Lecture 7 Three point cross

Lecture 7 Three point cross

Test cross A geneticist has two mutations on the X-chromosome: Cv+ = Normal vein cv = crossveinless V+ = Norm red eye v =Vermillion eye Construct pure-breeding stocks: Cv+ v/Cv+ v and cv V+/Y Normal vein crossveinless Vermillion norm red eye These are mated: F1= females = Cv+ v/ cv V+ males= Cv+ v/Y F1 females are mated to a double recessive male (cv v/Y) The following results are obtained in the F2: indep assortment linked loci Norm vein/Vermillion Crossveinless/Red eye Norm vein/norm eye Crossveinless/Vermillion total 380 120 1000 240 260 255 250 1000 Do these genes reside on the same or different chromosomes?

If on the same chromosome, what is the distance between them?
We first identify the parental and recombinant classes Then Recombinant frequency = #Recombinants/Total progeny F2: indep assortment linked loci Norm vein/Vermillion Crossveinless/Red eye Norm vein/norm eye Crossveinless/Vermillion total 380 120 1000

Mapping P Cv+--v x cv--V+ Cv+--v Y F1 Cv+--v x cv--v cv--V+ Y cv--v Cv+--v cv--v Cv+--v 390 Parental cv—V+ cv--v cv—V+ 370 Cv+--V+ cv--v Cv+--V+ 125 Recomb cv--v cv--v 115 Which of these are parental and which are recombinants? Map distance between the Cv+ and V+ genes is = 240 =24%

Orientation Cv+ V+ 24 Is This Correct? Cv+ V+ 24

Mapping Another mutation Ec+ (echinus eye) is isolated and recombination frequencies between this gene and V+ are determined P Ec+--v x ec--V+ Ec+--v Y F1 Ec+--v x ec--v ec--V+ Y ec--v Ec+--v ec--v Ec+--v 325 Parental ec--V+ ec--v 335 ec--V+ Ec+--V+ ec--v 165 Ec+--V+ Recomb 175 ec--v ec--v MU = / = 340/1000 = 34MU

Orientation Cv to V is 24MU V to Ec is 34 MU Cv+ V+ 24 Ec+ 34 34 Ec+ Cv+ V+ 24 Prediction1= Cv+ to Ec+ = 58 Prediction2 = Cv+ to Ec+ = 10

Mapping recombination frequencies between Ec+ (echinus eye) and V+ are: P Ec+--cv x ec--CV+ Ec+--cv Y F1 Ec+--cv x ec--cv ec--CV+ Y ec--v Ec+--v ec--v Ec+--cv 447 Parental ec--V+ ec--v 448 ec--CV+ Ec+--V+ ec--v 52 Ec+--CV+ Recomb 53 ec--cv ec--v MU = 52+53/1000 = 10.5 MU

Which is correct? Cv to V is 24MU V to Ec is 34 MU V to Ec is 10.5 MU Cv+ V+ 24 Ec+ 34 Cv+ V+ 24 34 Ec+ 10.5

Distance dependent accuracy
% recombinants Cv+---V+ 24 Ec+---V+ 34 Ec+---Cv Cv+ Ec+ 10.5 34 V+ 24 Cv+ V+ 24 34 Ec+ 10.5 =34.5 10.5 is close to 10 The map is not very accurate There is a small error in our results

What is going on? The map is not internally consistent?
Single cross-over ---Ec v Ec+--v parental Ec+--V+ Recomb ---ec V ec--v Recomb ec--V+ parental What if we get two crossovers between Ec+ and V+ A double cross-over ---Ec v----- Ec+--v parental ---ec V+----- ec--V+ parental Now the parental class is over counted because progeny with 2 crossovers are counted as parental class instead of recombinant class The recombinant class is under counted for the progeny with two crossovers

