**2. Genetic markers in plant breeding: Conceptions, types and application**

Genetic markers are the biological features that are determined by allelic forms of genes or ge‐ netic loci and can be transmitted from one generation to another, and thus they can be used as experimental probes or tags to keep track of an individual, a tissue, a cell, a nucleus, a chromo‐ some or a gene. Genetic markers used in genetics and plant breeding can be classified into two

© 2013 Jiang; licensee InTech. This is an open access article distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. © 2013 Jiang; licensee InTech. This is a paper distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

categories: classical markers and DNA markers (Xu, 2010). Classical markers include morpho‐ logical markers, cytological markers and biochemical markers. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods (south‐ ern blotting – nuclear acid hybridization, PCR – polymerase chain reaction, and DNA sequenc‐ ing) (Collard et al., 2005), such as RFLP, AFLP, RAPD, SSR, SNP, etc.

#### **2.1. Classical markers**

*Morphological markers:* Use of markers as an assisting tool to select the plants with desired traits had started in breeding long time ago. During the early history of plant breeding, the markers used mainly included visible traits, such as leaf shape, flower color, pubescence col‐ or, pod color, seed color, seed shape, hilum color, awn type and length, fruit shape, rind (exocarp) color and stripe, flesh color, stem length, etc. These morphological markers gener‐ ally represent genetic polymorphisms which are easily identified and manipulated. There‐ fore, they are usually used in construction of linkage maps by classical two- and/or threepoint tests. Some of these markers are linked with other agronomic traits and thus can be used as indirect selection criteria in practical breeding. In the green revolution, selection of semi-dwarfism in rice and wheat was one of the critical factors that contributed to the suc‐ cess of high-yielding cultivars. This could be considered as an example for successful use of morphological markers to modern breeding. In wheat breeding, the dwarfism governed by gene *Rht10* was introgressed into Taigu nuclear male-sterile wheat by backcrossing, and a tight linkage was generated between *Rht10* and the male-sterility gene *Ta1*. Then the dwarf‐ ism was used as the marker for identification and selection of the male-sterile plants in breeding populations (Liu, 1991). This is particularly helpful for implementation of recur‐ rent selection in wheat. However, morphological markers available are limited, and many of these markers are not associated with important economic traits (e.g. yield and quality) and even have undesirable effects on the development and growth of plants.

*Cytological markers:* In cytology, the structural features of chromosomes can be shown by chromosome karyotype and bands. The banding patterns, displayed in color, width, order and position, reveal the difference in distributions of euchromatin and heterochromatin. For instance, Q bands are produced by quinacrine hydrochloride, G bands are produced by Giemsa stain, and R bands are the reversed G bands. These chromosome landmarks are used not only for characterization of normal chromosomes and detection of chromosome mutation, but also widely used in physical mapping and linkage group identification. The physical maps based on morphological and cytological markers lay a foundation for genetic linkage mapping with the aid of molecular techniques. However, direct use of cytological markers has been very limited in genetic mapping and plant breeding.

*Biochemical/protein markers:* Protein markers may also be categorized into molecular markers though the latter are more referred to DNA markers. Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution (Xu, 2010). Therefore, iso‐ zyme markers can be genetically mapped onto chromosomes and then used as genetic markers to map other genes. They are also used in seed purity test and occasionally in plant breeding. There are only a small number of isozymes in most crop species and some of them can be identified only with a specific strain. Therefore, the use of enzyme markers is limited.

