{"paper_id":"47df7e8a-0c07-4cfc-bd4b-9134ca89fd66","body_text":"EasyKASP: a simple and fast tool for KASP primer designing | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF software EasyKASP: a simple and fast tool for KASP primer designing Jian Zhang, Jingjing Yang, Changlong Wen This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-7229783/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 19 Dec, 2025 Read the published version in BMC Bioinformatics → Version 1 posted 11 You are reading this latest preprint version Abstract 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 Figures Figure 1 Figure 2 Figure 3 Figure 4 Background 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. Implementation 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. Results 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 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 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. 3 A. 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. 3 B). 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. Discussion 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. Conclusions 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. Declarations 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. 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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. Additional Declarations No competing interests reported. Supplementary Files 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. Cite Share Download PDF Status: Published Journal Publication published 19 Dec, 2025 Read the published version in BMC Bioinformatics → Version 1 posted 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 Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. 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4\",\"display\":\"\",\"copyAsset\":false,\"role\":\"figure\",\"size\":4749155,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003e\\u003cstrong\\u003eGenotyping of InDel loci of different lengths using KASP technology\\u003c/strong\\u003e\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Figure4.jpg\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7229783/v1/1be0c953618446305e2ddd81.jpg\"},{\"id\":98814027,\"identity\":\"b744f32a-ef42-4bf2-8f8d-de3f1db2dd2f\",\"added_by\":\"auto\",\"created_at\":\"2025-12-22 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EasyKASP.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Additionalfile1.pdf\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7229783/v1/2e6643a2abd75cc5c369d04c.pdf\"},{\"id\":93074960,\"identity\":\"1b80889e-4265-4083-bae4-8add14fc86cf\",\"added_by\":\"auto\",\"created_at\":\"2025-10-08 18:42:31\",\"extension\":\"xls\",\"order_by\":2,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":141824,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdditional file 2: The program of EasyKASP installed in Excel.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Additionalfile2.xls\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7229783/v1/00c4261205736b6b1f82f4d3.xls\"},{\"id\":93075083,\"identity\":\"89661770-04a4-4f12-8772-33915193bb08\",\"added_by\":\"auto\",\"created_at\":\"2025-10-08 19:01:40\",\"extension\":\"xlsx\",\"order_by\":3,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":38836,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdditional file 3: Main parameters of 500 KASP primers.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Additionalfile3.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7229783/v1/fbe543a758f462384f9619e5.xlsx\"},{\"id\":93074974,\"identity\":\"69bd8ed2-acb2-4e81-ba1c-857c0019fa46\",\"added_by\":\"auto\",\"created_at\":\"2025-10-08 18:42:32\",\"extension\":\"xlsx\",\"order_by\":4,\"title\":\"\",\"display\":\"\",\"copyAsset\":false,\"role\":\"supplement\",\"size\":10327,\"visible\":true,\"origin\":\"\",\"legend\":\"\\u003cp\\u003eAdditional file 4: KASP primer of six InDels with different length of variations.\\u003c/p\\u003e\",\"description\":\"\",\"filename\":\"Additionalfile4.xlsx\",\"url\":\"https://assets-eu.researchsquare.com/files/rs-7229783/v1/8a7ddabc302f7e39accca5d8.xlsx\"}],\"financialInterests\":\"No competing interests reported.