Chapter 4: linkage and mapping Linkage: Cis and Trans Many genes are found on any one chromosome. Loci on the same chromosome are part of the same dsdn molecule Loci on the same chromosome are said to be physically linked. Without crossing over, linked alleles will always be transmitted to progeny together Slide 1 of Chapter 4 Dihybrids can be produced from either of two homozygote matings: BB x aabb, or aabb x abb If the loci are linked (i.e., on a single chromosome or linkage group), then the crosses can can be represented as: B / B x ab / ab,or [where the alleles on one side of the slash are on a single chromosome] ab / ab x b / b Slide 2 of Chapter 4 Parental vs. recombinant chromosomes/gametes The two alternative chromosomal configurations are deemed cis or coupling linkage [B and ab] and trans or repulsion linkage [ab and b] If chromosomes remain intact through meiosis, dihybrids will produce only two gametic genotypes when considering two linked genes B and ab if the parents were in cis linkage ab and b if the parents were in trans linkage Dihybrid Gamete genotypes: parental Parental gametes: gametes from dihybrid (abb) with same linkage arrangement as the homozygous parents For a dihybrid in cis linkage, cis gametic genotypes are parental: B and ab BB = B / B x aabb = ab / ab -> B / ab F1, For a dihybrid in trans linkage, trans gametic genotypes are parental: b and ab bb = b / b x aabb = ab / ab -> b / ab F1, Slide 3 of Chapter 4 Slide 4 of Chapter 4 Dihybrid gamete genotypes: recombinant Recombinant gametes: gametes from dihybrid (abb) with opposite linkage arrangement to that of the parents For a dihybrid in cis linkage, trans gametic genotypes are recombinant: b and ab For a dihybrid in trans linkage, cis gametic genotypes are recombinant: B and ab Without linkage, parental and recombinant gametes are equally abundant This results from independent segregation (assortment) of different homologous pairs Slide 5 of Chapter 4 Evidence of linkage: overabundance of parental gametes Linkage arrangement is not changed during somatic cell division (mitosis) If linkage arrangement is unchanged in meiosis, only parental gametes formed 50% of gametes are the same as those of one parent and 50 % of gametes are the same as the other parent Recombinant gametes non-existent Slide 6 of Chapter 4 CSS 1
Crossing over can create recombinant chromosomes Linkage arrangement can be altered if a crossover involving chromatids from different chromosomes occurs at a point between the two loci (see figure 4.5b) Cross-over is visible as a chiasma (plural=chiasmata,) in prophase I of meiosis Slide 7 of Chapter 4 One crossover generates two recombinant and two parental gametes cross over in every meiosis generates equal frequencies of parental and recombinant gametes 1 cross in 50 meiotic cells creates 2 recombinant and 198 parental gametes (see figure 4.7) So 2% crossing over rate (1/50 or 2/100) generates 1% recombinant gametes Recombination rate used as measure of genetic distance between gametes, expressed as centimorgans or cm. 1 cm = 1% recombination = 1 map unit Slide 8 of Chapter 4 Crossing over visulized as chiasmata Crossing over: key features Slide 9 of Chapter 4 Crossing over occurs in prophase I of meiosis Most bivalents (= chromosome pairs = paired homologs) have at least 1, but not more than 2 chiasmata One chiasma == visibile evidence of one cross over Some mechanism therefore acts upon each bivalent independently to ensure that each has at least one crossover. Chiasmata are instrumental in holding bivalents together as they migrate to the metaphase I plate Slide 10 of Chapter 4 Crossing over: key features II Crossovers can occur between sister chromatids of a single chromosome But these have no genetic effect since this represents reciprocal exchanges among identical DN molecules. Crossovers can also involve three or even all four of the chromatids of a bivalent) We will not focus on this, but simply accept that this is not important Crossing over: key features III Crossing over between two linked, heterozygous loci generates recombinant chromatids that become distinct chromosomes of the gametes. Crossing outside the region between two linked, heterozygous loci has no effect on the linkage arrangement of those loci Slide 11 of Chapter 4 Slide 12 of Chapter 4 CSS 2
