on length and sequence of the probe, location of SNP in the probe, and hybridization conditions (Table 2.1).
Table 2.1 Customized SNP array details in plant species.
| Crop | Species | Array fsize | References | Trait |
|---|---|---|---|---|
| Pigeonpea | (Cajanus cajan) | 62K | Singh et al. (2020) | Genetic diversity |
| Wheat | (Triticum) | 55K | Zhang et al. (2019) | Adult‐plant resistance to leaf and stripe rust |
| Apple | (Malus domestica) | 480K | Bianco et al. (2016) | Phenology, fruit quality, disease resistance, or drought tolerance |
| Apple | (Malus domestica) | 8K | Chagné et al. (2012) | Quantitative traits |
| Rice | (Oryza sativa) | 50K | Singh et al. (2015) | Genetic diversity |
| Rice | (Oryza sativa) | 44 K | Zhao et al. (2011) | Plant morphology, grain quality, plant development |
| Rye | (Secale cereale) | 5 K | Li et al. (2011) | Diversity analysis |
| Barley | (Hordeum vulgare) | 50 K | Bayer et al. (2017) | Evaluation and use of barley genetic resources |
| Sweet Cherry | (Prunus avium) | 6K | Peace et al. (2012) | Fruit taste |
| Pear | (Prunus) | 70K | Montanari et al. (2019) | Genetic diversity studies |
| Potato | (Solanum tuberosum) | 22K | Khlestkin et al. (2019) | Starch phosphorylation |
| Pear | (Pyrus) | 200K | Li et al. (2019) | Flowering time and candidate genes linked to the size of fruit |
| Walnut | (Juglans regia) | 700K | Marrano et al. (2019) | Genetic diversity of germplasm |
| Cotton | (Gossypium barbadense) | 63K | Kumar et al. (2019) | Fiber quality |
2.2.2.1.4 Sequencing‐based Platforms
The simplest method of genotyping is whole‐genome resequencing of genotypes followed by the reference‐based assembly for variant calling. But as the majority of the genome is noncoding, these noncoding regions are also reflected in the variant file generated by the mapping of the reads which in turn increases the complexity of analysis as well as interpretation. Reduced representation approaches like RAD‐seq and GBS methods have solved this issue as in spite of sequencing the whole genome, these methods are confined to coding/genic regions only. For this, we first digest the DNA with methylation‐sensitive restriction enzyme with the hypothesis that DNA is more methylated in the noncoding region hence the digestion will be limited to coding regions only. The restricted ends generated are then ligated with adapters and sequenced. Other widely used sequencing‐based technologies are exome sequencing (Exom‐seq) and double digest RAD‐seq (ddRAD‐seq).
Restriction Site‐Associated DNA (RAD‐seq)
Restriction site‐associated DNA (RAD) is a next‐generation sequencing‐based SNP genotyping method, which allows simultaneous discovery and scoring of thousands of SNP markers in hundreds of individuals. The basic principle behind RAD is the identification of genomic SNPs adjacent to enzyme restriction sites through sequencing. It requires genomic DNA, restriction enzymes, P1 adaptor containing Illumina sequencing forward primer and barcode information, and P2 adaptor containing reverse complement of the reverse amplification primer site. The RAD sequencing library is prepared by digesting genomic DNA of genotypes using restriction enzymes and P1 adaptor is ligated to the compatible ends of the fragments. The adaptor‐ligated fragments are subsequently pooled, randomly sheared, and a specific size fraction is selected following electrophoresis. The DNA fragments are then ligated with P2 adapter and sequenced using Illumina platform (Figure 2.3). The obtained RAD data are analyzed using bioinformatics tools like stack, PyRAD, dDocent, GATK HaplotypeCaller, GATK unifieldGenotyper, samtools mpileup, etc.
One enzyme digestion followed by random sharing causes high DNA loss during RAD sequencing. For organisms without a reference genome, a significant portion of the RAD‐Seq data has been discarded due to sequence read errors and the presence of variable sites. To overcome this problem, slight modification in the RAD sequencing protocol has been carried out. Use of two restriction enzymes instead of a single restriction enzyme and removal of the random sharing steps is adopted. The modified method is renamed as double digest RAD (ddRAD).
Genotyping‐by‐Sequencing (GBS)
Genotyping‐by‐sequencing (GBS) is also a next‐generation sequencing‐based SNP genotyping method that uses restriction enzymes to reduce the genome complexity. GBS only targets as little as 2.3% of a genome. This reduced representation can be achieved by digestion of genomic DNA with methylation‐sensitive restriction enzymes (REs). Methylation‐sensitive REs do not cut repetitive DNA because of its highly methylated in nature and hence digested fragments generated from low‐copy genomic regions. This helps in avoiding the repetitive regions and targeting low copy regions of the genome (Elshire et al. 2011). In GBS, genomic DNA is extracted and digested with methylation‐sensitive restriction enzymes. Illumina sequencing forward and reverse adaptors ligated to both the end of fragments. The forward adapter is enriched with barcode sequences which is helpful for multiplexing of the samples. Now
