Download - Gen Linkage Mapping

8/13/2019 Gen Linkage Mapping

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Linkage Mapping and Crossing-over


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Independent assortment vs linkage.

Independent assortment occurs when the genes for two different traits

or genetic markers are located on different chromosomes. Mendel was

lucky enough to have chosen such a configuration.

Assuming pea plants have approximately, !,!!! genes and seven

chromosomes, then each chromosome might carry around ,"!!genes. #hese genes are not expected to assort independently during

meiosis. In other words, there is a roughly "$ chance of the genes

%eing linked.


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Comparison of independent and linked genetic

markers.

&A

A

'

'

a

a

%

%( )

'

a %

A

Independent

#estcross*

a

a

%

%

'

a %

A

&a

a

%

%

a

A %

%a

a

'

%

+!$ parental

phenotypes

+!$ recom%inant

phenotypes

'

a %

A


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&A

A

a

a c

)

Linked genetic markers

#estcross*

cC

C

A C

a c

A C

a c

a

a c

c

&

A C

a c

A

a c

c

a c

Ca

reater than +!$

of progeny with parental

phenotype

less than +!$

of progeny with recom%inant

(henotype resulting from crossing over.

a

a c

c

#rans, or repulsion

Cis-configuration

or coupling


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&s/ s

)

#estcross*

In female flies

%%/

&

s/0 long wings

%/ 0gray %odies

s 0 short wings

% 0%lack %odies

s/ %/ s %

s/ %/

%

s/ %/

s/ %/

s

%s

%s

%s

%s

s/ %

%s %s

%s

%s

"$

"$

1$

1$

Long wing, gray %odies

Long wing, gray %odies short wing, %lack %odies


short wing, %lack %odies

short wing, gray %odies

Long wing, %lack %odies

(arental

com%inations

2ecom%inant

com%inations

)ruit flies

%/s


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&s/ s

)

% %/

&

s/0 long wings

%/ 0gray %odies

s 0 short wings

% 0%lack %odies

s/ %/s%

s/ %

s/ %/

s

%s

%s

%s

%s

%s

1$

1$


Long wing, gray %odies short wing, %lack %odies


short wing, %lack %odies

(arental

com%inations

2ecom%inant

com%inations

%/

s/ %

s %/

%s

%s

s/ %

"$

"$ short wing, gray %odies

Long wing, %lack %odies

s %/


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A C

a c

a

a c

c

A

A

C

C

All even num%er of exchanges %etween two segregating loci will yield

parental com%inations and thus go undetected.

In general the maximum fre9uency of recom%ination for two genes located

8n the same chromosome is +!$.

Two stranded double crossover


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A B

#wo-stranded dou%le crossover

#hree stranded dou%le crossover

)our-stranded dou%le crossover

:ou%le crossovers occur in the following ways

All

parental

p

p

r

r

p

p

r

r

A '

a %a %A %

All

2ecom

%inant

a '

A %

5

; p

; rec

; p

; rec


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If one looks at all the different possibilities of double crossovers one

arrives

at a similar conclusion, again at most 50 will be recombinant. !see

ne"t slide#.

6ven if crossovers did not occur %y chance %ut all the time, +!$ would still

%e the limit. 7imilarly, if one went through all the possi%ilities of triple

crossovers, one would again arrive at a theoretical maximum of +!$.etc.

8nly if nature had some kind of %ias toward four-stranded dou%le

crossovers, would the ratio come out to more than +!$


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C$%C&'(I$%) If over 50 of the gametes produced b* a + cross contain

parentalcombinations of genetic markers, this is an indication that genes are

linked. 3hen a large num%er of genes is analy5 average

num%er of all cross-overs per interval in a meiotic cell.


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If one assumes proportionality %etween the distance %etween two loci and the

average num%er of crossovers per chromatid then*

%umber of crossovers / !distance#,

3here ? is a proportionality constant. In that case one would also predict that the

map distances would also %e additive.

A CB

&$

recom

%ination

@$ recom%ination

&/@$ recom%ination

3hen distances are large B! - 5! map units the results are less then the

additivity would predict.

In that case dou%le or even num%ered crossovers occur almost as fre9uently

as single or uneven num%ered crossover. 6ven num%ered crossovers are not

phenotypically detected as recom%inant events, as the second event

apparently cancels the first.


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If p is the fre9uency for

one crossover in interval I

and 9 the fre9uency

)or interval two, the

fre9uency for a dou%le

crossover is p9 >" or

; x. ;,

7ince p or 9 are ;

each.

#he a%ility to identify theparental and the two

reciprocal dou%le

crossover

classes also allows one to

determine the order of theD genetic markers.


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Three-factor crosses

.If one uses three markersto map a chromosome it %ecomes possi%le todetect and uantif* double crossovers, and it %ecomes possible to orderthe markers relative to each other. In diploid organisms three factor

crosses are used in analogous fashion to two-factor crosses. #hat is,homo


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In a three-factor cross for three linked genes* how are the gametes formed4

1 2 3

x y a%c +!

A'c>a%c 5a%C>a%c D

A%C>a%c F+

a'c>a%c F!