Double crossovers Over large distance there will be a significant number of double crossovers that go undetected - the genetic distances are underestimated The solution is to include additional markers to greatly reduce the probability of undetected doubles: For instance with the intervening Cv+ marker the double crossovers can be separated: ---Ec Cv v----- Ec+--Cv+--v parental Ec+--cv--v db Recomb ---ec------cv V+----- ec--Cv+--V+ db Recomb ec--cv--V+ parental ---Ec Cv v----- Ec+--Cv+--v parental Ec+--cv--V+ s Recomb ---ec------cv V+----- ec--Cv+--v s Recomb ec--cv--V+ parental ---Ec Cv v----- Ec+--Cv+--v parental Ec+--CV+--V+ s Recomb ---ec------cv V+----- ec--cv--v s Recomb ec--cv--V+ parental

Three point cross Because of the problem of undetected double crossovers, geneticists try to map unknown genes to marker genes that are closely linked when constructing a detailed map. sc Scute Bristle v vermilion eye cv Crossveinless wing ec Echinus eye g Garnet eyes f forked bristles 9.1 10.5 24 11.2 10.9

This is one of the reasons behind a mapping technique known as
The Three-Point Testcross To map three genes with respect to one another, we can use three separate pair-wise crosses between heterozygotes OR A more efficient method is to perform a single cross using individuals heterozygous for the three genes

Three point crosses Here is a example involving three linked genes: Q: Is cut wings linked to vermilion and cross veinless and if so what is the gene order and distance between these three genes? v - vermilion eyes cv - crossveinless ct - cut wings To determine linkage, gene order and distance, we examine the data in pair-wise combinations When doing this, you must first identify the Parental and recombinant classes! P F1 F2

Vermilion, norm vein, norm wing Norm eye, crossvein, cutwing
Three point crosses v - vermilion eyes cv - crossveinless ct - cut wings P V+ cv ct x v Cv+ Ct+ V+ cv ct Y (normal eye, crossvein, cut) (vermilion, norm vein, norm wing) F1 v Cv+ Ct+ x v cv ct V+ cv ct Y F2 v cv ct v Cv+ Ct+ V+ cv ct v cv Ct+ V+ Cv+ ct V+ Cv+ Ct+ v Cv+ ct V+ cv Ct+ Phenotype Vermilion, norm vein, norm wing Norm eye, crossvein, cutwing Vermilion, crossvein, norm wing Norm eye, norm vein, cutwing Vermilion, crossvein, cutwing Norm eye, norm vein, norm wing Vermilion, norm vein, cutwing Norm eye, crossvein, norm wing 377 43 75 5 P R

Distance between v and cv
Vermilion, norm vein Norm eye, crossvein Vermilion, crossvein Norm eye, norm vein Vermilion, norm vein, norm wing Norm eye, crossvein, cutwing Vermilion, crossvein, norm wing Norm eye, norm vein, cutwing Vermilion, crossvein, cutwing Norm eye, norm vein, norm wing Vermilion, norm vein, cutwing Norm eye, crossvein, norm wing v cv ct v Cv+ Ct V+ cv ct v cv Ct+ 43 V+ Cv+ ct 43 v cv ct 75 V+ Cv+ Ct+ 75 v Cv+ ct 5 V+ cv Ct+ 5 v Cv+ V+ cv v cv V+ Cv+ v cv V+ Cv+ v Cv+ Parental v Cv V+ cv Recombinant V+ Cv v cv Recombinants/Total = 236/1000 = 23.6% The genes are linked!

Distance between ct and cv
Vermilion, norm vein, norm wing Norm eye, crossvein, cutwing Vermilion, crossvein, norm wing Norm eye, norm vein, cutwing Vermilion, crossvein, cutwing Norm eye, norm vein, norm wing Vermilion, norm vein, cutwing Norm eye, crossvein, norm wing norm vein, norm wing crossvein, cutwing crossvein, norm wing norm vein, cutwing v cv ct v Cv+ Ct+ Cv+ Ct+ 377 V+ cv ct cv ct 377 v cv Ct+ cv Ct+ 43 V+ Cv+ ct Cv+ ct 43 v cv ct cv ct 75 V+ Cv+ Ct+ Cv+ Ct+ 75 v Cv+ ct Cv+ ct 5 V+ cv Ct+ cv Ct+ 5 Parental Cv+ Ct cv ct Recombinant Cv+ ct 43+5 cv Ct 96/1000 = 9.6% The genes are linked!

