EasyKASP: a simple and fast tool for KASP primer designing

doi:10.21203/rs.3.rs-7229783/v1

Download PDF

software

EasyKASP: a simple and fast tool for KASP primer designing

https://doi.org/10.21203/rs.3.rs-7229783/v1

This work is licensed under a CC BY 4.0 License

Journal Publication

published 19 Dec, 2025

Read the published version in BMC Bioinformatics →

You are reading this latest preprint version

Background

Kompetitive Allele-Specific PCR (KASP) is a fluorescence-based, high-throughput and cost-effective genotyping technology, which has been widely used for detecting both single nucleotide polymorphisms (SNPs) and insertion-deletions (InDels) across various species. However, few software tools are available to automatically design KASP primers, especially for InDel variations.

Results

To address the need for efficient KASP primers design, we analyzed the sequencecharacteristics of KASP primers and developed a user-friendly program named EasyKASP on the Excel VBA platform. EasyKASP can design KASP primers for both SNP and InDel variations, with an average time of only 0.03 seconds per primer pair. A total of 80 SNP loci and 6 InDel loci with different length of variations were used to validate the KASP markers designed by EasyKASP, all of which successfully genotyped using KASP technology.

Conclusions

EasyKASP is a simple and rapid tool for KASP primer design, demonstrating broad applicability in KASP genotyping studies.

SNP

KASP primer design

EasyKASP

Excel VBA

With the rapid advancement of high-throughput genome sequencing technology, vast number of samples have been sequenced, leading to the identification of abundant whole-genome single-nucleotide polymorphism (SNP) variations [1]. As a third-generation molecular marker technology, SNPs have become as an ideal tool for genetic studies due to their widespread genomic distribution, high-throughput compatibility, genetic stability, and ease of standardization [2]. High-throughput sequencing technologies offer significant advantages over traditional SNP detection methods such as Sanger sequencing, CAPS, melting curve analysis, SNaPshot, TaqMan probes, and gene chip [3]. These conventional techniques, while widely used, often fail to meet the growing demand for cost-effective, rapid and large scale genotyping solutions [4].

Kompetitive Allele Specific PCR (KASP), developed by LGC Biosearch Technologies, combines PCR amplification with competitive allele-specific primer binding and fluorescence detection to enable accurate and cost-effective genotyping of target SNPs or InDel variations [5]. Compared to other genotyping technologies, KASP exhibits high analytical stability and accuracy, along wth great flexibility in the number of SNPs and samples that can be processed [6, 7]. It can also supports high-precision dual-allele genotyping for a limited number of target markers, including SNPs and InDels, across large segregating populations or natural germplasm collections [8]. KASP is compatible with low-, medium-, and high-throughput genotyping formats and can be integrated with automated platforms, making it suitable for a wide range of research and breeding applications [9].

Over the past five years, more than 800 studies have reported the development of KASP markers, establishing it as a benchmark genotyping technology worldwide [10]. In agricultural research, KASP has been widely applied for fine mapping of target genes, molecular-assisted breeding, germplasm resource identification, and genetic quality control of crop varieties [11–15]. In medical field, KASP has proven valuable for investigating molecular genetic mechanisms of diseases, mapping disease-associated genes, and screen for drug sensitivity and disease susceptibility loci [16]. Unlike conventional PCR primers, KASP primers consist of three oligonucleotides: two allele-specific forward primers, and one common reverse primer. The allele-specific primers are designed to match the two allelic variants of SNPs or InDels, with the 3’ ends located at the corresponding variant sites. Each specific primer includes 21-bp universal tail sequence at the 5’ ends. The common primer follows the same principles in designing regular PCR primers. The use of universal tail sequences eliminates the need to synthesize fluorescent probes for each variant, significantly reducing the reagent cost for KASP detection (http://www.lgcgenomics.com). Primer design is critical for the accuracy and efficiency of KASP genotyping. In addition to the general requirements for PCR primers——such as optimal primer length, GC content, Tm value, and avoidance of secondary structures [17], the common KASP primer should have higher Tm value than the two specific primers. Furthermore, the amplification products should be less than 100 bp to ensure efficient amplification. Since the SNP or InDel site defines the 3′ end of the allele-specific primers, the sequences available for primer design are inherently constrained.