#otal !!!


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:etermining the order of three linked markers.

#he dou%le crossover results in an interchange of the center marker. #hus,

#he recom%inant class with the lowest fre9uency indicates the identity of

central marker.

enotype Eum%er of progeny

A'C>a%c DF!

a%c>a%c DG+

A%c>a%c "+

a'C>a%c +!

A'c>a%c 5a%C>a%c D

A%C>a%c F+

a'c>a%c F!

(arental configuration all cis

7ingle crossover in interval I

7ingle crossover in interval II

:ou%le crossover* one in interval I

and one in interval II

#otal !!!


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7ince the recom%ination class A'c, a%C occurs with the lowest fre9uency

it must %e marker C>c that is located in the center, since marker C is

recom%inant in that class. #hat is the order of the markers must %e AC'.

#he linkage distances can now %e calculated as follows*

:istance %etween A and C* add fre9uencies of single and dou%le crossover

in

interval AC* A%c, a'C, is "+/+!/ 5/D>!!! 0 !. or !$.

Linkage distance in interval II, i.e. 'C* F+/F!/5/D>!!! 0 !.+ or +$.

#he dou%le crossovers are included in the calculation %ecause one of the 5

crossovers occurred in the interval.

! map units + map units

A,a C,c ',%

I t f


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Interference*

In a three factor testcross the o%served fre9uency of a

dou%le crossover was + out of a thousand or !.!!+. If the two crossovers

in a dou%le crossover had %een completely independent, the expected

fre9uency would have %een !.+ & !.0 !.!+. #he o%servation thatactually fewer dou%le crossovers occur than expected was called chromosome

interference or chiasma interference. #his is o%served in most dou%le crossovers. #his

is not to %e confused with chromatid interference.

#he degree of interference is measured %y the coefficient of coincidence*

Coefficient of coincidence 0 o%served dou%le crossover fre9uency

expected dou%le crossover fre9uency

Coefficient of interference 0 - coefficient of coincidence.

6xample one gives a coefficient of coincidence of !.!!+>!.!+0.DDD

Coeff. of interference 0 H !.DDD 0 !.F

A positive coefficient of interference %etween ! and indicates that the first crossover

interferes with a second.

In %acteria the coefficient of coincidence can %e greater than one negative interference

indicating that the occurrence of one crossover increases thero%a%ilit of a second.


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4ore reasons wh* recombination freuencies are not linearand additive over all

distances* ecombination freuencies differs between male and femaleindividuals,

the reason is not clear. (e"es can have different map distances for the same

chromosomeswith the same primary :EA se9uence. In :rosophila males there is

practically no crossing over occuring.

#he correlation %etween physical distance and genetic map distance can %reak down

within a chromosome as a result of changing chromatin structure, from euchromatin to

hetero chromatin. enetic distances appears much shorter in heterochromatin than in

euchromatin. 2emem%er, the closer the genes are the less recom%ination. Ksually

there is more heterochromatin in the vicinity of the centromere.

)or example in euchromatin the map distance may %e !" map units, the same stretch

of :EA will give rise to a recom%ination fre9uency of D map units in the heterochromatin

state.

=owever, generally in euchromatin the correlation %etween physical distance andgenetic map distance is relatively good.


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6xample II

:. melanogaster, curled> straight wings cu>cu/, e%ony>gray %ody

color e>e/ and scarlet> red eyes st>st/

enotype Eum%er of progeny

cu e st/> cu e st D

cu/e/ st> cu e st DG!

cu e st> cu e st 5"

cu/e/st/> cu e st D!

cu/e st> cu e st G1

cu e/st/> cu e st !+

cu e/st> cu e st 5cu/e st/> cu e st "

parental

7ingle crossover interval I

7ingle crossover interval II

dou%le crossover, per interval

!!!


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#he advantage of working in Neurospora.Neurosporacrassaspends a significant part of its life cycle the in haploidstate. 2ight after meiosis N.crassaundergoes one more mitotic division,su%se9uently spores can germinate and reproduce asexually %y mitotic

division of haploid cells to form mycelia. #hus the phenotype of thespores can %e assessed after meiosis, without any test crosses, since therecessive genes are not masked in the haploid state. )urthermore, in N.crassa the haploid products of meiosis ascospores are kept in linearorder within the tu%e like structure called ascus. #his order reflects theorder in which they were formed during meiosis.