Another example of biochemical markers used in plant breeding is high molecular weight glutenin subunit (HMW-GS) in wheat. Payne et al. (1987) discovered a correlation between the presence of certain HMW-GS and gluten strength, measured by the SDS-sedimentation volume test. On this basis, they designed a numeric scale to evaluate bread-making quality as a function of the described subunits (*Glu-1* quality score) (Payne et al., 1987; Rogers et al., 1989). Assuming the effect of the alleles to be additive, the Bread-making quality was pre‐ dicted by adding the scores of the alleles present in the particular line. It was established that the allelic variation at the *Glu-D1* locus have a greater influence on bread-making quali‐ ty than the variation at the others *Glu-1* loci. Subunit combination 5+10 for locus *Glu-D1* (*Glu-D1* 5+10) renders stronger dough than *Glu-D1* 2+12, largely due to the presence of an extra cysteine residue in the Dx-5 subunit compared to the Dx-2 subunit, which would pro‐ mote the formation of polymers with larger size distribution. Therefore, breeders may en‐ hance the bread-making quality in wheat by selecting subunit combination Glu-D1 5+10 instead of Glu-D1 2+12. Of course, the variation of bread-making quality among different varieties cannot be explained only by the variation in HMW-GS composition, because the low molecular weight glutinen subunit (LMW-GS) (as well as the gliadins in a smaller pro‐ portion) and their interactions with the HMW-GS also play an important role in the gluten strength and bread-making quality.

#### **2.2. DNA markers**

categories: classical markers and DNA markers (Xu, 2010). Classical markers include morpho‐ logical markers, cytological markers and biochemical markers. DNA markers have developed into many systems based on different polymorphism-detecting techniques or methods (south‐ ern blotting – nuclear acid hybridization, PCR – polymerase chain reaction, and DNA sequenc‐

*Morphological markers:* Use of markers as an assisting tool to select the plants with desired traits had started in breeding long time ago. During the early history of plant breeding, the markers used mainly included visible traits, such as leaf shape, flower color, pubescence col‐ or, pod color, seed color, seed shape, hilum color, awn type and length, fruit shape, rind (exocarp) color and stripe, flesh color, stem length, etc. These morphological markers gener‐ ally represent genetic polymorphisms which are easily identified and manipulated. There‐ fore, they are usually used in construction of linkage maps by classical two- and/or threepoint tests. Some of these markers are linked with other agronomic traits and thus can be used as indirect selection criteria in practical breeding. In the green revolution, selection of semi-dwarfism in rice and wheat was one of the critical factors that contributed to the suc‐ cess of high-yielding cultivars. This could be considered as an example for successful use of morphological markers to modern breeding. In wheat breeding, the dwarfism governed by gene *Rht10* was introgressed into Taigu nuclear male-sterile wheat by backcrossing, and a tight linkage was generated between *Rht10* and the male-sterility gene *Ta1*. Then the dwarf‐ ism was used as the marker for identification and selection of the male-sterile plants in breeding populations (Liu, 1991). This is particularly helpful for implementation of recur‐ rent selection in wheat. However, morphological markers available are limited, and many of these markers are not associated with important economic traits (e.g. yield and quality) and

ing) (Collard et al., 2005), such as RFLP, AFLP, RAPD, SSR, SNP, etc.

even have undesirable effects on the development and growth of plants.

markers has been very limited in genetic mapping and plant breeding.

*Cytological markers:* In cytology, the structural features of chromosomes can be shown by chromosome karyotype and bands. The banding patterns, displayed in color, width, order and position, reveal the difference in distributions of euchromatin and heterochromatin. For instance, Q bands are produced by quinacrine hydrochloride, G bands are produced by Giemsa stain, and R bands are the reversed G bands. These chromosome landmarks are used not only for characterization of normal chromosomes and detection of chromosome mutation, but also widely used in physical mapping and linkage group identification. The physical maps based on morphological and cytological markers lay a foundation for genetic linkage mapping with the aid of molecular techniques. However, direct use of cytological

*Biochemical/protein markers:* Protein markers may also be categorized into molecular markers though the latter are more referred to DNA markers. Isozymes are alternative forms or structural variants of an enzyme that have different molecular weights and electrophoretic mobility but have the same catalytic activity or function. Isozymes reflect the products of different alleles rather than different genes because the difference in electrophoretic mobility is caused by point mutation as a result of amino acid substitution (Xu, 2010). Therefore, iso‐