\",\"formattedTitle\":\"EasyKASP: a simple and fast tool for KASP primer designing\",\"fulltext\":[{\"header\":\"Background\",\"content\":\"\\u003cp\\u003eWith 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 [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR2\\\" class=\\\"CitationRef\\\"\\u003e2\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR3\\\" class=\\\"CitationRef\\\"\\u003e3\\u003c/span\\u003e]. These conventional techniques, while widely used, often fail to meet the growing demand for cost-effective, rapid and large scale genotyping solutions [\\u003cspan citationid=\\\"CR4\\\" class=\\\"CitationRef\\\"\\u003e4\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eKompetitive 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 [\\u003cspan citationid=\\\"CR5\\\" class=\\\"CitationRef\\\"\\u003e5\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR6\\\" class=\\\"CitationRef\\\"\\u003e6\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR7\\\" class=\\\"CitationRef\\\"\\u003e7\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR8\\\" class=\\\"CitationRef\\\"\\u003e8\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR9\\\" class=\\\"CitationRef\\\"\\u003e9\\u003c/span\\u003e].\\u003c/p\\u003e\\u003cp\\u003eOver the past five years, more than 800 studies have reported the development of KASP markers, establishing it as a benchmark genotyping technology worldwide [\\u003cspan citationid=\\\"CR10\\\" class=\\\"CitationRef\\\"\\u003e10\\u003c/span\\u003e]. 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 [\\u003cspan additionalcitationids=\\\"CR12 CR13 CR14\\\" citationid=\\\"CR11\\\" class=\\\"CitationRef\\\"\\u003e11\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR16\\\" class=\\\"CitationRef\\\"\\u003e16\\u003c/span\\u003e]. 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 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.lgcgenomics.com\\u003c/span\\u003e\\u003cspan address=\\\"http://www.lgcgenomics.com\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e). 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 [\\u003cspan citationid=\\\"CR17\\\" class=\\\"CitationRef\\\"\\u003e17\\u003c/span\\u003e], 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.\\u003c/p\\u003e\\u003cp\\u003eCurrently, several software tools have been developed for allele-specific PCR (AS-PCR) primer design, such as WAPS [\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], PolyMarker [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e], KASPspoon [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e] and FastPCR [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR22\\\" class=\\\"CitationRef\\\"\\u003e22\\u003c/span\\u003e]. Numerous tools have been developed using the Excel VBA platform, significantly accelerating data management and processing while enhancing workflow productivity [\\u003cspan additionalcitationids=\\\"CR24 CR25 CR26\\\" citationid=\\\"CR23\\\" class=\\\"CitationRef\\\"\\u003e23\\u003c/span\\u003e–\\u003cspan citationid=\\\"CR27\\\" class=\\\"CitationRef\\\"\\u003e27\\u003c/span\\u003e]. 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.\\u003c/p\\u003e\\n\\n\\u003cdiv id=\\\"Sec3\\\" class=\\\"Section2\\\"\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\n\\n\\n\"},{\"header\":\"Implementation\",\"content\":\"\\u003ch2\\u003eProgramming language and software design\\u003c/h2\\u003e\\u003cp\\u003eEasyKASP 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.\\u003c/p\\u003e\\u003cp\\u003eThe development of EasyKASP is structured into three components, as illustrated in Fig.\\u0026nbsp;\\u003cspan refid=\\\"Fig1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e. 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.\\u003c/p\\u003e\\u003ch3\\u003eSequence preparation\\u003c/h3\\u003e\\u003cp\\u003eEasyKASP 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.\\u003c/p\\u003e\\u003ch3\\u003eValidation of designed KASP primers\\u003c/h3\\u003e\\u003cp\\u003eOur previous studies have validated the success rate of EasyKASP in designing primers for SNP loci in cucumber and tomato [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR28\\\" class=\\\"CitationRef\\\"\\u003e28\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. To evaluate its application for Indel loci, six Indels with different length of variations in cucumber were selected for KASP primer design [\\u003cspan citationid=\\\"CR30\\\" class=\\\"CitationRef\\\"\\u003e30\\u003c/span\\u003e]. For SNP genotyping, genomic DNA was extracted from seedling of 96 hybrid varieties by SDS method with minor modification [\\u003cspan citationid=\\\"CR31\\\" class=\\\"CitationRef\\\"\\u003e31\\u003c/span\\u003e]. 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.\\u003c/p\\u003e\"},{\"header\":\"Results\",\"content\":\"\\u003cdiv id=\\\"Sec7\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eOptimal parameter of KASP primers\\u003c/h2\\u003e\\u003cp\\u003eTo 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.