Currently, several software tools have been developed for allele-specific PCR (AS-PCR) primer design, such as WAPS [18], PolyMarker [19], KASPspoon [20] and FastPCR [21]. However, some of these programs are no longer updated, and few are available and specifically designed for high-throughput KASP primers designing. Therefore, there is an urgent need for developing specialized software tailored to KASP primer design. Visual Basic for Applications (VBA) is an object-oriented programming language integrated into Microsoft Excel software, a widely used international spreadsheet software [22]. Numerous tools have been developed using the Excel VBA platform, significantly accelerating data management and processing while enhancing workflow productivity [23–27]. In primer design, DNA sequences can be treated as a text string in VBA, allowing parameters such as primer length and GC content to be easily calculated in Excel. The extensive function library of Excel provides robust support for developing customized algorithms for KASP primers. In this study, we developed a simple and user-friendly VBA-based tool named EasyKASP within Microsoft Excel. EasyKASP is freely accessible and facilitates the efficient design of KASP primers for both SNP and InDel loci. Moreover, its core functionality can be extended to accommodate diverse primer design requirements, highlighting its versatile and broad applicability in genotyping research.

Programming language and software design

EasyKASP leverages multiple character functions in Excel to analyze DNA sequences for KASP primer design. The FIND function scans the DNA sequence to locate the variation sites and identify repeated bases within primers. Functions such as MID, LEFT, and RIGHT are used to select and extract specific DNA segments as candidate primers, while the LEN function determines primer length. The Do...Loop structure in VBA automates repetitive tasks such as selecting candidate primers, calculating GC content, generating reverse complementary sequences, searching for simple sequence repeat (SSR) and consecutive complementary bases, and evaluating various primer quality parameters. Collectively, Excel’s comprehensive function library provides powerful and flexible platform for designing accurate and efficient KASP primers.

The development of EasyKASP is structured into three components, as illustrated in Fig. 1. The first part involves identifying the position of the SNP or InDel variation and its flanking sequences in the input sequence, verifying the base at the variation site, and determining the primer design direction based on the GC content and repeated bases within the 25-bp region surrounding the variation site. The second part focuses on selecting candidate primer sequences and evaluating their parameters. The Tm value, influenced by primer length and GC content, is a crucial parameter for KASP primers. Other factors such as base distribution, repeated bases, complementary bases, and SSR repeat units in the primer are also considered. The third part involves organizing and outputting the successfully designed primer sequences. Two specific KASP primers are appended with 21-bp universal adapters, while the common primer is reverse complemented. If primers are designed in the reverse direction, two specific primers are reverse complemented before 21-bp adapter addition, while the common primer remains unchanged. Finally, the KASP primers sequence are output to users. The open-source code and software interface of EasyKASP were provided in Additional file 1 and file 2, respectively.

Sequence preparation

EasyKASP requires only four columns of input information to initiate KASP primer design: Locous name, variation type (SNP or InDel), and at least 50 bp of flanking sequence. The target variation site is clearly indicated within square brackets, while other polymorphic sites in the sequence are denoted with ambiguous bases or small brackets. Notably, missing alleles of InDels should not be represented by any value or symbol.

Validation of designed KASP primers

Our previous studies have validated the success rate of EasyKASP in designing primers for SNP loci in cucumber and tomato [15, 28, 29]. To evaluate its application for Indel loci, six Indels with different length of variations in cucumber were selected for KASP primer design [30]. For SNP genotyping, genomic DNA was extracted from seedling of 96 hybrid varieties by SDS method with minor modification [31]. DNA concentration was detected by Nanodrop 2000 UV Spectrophotometer (Thermo Fisher Scientific, USA), with final concentrations exceeding 50 ng/µL. The KASP assay mix included two allele-specific primers and one common primer, each diluted to 10µM and mixed in a ratio of 2:2:5. For each sample, 3µL of DNA (20–30 ng/µL) and 0.014 µL of KASP assay mix were dispensed into a 384 well plate and dried in oven at 60℃ for 30 minutes. Then, 3µL of 1× Master mix (LGC genomics, UK) was added to each well. According to the KASP genotyping manual, PCR was performed with an initial denaturation at 94℃ for 15 minutes, followed by 10 touchdown cycles (94℃ for 20 seconds, 61 − 55℃ for 1 minute with a 0.6℃ decrease per cycle), and then 26 cycles of 94℃ for 20 second and 55℃ for 1minute. Endpoint fluorescence signals were detected using a PHERAstar (LGC Genomics Ltd, KBS-0021-001) with FAM (λex/em = 485/520 nm) and HEX (λex/em = 535/556 nm) channels. SNP genotypes were determined based on signal clusters: homozygous samples showed one dominant signal while heterozygous samples showed both FAM and HEX signals.

