Kompetitive Allele Specific PCR ( KASP ) : A Singleplex Genotyping Platform and Its Application

Single nucleotide polymorphism (SNP) can be detected by mining sequence databases or by using different singleplex or multiplex SNP genotyping platforms. Development of high-throughput genotyping molecular markers can be instrumental towards maximizing genetic gain. In this review we provide an overview of Kompetitive Allele Specific PCR (KASP) genotyping platform requirements and its application that might be helpful in KASP marker development. This literature further illustrates the possibilities to design KASP primers. Several research institutes routinely using KASP platform, producing in excess of humungous data points yearly for breeding cultivars and as well as for medical and commercial purposes. KASP genotyping technology offers cost effectiveness and high throughput molecular marker development platform. Conventional molecular markers can be converted into more robust and high throughput KASP markers. More than 2000 published references clearly show the popularity of KASP technology among the researchers.


Introduction
Development of molecular markers that predict the trait of interest with highest level of accuracy offers efficient tracking of superior genetic variations through breeding process bypassing phenotypic evaluation requirements.Most of the molecular markers developed for marker assisted selection (MAS) are actually diagnostic markers which forecast phenotype with varying degree of accuracy.Hence, the availability of more robust and reliable molecular markers have become the matter of need than merely a research theme.
In recent times, single nucleotide polymorphisms (SNPs) have been widely used as markers in MAS.SNPs have largely replaced simple sequence repeats (SSRs) in crop species that have been comprehensively sequenced, such as maize and they are expected to substitute other kinds of molecular markers in most of the species in the forthcoming times given the large scale use of next generation sequencing technologies for genotyping (Semagn et al., 2013).SNPs are abundant in genomes and they are amenable for high throughput detection.Beside this, low cost assay, locus specificity, simple documentation, co-dominant inheritance and relatively low genotyping error rates (Schlotterer, 2004), SNPs have emerged as powerful tools for genomic selection.In past, simple sequence repeat (SSR) markers facilitated the development of high density maps for rice (Mccouch et al., 2002), barley (Varshney et al., 2007), wheat (Somers et al., 2004), maize (Smith et al., 1997), and other crops (Varshney et al., 2005).At present SNPs are considered more informative as compared to SSRs (Xu & Crouch, 2008) because it is difficult to compare SSR data from different populations or platforms.Moreover, SSR motifs are  (Zhao et al., 2017) also reported the successful conversion of conventional CAPS marker into KASP assay to detect AhFAD2 mutation on peanut.Successful conversion of sequence-tagged site (STS) gel based marker for Lr21 into KASP assay has also been reported with high throughput, accurate, inexpensive and less laborious (Neelam et al., 2013).In prairie cordgrass (Spartine pectinata) validation of DNA polymorphism has also been documented by using KASP assay.Among 121 SNPs, the assay success rate was 78.5% with 26 assays failing development (Graves et al., 2016).From pigeonpea, successful conversion of 1616 (88.4%) out of 1827 SNPs into KASP assays has also been documented (Saxena et al., 2012).James Cockram and coworkers (Cockram et al., 2012) also reported 83% success conversion rate.KASP genotyping system used by Kusza and co-workers on 48 polymorphisms at different caprine loci and French Alpine and Saanen goat breeds found 94.81% assay success rate and found a high locus specific and accurate genotyping solution (Kusza et al., 2018).

KASP Requirements
Quality and quantity of DNA are the two major factors determining the KASP assay success.DNA extracted from leaf and/or seed can be used.The amount of DNA required differs according to genome size; more amount of DNA is needed for species having large genome size to ensure enough copies of the genome exist at the initiation of KASP.Concentration of the extracted DNA should be taken into account on 0.8-1.0%agarose gel electrophoresis and/or spectrophotometer.For every SNP assayed, KASP needs a 5-10 ng template DNA.Purity of DNA is of the most importance as the existence of contaminations in DNA can hinder PCR.Polyphenols and polysaccharides are most common contaminants which can affect PCR.Such contaminants can be removed by adding 10-phenanthroline, polyvinylpyrrolidone-40 (PVP-40), dierhlydithiocarbamic acid, sarcosyl and sorbitol (Zhang & Stewart, 2000).On the basis of research objectives, DNA can be extracted from one plant or bulk of different plants.For MAS in marker assisted re-current selection, DNA extracted from one plant per entry is preferable.As genotyping of each and every individual plant per accession is expensive and laborious, several reports have used bulked method (Reif et al., 2005, Warburton et al., 2010).For single cross hybrid, inbred lines, and bi-parental mapping populations, bulk method or DNA pooling (10-15 plants per sample prior to DNA extraction) is recommended for KASP genotyping.Generally, KASP works well for both methods, but genotype plots are often not sharper for bulk method than single plants.However, KASP does not work for open pollinated crops as it is not a quantitative assay.To address this issue, DNA should either be extracted from single plant or a minimum of 3-4 repeats are recommended of DNA bulk (Semagn et al., 2013).

