Marker assisted selection: a strategy for wheat improvement Bhakti Rana1, Preeti Rana 2, Manoj K. Yadav1, Sundeep Kumar1

Marker assisted selection
MAS is a breakthrough technology that changes the process of variety cultivation from traditional field based format to a laboratory format. It is the use of molecular markers to track the location of genes of interest in a breeding programme. MAS is a form of indirect selection and most widely used application of DNA markers. Once traits are mapped a closely linked marker may be used to screen large number of samples for rapid identification of progeny that carry desirable characteristics. MAS is one of the most widely used applications of molecular marker technologies and one that plant breeders have been quick to embrace. Biotechnology have provided additional tools that do not require the use of transgenic crops to revolutionize plant breeding progress in molecular genetics has resulted in the development of DNA tags and marker assisted selection strategies for cultivar development. Several molecular marker types are available and they each have their advantages and disadvantages. Restriction fragment length polymorphisms (RFLPs) were the first to be developed (some 15 years) and have been widely and successfully used to construct linkage maps of various species, including wheat. With the development of the polymerase chain reaction (PCR) technology, several marker types emerged. The first of those were random amplified polymorphic DNA (RAPD), which quickly gained popularity over RFLPs due to the simplicity and decreased costs of the assay. However, most researchers now realize the weaknesses of RAPDs and use them with much less frequency. Microsatellite markers or simple sequence repeats (SSRs) combine the power of RFLPs (codominant markers, reliable, specific genome location) with the ease of RAPDs and have the advantage of detecting higher levels of polymorphism. The amplified fragment length polymorphism (AFLP) approach takes advantage of the PCR technique to selectively amplify DNA fragments previously digested with one or two restriction enzymes (Hosington et al., FAO Document Repository). Later, microsatellite markers or SSRs (Simple Sequence Repeats) were developed, which took advantage over RAPD and RFLP. Playing with the number of selective bases of the primers and considering the number of amplification products per primer pair, this approach is certainly the most powerful in terms of polymorphisms identified per reaction.
The essential requirements for MAS in a plant-breeding program are as follows (Mohan et al. 1997):
(a) Marker(s) should co-segregate or be closely linked (1 cM or less) with the desired trait.
(b) An efficient means of screening large populations for the molecular markers should be available. A relatively easy analysis based on PCR technology is the best option.
(c) The screening technology should have high reproducibility across laboratories, be economical to use and user friendly.
RFLPs, RAPDs and AFLPs do not fit the first requirement. However, techniques are available to turn them into user-friendly markers. RFLP clones can be sequenced and primers designed to amplify the DNA fragments are shown by hybridization to be polymorphic. However, the resulting STS or SCAR does not always turn out to be polymorphic and further manipulations are needed if this is the case. The amplified fragment is usually digested with one or two restriction endonucleases to detect small length differences, or the fragment from two or more cultivars is cloned and sequenced again to create ASAs. ASAs are usually based on single nucleotide differences. RAPD and AFLP fragments can be isolated from the gel, cloned and sequenced to generate STSs or SCARs. Attempts to generate such markers for wheat are neither always successful nor easily achieved. SSRs, on the other hand, if tightly linked to genes of interest are probably the most attractive markers since no further manipulations are needed for implementation. Despite the large number of markers for wheat genes listed in Table 2 few of those markers are close enough to the genes of interest to be useful in breeding applications.
Breeders used molecular markers to increase the precision of selection for best trial combinations. Variety developed by MAS are not considered genetically modified organisms (GMOs) and accepted by local and international market. Molecular marker aided selection methods have resulted in significant improvement in breeding efficiency by reducing trial and error aspect of breeding process and by allowing for time and cost savings. Molecular marker systems will benefit from the constant increase in the integration of biotechnological production of segregating populations such as homozygous double haploids in wheat breeding cycles since, the major requirement of being co-dominant for molecular markers will disappear. Marker assisted selection (MAS) offers an opportunity to select desirable lines based on genotype rather than phenotype. Marker assisted selection is an invaluable tool for gene pyramiding (Bringing genes from different individuals together in one individual) and has been fairly successful for combining single gene traits.
Marker assisted selection (MAS) is based on the identification and use of markers, which are linked to the gene(s) controlling the trait of interest. By virtue of linkage, selection may be applied to the marker itself. The advantage consists in the opportunity of speeding up the application of the selection procedure. For instance, a character which is expressed only at the mature plant stage may be selected at the plantlet stage, if selection is applied to a molecular marker. Selection may be applied simultaneously to more than one character. Selection for a resistance gene may be carried out without needing to expose the plant to the pest, pathogen or deleterious agent. If linkage exists between a molecular marker and a quantitative trait locus (QTL), selection may become more efficient and rapid. The construction of detailed molecular and genetic maps of the genome of the species of interest is a prerequisite for most forms of MAS. However, the current cost of the application of these techniques is significant, and the choice of one cost technique rather than others may be dictated by factors. There are few examples of crop varieties in farmer’s fields, which have developed through MAS.