Distance between v and ct
v to ct Vermilion, norm wing Norm eye, cutwing Vermilion, norm wing Norm eye, cutwing Vermilion, cutwing Norm eye, norm wing Vermilion, cutwing Norm eye, norm wing Vermilion, norm vein, norm wing Norm eye, crossvein, cutwing Vermilion, crossvein, norm wing Norm eye, norm vein, cutwing Vermilion, crossvein, cutwing Norm eye, norm vein, norm wing Vermilion, norm vein, cutwing Norm eye, crossvein, norm wing v cv ct v Cv+ Ct+ v Ct+ 377 V+ cv ct V+ ct 377 v cv Ct+ v Ct+ 43 V+ Cv+ ct V+ ct 43 v cv ct v ct 75 V+ Cv+ Ct+ V+ Ct+ 75 v Cv+ ct v ct 5 V+ cv ct+ V+ Ct+ 5 Parental v Ct V+ ct Recombinant V+ Ct v ct 75+5 160/1000= 16% The genes are linked

Arranging the three genes
v cv 23.6 v ct 16 ct cv 9.6 v cv 23.6 ct 16 9.6 (25.6) The accurate map is: v cv ct 16 9.6

DCO Parental chromosomes v----Ct+-----cv+ & V+----ct----cv v----Ct+-----Cv+ V+----ct----cv v Ct+ Cv+ The parental homologs will pair in meiosisI. Crossing over will occur and a Double crossover produces: v Ct+ Cv+ V+ ct cv V+ ct cv v Ct+ Cv+ v ct Cv+ V+ Ct+ cv V+ ct cv Notice if one focuses on the v and cv markers, they will be scored as non-recombinant (parental). However if one also scores v-ct and ct-cv the double recombination event from which they arose can be detected. By including these double recombinants the map is internally consistent.

Three point cross Because of the problem of undetected double crossovers, geneticists try to map unknown genes to marker genes that are closely linked (LESS than 10 m.u.) when constructing a detailed map. sc Scute Bristle v vermilion eye cv Crossveinless wing ct Cut wing ec Echinus eye g Garnet eyes f forked bristles 9.1 10.5 9.6 16 11.2 10.9

Interference Interference: this is a phenomenon in which the occurrence of one crossover in a region influences the probability of another crossover occurring in that region. Interference is readily detected genetically. For example, we determined the following map for the genes v ct and cv. v m.u ct m.u cv Expected double crossovers = product of single crossovers The expected frequency of a double crossover is the product of the two frequencies of single crossovers: DCO= 0.16 x 0.096= Total progeny = 1000 Expected number of DCO is x 1000 = 15 Observed number of DCO = 10 The coefficient of coincidence is calculated by dividing the actual frequency of double recombinants by this expected frequency: c.o.c. = actual double recombinant frequency / expected double recombinant frequency Reduction is because of interference

Interference is often quantified by the following formula:
I= 1- observed frequency of doubles/ expected frequency of Doubles I= 1- 10/15 = 5/15 = 33% If actual frequency is the same as expected frequency then Interference is 1-1=0

xxxxxxx

P sc ec vg x Sc+ Ec+ Vg+ F1 Sc+ Ec+ Vg+ x sc ec vg F2
Another example Sc= scutellar bristle Ec= echinus rough eye Vg= vestigial wing P sc ec vg x Sc+ Ec+ Vg+ sc ec vg Sc+ Ec+ Vg+ F1 Sc+ Ec+ Vg+ x sc ec vg sc ec vg sc ec vg F2 sc ec vg sc ec vg Sc+ Ec+ Vg+ sc ec Vg+ Sc+ Ec+ vg sc Ec+ vg Sc+ ec Vg+ sc Ec+ Vg+ Sc+ ec vg 235 241 243 233 12 14 16 If these genes were on separate chromosomes, they should be assorting independently and all the classes should be equally frequent. ARE ALL THREE GENES ON THE SAME CHROMOSOME?

