**5. Molecular markers**

Genetic markers represent genetic differences between individual organisms or species. Generally, they do not represent the target genes themselves but act as signs or flags and they are used as chromosome landmarks to facilitate the introgression of chromosome re‐ gions with genes associated with economically important traits [19]. However, such markers themselves do not affect the phenotype of that trait of interest because they are located only near or are linked to genes controlling the target traits [31]. Various types of molecular markers are utilized to evaluate DNA polymorphism and are generally classified as either hybridization-based or polymerase chain reaction (PCR)-based markers [19]. DNA markers are useful particularly if they can reveal difference between individuals of the same species or different species [32, 33].

There are three types of genetic markers: morphological (or classical or visible) markers, which themselves are phenotypic traits or characters, biochemical markers which include allelic variants of enzymes called isozymes and DNA (or molecular) markers, which re‐ veal sites of variation in DNA [19]. Morphological markers are usually visually character‐ ized and include phenotypic characters such as flower colour, seed shape, growth habits or pigmentation [34]. However, these markers are limited in number so only small por‐ tion of the genome can be assayed for contribution towards complex characters. Also, the genes controlling morphological markers have pleiotropic effects on the characters under investigation; this eludes the actual location of genes due to distortion of segregation ra‐ tio. Isozyme markers are differences in enzymes that are detected by electrophoresis and specific staining. The number of useful protein markers is very small. Both morphological and biochemical markers are influenced by environmental factors and/or developmental stages of the plants [20, 16, 19, 34].

However, the properties to be considered desirable for ideal DNA markers for their use as DNA markers in MAS as suggested by authors in reference [32, 33] as: highly polymor‐ phic nature, codominant inheritance (distinguishing homozygous and heterozygous states of diploid organisms), quality and quantity of DNA required, frequent occurrence in ge‐ nome (reliability), selective neutral behaviour (the DNA sequences of any organism are neutral to environmental conditions or management practices), easy access (availability), easy and fast assay, high reproducibility and easy exchange of data between laboratories. However, it is not easy to find a molecular marker which meets all these criteria. Depend‐ ing on the type of study undertaken, a marker system can be identified that would fulfill at least a few of these criteria.

#### **5.1. Restricted Fragment Length Polymorphism (RFLP)**

obstacle for breeders as most pathogen exhibits a great variability for pathogenicity which mostly overcomes the resistance in the released cultivars. Breeders are thus continuously forced to look for new sources of resistances. The screening procedures to ascertain resist‐ ance is another setback as pathogenicity tests need to be reliable by exhibiting comparable and reproducible results [13]. The other constraint is whatever resistances detected with those tests should be efficient in controlling the target diseases in the field. Finally, methods usable by breeders for speeding up the breeding work should be developed. Genomics of *P. vulgaris* appear to be promising in discovering and tagging novel alleles [19, 31]. If closely linked to resistant genes, molecular markers such as Sequence Characterized Amplified Re‐ gion (SCAR), Simple Sequence Repeats (SSR), Restriction fragment length polymorphism (RFLP), Amplified fragment length polymorphism (AFLP) can enhance the efficiency of breeding programs especially in the so-called marker assisted selection (MAS) and can be used in initial and intermediate stages of the breeding process. The target traits can be ach‐ ieved indirectly using molecular markers closely linked to underlying genes or that have been developed from the actual gene sequences [32]. MAS can be used to simultaneously screen for resistance to diseases without affecting the growth of the plants. Selection for ge‐ netic markers linked with resistance genes and QTL can accelerate development of multiple resistant varieties and increase efficacy [20, 21]. The uses of MAS enable the introgression of resistance genes into a cultivar, decreases population size and ultimately reduce the time re‐

Genetic markers represent genetic differences between individual organisms or species. Generally, they do not represent the target genes themselves but act as signs or flags and they are used as chromosome landmarks to facilitate the introgression of chromosome re‐ gions with genes associated with economically important traits [19]. However, such markers themselves do not affect the phenotype of that trait of interest because they are located only near or are linked to genes controlling the target traits [31]. Various types of molecular markers are utilized to evaluate DNA polymorphism and are generally classified as either hybridization-based or polymerase chain reaction (PCR)-based markers [19]. DNA markers are useful particularly if they can reveal difference between individuals of the same species

There are three types of genetic markers: morphological (or classical or visible) markers, which themselves are phenotypic traits or characters, biochemical markers which include allelic variants of enzymes called isozymes and DNA (or molecular) markers, which re‐ veal sites of variation in DNA [19]. Morphological markers are usually visually character‐ ized and include phenotypic characters such as flower colour, seed shape, growth habits or pigmentation [34]. However, these markers are limited in number so only small por‐ tion of the genome can be assayed for contribution towards complex characters. Also, the genes controlling morphological markers have pleiotropic effects on the characters under investigation; this eludes the actual location of genes due to distortion of segregation ra‐

quired to develop a new variety.