KASP Primer Desiging
Usually KASP requires three primers.Two primers having one of each fluorophore (FAM or HEX) attached as a tail and common primer.Figure (2)(3)(4)(5) shows the possibilities to design KASP primer sets for SNP and InDel.

Sampling Assayed
DNA samples can be assayed in any 96, 384 or 1536-well micro-titter PCR plate.For 96-well plates, 5-6 µL of DNA is recommended while 2-2.5 µL of DNA is recommended for 384-and 1536-well plates.The genotyping must be carried out on at least 24 samples and two non-template control (NTC) to guarantee proper and clear clustering.

KASP Application
KASP method offers more flexibility than other multiplex methods in that it can be used either for many SNPs in few sample or vice versa (Semagn et al., 2013).KASP can be used for genotyping a wide range of species for several purposes.This genotyping technology has more than 2000 published references in applications including pharmaceuticals, disease and agriculture research fields.Study conducted by Rasheed et al. (2016), developed KASP assays for various economically important traits in wheat.Some of the reported KASP assay for agronomic traits, end-use-qulaity and pre-harvesting sprouting resistance are as follows, Ppd-B1, Ppd-D1, VRN-A1, VRN-B1, VRN-D1, Rht-B1, Rht-D1, TaCwi-5D, TaGS-D1, TaTGW6-3A, TaGASR-A1, TaSus2-2B, TaCKX-D1, TaMoc1-7A, TaDreb-B1, Glu-A1, Glu-B1, Glu-D1, Pina-D1, Pinb-D1, Pinb-B2, Ppo-A1, Ppo-D1, Psy-A1, Psy-B1, Psy-D1, Zds-A1, TaPHS1, TaSdr-B1, TaVp-1B, TaMFT-A1.International maize and wheat improvement center (CIMMYT) has been using KASP platform for the systematic allele mining of large set of germplasm for particular functional polymorphism and QTL mapping studies.Allele mining is favorable approach to dissect naturally occurring allelic variants at candidate genes govering important agronomic traits.A systematic allele mining of large germplasm collections for specific functional polymorphism is going on at CIMMYT courtesy KASP.QTL mapping includes identification of subset of markers that are significantly linked with one or more QTL, influencing the expression of the targeted traits.In maize, KASP assays for subset of SNPs around QTL has been developed for genotyping various fine mapping populations at CIMMYT.To validate minor QTL, marker assisted recurrent selection is a promising approach for accumulating desirable alleles from QTLs for traits governed by many genes.Plants having long juvenile phase, such as apple, which is a large woody perennial plant, are more ill-suited for traditional analysis that rely on high thoughput phenotypic selection for complex traits.Therefore, MAS gives an effective, substitute approach for pre-selecting favorable individuals for breeding tasks.SNP based markers are potential candidates for MAS.Seven SNPs near to QTLs for concentrations of soluble sugars and acids in apple have indicated markers for MAS that can assist in breeding for fruit quality (Ma et al., 2016).Haibo et al. (2018), constructed high-density genetic linkage map with 10,172 markers of apple, using SNP markers obtained through resitriction site-associated DNA sequencing (RADseq) and a segregating population of 350 seeldings from the cross of Honeycrisp and Qinguan.Thirty-three QTLs for carbon isotope composition in apple under both water stress and well-irrigated conditions were identified.Among the reported QTLs, three QTLs were stable for over 2 years under water stress on linkage groups LG8, LG15, and LG16, as validated by KASP assays.KASP assay based SNP marker, based on mapping of QTLs for δ 13 C.They further stated that, genotyping through this method produced similar results to those achieved with RADseq-based SNPs.Seed Biotechnology Center of the University of California, Davis, USA applied KASP genotyping technology on commercial basis to identify and selectively breed for pepper strains with SNPs linked with resistance to Phytophthora capsici, a root rot causing factor.
High throughout and quick turnaround times of KASP genotyping technology empowered the group to validate and publish their research work ahead of the field.This technology has also been used to investigate commercial egg laying chickens for polymorphisms in exons 2-6 of the gene encoding the eggshell protein ovocalyxin-32 (OCX32) which is associated to eggshell characteristics including firmness and thickness.This method provided quick detection of a larger number of SNPs not formerly reported.In a nut shell this single-plex genotyping technology can be applied to all genotypes after validation.KASP assay was recently used to screen SNPs in various neurological disorders like Alzheimer's disease (rs75932628 of TREM2) (Benitez et al., 2014), rs1476679 of ZCWPW1 gene (Allen et al., 2015), and both rs7412 and rs429358 for Apolipoprotein E genotyping (Keogh et al., 2017), Parkinson disease (Landoulsi et al., 2017), amyotrophic lateral sclerosis (Fogh et al., 2014), polyglutamine diseases (Bettencourt et al., 2016) and genetic generalized epilepsy (VRK2 rs2947349 (Epilepsies ILAECoC et al., 2014).