What about sc and ec? Are they linked not linked?
sc vg sc ec vg Sc+ Ec+ Vg sc ec Vg Sc+ Ec+ vg sc Ec+ vg 12 Sc+ ec Vg sc Ec+ Vg+ 14 Sc+ ec vg 16 sc ec Sc+ Ec+ sc Ec+ Sc+ ec 478 474 26 30 # recombinant/total progeny = 56/1008 =5.5% Sc and Ec are Linked sc ec What about Sc and Vg?

Are sc and vg linked/not linked???
To map them, we simply examine the pair-wise combinations and identify the parental and recombinant classes: To determine the distance between sc vg we remove ec sc vg sc ec vg Sc+ Ec+ Vg sc ec Vg Sc+ Ec+ vg sc Ec+ vg 12 Sc+ ec Vg sc Ec+ Vg+ 14 Sc+ ec vg 16 sc vg Sc+ Vg+ sc Vg+ Sc+ vg 247 255 257 249 # recombinant/total progeny = 506/1008 = 50% Therefore sc and vg are NOT LINKED! sc vg Sc and Ec are linked Sc and Vg are Not linked Prediction: Ec and Vg should not be linked

Are ec and vg linked? In theory they should not be.
From these observations what is the map distance between ec and vg? sc vg sc ec vg Sc+ Ec+ Vg sc ec Vg Sc+ Ec+ vg sc Ec+ vg 12 Sc+ ec Vg sc Ec+ Vg+ 14 Sc+ ec vg 16 ec vg Ec+ Vg+ ec Vg+ Ec+ vg 251 255 257 245 # recombinant/total progeny = 502/1008 = 50% Therefore ec and vg are NOT LINKED! sc ec vg

P sc ec vg x Sc+ Ec+ Vg+ F1 Sc+ Ec+ Vg+ x sc ec vg F2
Linked or unlinked? Sc= scutellar bristle Ec= echinus rough eye Vg= vestigial wing P sc ec vg x Sc+ Ec+ Vg+ sc ec vg Sc+ Ec+ Vg+ F1 Sc+ Ec+ Vg+ x sc ec vg sc ec vg sc ec vg F2 sc ec vg sc ec vg sc ec vg sc ec vg Sc+ Ec+ Vg+ sc ec Vg+ Sc+ Ec+ vg sc Ec+ vg Sc+ ec Vg+ sc Ec+ Vg+ Sc+ ec vg If these genes were on separate chromosomes, they should be assorting independently. Are all three assorting independently, are two assorting independently or are none assorting independently