Optimal parameter of KASP primers

To determine the characteristics and optimal ranges for KASP primers, we analyzed the main parameters of 500 KASP primers successfully used for SNP genotyping in our lab (Additional file 3). The analysis revealed no significant differences were observed in GC content, length, or Tm values between two specific primers. In contrast, the common primer exhibited lower GC content but greater length and higher Tm values than the specific primers (Fig. 2). This is attributed to the 21 bp adapter sequence added to the 5’end of specific primers. To minimize the total length of specific primers, the strand with higher GC content at the variant site was preferentially selected. Based on this analysis, we established the following formula for calculating the Tm value of KASP primers:

Tm = 72 + 37.4 × GC% − \(\:\frac{747}{\text{L}\text{E}\text{N}}\)

where LEN is the primer length and GC% is the GC content. Detailed parameters for KASP primer design are summarized in Table 1.

Table 1
Parameters and recommended ranges for KASP primers
Parameter	Recommended Range
Length	Specific primer: 21–27 bp
Length	Common primer: 22–32 bp
GC Content	Optimal: 30–60% Allowed: 20–70%
Tm Value	Specific primer: 57.5 ± 1.5°C
	Common primer: 60.5 ± 1.5°C
	Difference between specific primers: < 1°C Common primer Tm value should be 2°C higher than specific primers
Repeated Bases	No 6 consecutive repeated bases
	No 4 consecutive complementary bases
	No 3 or more SSR repeat units
Base Distribution	Evenly distributed; include at least 4 types of bases
Product Size	Amplicon length < 30 bp, Amplification product length < 100 bp
Primer 3’ End	No 4 consecutive G or C
Primer 3’ End	No 5 consecutive A or T

Efficiency evaluation of EasyKASP

EasyKASP, written in VBA, is simple and user-friendly. It requires Microsoft Excel on a PC but does not require an internet connection. After opening the xlsm file containing the program code, users must enable macros and prepare sequences in the format shown in Fig. 3A. Clicking the “Start” button initiates the primer design process. EasyKASP consumes minimal memory resources and demonstrates high efficiency designing primers for 20 variant sites in just 0.57 seconds, with an average of 0.03 seconds per locus. The results include FAM and HEX primer sequences (specific primers with 21-bp adapters, marked in blue and red, respectively) and the COM primer (common primer sequence) (Fig. 3B). A “Failure” result indicates that no suitable sequence could be designed as KASP primer.

Genotyping of InDel loci using KASP technology

EasyKASP has been used to design KASP markers for whole-genome SNP loci in 16 vegetables species [1], and 80 KASP primers were validated for variety identification in cucumber and tomato [15, 29]. To further verify its efficiency for InDel loci, six InDels in cucumber with variation lengths ranging from 1 bp to 50 bp were selected for KASP marker development (Additional file 4). Four InDels were located in functional genes: a 1-bp insertion in CsAPRR2 as controlling the white immature fruit [32], a 3-bp deletion in CsMLO1 associated with powdery mildew resistance [33], a 4-bp insertion in CsACS11 involved in sex determination [34], and 5-bp insertion in CsBADH responsible for fruit fragrance [35]. The other two InDels, a 34-bp insertion in chromosome 7 and a 50-bp deletion in chromosome 6, were identified in our lab for hybrid variety identification. KASP primers were successfully designed for all six InDels using EasyKASP, and genotypes were accurately determined using KASP technology (Fig. 4), demonstrating the tool’s applicability for both SNP and InDel loci.