124 Plant Breeding from Laboratories to Fields

**5. Molecular markers**

or different species [32, 33].

RFLPs are simply inherited naturally and are Mendelian characters. They have their origin in the DNA rearrangements that occur due to evolutionary processes, point mutations with‐ in the restriction enzyme recognition site sequences, insertions or deletions within the frag‐ ments, and unequal crossing over. The usefulness of these markers in improvement of common bean have been the assessment of genetic diversity as they are useful in detecting polymorphism among different lines and hence being used to determine how diverse the genome being assessed [35] and they have been found superior over isozymes for their bet‐ ter coverage of the genome and higher level of polymophiosm. These markers are useful in breeding for disease resistance when they are linked to disease resistant genes. For example, four RFLPs were found to be linked to *Are* gene for resistance to Anthracnose of common bean [36] and in this matter then the RFLPs can be used to breed for resistance.

#### **5.2. Microsatellite markers (SSR-Simple Sequence Repeats)**

They essentially belong to the repetitive DNA family. Fingerprints generated by these probes are also known as oligonucleotide fingerprints. The methodology has been derived from RFLP and specific fragments are visualized by hybridization with a labelled microsa‐ tellite probe. Microsatellites or short tandem repeats/simple sequence repeats consist of 1 to 6 bp long monomer sequence that is repeated several times. These loci contain tandem re‐ peats that vary in the number of repeated units between genotypes and are referred to as variable number of tandem repeats. Microsatellites thus form an ideal marker system creat‐ ing complex banding patterns by simultaneously detecting multiple DNA loci. Some of the prominent features of these markers are that they are dominant fingerprinting markers and codominant sequence tagged microsatellites markers. If many alleles exist in a population, the level of heterozygosity is high and they follow Mendelian inheritance.

These markers have been utilized in a variety of ways in bean improvement since they are linked to disease resistance genes and in diversity analysis [37]. The SSR markers have been used in diversity assessment in common bean because of their utilities like low costs, high efficiency, whole genome coverage, robustness and minimum DNA requirements [20, 37]. In addition these markers are preferred for use because of being highly polymorphic, co-domi‐ nant, being PCR based and easily detected [19, 37]. The SSR markers have been utilized in assessing the genetic structure and diversity among common beans [38]. In MAS some SSR markers have been identified to be linked to disease resistance genes as the case for Angular leaf spot genes where the primer *PV-atct001* was found to be linked to resistant allele to ALS [16, 39] and some markers have been used in Marker assisted backcrossing [29].

#### **5.3. Random Amplified Polymorphic DNA (RAPD)**

This procedure detects nucleotide sequence polymorphisms in DNA by using a single pri‐ mer of arbitrary nucleotide sequences. In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermocyclic amplification. On an average, each primer directs amplifica‐ tion of several discrete loci in the genome, making the assay useful for efficient screening of nucleotide sequence polymorphism between individuals.

These markers have been used in a variety of ways in genetic analysis. They have been used in gene pyramiding especially where conventional procedures couldn't solve the problem when there is epistasis between resistance genes to be pyramided [40, 41]. Some of the RAPD markers have been used to pyramid three rust resistance alleles *Up2, Ur-3* and *B-190* [42] with other epistatic resistance alleles from plant introduction collection [40, 43]. Similar‐ ly, pyramiding was also suggested in reference [44] where the two genes linked to *I* and *bc-3* genes for resistance to bean common mosaic virus disease and bean common mosaic ne‐ crosis virus disease respectively were incorporated in elite cultivar/line.

These markers have also been used in the assessment of genetic diversity of common bean. In reference [45] reported the potential of using RAPD markers as compared to RFLP, DAMD-PCR, ISSR and AFLP for assessing diversity of common bean and in this finding it shows that these markers were able to produce higher percentage of polymorphism than the others used hence being very useful in detecting polymorphism.

#### **5.4. Sequence Characterized Amplified Region (SCAR) markers**

In SCAR markers, the RAPD marker termini are sequenced and longer primers are designed (22–24 nucleotide bases long) for specific amplification of a particular locus [16]. The pres‐ ence or absence of the band indicates variation in sequence. These are better reproducible than RAPDs. SCARs are usually dominant markers, however, some of them can be convert‐ ed into codominant markers by digesting them with tetra cutting restriction enzymes and polymorphism can be deduced by using simple non denaturing gels to detect whether the products has different restriction sites for the different alleles. Compared to arbitrary pri‐ mers, SCARs exhibit several advantages in mapping studies (codominant SCARs are infor‐ mative for genetic mapping than dominant RAPDs), map-based cloning as they can be used to screen pooled genomic libraries by PCR, physical mapping, locus specificity, etc. SCARs also allow comparative mapping or homology studies among related species, thus making it an extremely adaptable concept in the near future [16, 19]. These markers have been widely used in breeding for disease resistance especially where the disease is controlled by domi‐ nant gene since these markers are dominant in nature. Different SCAR markers have been identified linked to resistance genes to many common bean diseases [46].

