marker-assisted selection in wheat - ictsd

Chapter 3 – Molecular markers for use in plant molecular breeding and germplasm evaluation 31IntroductionMolecular markers are valuable tools forthe classification of germplasm and inMAS. The purpose of this chapter is toprovide guidance in selecting appropriatemolecular marker systems based on theavailability of technological resources invarious species and to provide some examplesof MAS applications. One of the manybenefits of the increasing amount of DNAsequence information in many organisms isthe expanding opportunity for the developmentof new molecular markers. As the fullgenome sequence will not be available formost species of interest in the near future,it is important to find strategies for developingand using molecular markers whensequence resources are limited. This chapterdescribes several technologies that existfor developing molecular markers withoutDNA sequence information. It also drawson some examples from rice (Oryza sativaL.) to illustrate how molecular markerdevelopment was influenced by the additionof each layer of sequence information,culminating in the present status of rice asthe first crop with nearly complete genomesequence information.Molecular marker technologiesRestriction fragment lengthpolymorphismsRestriction fragment length polymorphisms(RFLPs) were the first DNA-based molecularmarkers. An application of Southernanalysis (Southern, 1975), RFLPs exploitthe ability of single stranded DNA to bind(hybridize) to DNA with a complementarysequence. RFLP markers detect variationin DNA sequences at the same loci indifferent individuals or accessions. Technically,RFLP technology involves thehybridization of cloned DNA to restrictionfragments of differing molecular weightsfrom restriction enzyme-digested genomicDNA. The digested DNA fragments aresize-separated on agarose gels by electrophoresisand transferred as denatured(single stranded) arrays of fragments tofilters through capillary action. The filtersare then incubated with specific labelledprobes (genes or anonymous fragmentsof single stranded DNA), washedand exposed to x-ray film. To identifypolymorphisms between individuals oraccessions, the genomic DNA extractedfrom each individual is digested with a seriesof restriction enzymes to find enzymesthat produce fragments (bands) that differin molecular weight between accessionsand can be distinguished by hybridizationwith a given probe. To ensure that probeshybridize to single fragments on a gel, theDNA used as a probe should be from asingle or low copy (non-repetitive) regionof the genome. Probes may represent genes(i.e. derived from complementary DNA[cDNA]) or they may represent anonymoussequences derived from genomic DNA.Genomic probes are generated by shearingor digesting DNA and cloning the fragmentsinto a plasmid vector that allows foramplification of the cloned fragment in asuitable host. To increase the frequency oflow copy clones in a genomic library, theDNA may be digested with a methylationsensitiveenzyme, such as PstI. Therepetitive regions of a genome are typicallyheavily methylated and thus producefragments >25 kb when digested with amethylation-sensitive enzyme. As a result,these fragments do not clone efficientlyinto plasmid vectors and consequentlyare effectively filtered out of the analysis.Thus, use of methylation-sensitive enzymesincreases the representation of unmethylatedand typically low copy gene sequences inRFLP analysis. Sharing of anonymous,