1. Lecture - 2 Establishment of plant genome mapping projects
Use of molecular markers in Plant Breeding

2. GENETIC MAPPING PHYSICAL MAPPING
GENOME MAPPING
GENETIC MAPPING
PHYSICAL MAPPING

3. GENOME MAPPING
Genetic mapping is based on the use of genetic techniques to construct maps showing the positions of genes and other sequence features on a genome.
Genetic techniques include cross-breeding experiments or,
Case of humans, the examination of family histories (pedigrees).
Physical mapping uses molecular biology techniques to examine DNA molecules directly in order to construct maps showing the positions of sequence features, including genes.

4. DNA MARKERS FOR GENETIC MAPPING
Mapped features that are not genes are called DNA markers. As with gene markers, a DNA marker must have at least two alleles to be useful. There are three types of DNA sequence feature that satisfy this requirement:
Restriction fragment length polymorphisms (RFLPs)
Simple sequence length polymorphisms (SSLPs), and
i) Minisatellites, also known as variable number of tandem repeats (VNTRs) in which the repeat unit is up to 25 bp in length;
ii) Microsatellites or simple tandem repeats (STRs), whose repeats are shorter, usually dinucleotide or tetranucleotide units.
Single nucleotide polymorphisms (SNPs).

5. Restriction fragment length polymorphisms (RFLP)

6. RFLP DETECTION

7. Restriction fragment length polymorphisms (RFLPs)

9. Pedigree based on RFLP analysis

11. Linkage analysis shows that the disease gene D lies between markers c and d.

12. RFLP
Distance between RFLP markers is also defined in recombination units or cM.

13. Amplified Fragment Length Polymorphism (AFLP)
AFLPs are differences in restriction fragment lengths caused by SNPs or INDELs that create or abolish restriction endonuclease recognition sites.
The AFLP technique is based on the selective PCR amplification of restriction fragments from a total digest of genomic DNA

14. RAPD (Random Amplified Polymorphic DNA)
RAPD markers are DNA fragments from PCR amplification of random segments of genomic DNA with single primer of arbitrary nucleotide sequence.
RAPD does not require any specific knowledge of the DNA sequence of the target organism
The identical 10-mer primers will or will not amplify a segment of DNA, depending on positions that are complementary to the primers' sequence.

15. RAPD (Random Amplified Polymorphic DNA)

16. Simple sequence length polymorphisms (SSLPs),
Unlike RFLPs, SSLPs can be multi-allelic as each SSLP can have a number of different length variants.

17. VNTRs - Minisatellites

18. VNTRs - Minisatellites

19. Microsatellites: simple tandem repeats (STRs)

20. Simple tandem repeats (STRs)

21. MAPPING TECHNIQUES Linkage analysis is the basis of genetic mapping.
The offspring usually co-inherit either A with B or a with b, and, in this case, the law of independent assortment is not valid.
Thus to test for linkage between the genes for two traits, certain types of matings are examined and observe whether or not the pattern of the combinations of traits exhibited by the offspring follows the law of independent assortment.
If not, the gene pairs for those traits must be linked, that is they must be on the same chromosome pair.

22. What types of matings can reveal that the genes for two traits are linked?
Only matings involving an individual who is heterozygous for both traits (genotype AaBb) reveal deviations from independent assortment and thus reveal linkage.
Moreover, the most obvious deviations occur in the test cross, a mating between a double heterozygote and a doubly recessive homozygote (genotype aabb).
Individuals with the genotype AaBb manifest both dominant phenotypes; those with the genotype aabb manifest both recessive phenotypes.

23. How do we estimate, from the offspring of a single family, the likelihood that two gene pairs are linked?
Recombination fraction
LOD score
Haldane mapping function

24. Recombination Frequency
Recombination fraction is a measure of the distance between two loci.
Two loci that show 1% recombination are defined as being 1 centimorgan (cM) apart on a genetic map.
1 map unit = 1 cM (centimorgan)
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
Genes with recombination frequencies less than 50 percent are on the same chromosome = linked

25. Calculation of Recombination Frequency
The percentage of recombinant progeny produced in a cross is called the recombination frequency, which is calculated as follows:

26. Recombination Frequency

27. Recombination fraction

28. LOD SCORE The LOD score is calculated as follows:
LOD = Z = Log10 probability of birth sequence with a given linkage
probability of birth sequence with no linkage
By convention, a LOD score greater than 2.5 is considered evidence for linkage.
On the other hand, a LOD score less than 2.5 is considered evidence to exclude linkage.