**2.1. Classical markers**

46 Plant Breeding from Laboratories to Fields

DNA markers are defined as a fragment of DNA revealing mutations/variations, which can be used to detect polymorphism between different genotypes or alleles of a gene for a par‐ ticular sequence of DNA in a population or gene pool. Such fragments are associated with a certain location within the genome and may be detected by means of certain molecular tech‐ nology. Simply speaking, DNA marker is a small region of DNA sequence showing poly‐ morphism (base deletion, insertion and substitution) between different individuals. There are two basic methods to detect the polymorphism: Southern blotting, a nuclear acid hybrid‐ ization technique (Southern 1975), and PCR, a polymerase chain reaction technique (Mullis, 1990). Using PCR and/or molecular hybridization followed by electrophoresis (e.g. PAGE – polyacrylamide gel electrophoresis, AGE – agarose gel electrophoresis, CE – capillary elec‐ trophoresis), the variation in DNA samples or polymorphism for a specific region of DNA sequence can be identified based on the product features, such as band size and mobility. In addition to Sothern blotting and PCR, more detection systems have been also developed. For instance, several new array chip techniques use DNA hybridization combined with la‐ beled nucleotides, and new sequencing techniques detect polymorphism by sequencing. DNA markers are also called molecular markers in many cases and play a major role in mo‐ lecular breeding. Therefore, molecular markers in this article are mainly referred to as DNA markers except specific definitions are given, although isozymes and protein markers are al‐ so molecular markers. Depending on application and species involved, ideal DNA markers for efficient use in marker-assisted breeding should meet the following criteria:


Since Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP) in human linkage mapping, substantial progress has been made in development and im‐ provement of molecular techniques that help to easily find markers of interest on a largescale, resulting in extensive and successful uses of DNA markers in human genetics, animal genetics and breeding, plant genetics and breeding, and germplasm characterization and management. Among the techniques that have been extensively used and are particularly promising for application to plant breeding, are the restriction fragment length polymor‐ phism (RFLP), amplified fragment length polymorphism (AFLP), random amplified poly‐ morphic DNA (RAPD), microsatellites or simple sequence repeat (SSR), and single nucleotide polymorphism (SNP). According to a causal similarity of SNPs with some of these marker systems and fundamental difference with several other marker systems, the molecular markers can also be classified into SNPs (due to sequence variation, e.g. RFLP) and non-SNPs (due to length variation, e.g. SSR) (Gupta et al., 2001). The marker techniques help in selection of multiple desired characters simultaneously using F2 and back-cross pop‐ ulations, near isogenic lines, doubled haploids and recombinant inbred lines. In view of page limitation, only five marker systems mentioned above are briefly addressed here ac‐ cording to published literatures. The details about the technical methods how to develop DNA markers and the procedures how to detect in practice have been described in the re‐ cently published reviews and books in this area (Farooq and Azam, 2002a, 2002b; Gupta et al., 2001; Semagn et al., 2006a; Xu, 2010).

*RFLP markers:* RFLP markers are the first generation of DNA markers and one of the impor‐ tant tools for plant genome mapping. They are a type of Southern-Boltting-based markers. In living organisms, mutation events (deletion and insertion) may occur at restriction sites or between adjacent restriction sites in the genome. Gain or loss of restriction sites resulting from base pair changes and insertions or deletions at restriction sites within the restriction fragments may cause differences in size of restriction fragments. These variations may cause alternation or elimination of the recognition sites for restriction enzymes. As a consequence, when homologous chromosomes are subjected to restriction enzyme digestion, different re‐ striction products are produced and can be detected by electrophoresis and DNA probing techniques.

so molecular markers. Depending on application and species involved, ideal DNA markers

