Linkage and crossing over
The tendency of genes or characters to be inherited together
because of their location on the same chromosome is called linkage. Based on
Mendel's work, several hybridization experiments were conducted on plants and
animals. But the results of certain dihybrid crosses in these experiments did
not follow the law of independent assortment.
In 1905, William Bateson, ER Saunders and Reginald Punnett
conducted experiments in sweet pea, Lathyrus
odoratus to confirm Mendel's dihybrid testcross. Here, Purple flower (B) is
dominant over the red flower (b) and long pollen (L) is dominant over round
pollen (l). They crossed true breeding plants having Purple flower with long
pollen (BBLL) and red flower with round pollen (bbll). All the F1 hybrids have Purple
flowers with long pollen (BbLl). A testcross between heterozygous Purple long
(BbLl) of F1 hybrid and double recessive parental stock red round (bbll)
did not result in ratio 9:3:3:1 or 1:1:1:1.
(A 1:1:1:1 ratio in a testcross of a dihybrid and a 9:3:3:1
ratio in a self of a dihybrid both reflect a gametic ratio of 1:1:1:1, which
shows the allele pairs are assorting independently and that the RF is 50
percent).
Here, Purple long and red round are parental forms and show
greater frequency 44 per cent each. Purple round and red long are recombinant
forms and show lesser frequency 6 per cent each. The dihybrid test cross ratio
obtained is 7:1:1:7 (Purple long: Purple round: red long: red round) and not
1:1:1:1. This indicates that the genes do not independently assort.
The explanation for this higher frequency of parental is
called linkage. The genes that are carried on the same chromosome will not
assort independently because of their tendency to remain linked together. This
is called linkage. The genes located on the same chromosomes that are inherited
together are known as linked genes.
It was Morgan (1910) who clearly proved and defined linkage
on the basis of his experiments in fruitfly Drosophila melanogaster. In 1911,
Morgan and Castle proposed chromosome theory of linkage. It states that
(i)
Linked
genes occur in the same chromosome.
(ii)
They
lie in a linear sequence in the chromosome.
(iii)
There
is a tendency to maintain the parental combination of genes except for occasional
crossovers.
(iv)
Strength
of the linkage between two genes is inversely proportional to the distance
between the two, i.e., two linked genes show higher frequency of crossing over
if the distance between them is higher and lower frequency if the distance is
small.
(v)
Linked
genes are those genes which occur on the same chromosome while unlinked genes
are the ones found on different chromosomes.
Linkage is of two types, complete
and incomplete.
Complete Linkage:
The genes located on the same
chromosome do not separate and are inherited together over the generations due
to the absence of crossing over. Complete linkage allows the combination of
parental traits to be inherited as such. It is rare but reported in male
Drosophila and some other heterogametic organisms.
Incomplete Linkage:
Genes present in the same
chromosome have a tendency to separate due to crossing over and hence produce
recombinant progeny besides the parental type. The number of recombinant
individuals is usually less than the number expected in independent assortment.
In independent assortment all the four types (two parental types and two
recombinant types) are each 25%. In case of linkage, each of the two parental
types is more than 25% while each of the recombinant types is less than 25%.
When there is an incomplete
linkage, new gene combinations are formed in the progeny or offsprings. This
occurs due to the formation of a chiasma or crossing over between the linked
genes.
Linkage Groups
A linkage group is a linearly
arranged group of linked genes which are normally inherited together except for
crossing over. It corresponds to a
chromosome which bears a linear sequence of genes linked and inherited
together. The number of linkage groups present in an individual corresponds to
number of chromosomes in its genome. It is known as principle of limitation of
linkage groups.
Fruit-fly
Drosophila melanogaster has four linkage groups (4 pairs of chromosomes),
human beings 23 linkage groups (23 pairs of chromosomes) and Escherichia coli one linkage group
Crossing Over
This is a phenomenon where genetic material is exchanged
between non-sister chromatids of homologous chromosomes which results in a new
gene combination. This process produces recombination of genes by interchanging
the corresponding segments between nonsister chromatids of homologous
chromosomes. A crossing over between linked genes allows their recombination
during meiosis. It helps the linked
genes to enter the gametes in combinations other than parental combinations due
to reciprocal exchange of genes along the pairs of homologous chromosomes.
Crossing over takes place in pachytene stage of prophase I
of meiosis. In pachytene stage, the bivalent chromosome becomes tetrad i.e.
with four chromatids. The adjacent nonsister chromatids are joined together at
certain points called chiasmata and crossing over occurs. At each chiasma, the
two nonsister chromatids break, exchange their segments and rejoin resulting in
crossing over.
Hence, out
of four chromatids the two adjacent chromatids are recombinants and other two
are original chromatids. Thus four types of gametes will be obtained.
