Genotyping by Sequencing for Crop Improvement. Группа авторов

including allele‐specific hybridization, (ii) enzymatic reactions including primer extension, and mini‐sequencing (Ding and Jin 2009). For making SNP arrays, the first step is the identification of genome‐wide SNPs by sequencing (preferably WGR) of a large diverse panel. The SNPs arrays may include SNPs from coding (genic) regions only and/or genome‐wide SNPs from other noncoding regions. SNPs are in silico validated with several custom tools and final filtered SNPs are identified. The oligonucleotide probes containing SNP alleles are designed and bound on a solid glass plate surface. SNP chips can be custom designed commercially from two widely used platforms: Affymetrics (www.affymetrics.com) as Axiom Affymetrics SNP Chips (Affymetrix/Thermo Fisher Axiom^®) or Illumina (https://www.illumina.com/science/technology/microarray.html) as Immunia Infinium assay (Illumina Infinium^®). Affymetrics SNP array relies on differential hybridization due to different melting temperatures for matched and mismatched SNPs binding to target DNA sequence. On the other hand, Illumina Infinium assay uses Illumina BeadArray technology that relies on primer extension to distinguish two SNP alleles. The Affymetrix SNP array uses 25‐mer for SNP calling while the Illumina BeadArray uses 50‐mer for target capture. In rice, a high‐resolution 44K Affymetrix array, 50K Infinium array, and 700K high‐density rice array are available for rice SNP genotyping (McCouch et al. 2010; Tung et al. 2010; Chen et al. 2013; McCouch et al. 2015). Additionally, high‐density SNP arrays have been developed for other crop plants such as maize (Ganal et al. 2011) and sunflower (Bachlava et al. 2012) as well as domestic animal species, including cattle (Gibbs et al. 2009; Matukumalli et al. 2009) and pig (Ramos et al. 2009). One major advantage of SNP arrays is the reproducibility of data points where GBS does have some shortcomings. However, the disadvantage is the less polymorphism as compared to GBS and WGR and detection of only alleles present in the array (Table 1.2).

Table 1.2 Comparison between different marker techniques commonly used in plant research.

	SSR	GBS	WGR	SNP array	KASP™
DNA quality	Moderate	High	High	High	High
PCR‐based	Yes	Yes	No	No	No
Allele detection	High	High	High	Low	Low
Polymorphism	High	High	High	Low	Low
Ease to use	Easy	Not easy	Not easy	Easy	Easy
Reproducibility	High	Low	High	High	High
Cost	Moderate	Low to moderate	High	High	moderate
Cost for analysis	High	High	High	Low	Low
Suitability for different approaches
Genetic diversity analysis	High	Moderate	High (cost concerns)	High	High
Bi‐parental QTL mapping	High	High	High	High	High
Genome wide association analysis	Moderate	High	High	High	Low
Genomic selection	Low	Moderate	High (cost concerns)	High	Low

      1.5.4 Kompetitive Allele‐Specific PCR (KASP^™)

KASP^™ is a trademark technology of KBiosciences (http://www.kbioscience.co.uk/) or LGC genomics (http://www.lgcgenomics.com) initially developed for in‐house genotyping, thereafter evolving as a benchmark technology for SNP genotyping. Any candidate SNP identified through GBS or WGR and associated with any important traits can be validated through KASP assay. Furthermore, any identified candidate SNP associated with a trait of interest can be readily converted into KASP assay to serve as a robust and cost‐effective marker to be used as a MAS tool for crop improvement. It works on the principle of competitive allele‐specific PCR permitting bi‐allelic scoring of SNP, insertion, and deletions (InDels) at a specific location in the genome (Figure 1.2). KASP genotyping reaction consists of DNA sample, KASP assay mix, and universal KASP master mix. Allele‐specific two forward primers and common reverse primer all unlabelled constitute KASP assay mix. Allele‐specific primers have a unique tail sequence complementary to FRET (fluorescence resonant energy transfer) cassette. Each allele harbors a tail sequence linked to different dyes (FAM™ and HEX™ dyes). KASP master mix has FRET cassettes in the quenched state, a passive reference dye (ROX™), and other components for PCR reaction. During the first round of reaction allele‐specific primer binds to template incorporating tail sequence in newly synthesized strands. In the next round of PCR, a complementary strand of the allele‐specific tail sequence is generated allowing the FRET cassette to bind enabling an unquenched state, and generating a fluorescent allele‐specific signal. In the case of a homozygous DNA sample, only one signal specific to the allele is seen and mixed signal is generated in the case of the heterozygous individual. It can be carried out in 96‐ to 1536‐well plate format. Application of KASP includes QC (quality control) analysis, QTL mapping, allele mining (Semagn et al. 2013), and MAS. However, it may not be a suitable platform for genome‐wide association mapping and genomic selection due to fewer data points. KASP markers have been utilized extensively for MAS in major crops like rice (Yang et al. 2019; Kang et al. 2019), wheat (Makhoul et al.