Genotyping by Sequencing for Crop Improvement. Группа авторов
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the developmental stage of the plant.
DNA markers show genetic differences that can be visualized by using a gel electrophoresis technique and staining ethidium bromide or hybridization with radioactive or colorimetric probes. Markers that can identify the difference between two individuals are referred to as polymorphic markers, whereas those that do not distinguish the individuals are called monomorphic markers. Based on how polymorphic markers can discriminate between individuals, they are described as codominant or dominant. Codominant markers indicate differences in size whereas dominant markers reveal differences based on their presence or absence. The different forms of a DNA marker in the form of band size on gels are known as marker “alleles.” Dominant marker has only two alleles whereas codominant markers may have many alleles.
1.3 Classes of Molecular Markers
Based on the method of their detection, DNA markers are broadly classified into three groups: (i) hybridization‐based, (ii) PCR‐based, and (iii) DNA sequence‐based molecular markers. Molecular markers have been discussed earlier in several reviews (Collard et al. 2005; Semagn et al. 2006; Gupta and Rustgi 2004) and book chapters (Mir et al. 2013; Singh and Singh 2015), which readers can also consult for more details. However, a brief description of each of these markers has been presented below.
1.3.1 Hybridization‐based Markers
1.3.1.1 Restriction Fragment Length Polymorphism (RFLP)
These are the first molecular markers used by Grodzicker et al. (1975) in adenovirus and Botstein et al. (1980) in human genome mapping. These were first used in plants by Helentjaris et al. (1986). In this type of marker, polymorphism is detected by cutting DNA into fragments by the use of restriction enzymes followed by hybridization of radioactively labeled DNA probes which are single or low copy DNA fragments and visualized by autoradiography. DNA probes could be genomic clones, cDNA clones, or even cloned genes. The RFLP markers show co‐dominance and are highly reliable in linkage analysis and breeding (Semagn et al. 2006). However, this technique requires a large quantity of DNA, labor‐intensive, relatively expensive, and hazardous. RFLP shows polymorphism in two different species if they differ due to point mutations, insertion/deletion, inversion, translocation, and duplication.
1.3.1.2 Diversity Array Technology (DArT™)
This is a high‐throughput DNA polymorphism analysis method which combines microarray and restriction‐based PCR methods. It is similar to AFLP where hybridization is used for the detection of polymorphism. It can able to provide a comprehensive genome coverage even in those organisms not having genome sequence information (Jaccoud et al. 2001). Diversity array technology (DArT) is a solid‐state open platform method for analyzing DNA polymorphism. DArT procedure includes (i) Generating a diversity panel and (ii) Genotyping using a diversity panel. The diversity panel is generated using a set of lines representing the breadth of variability in germplasm (~10 lines). An equal quantity of DNA from each representative line is pooled followed by restriction with two to three restriction endonucleases (REs) and ligation of RE‐specific adaptors. Later DNA fragments are amplified using adaptor complementary primers. The representation fragments are ligated to vector and transformed into Escherichia coli cells. The transformed cells with recombinant DNA are selected and amplified using M13 forward and reverse primer. The amplified DNA is isolated and purified. The purified DNA is coated onto polylysine‐coated glass slides to generate a diversity array.
For genotyping, the representation fragments of the target genotypes are prepared in the same as in the diversity panel. The DNA fragments are column purified and fluorescently labeled with two different dyes (Cy3 or Cy5). The labeled DNA fragments are used for hybridization onto the diversity array. Two representative panels – one labeled with Cy3 and another with Cy5 – can be hybridized simultaneously and hybridization signal intensities are measured for each spot. DArT, thus detects DNA polymorphism at several hundred genomic loci in a single array without relying on sequence information.
1.3.2 Polymerase Chain Reaction (PCR)‐based Markers
1.3.2.1 Simple‐Sequence Repeats (SSRs)
Simple‐sequence repeats (SSRs) (Litt and Luty 1989) are also known as microsatellites or short tandem repeats (STRs) or simple sequence length polymorphism (SSLP). These are widely used markers and are also referred to as the mother of all the markers. These are STRs, generally of one to eight nucleotide length. These are found dispersed throughout the genome and are hypervariable. These repeat regions are flanked with unique sequences that are highly conserved. The flanking unique sequences are used to design complementary primers which can be assayed with PCR. SSRs are highly polymorphic and codominant markers. These show polymorphism as a result of the variable number of repeat units. Before the era of genome sequencing, it was difficult to develop SSRs due to the extensive cost and labor involved in the identification of repeat regions and flanking unique sequences. However, with the availability of genome sequences of several organisms, the development of SSR has become very easy which involves in silico identification of STRs, designing of SSR from flanking unique sequences, and validation through experimentation. SSR markers have shown immense application in population genetic analysis, gene mapping, and cloning due to their abundance in the genome and high polymorphism, and very high reproducibility across labs. SSR‐based linkage maps have been developed in several important crop plants such as rice (Temnykh et al. 2000; McCouch et al. 2002; Orjuela et al. 2010), wheat (Roder et al. 1998), maize (Sharopova et al. 2002), potato (Milbourne et al. 1998), etc.
1.3.2.2 Sequence‐Tagged Sites (STSs)
Sequence‐tagged sites (STSs) were first developed for physical mapping of the human genome by Olsen et al. (1989). STS is the short unique sequences developed from polymorphic RFLP probe or AFLP fragment which is linked to desirable traits. The RFLP probes or AFLP fragments showing polymorphism are end‐sequenced and primers are designed to specifically amplify these fragments. STS markers are co‐dominant and highly reproducible. For example, STS markers have been developed for RFLP markers linked with bacterial blight resistance genes xa5, xa13, and Xa21 (Huang et al. 1997). One major limitation of these types of markers is the reduced polymorphism than the corresponding RFLP probe.
1.3.2.3 Randomly Amplified Polymorphic DNAs (RAPDs)
Williams et al. (1990) first developed these markers to amplify DNA without prior sequence information. In this type of marker, the arbitrary decamer sequences are used as primers at low annealing temperatures for DNA amplification. These markers are referred to as dominant markers because the polymorphism is determined based on the presence or absence of a particular amplified fragment. Polymorphism may also be due to varying brightness of bands at a particular locus due to copy number differences. These markers have been used for constructing linkage maps in several species (Hunt 1997; Laucou et al. 1998) and also for tagging genes of economic importance. However, due to the dominant nature, these may not be appropriate for genetic mapping and marker‐assisted selection (MAS). One major limitation of these markers is the lack of repeatability in certain cases. Variations of RAPD include AP‐PCR (arbitrarily primed PCR) and DAF (DNA amplification fingerprinting (Table 1.1).
Table 1.1 Details of the other important molecular markers.
| Marker | Description |
|---|---|
| Variable number tandem repeat (VNTR) or minisatellites | A short DNA sequence (10–100 bp) is present as tandem repeats and is a highly variable copy number |
| DNA amplification fingerprinting (DAF) | A variation of RAPD, where 4–5 bp single and arbitrary primer is used to detect polymorphism |