**•** Co-dominance in expression (so that heterozygotes can be distinguished from homozy‐

**•** High availability (un-restricted use) and suitability to be duplicated/multiplexed (so that

Since Botstein et al. (1980) first used DNA restriction fragment length polymorphism (RFLP) in human linkage mapping, substantial progress has been made in development and im‐ provement of molecular techniques that help to easily find markers of interest on a largescale, resulting in extensive and successful uses of DNA markers in human genetics, animal genetics and breeding, plant genetics and breeding, and germplasm characterization and management. Among the techniques that have been extensively used and are particularly promising for application to plant breeding, are the restriction fragment length polymor‐ phism (RFLP), amplified fragment length polymorphism (AFLP), random amplified poly‐ morphic DNA (RAPD), microsatellites or simple sequence repeat (SSR), and single nucleotide polymorphism (SNP). According to a causal similarity of SNPs with some of these marker systems and fundamental difference with several other marker systems, the molecular markers can also be classified into SNPs (due to sequence variation, e.g. RFLP) and non-SNPs (due to length variation, e.g. SSR) (Gupta et al., 2001). The marker techniques help in selection of multiple desired characters simultaneously using F2 and back-cross pop‐ ulations, near isogenic lines, doubled haploids and recombinant inbred lines. In view of page limitation, only five marker systems mentioned above are briefly addressed here ac‐ cording to published literatures. The details about the technical methods how to develop DNA markers and the procedures how to detect in practice have been described in the re‐ cently published reviews and books in this area (Farooq and Azam, 2002a, 2002b; Gupta et

*RFLP markers:* RFLP markers are the first generation of DNA markers and one of the impor‐ tant tools for plant genome mapping. They are a type of Southern-Boltting-based markers. In living organisms, mutation events (deletion and insertion) may occur at restriction sites or between adjacent restriction sites in the genome. Gain or loss of restriction sites resulting

for efficient use in marker-assisted breeding should meet the following criteria:

**•** Even distribution across the whole genome (not clustered in certain regions)

**•** Clear distinct allelic features (so that the different alleles can be easily identified)

**•** Low cost to use (or cost-efficient marker development and genotyping)

the data can be accumulated and shared between laboratories)

**•** Genome-specific in nature (especially with polyploids)

**•** High level of polymorphism

48 Plant Breeding from Laboratories to Fields

**•** Single copy and no pleiotropic effect

**•** Easy assay/detection and automation

**•** No detrimental effect on phenotype

al., 2001; Semagn et al., 2006a; Xu, 2010).

gotes)

RFLP markers are powerful tools for comparative and synteny mapping. Most RFLP mark‐ ers are co-dominant and locus-specific. RFLP genotyping is highly reproducible, and the methodology is simple and no special equipment is required. By using an improved RFLP technique, i.e., cleaved amplified polymorphism sequence (CAPS), also known as PCR-RFLP, high-throughput markers can be developed from RFLP probe sequences. Very few CAPS are developed from probe sequences, which are complex to interpret. Most CAPS are developed from SNPs found in other sequences followed by PCR and detection of restric‐ tion sites. CAPS technique consists of digesting a PCR-amplified fragment and detecting the polymorphism by the presence/absence of restriction sites (Konieczny and Ausubel, 1993). Another advantage of RFLP is that the sequence used as a probe need not be known. All that a researcher needs is a genomic clone that can be used to detect the polymorphism. Very few RFLPs have been sequenced to determine what sequence variation is responsible for the polymorphism. However, it may be problematic to interpret complex RFLP allelic systems in the absence of sequence information. RFLP analysis requires large amounts of high-quality DNA, has low genotyping throughput, and is very difficult to automate. Radio‐ active autography involving in genotyping and physical maintenance of RFLP probes limit its use and share between laboratories. RFLP markers were predominantly used in 1980s and 1990s, but since last decade fewer direct uses of RFLP markers in genetic research and plant breeding have been reported. Most plant breeders would think that RFLP is too labori‐ ous and demands too much pure DNA to be important for plant breeding. It was and is, however, central for various types of scientific studies.