The process of crossing over occurs in a sequence of following steps:
·
Synapsis
·
Duplication
of chromosomes
·
Crossing
over, Chiasmata formation and Terminalization
Synapsis:
The homologous chromosomes pair
lengthwise during prophase-I in meiosis. The pairing starts at one or more
points and proceeds along the whole length in a zipper fashion. The process of
pairing is called synapsis. The paired homologous chromosomes are called
bivalents. During synapsis, a molecular scaffold called synaptonemal complex
aligns the two homologous chromosomes side by side.
Duplication of chromosomes:
Synapsis is followed by the
duplication of chromosomes which changes the bivalent nature of chromosome to
four- stranded stage or tetravalent.
Crossing-over:
In pachytene, crossing over occurs.
Non-sister chromatids of homologous pair twist over each other due to action of
enzyme endonuclease. The chromatids get connected with each other at points known
as chiasmata. The crossing over can take place at several points. The number of
chiasmata formed is proportional to the length of chromatids. The genes at
distant loci undergo crossing-over but closely placed genes fail to cross-over
and exhibit linkage.
During diakinesis chiasmata move
towards the end of bivalent by a process called terminalization. Thus twisting
chromatids separate so that the homologous chromosomes are separated
completely.
At the end of meiosis, four types of
gametes are formed. Two will be of parent types and two will contain
chromosomes with recombination of genes formed during crossing- over.
Types of Crossing-over:
Single cross-over:
In this case, only one chiasma is
formed which leads to formation of single cross-over gametes. It is the most
common type of cross-over.
Double cross-over:
In double cross-over, two chiasmata
develop. These chiasmata may appear between the same chromatids or between
different chromatids. This type of crossing over forms double crossing-over
gametes.
Multiple cross-over:
Here, ‘more than two chiasmata are constituted. It may be further classified into triple (3 chiasmata), quadruple (4 chiasmata) and so on. Multiple crossing-over is of rare occurrence.
Factors influencing crossing-over are distance between the genes. More is the distance between two genes on same chromosome, higher will be the frequency of crossing over.
Significance of
crossing-over:
This process provides an inexhaustible store of genetic variability in sexually reproducing organisms.
Crossing over leads to the production of new combination of genes and provides basis for obtaining new varieties of plants and animals and is used by breeders.
It plays an important role in the process of evolution.
The crossing over frequency helps in the construction of genetic maps of the chromosomes.
It gives us the evidence for linear arrangement of linked genes in a chromosome.
Linkage
and recombination are phenomena that describe the inheritance of genes. A
linkage is a phenomenon where two or more linked genes are always inherited
together in the same combination for more than two generations. The
recombination frequency of the test cross progeny is always lower than 50%.
Therefore, if any two genes are completely linked, their recombination
frequency is almost 0%.
Cytological Evidence of
Crossing Over:
Morgan and his collaborators
established the genetic basis of crossing over and linkage and linear
arrangement of linked genes along chromosomes. This could not be demonstrated
cytologically since we can not observe the homologous chromosomes (being all
identical) under the microscope because of the following reasons.
(i) Crossing over occurs between
homologous chromosomes. Such chromosomes are alike in appearance and it is not
possible to distinguish between them in microscope.
(ii) During crossing over, the four
chromatids are intimately coiled around one another.
(iii) In living cells, crossing over cannot
be seen. In fixed and stained cells one cannot say that chromatids have
exchanged parts or not.
For nearly twenty years crossing over
remained only a working hypothesis. Finally the cytological evidence for the
occurrence of crossing over, was given by S. Stern on Drosophila and H.B.
Creigton and B.Mc Clintock on maize.
1. Stern’s Experiments or
Drosophila:
Stern discovered two varieties of
Drosophila. The strains contained
appropriate genetic markers and cytological markers, they were crossed and the
markers were analysed in the next generation
1.
One in which a part of Y chromosome had broken off and
became attached to the end of one of the X-chromosome.
2.
Other variety in
which one of the X-chromosomes was broken and
consisted of two approximately equal fragments.
These two X-chromosomes in the female fly could be distinguished not
only from each other, but also from normal X-chromosome under the microscope.
Usually in Drosophila normal fly has
red round (++) eyes. Two mutant genes were studied
1.
carnation (car) causing darkish red eyes which is
recessive to red (+) eye colour
2.
bar (B) causing narrow eyes and dominant to round (+)
eyes
Both are present in X chromosome. The
female fly is XX and male has XY chromosome.
In the
female fly, one of the two fragments of an X-chromosome carried mutant alleles
for carnation eye and barred eye (B is
dominant showing narrower eyes). The other X-chromosome, having a part of Y
attached, carried normal alleles of these two genes, so that the female
heterozygote had barred eyes and normal eye colour.
If no
crossing over takes place between the two genes in question, two types of
gametes i.e., car B and ++ will be produced from the female
flies. Crossing over will give two additional types of gametes i.e., car
+ and + B.
