The Pto gene in tomato confers resistance to races of Pseudomonas syringae pv. tomato that carry the avirulence gene avrPto. A yeast artificial chromosome clone that spans the Pto region was identified and used to probe a leaf complementary DNA (cDNA) library. A cDNA clone was isolated that represents a gene family, at least six members of which genetically cosegregate with Pto. When susceptible tomato plants were transformed with a cDNA from this family, they were resistant to the pathogen. Analysis of the amino acid sequence revealed similarity to serine-threonine protein kinases, suggesting a role for Pto in a signal transduction pathway.

https://science.sciencemag.org/content/sci/262/5138/1432.full.pdf

Map-based cloning of a protein kinase gene conferring disease resistance in tomato

Sequence characterized amplified regions (SCARs) were derived from eight random amplified polymorphic DNA (RAPD) markers linked to disease resistance genes in lettuce. SCARs are PCR-based markers that represent single, genetically defined loci that are identified by PCR amplification of genomic DNA with pairs of specific oligonucleotide primers; they may contain high-copy, dispersed genomic sequences within the amplified region. Amplified RAPD products were cloned and sequenced. The sequence was used to design 24-mer oligonucleotide primers for each end. All pairs of SCAR primers resulted in the amplification of single major bands the same size as the RAPD fragment cloned. Polymorphism was either retained as the presence or absence of amplification of the band or appeared as length polymorphisms that converted dominant RAPD loci into codominant SCAR markers. This study provided information on the molecular basis of RAPD markers. The amplified fragment contained no obvious repeated sequences beyond the primer sequence. Five out of eight pairs of SCAR primers amplified an alternate allele from both parents of the mapping population; therefore, the original RAPD polymorphism was likely due to mismatch at the primer sites.

Development of reliable PCR-based markers linked to downy mildew resistance genes in lettuce

Molecular genetic markers have been developed into powerful tools to analyse genetic relationships and genetic diversity. As an extension to the variety of existing techniques using polymorphic DNA markers, the Random Amplified Polymorphic DNA (RAPD) technique may be used in molecular ecology to determine taxonomic identity, assess kinship relationships, analyse mixed genome samples, and create specific probes. Main advantages of the RAPD technology include (i) suitability for work on anonymous genomes, (ii) applicability to problems where only limited quantities of DNA are available, (iii) efficiency and low expense.

https://onlinelibrary.wiley.com/doi/pdf/10.1111/j.1365-294X.1992.tb00155.x

Applications of random amplified polymorphic DNA (RAPD) in molecular ecology

Detection and analysis of genetic variation can help us to understand the molecular basis of various biological phenomena in plants. Since the entire plant kingdom cannot be covered under sequencing projects, molecular markers and their correlation to phenotypes provide us with requisite landmarks for elucidation of genetic variation. Genetic or DNA based marker techniques such as RFLP (restriction fragment length polymorphism), RAPD (random amplified polymorphic DNA), SSR (simple sequence repeats) and AFLP (amplified fragment length polymorphism) are routinely being used in ecological, evolutionary, taxonomical, phylogenic and genetic studies of plant sciences. These techniques are well established and their advantages as well as limitations have been realized. In recent years, a new class of advanced techniques has emerged, primarily derived from combination of earlier basic techniques. Advanced marker techniques tend to amalgamate advantageous features of several basic techniques. The newer methods also incorporate modifications in the methodology of basic techniques to increase the sensitivity and resolution to detect genetic discontinuity and distinctiveness. The advanced marker techniques also utilize newer class of DNA elements such as retrotransposons, mitochondrial and chloroplast based microsatellites, thereby revealing genetic variation through increased genome coverage. Techniques such as RAPD and AFLP are also being applied to cDNA-based templates to study patterns of gene expression and uncover the genetic basis of biological responses. The review details account of techniques used in identification of markers and their applicability in plant sciences.

/pdf/advances-in-molecular-marker-techniques-and-their-27nnkfjmsw.pdf

Advances in molecular marker techniques and their applications in plant sciences

Applications of genome mapping and marker-assisted selection (MAS) in crop improvement are reviewed. The following aspects are considered: a comparison of the choice of markers available for the generation of linkage maps (including amplified fragment length polymorphisms (AFLP); restriction fragment length polymorphisms (RFLP); randomly amplified polymorphic DNA (RAPD) and simple sequence repeats (SSR)); quantitative trait loci (QTL) analysis; use of molecular markers in the exploitation of hybrid vigour; physical genome mapping; map-based cloning and transposon tagging of agriculturally important genes; synteny in cereal genomes; and the use of MAS in breeding for disease and pest resistance.

Genome mapping, molecular markers and marker-assisted selection in crop plants

An approach to isolating DNA sequences that are linked to important plant genes is described. The strategy is based upon a recent modification of the polymerase chain reaction in which synthetic primers are used to amplify random sequences from genomic DNA. This technique, used in conjunction with near-isogenic lines (which differ only by the presence or absence of the target gene and a small region of surrounding DNA), leads to the rapid identification of sequences linked to the gene of interest. The feasibility of this method has been demonstrated by analyzing a pair of tomato near-isogenic lines that differ for a region on chromosome 5 that contains a gene (Pto) conferring resistance to Pseudomonas syringae pv. tomato. One hundred forty-four random primers were screened on these lines, and seven amplified products were identified that were present in one but not the other line. Of four products that were further investigated, three were confirmed by segregation analysis to be tightly linked to the Pto gene. Linked sequences identified by this method are useful for detecting the presence of the target gene in plant populations (e.g., in plant breeding) and, if very tightly linked, as starting points for a chromosome walk to isolate the gene. Since near-isogenic lines are a typical product of plant breeding and classical genetic studies, this method is applicable to a wide variety of species.

https://www.pnas.org/content/pnas/88/6/2336.full.pdf

John G. K. Williams

Papers

Rapid identification of markers linked to a Pseudomonas resistance gene in tomato by using random primers and near-isogenic lines.