*RAPD markers:* RAPD is a PCR-based marker system. In this system, the total genomic DNA of an individual is amplified by PCR using a single, short (usually about ten nucleotides/ bases) and random primer. The primer which binds to many different loci is used to amplify random sequences from a complex DNA template that is complementary to it (maybe in‐ cluding a limited number of mismatches). Amplification can take place during the PCR, if two hybridization sites are similar to one another (at least 3000 bp) and in opposite direc‐ tions. The amplified fragments generated by PCR depend on the length and size of both the primer and the target genome. The PCR products (up to 3 kb) are separated by agarose gel electrophoresis and imaged by ethidium bromide (EB) staining. Polymorphisms resulted from mutations or rearrangements either at or between the primer-binding sites are visible in the electrophoresis as the presence or absence of a particular RAPD band.

RAPD predominantly provides dominant markers. This system yields high levels of poly‐ morphism and is simple and easy to be conducted. First, neither DNA probes nor sequence information is required for the design of specific primers. Second, the procedure does not involve blotting or hybridization steps, and thus it is a quick, simple and efficient technique. Third, relatively small amounts of DNA (about 10 ng per reaction) are required and the pro‐ cedure can be automated, and higher levels of polymorphism also can be detected com‐ pared with RFLP. Fourth, no marker development is required, and the primers are nonspecies specific and can be universal. Fifth, the RAPD products of interest can be cloned, sequenced and then converted into or used to develop other types of PCR-based markers, such as sequence characterized amplified region (SCAR), single nucleotide polymorphism (SNP), etc. However, RAPD also has some limitations/disadvantages, such as low reprodu‐ cibility and incapability to detect allelic differences in heterozygotes.

*AFLP markers:* AFLPs are PCR-based markers, simply RFLPs visualized by selective PCR amplification of DNA restriction fragments. Technically, AFLP is based on the selective PCR amplification of restriction fragments from a total double-digest of genomic DNA under high stringency conditions, i.e., the combination of polymorphism at restriction sites and hy‐ bridization of arbitrary primers. Because of this AFLP is also called selective restriction frag‐ ment amplification (SRFA). An AFLP primer (17-21 nucleotides in length) consists of a synthetic adaptor sequence, the restriction endonuclease recognition sequence and an arbi‐ trary, non-degenerate 'selective' sequence (1-3 nucleotides). The primers used in this techni‐ que are capable of annealing perfectly to their target sequences (the adapter and restriction sites) as well as a small number of nucleotides adjacent to the restriction sites. The first step in AFLP involves restriction digestion of genomic DNA (about 500 ng) with two restriction enzymes, a rare cutter (6-bp recognition site, *EcoR*I, *Pts*I or *Hind*III) and a frequent cutter (4 bp recognition site, *Mse*I or *Taq*I). The adaptors are then ligated to both ends of the frag‐ ments to provide known sequences for PCR amplification. The double-stranded oligonucleotide adaptors are designed in such a way that the initial restriction site is not re‐ stored after ligation. Therefore, only the fragments which have been cut by the frequent cut‐ ter and rare cutter will be amplified. This property of AFLP makes it very reliable, robust and immune to small variations in PCR amplification parameters (e.g., thermal cycles, tem‐ plate concentration), and it also can produce a high marker density. The AFLP products can be separated in high-resolution electrophoresis systems. The fragments in gel-based or capil‐ lary DNA sequencers can be detected by dye-labeling primers radioactively or fluorescently. The number of bands produced can be manipulated by the number of selective nucleotides and the nucleotide motifs used.