KASP vs Amplifluor and TaqMan
The "Amplifluor" is another novel SNP genotyping system developed by Millipore, the company recently merged with Merck (http://www.merckmillipore.com).The basic principles of KASP and Amplifluor SPN genotyping are similar but the underlying chemistry and primer/probe assay has never been publicized by KBioscience Company for KASP (Khripin, 2006).In contrast to KASP, Milipore-Merck Company publicized the underlying chemistry, design and sequence of all components in Amplifluor SNP genotyping systems.A previous report confirms that KASP Master-mix can be used with self-developed Amplifluor system as well (Jatayev et al., 2017).The structure of universal probes (UPs) in KASP system, remains unrevealed.Although, two universal fluorescence resonance energy transfer (FRET) cassettes with fluorophores FAM and HEX are clearly mentioned on LGC Genomics web site (http://www.lgcgroup.com)but the information regarding type of quencher UPs is still unrevealed for KASP genotyping.At the moment, we only speculate the UPs general structures in both KASP and Amplifluor like system are similar, if not identical.Amplifluor like SNP genotyping system are considered 10-20 folds less expensive as compared to KASP as once-ordered stock of two UPs labelled with different dyes can used to analyze a number of SNPs.Although, Amplifluor like SNP genotyping system is considered less expensive, but the perfect optimization of UPs and associated well organized business and marketing makes KASP markers very popular amongst wider range of researchers (Jatayev et al., 2017).
KASP and TaqMan assays have common advanatgaes, i.e. less laborious, cost effective, more tolerance of variability in DNA quality and quantity.Both assays could be used for large sample numbers in much lesser time bypassing post-PCR handling (He et al., 2014).These properties are important to deal with larger scale genotyping.Beside this, KASP and TaqMan assays are appropriate to be used on any real-time PCR machine.TaqMan requires tagging of the SNP specific primers which increased the cost of assay.Therefore, KASP genotyping assay showed a cost saving option.The current KASP reaction costs five time cheaper than TaqMan.Furthermore, a study of SNP selection for soybean genotyping has stated that KASP assay cost 0.005 US$ as compared to 0.238 US$ for TaqMan (Yuan et al., 2014).Quite a large number of publications with reference to KASP analysis indicate a large interest of researchers in this technology.

KASP Drawbacks and Alternative SNP Genotying Techniques
Although KASP is a high throughput genotyping assay yet reports are there stating 0.7-1.6%error rate.KASP is also probably not suitable when the mutant allele is present in very low quantities.When there is less than 5% quantity of mutant allele as compared to wild type allele, then it is difficult to differentiate between the individual/sample containing mutant or wild type DNA.
To deal with the low quantity of mutant DNA, several other SNP genotyping techniques are available.CastPCR TM (http://www.lifetechnologies.com) is a technique which blocks the annealing and elongation steps of the allele that is not your target one (Roma et al., 2013).ddPCR TM is another technique (http://www.bio-rad.com),which enables the absolute quantitation of nucleic acids in a sample (Hindson et al., 2011).

Table 1 .
Minimum DNA quantity required for each sample for specific specie