#### **5.5. Sequence Tagged Sites (STS)**

These markers have been utilized in a variety of ways in bean improvement since they are linked to disease resistance genes and in diversity analysis [37]. The SSR markers have been used in diversity assessment in common bean because of their utilities like low costs, high efficiency, whole genome coverage, robustness and minimum DNA requirements [20, 37]. In addition these markers are preferred for use because of being highly polymorphic, co-domi‐ nant, being PCR based and easily detected [19, 37]. The SSR markers have been utilized in assessing the genetic structure and diversity among common beans [38]. In MAS some SSR markers have been identified to be linked to disease resistance genes as the case for Angular leaf spot genes where the primer *PV-atct001* was found to be linked to resistant allele to ALS

This procedure detects nucleotide sequence polymorphisms in DNA by using a single pri‐ mer of arbitrary nucleotide sequences. In this reaction, a single species of primer anneals to the genomic DNA at two different sites on complementary strands of DNA template. If these priming sites are within an amplifiable range of each other, a discrete DNA product is formed through thermocyclic amplification. On an average, each primer directs amplifica‐ tion of several discrete loci in the genome, making the assay useful for efficient screening of

These markers have been used in a variety of ways in genetic analysis. They have been used in gene pyramiding especially where conventional procedures couldn't solve the problem when there is epistasis between resistance genes to be pyramided [40, 41]. Some of the RAPD markers have been used to pyramid three rust resistance alleles *Up2, Ur-3* and *B-190* [42] with other epistatic resistance alleles from plant introduction collection [40, 43]. Similar‐ ly, pyramiding was also suggested in reference [44] where the two genes linked to *I* and *bc-3* genes for resistance to bean common mosaic virus disease and bean common mosaic ne‐

These markers have also been used in the assessment of genetic diversity of common bean. In reference [45] reported the potential of using RAPD markers as compared to RFLP, DAMD-PCR, ISSR and AFLP for assessing diversity of common bean and in this finding it shows that these markers were able to produce higher percentage of polymorphism than the

In SCAR markers, the RAPD marker termini are sequenced and longer primers are designed (22–24 nucleotide bases long) for specific amplification of a particular locus [16]. The pres‐ ence or absence of the band indicates variation in sequence. These are better reproducible than RAPDs. SCARs are usually dominant markers, however, some of them can be convert‐ ed into codominant markers by digesting them with tetra cutting restriction enzymes and polymorphism can be deduced by using simple non denaturing gels to detect whether the products has different restriction sites for the different alleles. Compared to arbitrary pri‐

[16, 39] and some markers have been used in Marker assisted backcrossing [29].

**5.3. Random Amplified Polymorphic DNA (RAPD)**

126 Plant Breeding from Laboratories to Fields

nucleotide sequence polymorphism between individuals.

crosis virus disease respectively were incorporated in elite cultivar/line.

others used hence being very useful in detecting polymorphism.

**5.4. Sequence Characterized Amplified Region (SCAR) markers**

RFLP probes specifically linked to a desired trait can be converted into polymerase chain re‐ action (PCR)-based Sequence-Tagged Sites (STS) markers based on nucleotide sequence of the probe giving polymorphic band pattern, to obtain specific amplicon. Using this techni‐ que, tedious hybridization procedures involved in RFLP analysis can be overcome. This ap‐ proach is extremely useful for studying the relationship between various species. When these markers are linked to some specific traits, for example the powdery mildew or stem rust resistance genes in barley, they can be easily integrated into plant breeding pro‐ grammes for MAS of the trait of interest [47].

#### **5.6. Amplified Fragment Length Polymorphism (AFLP)**

The technique based on the detection of genomic restriction fragments by PCR amplification and can be used for DNAs of any origin or complexity. The fingerprints are produced, with‐ out any prior knowledge of sequence, using a limited set of generic primers. The number of fragments detected in a single reaction can be 'tuned' by selection of specific primer sets. AFLP technique is reliable since stringent reaction conditions are used for primer annealing. This technique thus shows an ingenious combination of RFLP and PCR techniques and is extremely useful in detection of polymorphism between closely related genotypes. Due to their characteristics, these markers are useful in assessing diversity of common bean and in case the marker is linked to a trait of importance in common bean then it can be useful for MAS in selecting or screening genotypes for that particular trait [48, 49]. For example, AFLP studies conducted to determine genetic relatedness of two near-isogenic Teebus lines and Teebus of common bean to CBB resistance [44, 50]. These markers despite being useful, their analysis is too difficult and troublesome, for this they can be converted to other types of markers like SCAR or STS which is also a difficult thing to achieve.