A typical AFLP fingerprint (restriction fragment patterns generated by the technique) con‐ tains 50-100 amplified fragments, of which up to 80% may serve as genetic markers. In gen‐ eral, AFLP assays can be conducted using relatively small DNA samples (1-100 ng per individual). AFLP has a very high multiplex ratio and genotyping throughput, and is rela‐ tively reproducible across laboratories. Another advantage is that it does not require se‐ quence information or probe collection prior to generating the fingerprints, and a set of primers can be used for different species. This is especially useful when DNA markers are rare. However, AFLP assays have some limitations also. For instance, polymorphic informa‐ tion content for bi-allelic markers is low (the maximum is 0.5). High quality DNA is re‐ quired for complete restriction enzyme digestion. AFLP markers usually cluster densely in centromeric regions in some species with large genomes (e.g., barley and sunflower). In ad‐ dition, marker development is complicated and not cost-efficient, especially for locus-specif‐ ic markers. The applications of AFLP markers include biodiversity studies, analysis of germplasm collections, genotyping of individuals, identification of closely linked DNA markers, construction of genetic DNA marker maps, construction of physical maps, gene mapping, and transcript profiling.

Third, relatively small amounts of DNA (about 10 ng per reaction) are required and the pro‐ cedure can be automated, and higher levels of polymorphism also can be detected com‐ pared with RFLP. Fourth, no marker development is required, and the primers are nonspecies specific and can be universal. Fifth, the RAPD products of interest can be cloned, sequenced and then converted into or used to develop other types of PCR-based markers, such as sequence characterized amplified region (SCAR), single nucleotide polymorphism (SNP), etc. However, RAPD also has some limitations/disadvantages, such as low reprodu‐

*AFLP markers:* AFLPs are PCR-based markers, simply RFLPs visualized by selective PCR amplification of DNA restriction fragments. Technically, AFLP is based on the selective PCR amplification of restriction fragments from a total double-digest of genomic DNA under high stringency conditions, i.e., the combination of polymorphism at restriction sites and hy‐ bridization of arbitrary primers. Because of this AFLP is also called selective restriction frag‐ ment amplification (SRFA). An AFLP primer (17-21 nucleotides in length) consists of a synthetic adaptor sequence, the restriction endonuclease recognition sequence and an arbi‐ trary, non-degenerate 'selective' sequence (1-3 nucleotides). The primers used in this techni‐ que are capable of annealing perfectly to their target sequences (the adapter and restriction sites) as well as a small number of nucleotides adjacent to the restriction sites. The first step in AFLP involves restriction digestion of genomic DNA (about 500 ng) with two restriction enzymes, a rare cutter (6-bp recognition site, *EcoR*I, *Pts*I or *Hind*III) and a frequent cutter (4 bp recognition site, *Mse*I or *Taq*I). The adaptors are then ligated to both ends of the frag‐ ments to provide known sequences for PCR amplification. The double-stranded oligonucleotide adaptors are designed in such a way that the initial restriction site is not re‐ stored after ligation. Therefore, only the fragments which have been cut by the frequent cut‐ ter and rare cutter will be amplified. This property of AFLP makes it very reliable, robust and immune to small variations in PCR amplification parameters (e.g., thermal cycles, tem‐ plate concentration), and it also can produce a high marker density. The AFLP products can be separated in high-resolution electrophoresis systems. The fragments in gel-based or capil‐ lary DNA sequencers can be detected by dye-labeling primers radioactively or fluorescently. The number of bands produced can be manipulated by the number of selective nucleotides

A typical AFLP fingerprint (restriction fragment patterns generated by the technique) con‐ tains 50-100 amplified fragments, of which up to 80% may serve as genetic markers. In gen‐ eral, AFLP assays can be conducted using relatively small DNA samples (1-100 ng per individual). AFLP has a very high multiplex ratio and genotyping throughput, and is rela‐ tively reproducible across laboratories. Another advantage is that it does not require se‐ quence information or probe collection prior to generating the fingerprints, and a set of primers can be used for different species. This is especially useful when DNA markers are rare. However, AFLP assays have some limitations also. For instance, polymorphic informa‐ tion content for bi-allelic markers is low (the maximum is 0.5). High quality DNA is re‐ quired for complete restriction enzyme digestion. AFLP markers usually cluster densely in centromeric regions in some species with large genomes (e.g., barley and sunflower). In ad‐

cibility and incapability to detect allelic differences in heterozygotes.

and the nucleotide motifs used.

