**4.5. KASPar (KBioscience, UK)**

germplasm with respect to fruit shape and color, and observed polymorphic information

It includes whole genome amplification followed by hybridization to oligonucleotide probe attachedtoabead,extension,anddetectionoffluorescencebyiScanReader.Theassayconsiders upto fourmillionSNPs ina single sample run, or evenupto severalhundredthousandmultiple samples in the same array. The chemistry involves incubation of samples on bead chip where they anneal to locus-specific 50-mers covalently linked to beads followed by allele-specific single-base extension, fluorescent staining, signal amplification, scanning in a dual-color channel reader, and analysis. This technology is advantageous as one can use a premade array that is easily available commercially for selected species. Hamilton et al. [26] identified 69,011 high confidence SNPs from six potato cultivars and used for genotyping with the Infinium platform. A total of 96 ofthese SNPs were used to assess allelic diversity in 248 germplasms and found 82 informative SNPs for subsequent analyses. In 2012, Felcher et al. [166] reported "Infinium 8303 Potato Array" comprising of 8303 functional markers which includes 3018 from candidate genes of interest by utilizing the transcriptome data from Hamilton et al. [26]. These were used for the genotyping and development of linkage maps. In tomato, a large-scale SNP genotyping array using 8784 SNPs were obtained from transcriptome sequencing [30] and later

This method involves a single-base extension assay and tag array technology. It starts with a multiplexed SNP-specific PCR followed by a primer extension reaction using tagged primers and fluorescent-labeled nucleotide terminators, i.e., ddNTPs. The products are captured on a tag array, which is then scanned to detect the hybridized extension primers and produce calls. It allows the processing of up to three million genotypes in 384 samples at a time. This genotyping system combines solid-phase primer extension assay and universal tags for SNP genotyping. The instrument allows processing of 4,600–3,000,000 genotypes per day [167].

The GeneChip assays are based on allelic discrimination by the direct hybridization of genomic DNA to arrays containing locus- and allele-specific oligonucleotides (25 mers). Genomic DNA is digested with a restriction endonuclease and ligated to adaptors, which are then amplified by PCR using a single universal primer thereby creating a reduced representation of the genome [168]. These PCR amplicons are fragmented, end-labeled, and hybridized. The fluorescence signal is recorded by the GeneChip 3000 scanner (Affymetrix). The hybridization scanning is evaluated as positive and negative signals. Hill et al. [42] developed a GeneChip® array for analysis of polymorphism and expression in Capsicum. The array was designed from 30,815 unigenes, and hybridization was performed using genomic DNA of 40 diverse lines of *C. annum*. They detected 33,401 single-position polymorphisms within 13,323 unigenes. A total of 251 highly informative markers across these *C. annuum* lines were found. Also, a region of 8.7 cM was detected around Pun1 locus in nonpungent line that showed no polymorphism.

content values ranged from 0.29 to 0.5 with a mean value of 0.43.

used for construction of a high-density linkage map of tomato [70].

**4.3. SNPStream (Beckman coulter)**

**4.4. GeneChip (Affymetrix, USA)**

**4.2. BeadChip-based Infinium assay (Illumina)**

268 Next Generation Sequencing - Advances, Applications and Challenges

The KBioscience-competitive allele-specific PCR (KASPar) is a simple, cost-effective, and flexible way for determining both SNP and InDel in genotypes. It is a custom-based technology that covers 96-1536‐well plate formats like Illumina's GGGT. It relies on the discrimination power of a novel form of competitive allele-specific PCR to determine the alleles at a specific locus. The improvement has been made by incorporating a 5′–3′ exonuclease cleaved *Taq* DNA polymerase (the engineered *Taq* increases its discrimination power) and a homogeneous fluorescence resonance energy transfer (FRET) detection system, which makes this technology more competent among the genotyping platforms. From the pepper transcriptome sequence data, Ashrafi et al. [41] identified a large number of SNPs. A subset of them was validated by KASPar assay and identified 78 polymorphic SNPs.