\\u0026nbsp;\\u003cspan refid=\\\"Fig2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). This is attributed to the 21 bp adapter sequence added to the 5\\u0026rsquo;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:\\u003c/p\\u003e\\u003cp\\u003eTm\\u0026thinsp;=\\u0026thinsp;72\\u0026thinsp;+\\u0026thinsp;37.4 \\u0026times; GC% \\u0026minus; \\u003cspan class=\\\"InlineEquation\\\"\\u003e\\u003cspan class=\\\"mathinline\\\"\\u003e\\\\(\\\\:\\\\frac{747}{\\\\text{L}\\\\text{E}\\\\text{N}}\\\\)\\u003c/span\\u003e\\u003c/span\\u003e\\u003c/p\\u003e\\u003cp\\u003ewhere LEN is the primer length and GC% is the GC content. Detailed parameters for KASP primer design are summarized in Table\\u0026nbsp;\\u003cspan refid=\\\"Tab1\\\" class=\\\"InternalRef\\\"\\u003e1\\u003c/span\\u003e.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab1\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 1\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eParameters and recommended ranges for KASP primers\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"2\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eParameter\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eRecommended Range\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eLength\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSpecific primer: 21\\u0026ndash;27 bp\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCommon primer: 22\\u0026ndash;32 bp\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eGC Content\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eOptimal: 30\\u0026ndash;60%\\u003c/p\\u003e\\u003cp\\u003eAllowed: 20\\u0026ndash;70%\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eTm Value\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eSpecific primer: 57.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eCommon primer: 60.5\\u0026thinsp;\\u0026plusmn;\\u0026thinsp;1.5\\u0026deg;C\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eDifference between specific primers: \\u0026lt; 1\\u0026deg;C\\u003c/p\\u003e\\u003cp\\u003eCommon primer Tm value should be 2\\u0026deg;C higher than specific primers\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"2\\\" rowspan=\\\"3\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eRepeated Bases\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo 6 consecutive repeated bases\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo 4 consecutive complementary bases\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo 3 or more SSR repeat units\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eBase Distribution\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eEvenly distributed; include at least 4 types of bases\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003eProduct Size\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eAmplicon length\\u0026thinsp;\\u0026lt;\\u0026thinsp;30 bp,\\u003c/p\\u003e\\u003cp\\u003eAmplification product length\\u0026thinsp;\\u0026lt;\\u0026thinsp;100 bp\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\" morerows=\\\"1\\\" rowspan=\\\"2\\\"\\u003e\\u003cp\\u003e\\u003cb\\u003ePrimer 3\\u0026rsquo; End\\u003c/b\\u003e\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo 4 consecutive G or C\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo 5 consecutive A or T\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec8\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eEfficiency evaluation of EasyKASP\\u003c/h2\\u003e\\u003cp\\u003eEasyKASP, 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.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eA. Clicking the \\u0026ldquo;Start\\u0026rdquo; 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.\\u0026nbsp;\\u003cspan refid=\\\"Fig3\\\" class=\\\"InternalRef\\\"\\u003e3\\u003c/span\\u003eB). A \\u0026ldquo;Failure\\u0026rdquo; result indicates that no suitable sequence could be designed as KASP primer.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\n\\u003ch3\\u003eGenotyping of InDel loci using KASP technology\\u003c/h3\\u003e\\n\\u003cp\\u003eEasyKASP has been used to design KASP markers for whole-genome SNP loci in 16 vegetables species [\\u003cspan citationid=\\\"CR1\\\" class=\\\"CitationRef\\\"\\u003e1\\u003c/span\\u003e], and 80 KASP primers were validated for variety identification in cucumber and tomato [\\u003cspan citationid=\\\"CR15\\\" class=\\\"CitationRef\\\"\\u003e15\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR29\\\" class=\\\"CitationRef\\\"\\u003e29\\u003c/span\\u003e]. 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 \\u003cem\\u003eCsAPRR2\\u003c/em\\u003e as controlling the white immature fruit [\\u003cspan citationid=\\\"CR32\\\" class=\\\"CitationRef\\\"\\u003e32\\u003c/span\\u003e], a 3-bp deletion in \\u003cem\\u003eCsMLO1\\u003c/em\\u003e associated with powdery mildew resistance [\\u003cspan citationid=\\\"CR33\\\" class=\\\"CitationRef\\\"\\u003e33\\u003c/span\\u003e], a 4-bp insertion in \\u003cem\\u003eCsACS11\\u003c/em\\u003e involved in sex determination [\\u003cspan citationid=\\\"CR34\\\" class=\\\"CitationRef\\\"\\u003e34\\u003c/span\\u003e], and 5-bp insertion in \\u003cem\\u003eCsBADH\\u003c/em\\u003e responsible for fruit fragrance [\\u003cspan citationid=\\\"CR35\\\" class=\\\"CitationRef\\\"\\u003e35\\u003c/span\\u003e]. 