==========

Meiosis consists of two divisions, meiosis I and II, by which a diploid cell produces four haploid daughters. Reduction in ploidy occurs at meiosis I, when homologous chromosomes (homologs) disjoin. This event is prepared during meiotic prophase, when homologs recognize each other and form stable pairs (bivalents) that can line up in the metaphase I spindle. In most eukaryotes, including mouse and yeast, both the recognition of homologs and the formation of stable bivalents depend on recombinational interactions between homologs (reviewed in ref. 1). For this process, the meiotic prophase cell actively induces DNA double-strand breaks (DSBs) and repairs them by homologous recombination, using preferably a nonsister chromatid of the homolog as template (2). In species such as yeast and mouse, most interhomolog recombinational interactions are not resolved as reciprocal exchanges [crossovers (COs)] and probably serve homolog recognition and alignment (3, 4). A small proportion, however, yields COs, which become cytologically visible as chiasmata and are essential for the stable connection of homologs. COs are not randomly distributed among and along bivalents; every bivalent forms at least one CO (obligate CO), and, if multiple COs occur, they are more evenly spaced along the bivalent than would be expected if they were randomly placed. This phenomenon was originally detected genetically by the finding that the frequency of double recombinants involving a pair of adjacent or nearby intervals was lower than the frequency expected from recombinant frequencies for each of those intervals (reviewed in refs. 5 and 6). Interference has also been analyzed cytologically, from spatial distributions of chiasmata (7, 8) or recombination complexes along chromosomes during meiotic prophase, when recombination is in progress (9). How interference is imposed is not known. Concomitantly with meiotic recombination, the sister chromatids of each chromosome form a common axis, the axial element (AE), and the AEs of homologs align. Then, numerous transverse filaments connect the AEs of homologs, and a zipper-like structure, the synaptonemal complex (SC), is formed between the homologs (1). Protein complexes that mediate, and mark the sites of, recombination have been localized to AEs or SCs by both EM and immunocytology (reviewed in refs. 10 and 11). These studies (9, 12), together with molecular genetic analyses (13, 14), have elicited several specific questions regarding the imposition of interference: At which step in meiotic recombination is interference first detectable? Is the level of interference the same among recombination complexes representing early and late steps in meiotic recombination? Does the SC contribute to interference? We have analyzed these questions in the mouse by examining how protein complexes that are thought to mark intermediate and late events in meiotic recombination are distributed along SCs in two stages of meiotic prophase. In mouse, many recombination-related proteins have been identified, and the meiotic time courses of immunofluorescent foci containing these proteins have been described (15, 16). The mouse transverse filament protein SYCP1 is also known (17, 18), and SYCP1-deficient mice have been constructed (19). We have analyzed the distributions of four types of foci along mouse SCs or AEs in wild-type and/or Sycp1–/– strains: (i) MLH1 foci, which occur during pachytene and specifically mark the sites of COs (9, 20); (ii and iii) MSH4 and replication protein A (RPA) foci, which appear earlier, during zygotene, and were analyzed here at late zygotene. In mouse, these foci outnumber the prospective COs. However, a subset of them likely matures into MLH1 foci and then into COs, because early MLH1 foci colocalize with MSH4 (16, 21) but then lose MSH4 at later stages; (iv) because Sycp1–/– strains do not form MLH1 foci (19), we analyzed {gamma}H2AX signals in Sycp1–/– pachytene spermatocytes. In wild-type meiosis, {gamma}H2AX signals occur from leptotene until pachytene (22). Based on their timing and other evidence (reviewed in refs. 13 and 23), MSH4 and RPA foci likely mark early intermediate stages of recombination involving strand exchange, whereas MLH1 foci likely mark the latest stages, e.g., conversion of double Holliday junctions to COs. {gamma}H2AX signals mark various DNA lesions, including DSBs (24); in Sycp1–/– pachytene, they probably represent (perhaps diverse) unresolved recombination intermediates (19). For the detection of genetic interference, the coefficient of coincidence (CC) is often used. However, CC is problematic as a measure for the level of interference because it is not based on the precise positions of genetic exchanges but instead is based on the frequencies of recombinants for genetic markers that delimit two adjacent or nearby chromosomal intervals.