Wide application of EasyKASP in KASP genotyping

With the continuous advances in gene function research, phenotype prediction and selection based on genotype has become feasible [36]. Numberous SNP and InDel variations have been identified from whole-genome resequencing data. SNPs are widely used and actively developed as molecular markers in genetic studies [37]. Among SNP genotyping methods, KASP offers flexibility, high accuracy, cost-effectiveness, and efficiency [38]. Primer design software such as Primer3 and Oligo have simplified primer design [39], but few tools are available for high-throughput KASP primer design. VBA, embedded in Excel, provides powerful functions and loop structures for character recognition and statistical analysis, meeting the requirements for KASP primer design [40]. In this study, we developed EasyKASP, a VBA-based Excel program for designing KASP primers. A key criterion for primer design software is user-friendly interface that minimizes user effort [41, 42]. EasyKASP is easy to install and use, running on any computer with Excel software without an internet connection or parameter selection. With a single click, users can perform batch and automated KASP primer design. EasyKASP efficiently designs KASP primers in 0.03 seconds per pair and supports InDel variantions. Users can also customize primer design direction and amplicon length by adding invalid bases or deleting raw bases in the sequence.

Comparison of EasyKASP with existing tools

We compared EasyKASP with other software tools for KASP primer design (Table 2). The main differences lie in efficiency and the ability to design primers for InDels. Kraken, a commercial software developed by LGC Limited, is not free. WASP (https://bioinfo.biotec.or.th/WASP) is a web-based software for AS-PCR primer design[18], but it introduces an additional deliberate mismatch at the penultimate base of specific primers when designing KASP primers, which may reduce PCR efficiency and cause genotyping errors. PolyMarker (http://www.polymarker.info) generates SNP markers for hexaploid wheat using local alignments and standard primer design tools to test the viability of primers [19]. KASPspoon is an in silico PCR analysis tool for high-throughput SNP genotyping [20], but both tools only search for primers on one side of the polymorphic site and do not add tails sequence. FastPCR (http://primerdigital.com/tools/) is a Java-based tool for designing allele-specific PCR primers [21], but it provides numerous similar primer sequences, making it difficult to select optimal primers. EasyKASP analyzes flanking sequences and candidate primers to provided users with an optimal primer sequence. It is free, high-throughput, requires no local installation and automatically adds tail sequences for both SNP and InDel loci.

Table 2
Comparison of EasyKASP with other published software in KASP primer design
Comparison	EasyKASP	Kraken	WASP	PolyMarker	KASPspoon	FastPCR
Free to use	Yes	No	Yes	Yes	Yes	No
Local installation	No	Yes	No	No	Yes	No
Web-based	No	No	Yes	Yes	Yes	Yes
High-throughput	Yes	No	No	Yes	No	Yes
Design primer for InDels	Yes	Yes	No	No	No	Yes
Table-formatted output	Yes	Yes	No	Yes	No	Yes
User-friendly	Yes	No	No	No	No	No

Considerations in KASP primer design

Not all genetic variations are suitable for KASP assays, and not all KASP primers successfully yield genotyping results. PCR primers must be single-copy in the genome to avoid the amplification of nonspecific amplification [43]. Since KASP genotyping relies on fluorescence signals rather than product length, the sequences available for KASP primer design are highly limited. Allele-specific primers can only be selected from 20–30 bp upstream or downstream of the SNP/InDel site, and amplicon lengths typically range from 50 to 100 bp. Flanking sequence alignment or primer validation is necessary to ensure PCR product uniqueness. Additionally, non-target variations in flanking sequences must be considered. The quality of the reference genome and sequencing errors may also affect sequence authenticity. Undiscovered variations may exist in immediate proximity to target SNP or InDel loci, which may influence the result of KASP genotyping. For critical genetic variations, Sanger sequencing of the variant and its flanking region is recommended before designing KASP primers.

KASP is a high-throughput, fluorescence-based genotyping technology, yet accessible tools for designing KASP primers remain limited. To address this gap, we developed EasyKASP, a free and user-friendly tool on the Excel VBA platform that enables high-throughput design of KASP primers for both SNP and InDel variations. This implementation demonstrates the robust capability of Excel VBA for molecular assay design, with promising potential for adaptation to other PCR-based primer development applications.

Availability and requirements

Project name: EasyKASP

Project home page: None

Operating system: Windows

Programming language: Visual Basic for Applications

Other requirements: Microsoft Office 2007 or higher

License: EasyKASP is licensed under the GNU general public license.

Any restrictions to use by non-academics: None.

Ethics approval and consent to participate

Not applicable.