Such
females were crossed with male flies having recessive alleles for both these
genes (car, +). Two
types of sperms will be produced, the Y bearing and the X bearing. The Y sperm has no genetic markers of
relevance here and the X sperm carried the car and + alleles.
Due to
fertilization of two types of non-crossover and the other two types of
crossover gametes by male gametes carrying X-chromosome (car +), four
kinds of female flies will be produced. Another four kinds of male flies will
be produced due to fertilization by Y carrying male gametes.
If no recombination occurred – no exchange of chromosome part - two types of progeny
1.
carnation eye with bar shape
2.
red eye with round eye shape – wild type
Two classes of recombinants –
1.
red eye with bar shape
2.
carnation eye with round shape
The flies which are classified as
crossovers on the basis of phenotype i.e., carnation (with normal eye shape)
and barred (with normal eye colour) were studied cytologically Here the
carnation flies had a complete X chromosome and bar flies had a shorter piece
of X chromosome to which a piece of Y was attached. This happened due to physical exchange
between homologous chromosomes.
Thus, cytological basis of crossing
over was established by distinguishing chromosomes under microscope.
2. Creighton and Mc Clintock’s
Experiments on Maize:
Similar demonstration of cytological
crossing over was demonstrated in maize. They observed a corn plant which had a
pair of chromosome 9, one was normal and another had a translocated piece of
another chromosome (from chromosome 8) at one end which gave it a knob like
appearance.
The normal chromosome carried ‘c’ for
colourless endosperm and Wx for starchy endosperm (amylose and amylopectin). Other
knobbed chromosome had alleles ‘C’ for coloured and wx for waxy endosperm
(amylopectin only).
Cw
was carried on the knobbed
chromosome and cWx on the knobless chromosome, (c =
recessive for colourless seed; w = recessive for waxy
endosperm). During meiosis crossing over occurs between the two loci. The two types of recombinants showed
specific cytological features (the knob and the extra piece) while no such
exchange of physical markers were observed on the parental type of progeny.
Molecular mechanism of crossing over
Various
models to explain homologous genetic recombination have been proposed based
primarily on genetic observation in bacteria and fungi. In 1964 Robin Holliday
proposed a model to explain the molecular process involved during the exchange
of DNA between two homologous double stranded DNA molecules.
Holliday Junction Model:
The key steps of this model are as follows:
First
of all, pairing or alignment or synapsis between two homologous DNA duplexes
takes place. Their sequences are perfectly indentical except that they may
contain small regions of different genes called alleles.
Then
breaks or nick occur at identical sites in one DNA strand of both homologous
DNA duplexes precisely at the same point. The broken ends of strands then
invade the opposite complementary strands creating short heteroduplex regions
(because of different alleles). This process is called strand invasion. This
crossover structure formed is called Holiday junction.
Holliday
junction moves in lateral direction. During this process a DNA strand is
progressively exchanged with a DNA strand of the other helix. This lateral
migration of Holliday junction is called branch migration. The original base
pairs are broken in parental molecules and new base pairs are formed in
recombined strands.
If
the two molecules have alternate alleles, A/a, B/b and C/c then the exchange of
DNA strands during branch migration produces double strand regions which are
not identical. These mismatched regions are called heteroduplexes. During
branch migration, the heteroduplex region is elongated.
Breakage
and subsequent reunion lead to formation of this joint molecule composed of
four interlocked strands of DNA.
The
key feature of Holliday junction is the cleavage or cutting across the
crossover point which resolves or separates the recombined molecules. To expose
two cut sites, the Holliday junction is rotated by 180° to form a square planar
structure. Resolution of Holliday junction occurs by cutting the DNA strands at
the site of cross and re-joining them.
Resolution
occurs in one of the two ways. This gives rise to two classes of DNA products.
The cut site 1 cleaves the two strands which were initially broken at the start
of recombination process (Invading strands are cut). The resolution produces
two non-recombinant molecules as only exchange of alleles has taken place in
the middle region of duplex B/b and b/B. Thus a patch of hybrid DNA is formed.
These molecules are known as patch products.
The
cut site 2 cleaves (Non-invading strands are cut) and re-joins two duplexes in
such a way that flanking or peripheral genes are exchanged. Here DNA is
reciprocally recombined. Crossing over occurs between A and C genes.
Gene mapping
Genes
are arranged linearly in a chromosome. The point in a chromosome where the gene
is located is called locus. The diagrammatic representation of location and
arrangement of genes and relative distance between linked genes of a chromosome
is called linkage or genetic map.
Constructing Genetic Maps
When two genes are close together on the same chromosome, they do not assort independently and are said to be linked. Genes located on different chromosomes are not linked and they assort independently and have a recombination frequency of 50%. Linked genes have a recombination frequency less than 50%.