50 Plant Breeding from Laboratories to Fields

*SSR markers:* SSRs, also called microsatellites, short tandem repeats (STRs) or sequence-tag‐ ged microsatellite sites (STMS), are PCR-based markers. They are randomly tandem repeats of short nucleotide motifs (2-6 bp/nucleotides long). Di-, tri- and tetra-nucleotide repeats, e.g. (GT)n, (AAT)n and (GATA)n, are widely distributed throughout the genomes of plants and animals. The copy number of these repeats varies among individuals and is a source of polymorphism in plants. Because the DNA sequences flanking microsatellite regions are usually conserved, primers specific for these regions are designed for use in the PCR reac‐ tion. One of the most important attributes of microsatellite loci is their high level of allelic variation, thus making them valuable genetic markers. The unique sequences bordering the SSR motifs provide templates for specific primers to amplify the SSR alleles via PCR. SSR loci are individually amplified by PCR using pairs of oligonucleotide primers specific to unique DNA sequences flanking the SSR sequence. The PCR-amplified products can be sep‐ arated in high-resolution electrophoresis systems (e.g. AGE and PAGE) and the bands can be visually recorded by fluorescent labeling or silver-staining.

SSR markers are characterized by their hyper-variability, reproducibility, co-dominant na‐ ture, locus-specificity, and random genome-wide distribution in most cases. The advantages of SSR markers include that they can be readily analyzed by PCR and easily detected by PAGE or AGE. SSR markers can be multiplexed, have high throughput genotyping and can be automated. SSR assays require only very small DNA samples (~100 ng per individual) and low start-up costs for manual assay methods. However, SSR technique requires nucleo‐ tide information for primer design, labor-intensive marker development process and high start-up costs for automated detections. Since the 1990s SSR markers have been extensively used in constructing genetic linkage maps, QTL mapping, marker-assisted selection and germplasm analysis in plants. In many species, plenty of breeder-friendly SSR markers have been developed and are available for breeders. For instance, there are over 35,000 SSR mark‐ ers developed and mapped onto all 20 linkage groups in soybean, and this information is available for the public (Song et al., 2010).

*SNP markers:* An SNP is a single nucleotide base difference between two DNA sequences or individuals. SNPs can be categorized according to nucleotide substitutions either as transi‐ tions (C/T or G/A) or transversions (C/G, A/T, C/A or T/G). In practice, single base variants in cDNA (mRNA) are considered to be SNPs as are single base insertions and deletions (in‐ dels) in the genome. SNPs provide the ultimate/simplest form of molecular markers as a sin‐ gle nucleotide base is the smallest unit of inheritance, and thus they can provide maximum markers. SNPs occur very commonly in animals and plants. Typically, SNP frequencies are in a range of one SNP every 100-300 bp in plants (Edwards et al., 2007; Xu, 2010). SNPs may present within coding sequences of genes, non-coding regions of genes or in the intergenic regions between genes at different frequencies in different chromosome regions.

Based on various methods of allelic discrimination and detection platforms, many SNP geno‐ typing methods have been developed. A convenient method for detecting SNPs is RFLP (SNP-RFLP) or by using the CAPS marker technique. If one allele contains a recognition site for a restriction enzyme while the other does not, digestion of the two alleles will produce different fragments in length. A simple procedure is to analyze the sequence data stored in the major da‐ tabases and identify SNPs. Four alleles can be identified when the complete base sequence of a segment of DNA is considered and these are represented by A, T, G and C at each SNP locus in that segment. There are several SNP genotyping assays, such as allele-specific hybridization, primer extension, oligonucleotide ligation and invasive cleavage based on the molecular mechanisms (Sobrino et al., 2005), and different detection methods to analyze the products of each type of allelic discrimination reaction, such as gel electrophoresis, mass spectrophotome‐ try, chromatography, fluorescence polarization, arrays or chips, etc. At the present, SNPs are also widely detected by sequencing. Detailed procedures are described in the review by Gup‐ ta at el. (2001) and the book Molecular Plant Breeding by Xu (2010).

