1. 4 Gene Linkage and Genetic Mapping

2. Mendel’s Laws: Chromosomes
Locus = physical location of a gene on a chromosome
Homologous pairs of chromosomes often contain alternative forms of a given gene = alleles
Different alleles of the same gene segregate at meiosis I
Alleles of different genes assort independently in gametes
Genes on the same chromosome exhibit linkage: inherited together

3. Gene Mapping
Gene mapping determines the order of genes and the relative distances between them in map units
1 map unit = 1 cM (centimorgan)
In double heterozyote:
Cis configuration = mutant alleles of both genes are on the same chromosome = ab/AB
Trans configuration = mutant alleles are on different homologues of the same chromosome = Ab/aB

4. Gene Mapping Gene mapping methods use recombination
frequencies between alleles in order to determine the relative distances between them
Recombination frequencies between genes are inversely proportional to their distance apart
Distance measurement: 1 map unit = 1 percent recombination (true for short distances)

5. Fig. 4.6

6. Gene Mapping
Genes with recombination frequencies less than 50 percent are on the same chromosome = linked)
Linkage group = all known genes on a chromosome
Two genes that undergo independent assortment have recombination frequency of 50 percent and are located on nonhomologous chromosomes or far apart on the same chromosome = unlinked
Fig. 4.7

7. Recombination
Recombination between linked genes occurs at the same frequency whether alleles are in cis or trans configuration
Recombination frequency is specific for a particular pair of genes
Recombination frequency increases with increasing distances between genes
No matter how far apart two genes may be, the maximum frequency of recombination between any two genes is 50 percent.

8. Gene Mapping
Recombination results from crossing-over between linked alleles.
Recombination changes the allelic arrangement on homologous chromosomes
Fig. 4.4

9. Genetic Mapping
The map distance (cM) between two genes equals one half the average number of crossovers in that region per meiotic cell
The recombination frequency between two genes indicates how much recombination is actually observed in a particular experiment; it is a measure of recombination
Over an interval so short that multiple crossovers are precluded (~ 10 percent recombination or less), the map distance equals the recombination frequency because all crossovers result in recombinant gametes.
Genetic map = linkage map = chromosome map

10. Gene Mapping: Crossing Over
Two exchanges taking place between genes, and both involving the same pair of chromatids, result in a nonrecombinant chromosomes
Fig. 4.13

11. Gene Mapping: Crossing Over
Crossovers which occur outside the region between two genes will not alter their arrangement
The result of double crossovers between two
genes is indistinguishable from independent assortment of the genes
Crossovers involving three pairs of alleles specify gene order = linear sequence of genes

12. Fig. 4.12

13. Genetic vs. Physical Distance
Map distances based on recombination frequencies are not a direct measurement of physical distance along a chromosome
Recombination “hot spots” overestimate physical length
Low rates in heterochromatin and centromeres underestimate actual physical length

14. Fig. 4.11

15. Gene Mapping
Mapping function: the relation between genetic map distance and the frequency of recombination
Chromosome interference: crossovers in one region decrease the probability of a second crossover close by
Coefficient of coincidence = observed number of double recombinants divided by the expected number
Interference = 1-Coefficient of coincidence

16. Mapping Genes in Human Pedigrees
Methods of recombinant DNA technology are used to map human chromosomes and locate genes
Genes can then be cloned to determine structure and function
Human pedigrees and DNA mapping are used to identify dominant and recessive disease genes
Polymorphic DNA sequences are used in human genetic mapping.

17. Genetic Polymorphisms
The presence in a population of two or more relatively common forms of a gene or a chromosome is called polymorphism
Changes in DNA fragment length produced by presence or absence of the cleavage sites in DNA molecules are known as restriction fragment length polymorphism (RFLP)
A prevalent type of polymorphism is a single base pair difference, simple-nucleotide polymorphism (SNP)
A genetic polymorphism resulting from a tandemly repeated short DNA sequence is called a simple sequence repeat (SSR)

18. RFLPs
Restriction endonucleases are used to map genes as they produce a unique set of fragments for a gene
There are more than 200 restriction endonucleases in use, and each recognizes a specific sequence of DNA bases
EcoR1 cuts double-stranded DNA at the sequence
5’-GAATTC-3’ wherever it occurs

19. Fig. 4.18

20. RFLPs
Differences in DNA sequence generate different recognition sequences and DNA cleavage sites for specific restriction enzymes
Two different genes will produce different fragment patterns when cut with the same restriction enzyme due to differences in DNA sequence
Fig. 4.19

21. SNPs
Single-nucleotide polymorphisms (SNPs) are abundant in the human genome
Rare mutants of virtually every nucleotide can probably be found, but rare variants are not generally useful for family studies of heritable variation in susceptibility to disease
For this reason, in order for a difference in nucleotide sequence to be considered as an SNP, the less-frequent base must have a frequency of greater than about 5% in the human population.
By this definition, the density of SNPs in the human genome averages about one per 1300 bp

22. SSRs
A third type of DNA polymorphism results from differences in the number of copies of a short DNA sequence that may be repeated many times in tandem at a particular site in a chromosome
When a DNA molecule is cleaved with a restriction endonuclease that cleaves at sites flanking the tandem repeat, the size of the DNA fragment produced is determined by the number of repeats present in the molecule
There is an average of one SSR per 2 kb of human DNA

23. Mapping Genes in Human Pedigrees
One source of the utility of SNPs and SSRs in human genetic mapping is their high density across the genome
Additional source of utility of SSRs in genetic mapping is the large number of alleles that can be present in any human population.