Another method to solve a three point cross
P V+ cv ct x v Cv+ Ct+ V+ cv ct Y (normal eye, crossvein, cut) (vermilion, norm vein, norm wing) F1 v Cv+ Ct+ x v cv ct V+ cv ct Y Solving three-point crosses Identify the two parental combinations of alleles in the F2 progeny F2 v cv ct v Cv+ Ct+ 377 V+ cv ct 377 v cv Ct+ 43 V+ Cv+ ct 43 v cv ct 75 V+ Cv+ Ct+ 75 v Cv+ ct 5 V+ cv Ct+ 5 Parent v Cv+ Ct+ & V+ cv ct

Solving three-point crosses
Identify the two parental combinations of alleles (v Cv+ Ct+ & V+ cv ct) 2. The two most rare classes represent the product of double crossover. DCO v Cv+ ct & V+ cv Ct+ F2 v cv ct v Cv+ Ct+ 377 V+ cv ct 377 v cv Ct+ 43 V+ Cv+ ct 43 v cv ct 75 V+ Cv+ Ct+ 75 v Cv+ ct 5 V+ cv Ct+ 5 3. With this knowledge, you can establish a gene order in which a double cross produces the allelic combination observed in the most rare class. There are three possible relative order of the three genes in the parent:

Parent v Cv+ Ct+ & V+ cv ct
vermillion red normal vein crossveinless normal wing cut wing DCO v Cv+ ct & V+ cv Ct+ cut wing normal wing There are three possible gene orders for the parental combination **basically we want to know which of the three is in the middle** vermillion ----crossvein----cut wing vermillion----cut wing----crossvein cut wing----vermillion----crossvein You are driving along Rte1 and you are told that there are three towns along this route- San Francisco, Half moon bay and Santa Cruz. You have no idea which town you will encounter first, second and last. How many possible orders are there? San Francisco----Santa Cruz----Half moon bay San Francisco----Half moon bay----Santa Cruz Half moon bay----San Francisco----Santa Cruz

Parent v, Cv+, Ct+ & V+, cv, ct
vermillion red normal vein crossveinless normal wing cut wing F1 v, Cv+, Ct+ x v cv ct V+, cv, ct Y Observed DCO v, Cv+, ct & V+, cv, Ct+ There are three possible gene orders for the parental combination predicted DCO v----cv----Ct+ V+---Cv+---ct v----ct----Cv+ * V+---Ct+---cv Ct+----V+---Cv+ ct-----v----cv v----Cv+----Ct+ x x V+---cv-----ct OR v----Ct+----Cv+ x x V+---ct-----cv Ct+----v----Cv+ x x ct-----V+---cv Each relative order in the parent will give a different allelic combination after a double crossover!

3. Once the parental chromosomes are identified and the order is established, the non-recombinants, single recombinants and double recombinants can be identified v, Cv+, Ct+ V+, cv, ct Gene Order v----ct----cv REWRITE THE COMBINATION IN THE PARENTS v---Ct+---Cv+ and V+---ct---cv v Ct+ Cv+ V+ ct cv CO1 CO2 v cv ct v, Cv+, Ct V+, cv, ct v, cv, Ct V+, Cv+, ct 43 v, cv, ct V+, Cv+, Ct v, Cv+, ct 5 V+, cv, Ct+ 5 v—Ct+—Cv+ V+—ct—cv v—Ct+—cv V+—ct—Cv+ v—ct—cv V+—Ct+—Cv+ v—ct—Cv+ V+—Ct+—cv P SCO-II SCO-I DCO

Now the non-recombinants, single recombinants, and double recombinants are readily identified
Recomb freq in region I = = 160/1000 SCOI DCO Recomb freq in region II = =96/1000 SCOII DCO Now the DCO are not ignored. With this information one can easily determine the map distance between any of the three genes v m.u ct m.u cv

Now the non-recombinants, single recombinants, and double recombinants are readily identified
Parental input: (As a check that you have not made a mistake, reciprocal classes should be equally frequent) With this information one can easily determine the map distance between any of the three genes:

Lecture 7 Three point cross

Similar presentations

Presentation on theme: "Lecture 7 Three point cross"— Presentation transcript:

Similar presentations

About project

Feedback

Log in

Auth with social network:

Lecture 7 Three point cross

Similar presentations

Presentation on theme: "Lecture 7 Three point cross"— Presentation transcript:

Similar presentations

About project

Feedback