SNPs are co-dominant markers, often linked to genes and present in the simplest/ultimate form for polymorphism, and thus they have become very attractive and potential genetic markers in genetic study and breeding. Moreover, SNPs can be very easily automated and quickly detected, with a high efficiency for detection of polymorphism. Therefore, it can be expected that SNPs will be increasingly used for various purposes, particularly as whole DNA sequences become available for more and more species (e.g., rice, soybean, maize, etc.). However, high costs for start-up or marker development, high-quality DNA required and high technical/equipment demands limit, to some extent, the application of SNPs in some laboratories and practical breeding programs.

The features of the widely used DNA markers discussed above are compared in Table 1. The advantages or disadvantages of a marker system are relevant largely to the purposes of re‐ search, available genetic resources or databases, equipment and facilities, funding and per‐ sonnel resources, etc. The choice and use of DNA markers in research and breeding is still a challenge for plant breeders. A number of factors need to be considered when a breeder chooses one or more molecular marker types (Semagn et al., 2006a). A breeder should make an appropriate choice that best meets the requirements according to the conditions and re‐ sources available for the breeding program.



Based on various methods of allelic discrimination and detection platforms, many SNP geno‐ typing methods have been developed. A convenient method for detecting SNPs is RFLP (SNP-RFLP) or by using the CAPS marker technique. If one allele contains a recognition site for a restriction enzyme while the other does not, digestion of the two alleles will produce different fragments in length. A simple procedure is to analyze the sequence data stored in the major da‐ tabases and identify SNPs. Four alleles can be identified when the complete base sequence of a segment of DNA is considered and these are represented by A, T, G and C at each SNP locus in that segment. There are several SNP genotyping assays, such as allele-specific hybridization, primer extension, oligonucleotide ligation and invasive cleavage based on the molecular mechanisms (Sobrino et al., 2005), and different detection methods to analyze the products of each type of allelic discrimination reaction, such as gel electrophoresis, mass spectrophotome‐ try, chromatography, fluorescence polarization, arrays or chips, etc. At the present, SNPs are also widely detected by sequencing. Detailed procedures are described in the review by Gup‐

SNPs are co-dominant markers, often linked to genes and present in the simplest/ultimate form for polymorphism, and thus they have become very attractive and potential genetic markers in genetic study and breeding. Moreover, SNPs can be very easily automated and quickly detected, with a high efficiency for detection of polymorphism. Therefore, it can be expected that SNPs will be increasingly used for various purposes, particularly as whole DNA sequences become available for more and more species (e.g., rice, soybean, maize, etc.). However, high costs for start-up or marker development, high-quality DNA required and high technical/equipment demands limit, to some extent, the application of SNPs in

The features of the widely used DNA markers discussed above are compared in Table 1. The advantages or disadvantages of a marker system are relevant largely to the purposes of re‐ search, available genetic resources or databases, equipment and facilities, funding and per‐ sonnel resources, etc. The choice and use of DNA markers in research and breeding is still a challenge for plant breeders. A number of factors need to be considered when a breeder chooses one or more molecular marker types (Semagn et al., 2006a). A breeder should make an appropriate choice that best meets the requirements according to the conditions and re‐

**RFLP RAPD AFLP SSR SNP**

dominant

high

Whole genome Whole genome Whole genome Whole genome

Very high

Co-dominant Co-dominant

Genomic abundance High High High Moderate to

Expression/inheritance Co-dominant Dominant Dominant / co-

ta at el. (2001) and the book Molecular Plant Breeding by Xu (2010).

some laboratories and practical breeding programs.

52 Plant Breeding from Laboratories to Fields

sources available for the breeding program.

region

Genomic coverage Low copy coding

**Feature and description**

> **Table 1.** Comparison of most widely used DNA marker systems in plants; Adapted from Collard et al. (2005), Semagn et al. (2006a), Xu (2010), and others.