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.\\u0026nbsp;\\u003cspan refid=\\\"Fig4\\\" class=\\\"InternalRef\\\"\\u003e4\\u003c/span\\u003e), demonstrating the tool\\u0026rsquo;s applicability for both SNP and InDel loci.\\u003c/p\\u003e\\u003cp\\u003e\\u003c/p\\u003e\"},{\"header\":\"Discussion\",\"content\":\"\\u003cdiv id=\\\"Sec11\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eWide application of EasyKASP in KASP genotyping\\u003c/h2\\u003e\\u003cp\\u003eWith the continuous advances in gene function research, phenotype prediction and selection based on genotype has become feasible [\\u003cspan citationid=\\\"CR36\\\" class=\\\"CitationRef\\\"\\u003e36\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR37\\\" class=\\\"CitationRef\\\"\\u003e37\\u003c/span\\u003e]. Among SNP genotyping methods, KASP offers flexibility, high accuracy, cost-effectiveness, and efficiency [\\u003cspan citationid=\\\"CR38\\\" class=\\\"CitationRef\\\"\\u003e38\\u003c/span\\u003e]. Primer design software such as Primer3 and Oligo have simplified primer design [\\u003cspan citationid=\\\"CR39\\\" class=\\\"CitationRef\\\"\\u003e39\\u003c/span\\u003e], 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 [\\u003cspan citationid=\\\"CR40\\\" class=\\\"CitationRef\\\"\\u003e40\\u003c/span\\u003e]. 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 [\\u003cspan citationid=\\\"CR41\\\" class=\\\"CitationRef\\\"\\u003e41\\u003c/span\\u003e, \\u003cspan citationid=\\\"CR42\\\" class=\\\"CitationRef\\\"\\u003e42\\u003c/span\\u003e]. 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.\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec12\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eComparison of EasyKASP with existing tools\\u003c/h2\\u003e\\u003cp\\u003eWe compared EasyKASP with other software tools for KASP primer design (Table\\u0026nbsp;\\u003cspan refid=\\\"Tab2\\\" class=\\\"InternalRef\\\"\\u003e2\\u003c/span\\u003e). 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 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttps://bioinfo.biotec.or.th/WASP\\u003c/span\\u003e\\u003cspan address=\\\"https://bioinfo.biotec.or.th/WASP\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) is a web-based software for AS-PCR primer design[\\u003cspan citationid=\\\"CR18\\\" class=\\\"CitationRef\\\"\\u003e18\\u003c/span\\u003e], 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 (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://www.polymarker.info\\u003c/span\\u003e\\u003cspan address=\\\"http://www.polymarker.info\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) generates SNP markers for hexaploid wheat using local alignments and standard primer design tools to test the viability of primers [\\u003cspan citationid=\\\"CR19\\\" class=\\\"CitationRef\\\"\\u003e19\\u003c/span\\u003e]. KASPspoon is an in silico PCR analysis tool for high-throughput SNP genotyping [\\u003cspan citationid=\\\"CR20\\\" class=\\\"CitationRef\\\"\\u003e20\\u003c/span\\u003e], but both tools only search for primers on one side of the polymorphic site and do not add tails sequence. FastPCR (\\u003cspan class=\\\"ExternalRef\\\"\\u003e\\u003cspan class=\\\"RefSource\\\"\\u003ehttp://primerdigital.com/tools/\\u003c/span\\u003e\\u003cspan address=\\\"http://primerdigital.com/tools/\\\" targettype=\\\"URL\\\" class=\\\"RefTarget\\\"\\u003e\\u003c/span\\u003e\\u003c/span\\u003e) is a Java-based tool for designing allele-specific PCR primers [\\u003cspan citationid=\\\"CR21\\\" class=\\\"CitationRef\\\"\\u003e21\\u003c/span\\u003e], 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.\\u003c/p\\u003e\\u003cp\\u003e\\u003cdiv class=\\\"gridtable\\\"\\u003e\\u003ctable float=\\\"Yes\\\" id=\\\"Tab2\\\" border=\\\"1\\\"\\u003e\\u003ccaption language=\\\"En\\\"\\u003e\\u003cdiv class=\\\"CaptionNumber\\\"\\u003eTable 2\\u003c/div\\u003e\\u003cdiv class=\\\"CaptionContent\\\"\\u003e\\u003cp\\u003eComparison of EasyKASP with other published software in KASP primer design\\u003c/p\\u003e\\u003c/div\\u003e\\u003c/caption\\u003e\\u003ccolgroup cols=\\\"7\\\"\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c1\\\" colnum=\\\"1\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c2\\\" colnum=\\\"2\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c3\\\" colnum=\\\"3\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c4\\\" colnum=\\\"4\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c5\\\" colnum=\\\"5\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c6\\\" colnum=\\\"6\\\"\\u003e\\u003c/div\\u003e\\u003cdiv