The document summarizes the process of map-based cloning, also called positional cloning. It describes how researchers used genetic mapping and linkage analysis to clone the Pto gene in tomato, which provides resistance to bacterial speck disease. The key steps included identifying a molecular marker closely linked to the Pto gene, screening a YAC library to find a clone containing the gene region, identifying candidate genes on the clone, and ultimately transforming plants to show that one candidate gene, CD186, rescued the wild-type phenotype when introduced into susceptible plants.

1. Map based cloning
Presented by :
Chaudhary Ankitkumar R.
Submitted to :
Dr. Vipul Parikh
Department of Genetics & Plant Breeding
N .M . College of Agriculture
N. A. U., Navsari
Reg. No. : 1010119005

2. Map based cloning
• Map-based cloning or positional cloning is the process to
recognize the genetic basis of a mutant phenotype with the
help of linkage to markers whose physical location in the
genome is known.
• Map-based cloning using the genetic relationship between a
gene and a marker as the basis for beginning a search for a
gene

3. • A genetic map is constructed on the basis of recombination
events between two non-sister chromatids of each pair of
homologous chromosomes during meiosis.
• Alfred H. Sturtevant presented the first concept of a genetic
map in (1913) by ordering five sex-linked characters of
Drosophila on the Y chromosome in a linear fashion.
Map based cloning

4. • The principles of genetic mapping and linkage analyses are
used even today in much similar way but Now the whole
genomes are being sequenced with greater speed than ever
before. With the availability of whole genome sequences,
Map-Based cloning and functional genetic studies in model
plant systems have become easier and provide fundamental
knowledge for understanding plant growth and environmental
response
Map based cloning

5. Map-based Cloning
• Principles of Map-based or Positional Cloning
• The first step of map-based or positional cloning is to identify a
molecular marker that lies close to you gene of interest.
• The next step is to saturate the region around that original
molecular marker with other markers. At this point you are
looking for a one that rarely shows recombination with your gene.
At this stage, the population size could increase to 300-600
individuals.
• The next step is to screen a large insert genomic library (BAC or
YAC) with your marker to isolate clones that hybridize to your
molecular marker. Once you identify the initial markers that map
are near (or better yet) flank your gene and a clone to which the
markers hybridize.

7. Map-based Cloning
Experimental design and
mapping populations
Marker identification
genotyping
Phenotyping fine mapping
Candidate gene analysis
Gene confirmation

8. Steps Involved in Map-based Cloning
• Identify a cosegregating marker in a "large" mapping population
Identify a cosegregating marker in a "large" mapping population
• Perform high density molecular mapping
Find a YAC or BAC clone to which the marker probe hybridizes
Chromosome walking
Chromosome landing

9. • Determine that the gene is on the YAC or BAC clone
• Find an appropriate recombinant genotype
• Identify a candidate gene on the YAC or BAC clone
• Search for a c DNA clone

10. • Perform genetic complementation to rescue the wild-type phenotype
• Transform a plant without the gene and look for phenotypic rescue
• Sequence the gene and determine if the function is known
Determine the molecular sequence and compare it against a
sequence database

12. Map-based Cloning
Example of Map-based Cloning

13. • Recently map-based cloning was employed to clone the first
plant resistance gene that follows the gene-for-gene
interaction.
• The tomato gene that was cloned was Pto and it provides
resistance against bacterial speck disease of tomato caused
by Pseudomonas syringae pv. tomato.
• This pathogen expresses the avirulence gene avrPto that
interacts with Pto to provide resistance in tomato. This gene
was cloned by Martin et al. [Science (1993) 262:1432]..

14. • The following is the list of the various steps used to clone
the Pto gene.
1) A genetic population of 251 F2 plants was screened with DNA
probes, and the locus TG538 cosegregated with Pto.
2) A YAC library was screened with the TG538 probe, and the clone
PTY538-1 was identified.
3) Primers that marked the end of the 400 kb clone were created, and the
population was screened. The left end of the PTY538-1 (PCR marker
PTY538-1L was 1.8 cM from Pto, and the right end of the clone
(PTY538-1R) cosegregated with Pto. The right end of the clone
maybe to left or right of Pto, so it is important to determine if the
YAC clone spans the Pto gene.

15. 4) 1300 plants from F2, F3 and cultivars were screened, and one plant
that contains the Pto allele at the TG538 and the PTY538-1R pto allele
was found. For this result to occur, Pto must be located on the YAC
clone PTY538-1.
5) To find a candidate clone on the YAC clone, DNA from the PTY538-
1 clone was hybridized to a leaf cDNA library. 200 clones were found
and 30 analyzed. A population of 50 plants with recombination in
the Pto region was screened. cDNA clone CD127 cosegregated
with Pto.

16. 6) Southern hybridization with CD127 detected many polymorphic
fragments, the clone could be detecting exons or a multigene
family. 14 other cross hybridizing clones were found that all
represented six different classes of related genes. Analyses of
PTY538-1 detected all (but one) of the genomic fragments that
were detected with Southern hybridization. It was concluded that
PTY538-1 contained a multigene family.
7) To determine if the cDNAs contained the Pto gene,
transformation vectors were created with CD127 and CD186. Two
pPTC8 resistant plants were found. No pPTC5 resistant plants were
found.

17. 8) One resistant plant (genotype=Pto/Pto) was crossed to a
susceptible cultivar (genotype= pto/pto). Of 22 plants 9 contained
the CD186 sequence and were resistant to the bacterial speck
pathogen, and the 13 without CD186 were susceptible to
bacterial speck.
9) The clone CD186 was sequenced, and a 321 amino acid
opening reading frame was found. The sequence of the open
reading frame was similar to a serine-threonine protein kinase.