29. LOD Score Analysis The likelihood ratio is defined by :-
L(pedigree| = x)
L(pedigree| = 0.50)
where  represents the recombination fraction, where 0  x  0.49.
L.R. =
The LOD score (z) is the log10 (L.R.)

30. Method to evaluate the statistical significance of results.
Maximum-likelihood analysis, which estimates the “most likely” value of the recombination fraction Ø as well as the odds in favour of linkage versus nonlinkage.
Given by Conditional probability L (data 1 Ø), which is the likelihood of obtaining the data if the genes are linked and have a recombination fraction of Ø.
Likelihood of obtaining one recombinant and seven non-recombinants when the recombination fraction is Ø is proportional to Ø1(1–Ø)7,
Where:
Ø is, by definition, the probability of obtaining a recombinant ,
(1 – Ø) is the probability of obtaining a nonrecombinant.

31. Mapping function
The genetic distance between locus A and locus B is defined as the average number of crossovers occurring in the interval AB.
Mapping function is use to translate recombination fractions into genetic distances.
In 1919 the British geneticist J. B. S. Haldane proposed such Mapping function.
Haldane defined the genetic distance, x between two loci as the average number of crossovers per meiosis in the interval between the two loci.

32. What is Haldane ’s mapping function ?
Assumptions: crossovers occurred at random along the chromosome and that the probability of a crossover at one position along the chromosome was independent of the probability of a crossover at another position.
Using these assumptions, he derived the following relationship between
Ø, the recombination fraction and
x , the genetic distance (in morgans):
Ø= 1/2 (1-e-2x) or equivalently,
X= -1/2 ln (1-2Ø)

33. Genetic distance between two loci increases, the recombination fraction approaches a limiting value of 0.5.
Cytological observations of meiosis indicate that the average number of crossovers undergone by the chromosome pairs of a germ-line cell during meiosis is 33.
Therefore, the average genetic length of a human chromosome is about 1.4 morgans, or about 140 centimorgans.

34. Integration of MAP

35. LIMITATIONS
A map generated by genetic techniques is rarely sufficient for directing the sequencing phase of a genome project. This is for two reasons:
The resolution of a genetic map depends on the number of crossovers that have been scored .
Genes that are several tens of kb apart may appear at the same position on the genetic map.
Genetic maps have limited accuracy .
Presence of recombination hotspots means that crossovers are more likely to occur at some points rather than at others.
Physical mapping techniques has been developed to address this problem.

36. PHYSICAL MAPPING

37. Physical mapping Actual physical distances
Units in base-pairs
Contigs of large DNA fragments
Large insert DNA libraries (BACs, PACs, etc)
Restriction fragment fingerprinting
Minimum tiling set to cover entire genome
Correlation of genetic and physical maps
Genetic marker screening
EST screening
BAC-end sequencing
FISH

38. Physical Mapping Systems (like a Filing system of clones)
Yeast Artificial Chromosomes (YACs) kb
Bacteriophage P1 90 kb
Cosmids 40 kb
Bacteriophage l 9-23 kb
Plasmids (2-6 kb)

39. Contigs Contigs are groups of overlapping pieces of chromosomal DNA
Make contiguous clones
For sequencing one wants to create “minimum tiling path”
Contig of smallest number of inserts that covers a region of the chromosome
The overlapping pieces of DNA that span a region of the chromosome are known as “contigs,” a term that derives from “contiguous,” as in, producing a contiguous series of DNA fragments. For efficient sequencing, the goal is to start from the smallest number of DNA fragments that form a contig covering the region of the chromosome that you want to sequence. This set of fragments is known as the “minimum tiling path.”
genomic DNA
contig
minimum
tiling path

40. PHYSICAL MAPPING
Restriction mapping, which locates the relative positions on a DNA molecule of the recognition sequences for restriction endonucleases;
Fluorescent in situ hybridization (FISH), in which marker locations are mapped by hybridizing a probe containing the marker to intact chromosomes;
Sequence tagged site (STS) mapping, in which the positions of short sequences are mapped by PCR and/or hybridization analysis of genome fragments.