align=\\\"left\\\" class=\\\"colspec\\\" colname=\\\"c7\\\" colnum=\\\"7\\\"\\u003e\\u003c/div\\u003e\\u003cthead\\u003e\\u003ctr\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eComparison\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eEasyKASP\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eKraken\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eWASP\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003ePolyMarker\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eKASPspoon\\u003c/p\\u003e\\u003c/th\\u003e\\u003cth align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eFastPCR\\u003c/p\\u003e\\u003c/th\\u003e\\u003c/tr\\u003e\\u003c/thead\\u003e\\u003ctbody\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eFree to use\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eLocal installation\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eWeb-based\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eHigh-throughput\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eDesign primer for InDels\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eTable-formatted output\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003ctr\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c1\\\"\\u003e\\u003cp\\u003eUser-friendly\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c2\\\"\\u003e\\u003cp\\u003eYes\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c3\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c4\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c5\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c6\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003ctd align=\\\"left\\\" colname=\\\"c7\\\"\\u003e\\u003cp\\u003eNo\\u003c/p\\u003e\\u003c/td\\u003e\\u003c/tr\\u003e\\u003c/tbody\\u003e\\u003c/colgroup\\u003e\\u003c/table\\u003e\\u003c/div\\u003e\\u003c/p\\u003e\\u003c/div\\u003e\\u003cdiv id=\\\"Sec13\\\" class=\\\"Section2\\\"\\u003e\\u003ch2\\u003eConsiderations in KASP primer design\\u003c/h2\\u003e\\u003cp\\u003eNot 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 [\\u003cspan citationid=\\\"CR43\\\" class=\\\"CitationRef\\\"\\u003e43\\u003c/span\\u003e]. 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\\u0026ndash;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.\\u003c/p\\u003e\\u003c/div\\u003e\"},{\"header\":\"Conclusions\",\"content\":\"\\u003cp\\u003eKASP 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.\\u003c/p\\u003e\"},{\"header\":\"Declarations\",\"content\":\"\\u003cp\\u003e\\u003cstrong\\u003eAvailability and requirements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eProject name: EasyKASP\\u003c/p\\u003e\\n\\u003cp\\u003eProject home page: None\\u003c/p\\u003e\\n\\u003cp\\u003eOperating system: Windows\\u003c/p\\u003e\\n\\u003cp\\u003eProgramming language: Visual Basic for Applications\\u003c/p\\u003e\\n\\u003cp\\u003eOther requirements: Microsoft Office 2007 or higher\\u003c/p\\u003e\\n\\u003cp\\u003eLicense: EasyKASP is licensed under the GNU general public license.\\u003c/p\\u003e\\n\\u003cp\\u003eAny restrictions to use by non-academics: None.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eEthics approval and consent to participate\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConsent for publication\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eNot applicable.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAvailability of data and materials\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe SNP loci and KASP primers designed during the current study are available in the VegSNPDB (http://www.vegsnpdb.cn/)\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eCompeting interests\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors declare that they have no competing interests.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eFunding\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThis 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).\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAuthor contributions\\u0026nbsp;\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eCW 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.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAcknowledgements\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eThe authors express their gratitude to Naveed Shahzad Amir (China Pakistan Economic Corridor Secretariat, Pakistan) for refining the language of the article.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eAdditional Information\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003ePublisher’s note\\u0026nbsp;\\u003c/strong\\u003eSpringer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.\\u003c/p\\u003e\"},{\"header\":\"References\",\"content\":\"\\u003col\\u003e\\n\\u003cli\\u003eYang 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. 