur between non-sister chromatids of homologous chromosomes.
*Progeny output -Results in a mixture of both parental and recombinant phenotypes, with the frequency of recombination directly proportional to the physical distance between the genes.
Coupling phase & Repulsion phase of linkage
Coupling phase:
Definition: When two dominant alleles of linked genes reside on the same homologous chromosome, it is known as the coupling phase of linkage.
Genetic effect: The coupling arrangement of alleles tends to keep parental combinations together which resulted in a higher frequency of non-recombinant /dominant parental phenotype in offsprings in higher than expectations.
Repulsion phase:
Definition: When a dominant allele of one gene and a recessive allele of a second gene resides on the same homologous chromosome, it is known as the repulsion phase of linkage.
Genetic effect:: This arrangement tends to produce higher frequencies of recombinant/ non-parental phenotypes in the offspring compared to independent assortment.
Bateson and Punnet's experiment ,1906:
Bateson and Punnet worked with sweet pea which are presented below:
According to the Mandelian principal the phenotypic ratio will be 4/16: 4/16 : 4/16:4/16 or 1:1:1:1 and a frequency of 25% of each classi.e , parental frequency 25%+25%=50% and similarly recombinant frequency is also 25%+25%=50% in F2 generation.
Moreover,the frequency of parental progeny turned 43.75%+43.75%= 87.50%. Simultaneously, frequency of recombination appears 6.25%+6.25%=12.50% . The frequency of recombination less than 50% implies that the genes are linked on the same chromosome. Bateson and Punnet concluded that as gene P and L were from same parent so show the tendency to remain together in progenies and they termed the arrangement as cis- configuration or coupling phase.
Linkage Group:
Linkage Group refers to a group of genes which are present in one chromosome. It means the genes present in one chromosome constitute the linkage group of that individual.
The maximum number of a linkage group is equal to the haploid number of that organism. e.g., the linkage group of Alliam cepa is 8 ( 2n = 16), and in Drosophila melanogesture 4(2n = 8).
CROSSING OVER
Cytological basis of crossing over :
In 1931,C.Stern ( using Drosophila) and H.S. Creighton and B.McClintock (using maize) independently provided empirical proof for the cytological basis of crossing over.By utlizing cytological markers----specially , structrully altered chromosomes resulting from chromosomal aberrations---- they were able to visually distinguish homologous partners and correlate physical exchange with genetic recombination.
Coincidence: The simultaneous occurrence of two or more crossovers within the same region of a pair of homologous chromosomes, resulting in a double Cross Over product is called Coincidence.
It is estimated by the following formula:
observed frequency of DCO
CoC=----------------------------------------------------
expected frequency of DCO
Hence, the ratio between the observed and expected frequencies of double cross over is called Coefficient of Coincidence.
Interference: Interference is the biological phenomenon in genetics where a crossing -over event in one region of a chromosome reduces or prevents the occurrence of additional crossing -over events in adjacent or neighbouring segments of that chromosome.
Positive Interference: The tendency of one cross over to prevent anothar cross over from occurring in its vicinity.
Negative Interference: When one cross over enhances the chances of another cross over in the adjacent region.
Calculation:
Coefficient of interference = 1- CoC
Three Point Test Cross & Gene Mapping
A Three point testcross is a fundamental genetic mapping technique used to determine the linear order and calculate the map distances/ recombination frequencies among three linked genes on a single chromosome.
Problems:
1. In a three-point testcross (CshWx/cShwx x cshwx/cshwx following data was obtained:
Calculate the recombination values and prepare a linkage map showing relative distances and linear order between the genes.
Also calculate Coefficient of Coincidence and interference.
Solution
Step I : Determination of Gene Order
(1) Identification of Parental classes(Non-recombinsnts):
The highest frequencies shown in the given data is:
CshWx = 2777
cShwx= 2708
-------------------------------
Total = 5485
So, these are the parental classes.
(2) Identification of the Double Cross Over (DOC) classes:
CShWx = 4
cshwx = 3
--------------------------------
Total = 7
These classes show lowest frequencies,hence , these are the DCO classes.
(3) Determination of Gene Order:
By comparing alleles of DCo with the parental classes configuration it has been observed that alleles of gene Sh / sh flipped relative to the parents indicates the gene Sh/sh is placed in between middle C/c and Wx/wx genes.
Here, C (c) and WX (wx) remain with their original parental partners, while Sh (sh) has switched position .
Therefore, Sh is in the middle and the gene sequences will be :
C_______sh______WX
c_______Sh_______wx
Step II : Calculation of Recombination Frequencies/ Map Distance:
(1)Recombination Frequencies between C--Sh:
116+123+4+3
= -----------------------------x100
7000
= 3.51%
(2) Recombination Frequencies between Sh--Wx
643+626+4+3
=-------------------------------x100
7000
= 18.23%
(3) Recombination Frequencies between C--Wx
116+123+643+626+2(4+3)
=--------------------------------------------------x100
7000
= 21.74%
Since, 1map unit(mu) or cM (Centimorgan) equals to 1% recombination,then
Distance between C & Sh = 3.51cM ,
Distance between Sh & Wx = 18.23 cM ,and
Distance between C & Wx = 21.74 cM
Gene Map:
<-- 3.51 cM ---> <- 18.23 cM-->
C_________________Sh_______________Wx
<------------------------21.74cM----------------->
Fig: A linkage map prepared from results obtained from given data in the problem.
