Genotyping by Sequencing for Crop Improvement. Группа авторов

A variation of RAPD, where 18–32 bp long single and arbitrary primer is used to detect polymorphism Inter‐simple sequence repeat (ISSR) Primers are designed based on the repeat region of microsatellites. These primers are used to amplify the region between two microsatellites. The stretches of unique DNA in between or flanking the SSRs are amplified. A single SSR‐based primer is used to prime PCR Selective amplification of microsatellite polymorphic loci (SAMPL) A modification of ISSR, where SSR‐based primer is used along with AFLP primer. The template is identical to the AFLP template and the rare cutter primer is replaced by SSR‐based primer Cleaved amplified polymorphic sequences (CAPS) These markers are also called PCR‐RFLP, where amplified PCR product is digested with endonucleases to reveal polymorphism. These are used when PCR product does not show polymorphism and restriction enzyme site present in amplified PCR product may detect polymorphism Derived cleaved amplified polymorphic sequences (dCAPS) A variation of CAPS, where a primer containing one or more mismatches to template DNA is used to create a restriction enzyme recognition site in one allele but not in another due to the presence of SNP. Thus, obtained PCR product is subjected to restriction enzyme digestion to find the presence or absence of the SNP Single‐strand conformational polymorphism (SSCP) DNA fragments of size ranging from 200 to 800 bp were amplified by PCR using specific primers (20–25 bp), followed by gel‐electrophoresis of single‐strand DNA to detect nucleotide sequence variation. The method is based on a principle that the secondary structure of single‐strand DNA molecule changes significantly if it harbors mutation. This method detects nucleotide variation without sequencing a DNA sample Denaturing/temperature gradient gel electrophoresis (DGGE, TGGE) These methods reveal polymorphism due to differential movement of the same genomic double‐stranded region with different base‐pair composition. As an example, the AT‐rich region would have a lower melting temperature than the GC‐rich region Target region amplification polymorphism (TRAP) This method employs primers designed from the EST database for detecting polymorphism around a selected candidate gene. This includes two primers of 18 bp, one of which is designed from targeted EST and the other is an arbitrary primer

      1.3.2.4 Sequence Characterized Amplified Regions (SCARs)

      These markers overcome the limitation of RAPDs. In this case, the RAPD fragments that are linked to a gene of interest are cloned and sequenced. Based on the terminal sequences, longer primers (20 mer) are designed. These SCAR primers more specifically amplify a particular locus. These are similar to STS markers in design and application. The presence or absence of the band indicates variation in sequences. The SCAR markers thus are dominant in nature. These, however, can be converted to codominant markers in certain cases by digesting the amplified fragment with tetranucleotide recognizing restriction enzymes. There are several examples where the RAPD markers linked to the gene of importance have been converted to SCAR markers (Joshi et al. 1999; Liu et al. 1999; Kasai et al. 2000; Akkurt et al. 2007; Chao et al. 2018).

      1.3.2.5 Amplified Fragment Length Polymorphism (AFLP)

      This marker technique was developed by Vos et al. (1995) and is patented by Keygene (www.keygene.com). In this technique, DNA is cut into fragments by a combination of restriction enzymes which are frequent (four bases) and rare (six bases) cutters that generate restriction overhangs on both sides of fragments. This is followed by the annealing of double‐stranded oligonucleotide adapters of a few oligonucleotide bases with respective restriction overhangs. The oligonucleotide adapters are designed in such a way that the original restriction sites are not reinstated and also provide the PCR amplification sites. The fragments are PCR amplified and visualized on agarose gel. This method produces many restriction fragments enabling the polymorphism detection. The number of amplified DNA fragments can be controlled by selecting different number or composition of bases in the adapters. The stringent reaction conditions used for primer annealing make this technique more reliable. This method is a combination of both RFLP and PCR techniques and is extremely useful in the detection of polymorphism between closely related genotypes. Like RAPD, AFLP is a dominant marker and is not preferred for genetic mapping studies and MAS. AFLP maps have been constructed in several species and integrated into already existing RFLP maps e.g. tomato (Haanstra et al. 1999), rice (Cho et al. 1997), and wheat (Lotti et al. 2000).

      1.3.2.6 Expressed Sequence Tags (ESTs)

      These markers are developed by end sequencing (generally 200–300 bp) of random cDNA clones. The sequence thus obtained is referred to as expressed sequence tags (ESTs). A large number of ESTs have been synthesized in several crop plants and are available in the EST database at NCBI (https://www.ncbi.nlm. nih.gov/dbEST/). These markers were originally developed to identify gene transcripts and have played important role in the identification of several genes and the development of markers such as RFLP, SSR, SNPs, CAPS, etc. (Semagn et al. 2006). However, EST‐based SSRs show less polymorphism as compared to genomic DNA‐based SSRs. Since EST markers are from expressed sequence regions, these are highly conserved among the species and can be used for synteny mapping. Most of these could also be functional genes. A large number of EST markers have been used in rice for developing a high‐density linkage map (Harushima et al. 1998) and for chromosome bin mapping in wheat using deletion stocks (Qi et al. 2003). In addition to these, several other molecular marker variants have been developed. The description of those markers is presented in Table 1.1.

1.4 Sequencing‐based Markers

      1.4.1 Single‐Nucleotide Polymorphisms (SNPs)

      Single‐nucleotide polymorphisms (SNPs) are more abundant resulted from single‐base pair variations. These are evenly distributed in a whole genome that can tag almost any gene or locus of a genome (Brookes 1999). However, the distribution of SNPs varies among species with 1 SNP per 60–120 bp in maize (Ching et al. 2002) and 1 SNP per 1000 bp in humans (Sachidanandam et al. 2001). SNPs are more prevalent in the noncoding region. In the coding region, SNPs could be synonymous or nonsynonymous. In synonymous SNPs, there is no change in the amino acid resulting in no phenotypic differences. However, phenotypic differences could be produced due to modified mRNA splicing (Richard and Beckman 1995). In nonsynonymous SNPs, change in amino acid results in phenotypic differences. SNPs are mostly bi‐allelic and cause polymorphism due to nucleotide base substitution. The two types of nucleotide base substitutions result in SNPs. A transition substitution occurs between purines (A, G) or between pyrimidines (C, T). This type of substitution constitutes two‐thirds of all SNPs. A transversion substitution occurs between a purine and pyrimidine. SNPs can be detected by the alignment of the similar genomic region of two different species. The SNPs have only two alleles compared to typical multiallele SSLP; however, this disadvantage can be compensated by using the high density of SNPs.

      1.4.2 Identification of SNP in a Pregenomic Era

      Initially, identification of SNP markers was laborious and expensive and involved allele‐specific sequencing (Ganal et al. 2009). This includes sequencing of unigene‐derived amplicons using Sanger’s method from two or more than two lines. In an experiment, about 350 bp of the RFLP clone, A‐519