Consent for publication

Not applicable.

Availability of data and materials

The SNP loci and KASP primers designed during the current study are available in the VegSNPDB (http://www.vegsnpdb.cn/)

Competing interests

The authors declare that they have no competing interests.

Funding

This research was financially supported in part by grants from Biological Breeding-National Science and Technology Major Project (NK2022090404, 2022ZD0401903), Beijing Academy of Agricultural and Forestry Sciences (QNJJ202327, KJCX20230301, KJCX20230308).

Author contributions

CW and JZ designed this research. JZ and JY performed this research. JZ analyzed the data and wrote this manuscript. All authors have read and approved the final manuscript.

Acknowledgements

The authors express their gratitude to Naveed Shahzad Amir (China Pakistan Economic Corridor Secretariat, Pakistan) for reﬁning the language of the article.

Additional Information

Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Yang J, Zhang J, Du H, Zhao H, Li H, Xu Y, et al. The vegetable SNP database: An integrated resource for plant breeders and scientists. Genomics. 2022;114:110348.
Tian H-L, Wang F-G, Zhao J-R, Yi H-M, Wang L, Wang R, et al. Development of maizeSNP3072, a high-throughput compatible SNP array, for DNA fingerprinting identification of Chinese maize varieties. Mol Breed. 2015;35:136.
Yang S, Gill RA, Zaman QU, Ulhassan Z, Zhou W. Insights on SNP types, detection methods and their utilization in Brassica species: Recent progress and future perspectives. J Biotechnol. 2020;324:11–20.
Semagn K, Babu R, Hearne S, Olsen M. Single nucleotide polymorphism genotyping using Kompetitive Allele Specific PCR (KASP): overview of the technology and its application in crop improvement. Mol Breed. 2014;33:1–14.
Dipta B, Sood S, Mangal V, Bhardwaj V, Thakur AK, Kumar V, et al. KASP: a high-throughput genotyping system and its applications in major crop plants for biotic and abiotic stress tolerance. Mol Biol Rep. 2024;51:508.
Zhao Y, Chen W, Cui Y, Sang X, Lu J, Jing H, et al. Detection of candidate genes and development of KASP markers for Verticillium wilt resistance by combining genome-wide association study, QTL-seq and transcriptome sequencing in cotton. Theor Appl Genet. 2021;134:1063–81.
Grewal S, Coombes B, Joynson R, Hall A, Fellers J, Yang C-Y, et al. Chromosome-specific KASP markers for detecting Amblyopyrum muticum segments in wheat introgression lines. Plant Genome. 2022;15:e20193.
Chen X, Huang L, Fan J, Yan S, Zhou G, Zhang J. KASP-IEva: an intelligent typing evaluation model for KASP primers. Front Plant Sci. 2023;14:1293599.
Wu J, Liu S, Wang Q, Zeng Q, Mu J, Huang S, et al. Rapid identification of an adult plant stripe rust resistance gene in hexaploid wheat by high-throughput SNP array genotyping of pooled extremes. Theor Appl Genet. 2018;131:43–58.
Chang C-C, Silva BBI, Huang H-Y, Tsai C-Y, Flores RJD, Tayo LL, et al. Development and Validation of KASP Assays for the Genotyping of Racing Performance-Associated Single Nucleotide Polymorphisms in Pigeons. Genes (Basel). 2021;12.
Sandhu N, Singh J, Ankush AP, Augustine G, Raigar OP, Verma VK, et al. Development of Novel KASP Markers for Improved Germination in Deep-Sown Direct Seeded Rice. Rice (N Y). 2024;17:33.
Jia Q, Zhou M, Xiong Y, Wang J, Xu D, Zhang H, et al. Development of KASP markers assisted with soybean drought tolerance in the germination stage based on GWAS. Front Plant Sci. 2024;15:1352379.
Xi X, Gutierrez B, Zha Q, Yin X, Sun P, Jiang A. Optimization of In Vitro Embryo Rescue and Development of a Kompetitive Allele-Specific PCR (KASP) Marker Related to Stenospermocarpic Seedlessness in Grape (Vitis vinifera L.). Int J Mol Sci. 2023;24.
Xiao Y, Wu L, Wang B, Zhang M, Pan Q, Xian L, et al. Development and application of Key Allele-Specific PCR (KASP) molecular markers for assessing apple fruit crispness. Mol Breed. 2024;44:71.
Zhang J, Ren J, Yang J, Fu S, Zhang X, Xia C, et al. Evaluation of SNP fingerprinting for variety identification of tomato by DUS testing. Agric Commun. 2023;1:100006.
Cabrera S, Bebia V, López-Gil C, Luzarraga-Aznar A, Denizli M, Salazar-Huayna L, et al. Molecular classification improves preoperative risk assessment of endometrial cancer. Gynecol Oncol. 2024;189:56–63.
Thornton B, Basu C. Rapid and simple method of qPCR primer design. Methods Mol Biol. 2015;1275:173–9.
Wangkumhang P, Chaichoompu K, Ngamphiw C, Ruangrit U, Chanprasert J, Assawamakin A, et al. WASP: a Web-based Allele-Specific PCR assay designing tool for detecting SNPs and mutations. BMC Genomics. 2007;8:275.
Ramirez-Gonzalez RH, Uauy C, Caccamo M. PolyMarker: A fast polyploid primer design pipeline. Bioinformatics. 2015;31:2038–9.