Coefficient of coincidence
Observed DCO
CoC=---------------------------------------------x100
Expected DCO
4+3
= -----------------------------------------
0.0351 x 0.1823 x 7000
7
= -----------------------------------
44.79
= 0.1562
Interference
Interference= 1-- 0.1562
= 0.8437
or , 84.37% indicating that about 84.37% of expected DCOs were prevented due to positive interference.
2. In a three-point testcross (ABC/abc x abc/abc), following data are obtained ( only phenotypes are given)
ABC .....230
abc.......240
aBc........96
AbC......104
ABc......138
abC......142
aBC........12
Abc........08
_______________________________
Total. 970
(a) Calculate the recombination values and prepare a linkage map showing relative distances and linear order between the genes.
(b) Calculate Coefficient of Coincidence and interference.
Solution:
Determination of Gene order:
Highest frequency:
ABC.......230
abc........240
----------------------------------------
Total= 470
Lowest frequency:
aBC......12
Abc......08
-----------------------------------------
Total = 20
Therefore, Parental classes( non- recombinant) = ABC & abc
Double Cross Overs = aBC & Abc
Comparison of parental classes and DCOs:
Calculation of Recombination Frequencies /Map Distance :(1) Recombination Frequencies between B--A
96+104+12+8
= --------------------------------------x100
970
22.68%
(2) Recombination Frequencies between A--C
138+142+12+8
=----------------------------------------x 100
970
= 30.92%
(3) Recombination Frequencies between B--C
96+104+138+142+2(12+8)
=------------------------------------------------x100
970
= 53.60%
Since, 1 mu or 1 cM = 1% Recombination Frequencies, therefore,
Distance between B--A = 22.68 cM
Distance between A--C = 30.92 cM
Distance between B--C= 53. 60 cM
Gene Map:
<---22.68 cM--> <----30.92--cM--->
B A C
-----------------------------------------------------
<--------------53.60 cM---------------------->
Or,
<---30.92 cM------> <-----22.68cM---->
C A B
-----------------------------------------------------
<----------------------53.60 cM --------------->
Coefficient of Coincidence:
Observed DCO
CoC =------------------------------------------
Expected DCO
12+8
= -------------------------------------------
0.2268 x 0.3092 X 970
20
= ------------------------------------
68.02
= ~0.2940
Interference :
1 - CoC = 1-- 0.2940
= 0.7060
Or, 70.60% , indicating that about 70.60% of expected DCOs were prevented due to positive interference.
Unit III
Chromosomal aberration
Definition:
A chromosomal aberration is a structural alteration or mutation in a chromosome resulting from breakage and rearrangement of its segments.
or,
Chromosomal aberrations refer to structural mutations in chromosomes caused by segment breakage followed by abnormal reunion or rearrangement.
Historical Significance:
Harriet Creighton and Barbara McClintock, 1931, first cytologically demonstrate the rearrangement and crossing over in maize (Zea mays) during meiotic chromosome pairing.
Classification of Chromosomal Aberrations
Chromosomal Aberrations are divided into two types based on whether the total amount of genetic material / chromatin is altered .
Type I: Quantative Alterations ( Gain or Loss of Chromatin)
These alterations change the total amount of genetic material in the chromosome.
1. Deletion or Deficiency:
Loss of a chromosome segment ,resulting in a reduction of chromatin and genetic information.
2.Duplication:
Addition or reputation of a chromosome segment, resulting in an excess of chromatin.
Type II: Qualitative Alteration ( Rearrangement of Chromatin)
These alterations involve structural rearrangement without altering the total amount of chromatin.
3.Inversion:
A chromosomal segments breaks, rotates by 180° ,and reunited in reverse orientation within the same chromosome.
4.Translocation:
Detachment of a chromosomal segment and its transfer or attachment to a non - homologous chromosome.
Deletion
Types of Chromosomal Deletion
It is classified into two types based on the location of the break:
1. Terminal Deletion:
Mechanism: Occurs near the end of a chromosome and involves a single break.
Result: The terminal portion containing the telomere is lost .
2. Intercalary ( Interstitial) Deletion:
Mechanism: Occurs within the body of a chromosome and involves two breaks on either side of the lost segment.
Result: The intermediate segment is removed,and the remaining ends rejoin.
Meiotic behaviour due to deletion
During synapsis in meiosis , homologous chromosomes must align allele- for- allele. When one chromosome has a deletion, pairing occurs in specific ways:
1. In Terminal Deletion:
• The homologous region of the normal chromosome pairs with the intact portion of the deficient chromosome.
•The unpaired portin corresponding to the deleted segment remains un- synapsed ( unpaired) at the end.
2.In Intercalary ( Interstitial) Deletion:
• To achive allele- to-allele pairing between the remaining homologous regions, the extra segment of the normal ( non- deleted) chromosome bulges outward, forming a deletion loop.
Effect of deletion on Inheritance:
When a dominant allele is deleted the remaining recessive allele appears to behave as a dominant allele in hemizygous situation due to the absence of the dominant functional counterpart.This behaviour of recessive allele is called as pseudodominance.
Example:
(i) In Drosophila melanogesture deletion of the wild-type Notch or white-eye region allows the underlying recessive mutant alleles to express visually.
(ii) In Maize (Zea mays) deficiency leads to the unexpected expression of recessive seed coat colours or endosperm traits.
Example of Deletion in human being
Cri-du-Chat ( Cry of the cat) Syndrome
Cause: A partial deletion of the short arm ( p arm) of chromosome 5 ( 5p-).
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