Genetic resistance in wheat against diseases like leaf rust, stripe rust, stem rust, spot blotch, hill bunt etc., is generally governed by one, two or three genes and these genes can be tagged with any of the above DNA markers, specially which are based on PCR technology. In wheat, RFLPs have been used to map seed storage protein loci, loci associated with protein flour colour, cultivar identification, vernalization and frost resistance gene, intrachromosomal mapping of genes for dwarfing and vernalization, resistance to preharvest sprouting, quantitative trait loci (QTL) controlling tissue culture response, nematode resistance and milling yield. PCR based markers have been useful for characterization of genes for resistance against common bunt, powdery mildew, leaf rust resistance against hessian fly and Russsian wheat aphid. RFLP, DNA sequencing, and a number of PCR-based markers are being used extensively for reconstructing phylogenies of various species. The techniques are speculated to provide path breaking information regarding the fine time scale on which closely related species have diverged and what sort of genetic variations are associated with species formation. Efforts are being made for studying the genetic variation in plants to understand their evolution from wild progenitors and to classify them into appropriate groups (Jeffrey 1995). RFLP markers have proved their importance as markers for gene tagging locating and manipulating quantitative trait loci (QTL), in evolutionary studies for deducing the relationship between the hexaploid genome of bread wheat and its ancestors (Gill 1991). Specific markers like STMS (Sequence-tagged microsatellite markers) ALPs (Amplicon length polymorphisms) or STS markers have proved to be extremely valuable in the analysis of gene pool variation of crops during the process of cultivar development and classification of germplasm.
Wheat biotechnological research have been relatively slow, due to its ploidy level, the size and complexity of its genome, the very high percentage of repetitive sequences and low level of polymorphism (Table 1). Lack of genetic polymorphism in crops like wheat and soybeans and the consequent problems to identify molecular markers have been a major limitation to the impact of marker assisted selection (MAS) in wheat breeding. However, the identification of a high number of polymorphism in Single Sequence Repeats (SSR) should therefore, greatly enhance the potential to find molecular markers in wheat.
RAPDs emerged as a convenient and effective technique for tracing alien chromosome segments in translocation lines (Williams et al. 1990). RAPD markers provide a useful alternative to RFLP analysis for screening markers linked to a single trait within near isogenic lines and bulked segregants. He et al. (1992) reported the development of a DNA polymorphism detection method by combining RAPD with DGGE (denaturing gradient gel elecrophoresis) for pedigree analysis and fingerprinting of wheat cultivars. RAPD markers can be converted to more user-friendly Sequence Characterized Amplified Region (SCAR) markers, which display a less complex banding pattern. SCAR markers linked to resistance genes against fungal pathogens have been characterized in combination with RAPD and RFLP (Procunier et al.1997; Myburg et al. 1998; Liu et al. 1999). In recent years, RAPD and other PCR based markers like Sequence Characterized Amplified Regions (SCAR), Sequence Tagged Sites (STS) and Differential Display Reverse Transcriptase PCR (DDRT-PCR) are increasingly being used for identification of desirable traits in wheat and related genera. These markers have been used in particular for disease resistance against viral and fungal pathogens and also for insect and nematode pests and have the potential of pyramiding of resistance genes for effective breeding programs. PCR based markers have been extensively characterized for genes of resistance against common bunt, Tilletia tritici (Demeke et al. 1996), powdery mildew, Erysiphe graminis (Hartl et al.; 1995; Qi et al. 1996), leaf rust, Puccinia recondita (Dedryver et al. 1996; Feuillet et al. 1995; Seyfarth et al. 1999), resistance against Hessian fly, Mayetiola destructor (Dweikat et al. 1994) and Russian wheat aphid, Diuraphis noxia (Myburg et al. 1998; Venter and Botha, 2000). SSRs or Microsatellites are more promising molecular markers for the identification and differentiation of genotypes within a species. The high level of polymorphism and easy handling has made microsatellites extremely useful for different applications in wheat breeding (Devos et al. 1995; Roder et al. 1995; Bryan et al. 1997; Korzun et al. 1997; Roy et al. 1999.). Microsatellites have also been used to identify resistance genes like Pm6 from Triticum timopheevii (Tao et al. 1999) and Yr15 from breadwheat (Chague et al. 1999) (Table 2).