Alsamman AM, Ibrahim SD, Hamwieh A. KASPspoon: an in vitro and in silico PCR analysis tool for high-throughput SNP genotyping. Bioinformatics. 2019;35:3187–90.
Kalendar R, Shustov A V, Akhmetollayev I, Kairov U. Designing Allele-Specific Competitive-Extension PCR-Based Assays for High-Throughput Genotyping and Gene Characterization. Front Mol Biosci. 2022;9:773956.
Bauzon J, Murphy C, Wahi-Gururaj S. Using macros in microsoft excel to facilitate cleaning of research data. J community Hosp Intern Med Perspect. 2021;11:653–7.
Fasoula S, Zisi C, Gika H, Pappa-Louisi A, Nikitas P. Retention prediction and separation optimization under multilinear gradient elution in liquid chromatography with Microsoft Excel macros. J Chromatogr A. 2015;1395:109–15.
Lai Z, Liang Z, Yan L, Qian X, Jiang H, Zhong W. Determination of modification sites and relative quantitation in large protein conjugation via automated data processing. J Pharm Biomed Anal. 2021;198:113995.
Suárez-Navarro JA, Gascó C, Alonso MM, Blanco-Varela MT, Lanzon M, Puertas F. Use of Genie 2000 and Excel VBA to correct for γ-ray interference in the determination of NORM building material activity concentrations. Appl Radiat Isot Incl data, Instrum methods use Agric Ind Med. 2018;142:1–7.
Hu W, Pan L, Chen H, Tang M. VBA-AMF: A VBA Program Based on the Magnified Intersections Method for Quantitative Recording of Root Colonization by Arbuscular Mycorrhizal Fungi. Indian J Microbiol. 2020;60:374–8.
Cao X, He W, He W, Shi Y, An T, Wang X, et al. EMMTE: An Excel VBA tool for source apportionment of nitrate based on the stable isotope mixing model. Sci Total Environ. 2023;868:161728.
Zhang J, Yang J, Wen C. A New SNP Genotyping Technology by Target SNP-Seq BT - Plant Genotyping: Methods and Protocols. In: Shavrukov Y, editor. New York, NY: Springer US; 2023. p. 365–71.
Zhang J, Yang J, Fu S, Ren J, Zhang XF, Xia C, et al. Comparison of DUS testing and SNP fingerprinting for variety identification in cucumber. Hortic Plant Journal. 2022;8:8.
Yang J, Meng P, Mi H, Wang X, Yang J, Fu S, et al. The development of ideal insertion and deletion (InDel) markers and initial indel map variation in cucumber using re-sequenced data. BMC Genomics. 2025;26:391.
Xia Y, Chen F, Du Y, Liu C, Bu G, Xin Y, et al. A modified SDS-based DNA extraction method from raw soybean. Biosci Rep. 2019;39.
Tang H-Y, Dong X, Wang J-K, Xia J-H, Xie F, Zhang Y, et al. Fine Mapping and Candidate Gene Prediction for White Immature Fruit Skin in Cucumber (Cucumis sativus L.). Int J Mol Sci. 2018;19.
Jingtao N, Yunli W, Huanle H, Chunli G, Wenying Z, Jian P, et al. Loss-of-Function Mutations in CsMLO1 Confer Durable Powdery Mildew Resistance in Cucumber (Cucumis sativus L.). Front Plant Sci. 2015;6:1155-.
Boualem A, Troadec C, Camps C, Lemhemdi A, Morin H, Sari M-A, et al. A cucurbit androecy gene reveals how unisexual flowers develop and dioecy emerges. Science. 2015;350:688–91.
Yundaeng C, Somta P, Tangphatsornruang S, Chankaew S, Srinives P. A single base substitution in BADH/AMADH is responsible for fragrance in cucumber (Cucumis sativus L.), and development of SNAP markers for the fragrance. Theor Appl Genet. 2015;128:1881–92.
Crossa J, Montesinos-López OA, Pérez-Rodríguez P, Costa-Neto G, Fritsche-Neto R, Ortiz R, et al. Genome and Environment Based Prediction Models and Methods of Complex Traits Incorporating Genotype × Environment Interaction. Methods Mol Biol. 2022;2467:245–83.
Khassanova G, Khalbayeva S, Serikbay D, Mazkirat S, Bulatova K, Utebayev M, et al. SNP Genotyping with Amplifluor-Like Method. Methods Mol Biol. 2023;2638:201–19.
Jatayev S, Kurishbayev A, Zotova L, Khasanova G, Serikbay D, Zhubatkanov A, et al. Advantages of Amplifluor-like SNP markers over KASP in plant genotyping. BMC Plant Biol. 2017;17 Suppl 2:254.
Kõressaar T, Lepamets M, Kaplinski L, Raime K, Andreson R, Remm M. Primer3_masker: integrating masking of template sequence with primer design software. Bioinformatics. 2018;34:1937–8.
Peakall R, Smouse PE. GenAlEx 6.5: genetic analysis in Excel. Population genetic software for teaching and research--an update. Bioinformatics. 2012;28:2537–9.
Divate M, Cheung E. GUAVA: A Graphical User Interface for the Analysis and Visualization of ATAC-seq Data. Front Genet. 2018;9:250.
Ergun MA, Cinal O, Bakışlı B, Emül AA, Baysan M. COSAP: Comparative Sequencing Analysis Platform. BMC Bioinformatics. 2024;25:130.
Fautt C, Hockett KL, Couradeau E. Evaluation of the taxonomic accuracy and pathogenicity prediction power of 16 primer sets amplifying single copy marker genes in the Pseudomonas syringae species complex. Mol Plant Pathol. 2023;24:989–98.