The number of genetic recombinants produced is characteristic of the two linked genes involved. Genetic experiments can be used in genetic mapping.
Unlinked genes assort independently. The best cross to use to test for linkage is the testcross, a cross of an individual with another individual homozygous recessive for all genes involved. In a testcross between a+ /a b+ /b and a/a b/b, where genes a and b are unlinked, progeny phenotypic ratio will be 1:1:1:1. A significant deviation from this ratio (more parental types and few recombinant types) indicates that the two genes do not assort independently and that they are linked. The chi-square test can be used to find the significant deviation.
In an individual that is doubly heterozygous for the w and m alleles, for example, the alleles can be arranged in two ways:
In the arrangement on the left, the two wild-type alleles are on one homolog and the two recessive mutant alleles are on the other homolog, an arrangement called coupling (or the cis configuration). Crossing-over between the two loci produces w+m and wm+ recombinants.
In the arrangement on the right, each homolog carries the wild-type allele of one gene and the mutant allele of the other gene, an arrangement called repulsion (or the trans configuration). Crossing-over between the two genes produces w+m+ and wm recombinants.
The recombination frequency for two linked genes is the same, regardless of whether the alleles of the two genes involved are in coupling or in repulsion. Although the actual phenotypes of the recombinant classes are different for the two arrangements, the percentage of recombinants among the total progeny will be the same in each case.
In 1913, a student of Morgan’s, Alfred Sturtevant, determined that recombination frequencies could be used as a quantitative measure of the genetic distance between two genes on a genetic map. The genetic distance between genes is measured in map units (mu),where 1 map unit is defined as the interval in which 1 percent crossing-over takes place. The map unit is also called a centimorgan (cM), a term named by Sturtevant in honor of Morgan.
The unit of genetic map is Morgan or centimorgan. When the percentage of crossing over between two linked genes is 1 per cent, then the map distance between the linked genes is one morgan.
There is a greater probability of occurrence of crossing over, when the two genes are farther apart in a chromatid. The probability of crossing over between two genes is directly proportional to the distance between them.
When two genes are nearer, the probability of occurrence of crossing over between them is limited.
Gene Mapping with Two-Point Testcrosses
Testcrosses are used for mapping because the homozygous recessive parent produces only one type of gamete, with alleles that are recessive to the alleles in gametes produced by the heterozygous parent. So in a testcross we use one parent that is heterozygous for the genes being mapped and another parent that has the recessive alleles for those genes.
A two-point testcross should yield a pair of parental types that occur with about equal frequency and a pair of recombinant types that also occur with about equal frequency.
The following formula is used to calculate the recombination frequency:
Number of recombinants X 100 = Recombination Frequency = Map Units
Total number of testcross progeny
The recombination frequency is used directly as an estimate of map units.
The two-point method of mapping is most accurate when the two genes examined are close together; when genes are far apart, there are inaccuracies.
Gene Mapping with Three-Point Testcrosses
Genetic maps can be built by using a series of two-point testcrosses. Still more complex type of mapping analysis for three linked genes can be done using a three-point testcross. In diploid organisms, the three-point testcross is a cross of a triple heterozygote with a triply homozygous recessive.
Uses of gene mapping
1. It is useful to determine the location, arrangement and linkage of genes in a chromosome.
2. It is useful to predict the results of dihybrid and trihybrid crosses.
Uses of gene mapping
1. It
is useful to determine the location, arrangement and linkage of genes in a
chromosome.
2. It
is useful to predict the results of dihybrid and trihybrid crosses.
Interference and Coincidence:
Besides single crossing over, having
only one chiasma, there may be double or multiple crossing over. It has been
discovered by H.J.Muller (1911) that when there are two double cross-overs
(suppose a and b) then one cross-over (a) tries to prevent the formation of
other cross over (b) This tendency of one cross-over to interfere with the
other cross over is termed as interference. Suppose frequency of ‘a’ crossover
is 10 and frequency of ‘b’ cross-over is 12, then their total frequency will
not be 10+12 = 22 as required but will be less than 22 due to interference.
When the two things happen the same
time and at the same place, they then coincide or intermix and this occurrence
may be considered coincidence. This coincidence refers to the occurrence of two
or more distinct cross-over (double or multiple) at about the same time in the
same chromosomal region. Double cross-overs are the result of coming together
(coincidence) of two single cross-overs.
When doubles occur in regular
expected ratio, coincidence is said to be 100%, whereas interference will be
nil. But when there are no doubles (coincidence) the interference is nil i.e.,
coincidence is inversely proportional to the interference.
According to Muller (1916) the
coefficient of coincidence is the ratio between the observed and expected
frequencies of double cross overs.
Coefficient of coincidence = Actual
number of double cross-overs/Expected number of double cross-overs.
