Banoth Madhu: Map based gene cloning in plant. In the process of map-based cloning, one starts with a mutant and eventually identifies the gene responsible for the altered phenotype, allowing the plant to tell you what genes are important in the physiological process of interest and using the genetic relationship between a gene and a marker as the basis for beginning a search for a gene

1. Map Based Gene Cloning In Plant
Banoth Madhu
Ph.D Research Scholar
Genetics and Plant Breeding
Tamil Nadu Agricultural University, Coimbatore

2. Map Based Gene Cloning In Plant
Map-based or positional cloning
 using the genetic relationship between a gene and a marker as the basis for beginning a search for a
gene
Chromosome walking
 moving toward a gene by using a probe for a marker near a gene to select a genomic clone near
the gene and moving toward the gene by repeatedly selecting for overlapping clones until you
have a clone that contains the gene
 original approach used to find a large insert clone that contains your gene of interest; replaced by
high density mapping

3. Chromosome landing
 using high density mapping in the region of a gene to find markers that co-segregate with the
gene of interest
 this marker used to select a genomic clone that contains the gene
Co-segregation
 means that whenever one allele of your gene is expressed, the markers associated with that allele
are also present. In other words, recombination is not seen between your gene and the markers.
Plant transformation
 DNA fragments between the flanking markers are cloned and introduced into a genotype mutant
for your gene by a genetic engineering technique.
Open Reading Frame (ORF)
 sequences that most likely will encode a gene product (know gene of interest).

4. Steps Involved in Map-based Cloning
1. Identify a marker tightly linked to your gene in a "large" mapping population
2. Find a YAC or BAC clone to which the marker probe hybridizes
3. Create new markers from the large-insert clone and determine if they co-segregate with your gene
4. If necessary, re-screen the large-insert genomic library for other clones and search for co-
segregating markers Identify a candidate gene from large-insert clone whose markers co-segregate
with the gene
5. Perform genetic complementation (transformation) to rescue the wild-type phenotype
6. Sequence the gene and determine if the function is known

6. Example: Cloning of the tomato Pto gene
 Provides resistance against bacterial speck disease
 Disease caused by Pseudomonas syringa pv. tomato
 Pathogen expresses the avirulence gene avrPto
 Gene-for-gene interaction between Pto and avrPto
Martin et al.(1993)

12. Application of Map-based Cloning
1. Cloning of disease resistance genes (Lr34,Sr2-wheat, RP1-Maize, Pto-Tomato, Mla-Barley, Bs2-
Pepper, Npr-arabidopsis)
2. Chromosomal location, Transposon or molecular tagging and mapping of disease resistance genes
3. Sequencing, Mutagenesis, Genotyping or heterologous expression of protein
4. Identifying rarely mutation implicated in simple mendelian disease