No competing interests reported.

Additionalfile1.pdf
Additional files Additional file 1: The source code of EasyKASP.
Additionalfile2.xls
Additional file 2: The program of EasyKASP installed in Excel.
Additionalfile3.xlsx
Additional file 3: Main parameters of 500 KASP primers.
Additionalfile4.xlsx
Additional file 4: KASP primer of six InDels with different length of variations.

Download PDF

Journal Publication

published 19 Dec, 2025

Read the published version in BMC Bioinformatics →

Editorial decision: Revision requested
10 Oct, 2025
Reviews received at journal
09 Oct, 2025
Reviews received at journal
07 Oct, 2025
Reviewers agreed at journal
06 Oct, 2025
Reviewers agreed at journal
26 Sep, 2025
Reviewers agreed at journal
26 Sep, 2025
Reviewers invited by journal
26 Sep, 2025
Editor assigned by journal
13 Sep, 2025
Editor invited by journal
11 Sep, 2025
Submission checks completed at journal
10 Sep, 2025
First submitted to journal
10 Sep, 2025

You are reading this latest preprint version

EasyKASP: a simple and fast tool for KASP primer designing

Status:

Journal Publication

Version 1

Abstract

Figures

Background

Implementation

Programming language and software design

Sequence preparation

Validation of designed KASP primers

Results

Optimal parameter of KASP primers

Efficiency evaluation of EasyKASP

Genotyping of InDel loci using KASP technology

Discussion

Wide application of EasyKASP in KASP genotyping

Comparison of EasyKASP with existing tools

Considerations in KASP primer design

Conclusions

Declarations

References

Additional Declarations

Supplementary Files

Status:

Journal Publication

Version 1