In Neurosporathe products of meiosis can %e phenotypically analy


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If crossing over occurs %efore chromosome


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If crossing over occurs %efore chromosome

replication*

A '

a %

A %

a '

A %

A %

a '

a '

!!$ recom%inant

A '

a %

A '

a %

a '

A %

A '

a %

5+$(arental

5+$ parental

+! $recom%inant

If crossing over occurs after chromosomereplication*


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)irst divisional segregation pattern


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A

a

a

A

a

A

a

tedrad

Meiosis I

A

Meiosis II MitosisA

A

a

a

All A

All a

A

a

A

a

)irst divisional segregation pattern

Alleles are segregated into different nuclei

In first division

second division segregation pattern

Alleles are segregated into different nuclei in 5nddiv.

tedrad

Meiosis IMeiosis II

5A ascospores

5A

5a

5a

As well as opposite


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A

A

A

A

A

A

A

A

a

a

a

a

a

a

a

a

#hese four second-division segregation ascus patterns are e9uivalent.

they all reflect the same event, that is a single crossover relative to the

centromere .

ascospores


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a %

Meiosis I

A '

Meiosis IIMitosisA '

A '

a %

a %

All A'

All a%

A '

A '

a %

a %

A '

A '

a %

a %

(arental ditype ascus pattern, no crossover

a '

A %

A '

a %

Meiosis I

Meiosis II5A'

5a'

5a%

5A%

a %

A '

A '

A %

a '

a %

A '

A %

a '

a %

#etratype ascus pattern* single crossover

ascospores


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If tedrads are ordered in the ascus and recovered> dissected without

distur%ing the order, as in E crassa, %ut not in 7. cerevisiae

it %ecomes possi%le to order the two markers relative to the centromere.

#he recombination freuenc* from a gene to the centeromere is 6 the

freuenc* of asci that e"hibit second7division segeragation patterns

for the alleles of the gene%ecause only half of the chromatids are involved in

the crossover.

#he map distance from centromere to gene A 0

!.+xnum%er of asci with second division segregation pattern&!!

total num%er of asci

some %ooks say !.+ %ecause only half of the single crossovers are productive.


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a '

A %

A %

a '

Eon-parental ditype, four strand dou%le crossover

#his ditype will %e rare in the case of linked genes %ut

will %e as fre9uent as parental ditype for independently

assorting genes.

"A%

"a'

A '

a %

#he pattern for a two stranded dou%le crossover %etween the two loci looks

again like the parental ditype. #he relative fre9uencies of parental ditype ,tetratype and nonparental ditype asci can %e used to calculate the linkage

distance %etween the two loci.

In ordered tetrad data, the centromere can %e used as a marker. #his is done

%y determining whether each ascus is the result of first or second division

segregation.


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#ype of ascus pattern


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7pore pair 5 D "


A' A' A' A' A%

5 A' A% a% a' A%

D a% a' A' A% a'

" a% a% a% a% a'

#otal num%er of asci* 5 "G DG 5 !

(: ## ## E(:

):A ):A 7:A 7:A

):' 7:' 7:' ):'

#ype*

:ivision segregation pattern*

(:, parental ditype

##, tetratype, single cross over %etween two of the markers.

7: second division segregation pattern* cross-over %etween centromere and the two

markers,

):* first division segregation pattern, no crossover %etween that marker and the

centromere. E(:, non parental ditype, four-stranded dou%le cross-over.



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7pore pair 5 D "


A' A' A' A'

5 A' A% a% a'

D a% a' A' A%

" a% a% a% a%

#otal num%er of asci* 5 "G DG 5

I Eot alll patterns are e9ual in their fre9euncy, so genes A and ' are

linked. i.e. on the same chromosome.

II Ascus pattern t*pe oneis most fre9uent and represent the parental ditype, and first division

segregation, no crossover occurred.

III Ascus pattern t*pe fouris least fre9uent* therefore a two stranded dou%le crossover gives rise

to that pattern.

I udging %y there intermediate fre9uency, ascus pattern type 5 and D are

most likely the result of a single crossover.


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Map distance 0 !!x!.+x N of asci w> 5nd

div. segr patterns> total N of asci*

=ence, distance A,' 0 !! x !.+"G/5>5!!05.+ map units

:istance cetromere, A* 0!!x!.+DG/5>5!!0! map units.

7pore pair 5 D "


A' A' A' A'

5 A' A% a% a'

D a% a' A' A%

" a% a% a% a%

#otal num%er of asci* 5 "G DG 5


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I #ype 5 displays a second division segregation patter with respect to allele

', and first division segreg. pattern with respect to A alleles, indicating that

a crossover occurred %etween markers A and ', %ut not %etween the markers

and the centromere. #hus the fre9uency represents interval A'.

I #ype D represents a second division segregation for %oth A and

' loci, indicating a single crossover %etween %oth , A and ' loci, and the

centromere, %ut not %etween A and ', since that would have re9uired a

dou%le crossover. #hus the centromere has to %e one the side of either A, or '.

II udging %y the fre9uency, #ype " must result from a two stranded dou%le

Crossover. If the centromere were in the middle, the pattern would

have to %e parental, having a second division segregation %etween the

centromere and A, %ut not %etween the centromere and ', indicates that

the dou%le crossover happened in intervals centromere and A, and A'.#hus the order must %e centromere, A,'


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89

%89

TT


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TT

(9(

TT


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3hen taking all the different crossover patterns into account, including multiplecrossovers then the formula %ecomes

map distance 0.5 !single crossovers#:total ; < " 0.5 !doubles#:total ;

= " 0.5 !triples#:total ; > " 0.5!uadruples#:totaletc.

#he factors !.+, , .+ is a matter of how many times does the crossover type affect

the given interval in a tetrad. 6g. A single exchange affects half of the strands in that

interval , a dou%le exchange affects


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is parental

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