24. Mapping Genes in Human Pedigrees
Human pedigrees can be analyzed for the inheritance pattern of different alleles of a gene based on differences in SSRs and SNPs
Restriction enzyme cleavage of polymorphic alleles that are different in RFLP pattern produces different size fragments by gel electrophoresis

25. Fig. 4.21

26. Tetrad Analysis
In some species of fungi, each meiotic tetrad is contained in a sac-like structure, called an ascus
Each product of meiosis is an ascospore, and all of the ascospores formed from one meiotic cell remain together in the ascus
Fig. 4.22

27. Tetrad Analysis
Several features of ascus-producing organisms are especially useful for genetic analysis:
They are haploid, so the genotype is expressed directly in the phenotype
They produce very large numbers of progeny
Their life cycles tend to be short

28. Ordered and Unordered Tetrads
Organisms like Saccharomyces cerevisiae, produce unordered tetrads: the meiotic products are not arranged in any particular order in the ascus
Unordered tetrads have no relation to the geometry of meiosis.
Bread molds of the genus Neurospora have the meiotic products arranged in a definite order directly related to the planes of the meiotic divisions—ordered tetrads
The geometry of meiosis is revealed in ordered tetrads

29. Tetrad Analysis
In tetrads when two pairs of alleles are segregating, three patterns of segregation are possible
parental ditype (PD) = two parental genotypes
nonparental ditype (NPD) = only recombinant combinations
tetratype (TT) = all four genotypes observed
Fig. 4.24

30. Tetrad Analysis
The existence of TT for linked genes demonstrates two important features of crossing-over
The exchange of segments between parental chromatids takes place in prophase I, after the chromosomes have duplicated
The exchange process consists of the breaking and rejoining of the two chromatids, resulting in the reciprocal exchange of equal and corresponding segments

31. Tetrad Analysis
When genes are unlinked, the parental ditype tetrads and the nonparental ditype tetrads are expected in equal frequencies: PD = NPD
Linkage is indicated when nonparental ditype tetrads appear with a much lower frequency than parental ditype tetrads: PD » NPD
Map distance between two genes that are sufficiently close that double and higher levels of crossing-over can be neglected, equals
1/2 x (Number TT / Total number of tetrads) x 100

32. Neurospora: Ordered Tetrads
Ordered asci also can be classified as PD, NPD, or TT with respect to two pairs of alleles, which makes it possible to assess the degree of linkage between the genes
The fact that the arrangement of meiotic products is ordered also makes it possible to determine
the recombination frequency between any particular gene and its centromere

33. Fig. 4.26

34. Tetrad Analysis: Ordered Tetrads
Homologous centromeres of parental chromosomes separate at the first meiotic division
The centromeres of sister chromatids separate at the second meiotic division
When there is no crossover between the gene and centromere, the alleles segregate in meiosis I
A crossover between the gene and the centromere delays segregation alleles until meiosis II

35. Tetrad Analysis: Ordered Tetrads
The map distance between the gene and its centromere equals
1/2 x (Number of asci with second division segregation/ Total number of asci) x 100
This formula is valid when the gene is close enough to the centromere and there are no multiple crossovers

36. Fig top

37. Fig bottom

38. Gene Conversion
Most asci from heterozygous Aa diploids demonstrate normal Mendelian segregation and contain ratios of
2A : 2a in four-spored asci, or 4A : 4a in eight-spored asci
Occasionally, aberrant ratios are also found, such as
3A : 1a or 1A : 3a and 5A : 3a or 3A : 5a
The aberrant asci are said to result from gene conversion because it appears as if one allele has “converted” the other allele into a form like itself

39. Gene Conversion
Gene conversion is frequently accompanied by recombination between genetic markers on either side of the conversion event, even when the flanking markers are tightly linked
Gene conversion results from a normal DNA repair process in the cell known as mismatch repair
Gene conversion suggests a molecular mechanism of recombination

40. Fig. 4.29

41. Recombination
Recombination is initiated by a double-stranded break in DNA
The size of the gap is increased by nuclease digestion of the broken ends
These gaps are repaired using the unbroken homologous DNA molecule as a template
The repair process can result in crossovers that yield chiasmata between nonsister chromatids

42. Recombination: Holliday Model
The nicked strands unwind, switch partners, forming a short heteroduplex region with one strand and a looped-out region of the other strand called a D loop
The juxtaposed free ends are joined together, further unwinding and exchange of pairing partners increase the length of heteroduplex region—process of branch migration

43. Recombination: Holliday Model
One of two ways to resolve the resulting structure, known as a Holliday junction, leads to recombination, the other does not
The breakage and rejoining is an enzymatic function carried out by an enzyme called the Holliday junction-resolving enzyme

44. Fig. 4.31