41. The basic methodology for restriction mapping

43. Restriction mapping
partial restriction

44. Physical maps
Physical maps can be generated by aligning the restriction maps of specific pieces of cloned genomic DNA (for instance, in YAC or BAC vectors) along the chromosomes.
These maps are extremely useful for the purpose of map-based gene cloning.

45. Fluorescent in situ hybridization (FISH)
FISH enables the position of a marker on a chromosome or extended DNA molecule to be directly visualized
In FISH, the marker is a DNA sequence that is visualized by hybridization with a fluorescent probe.
In situ hybridization intact chromosome is examined by probing it with a labeled DNA molecule.

46. In situ hybridization with radioactive or fluorescent probes
The position on the chromosome at which hybridization occurs provides information about the map location of the DNA sequence used as the probe
DNA in the chromosome is made single stranded (‘denatured’).
The standard method for denaturing chromosomal DNA without destroying the morphology of the chromosome is to dry the preparation onto a glass microscope slide and then treat with formamide.

47. Can distinguish chromosomes by “painting” – using DNA hybridization + fluorescent probes – during mitosis

48. FISH

49. FISH 16 16
DNA appears as a yellow band on chromosome16, thus locating this particular simple sequence to one site in the genome.

50. Sequence tagged site (STS) mapping
A sequence tagged site or STS is simply a short DNA sequence, generally between 100 and 500 bp in length, that is easily recognizable and occurs only once in the chromosome or genome being studied.
To map a set of STSs, a collection of overlapping DNA fragments from a single chromosome or from the entire genome is needed.

51. STS mapping

52. STS mapping
The data from which the map will be derived are obtained by determining which fragments contain which STSs.
The chances of two STSs being present on the same fragment will, of course, depend on how close together they are in the genome.
The data can therefore be used to calculate the distance between two markers.
Each map distance is based on the frequency at which breaks occur between two markers

53. Genetic V/s 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

54. Genetic vs. Physical Distance

55. Genetic and physical maps may differ in relative distances and even in the position of genes on a chromosome.

56. Map-based sequencing
The first method for assembling short, sequenced fragments into a whole-genome sequence, called a map-based approach,
Requires the initial creation of detailed genetic and physical maps of the genome,
It provide known locations of genetic markers (restriction sites, other genes, or known DNA sequences) at regularly spaced intervals along each chromosome.

57. Map-based sequencing

58. Molecular marker applications in plant breeding
Molecular markers are used in plant breeding, taxonomy, physiology, embryology, genetics, evolution, genetic engineering etc
Some of applications in plant breeding are:
1. Marker-assisted selection
→ It replaces evaluation of a trait (difficult or expensive).
→ It is not influenced by growth stage and environmental factors.
→ It can increase efficiency of selection for low-heritability.
2. Genetic diversity analysis
→ Plant breeding is dependant of genetic variation
→ Genetic diversity is measured by genetic distance (GD) or genetic similarity (GS = 1 - GD)
→ It is used to assess variation over time, protection of intellectual property rights, registration of germplasm, and evaluation of new sources of germplasm for their potential to increase genetic diversity
→ It is superior to morphological, pedigree, heterosis, and biochemical
Cont...

59. ...Cont 3. Genetic mapping → Markers can be used for map construction.
→ It predicts the linear arrangement of markers on a chromosome and the recombination frequency.
4. Manipulating traits controlled by a few major loci
→ To map and transfer the gene of interest
5. Authentication or DNA fingerprinting
→ For diagnosis, protection property right and quality controlling
6. Phylogeny and evolution
Perform phylogenic analysis on a species by comparing the presence/absence of various markers in their genome.
For taxonomic classification to determine the primary, secondary or tertiary genepool of the system

60. Summary Plant breeding depends on genetic variation and selection.
Selection is difficult and assisted by morphological, biochemical and molecular markers.
Molecular markers are DNA marker, polymorphic, independent of environmental factors, easy, fast, co-dominant and reproducible but it is costly.
The two forms of molecular techniques (hybridization and PCR methods).
Many forms of molecular markers
→ RFLP, RAPD, VNTR, SSR, AFLP …….
Molecular markers are used in diversity analysis, parent selection, germplasm characterization, identification, genetic fingerprinting, genetic diagnostics, genome organization and phylogenic analysis.