No genetically evident recombination Ineffective cross overs I. Crossover between loci where one is homozygous Slide 13 of Chapter 4 Crossing over: key features IV Crossing over between linked loci does NOT generate recombinant chromosomes if either or both of the loci are homozygous In other words if both homologs are carrying the same allele at either loci Genetically evident crossing over occurs between linked, heterozygous loci Involving one chromatid from one homolog, and a chromatid from the other homolog. Double crossovers can re-establish the parental linkage arrangements Slide 14 of Chapter 4 Crossing over : probability The probability of between-locus crossing over differs from one pair of loci to the next. In general, rates of crossing over are correlated with the number of base pairs that separate two loci The probability of crossing over between two loci will equal the probability of recombinant (non-parental) chromosomes among those produced by meiosis of a dihybrid. The probability of recombinant chromosomes is experimentally estimated by assessing the genotypes of a large sample of gametes from a dihybrid. Slide 15 of Chapter 4 Meiotic assays enable estimates of the probability of crossing over Use a random sample of the gametes from the dihybrid in a cross with a second parent of known genotype Test cross- the second parent is the double recessive homozygote (aabb) F2- the second parent is the dihybrid itself. Recombinant inbred lines nther/pollen culture or induced parthenogenisis of eggs followed by chromosome doubling Linkage arrangement (cis/trans) does not influence outcome of meiotic Slide 16 of Chapter 4 assay experiments Gamete (nther) Culture to Produce Inbred Lines ssuming no Pollen x aa linkage Completely homozygous diploid plants= recombinant inbred lines (RILs) ¼ BB ¼ aabb ¼ bb ¼ aabb. Callus induction ¼ B ¼ ab ¼ b ¼ ab Callus (N) transfer to shoot induction media Shoots (haploid) colchicine to double chromosome #, then transfer to rooting medium transplant to pots Slide 17 of Chapter 4 Plantlets (haploid) Genetic Maps: distance between loci Probability of crossing over is equated with map distance Units on genetic maps are centimorgans bbreviated cm One cm theoretically equals a 1% probability of crossing over between two loci. Relative frequency of recombinant gametes is used to estimate cm distances. Slide 18 of Chapter 4 CSS 3
Genetic maps: experimental considerations The minimum recombination frequency, or centimorgans(cm) between two loci is zero. Complete linkage- parental linkage arrangements not altered The maximum recombination frequency for two loci is 50% If every meiotic cell had a crossover between two loci, the frequency of recombinants would be 50% Experiments with only two heterozygous loci will have an upper limit of 50 cm as their estimates of distance between the two loci Slide 19 of Chapter 4 Map units are more or less additive when considering three or more adjacent loci If the order of three linked loci is B C, then the map units separating and C will roughly equal the sum of the map units for the B and BC intervals. That fact enables the ordering of adjacent loci Linear order of loci on a linkage group can be determined by a series of two point linkage analyses Can involve many different crosses leading to a map Slide 20 of Chapter 4 Multiple two point linkage experiments lead to a concensus map Requirements for a two point linkage test Mutants for each locus with high expressivities such that phenotypes clearly indicate genotypic class So Ww and WW are always round, not wrinkled, and ww is always wrinkled Irrespective of genetic and external environments Dihybrid for the targeted loci For a multi-locus gene map you need: Segregation data from a hybrid that is heterozygous for all targeted loci Not practical since mutants generally found in different parent stocks Or, segregation data from pairs of two point linkage tests where many loci are involved in two or more tests Slide 21 of Chapter 4 Concensus gene maps Hypothetical: mutants at the, B, C, D, and E loci exist in the following genotypes: 1) aabbccdd, 2) bbccdd, 3) BBccDD, 4) BBCCdd Create hybrids for instance, 1 x 2 (dihybrid for and B loci); 2 x 3 (dihybrid for B and C loci); 3 x 4 (dihybrid for C and D loci) Conduct two point linkage tests i..e, cross each to a double recessive homozygote for its critical loci Establish map unit distance between pairs of loci Deduce order of linked loci Slide 22 of Chapter 4 Coupling phase parents and 2-point linkage Repulsion phase parents and 2-point linkage y and rb are recessive alleles at two loci, + s are dominant cv and rb are recessive alleles at two loci, + s are dominant F1: y rb / + + F1: + rb / cv + Progeny: Progeny: + + 460 + rb 460 y rb 465 parental sum= 925 cv + 478 parental sum= 938 + rb 40 + + 28 y + 35 recombinant sum= 75 cv rb 34 recombinant sum= 62 75/1000=0.075 recombination freq=7.5 cm Slide 23 of Chapter 4 62/1000=0.062 recombination freq=6.2 cm Slide 24 of Chapter 4 CSS 4
Map assembly y <-> rb is 7.5cM rb <-> cv is 6.2cM all three are in one linkage group, what is the order? which is in the middle y <-> cv is 13.3 cm (data not shown), so order is: y 7.5 cm rb 6.2 cm cv This is a genetic map! Slide 25 of Chapter 4 CSS 5