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Mol Plant Pathol. 2023;24:989\\u0026ndash;98.\\u003c/li\\u003e\\n\\u003c/ol\\u003e\"}],\"fulltextSource\":\"\",\"fullText\":\"\",\"funders\":[],\"hasAdminPriorityOnWorkflow\":false,\"hasManuscriptDocX\":true,\"hasOptedInToPreprint\":true,\"hasPassedJournalQc\":\"\",\"hasAnyPriority\":false,\"hideJournal\":false,\"highlight\":\"\",\"institution\":\"\",\"isAcceptedByJournal\":true,\"isAuthorSuppliedPdf\":false,\"isDeskRejected\":\"\",\"isHiddenFromSearch\":false,\"isInQc\":false,\"isInWorkflow\":false,\"isPdf\":false,\"isPdfUpToDate\":true,\"isWithdrawnOrRetracted\":false,\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-bioinformatics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"binf\",\"sideBox\":\"Learn more about [BMC Bioinformatics](http://bmcbioinformatics.biomedcentral.com/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/binf\",\"title\":\"BMC Bioinformatics\",\"twitterHandle\":\"@BMC_Bioinformatics\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true},\"keywords\":\"SNP, KASP primer design, EasyKASP, Excel VBA\",\"lastPublishedDoi\":\"10.21203/rs.3.rs-7229783/v1\",\"lastPublishedDoiUrl\":\"https://doi.org/10.21203/rs.3.rs-7229783/v1\",\"license\":{\"name\":\"CC BY 4.0\",\"url\":\"https://creativecommons.org/licenses/by/4.0/\"},\"manuscriptAbstract\":\"\\u003cp\\u003e\\u003cstrong\\u003eBackground\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eKompetitive 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.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eResults\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eTo 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.\\u003c/p\\u003e\\n\\u003cp\\u003e\\u003cstrong\\u003eConclusions\\u003c/strong\\u003e\\u003c/p\\u003e\\n\\u003cp\\u003eEasyKASP is a simple and rapid tool for KASP primer design, demonstrating broad applicability in KASP genotyping studies.\\u003c/p\\u003e\",\"manuscriptTitle\":\"EasyKASP: a simple and fast tool for KASP primer designing\",\"msid\":\"\",\"msnumber\":\"\",\"nonDraftVersions\":[{\"code\":1,\"date\":\"2025-10-08 18:42:23\",\"doi\":\"10.21203/rs.3.rs-7229783/v1\",\"editorialEvents\":[{\"type\":\"communityComments\",\"content\":0},{\"type\":\"decision\",\"content\":\"Revision requested\",\"date\":\"2025-10-10T08:18:59+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-10-09T16:56:22+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"editorInvitedReview\",\"content\":\"\",\"date\":\"2025-10-07T10:28:34+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"64918409111685821813382827978519243936\",\"date\":\"2025-10-06T04:37:48+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"124113223776925379892934411226029646573\",\"date\":\"2025-09-26T13:53:48+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewerAgreed\",\"content\":\"45790524521003751081724776498300906742\",\"date\":\"2025-09-26T11:51:50+00:00\",\"index\":\"hide\",\"fulltext\":\"\"},{\"type\":\"reviewersInvited\",\"content\":\"\",\"date\":\"2025-09-26T11:45:25+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorAssigned\",\"content\":\"\",\"date\":\"2025-09-13T11:08:21+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"editorInvited\",\"content\":\"\",\"date\":\"2025-09-11T10:59:09+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"checksComplete\",\"content\":\"\",\"date\":\"2025-09-10T12:43:16+00:00\",\"index\":\"\",\"fulltext\":\"\"},{\"type\":\"submitted\",\"content\":\"BMC Bioinformatics\",\"date\":\"2025-09-10T12:39:59+00:00\",\"index\":\"\",\"fulltext\":\"\"}],\"status\":\"published\",\"journal\":{\"display\":true,\"email\":\"info@researchsquare.com\",\"identity\":\"bmc-bioinformatics\",\"isNatureJournal\":false,\"hasQc\":true,\"allowDirectSubmit\":false,\"externalIdentity\":\"binf\",\"sideBox\":\"Learn more about [BMC Bioinformatics](http://bmcbioinformatics.biomedcentral.com/)\",\"snPcode\":\"\",\"submissionUrl\":\"https://www.editorialmanager.com/binf\",\"title\":\"BMC Bioinformatics\",\"twitterHandle\":\"@BMC_Bioinformatics\",\"acdcEnabled\":true,\"dfaEnabled\":false,\"editorialSystem\":\"em\",\"reportingPortfolio\":\"BMC Series\",\"inReviewEnabled\":true,\"inReviewRevisionsEnabled\":true}}],\"origin\":\"\",\"ownerIdentity\":\"66166ab2-396f-41bd-a500-d1c46d126a72\",\"owner\":[],\"postedDate\":\"October 8th, 2025\",\"published\":true,\"recentEditorialEvents\":[],\"rejectedJournal\":[],\"revision\":\"\",\"amendment\":\"\",\"status\":\"published-in-journal\",\"subjectAreas\":[],\"tags\":[],\"updatedAt\":\"2025-12-22T16:02:55+00:00\",\"versionOfRecord\":{\"articleIdentity\":\"rs-7229783\",\"link\":\"https://doi.org/10.1186/s12859-025-06322-x\",\"journal\":{\"identity\":\"bmc-bioinformatics\",\"isVorOnly\":false,\"title\":\"BMC Bioinformatics\"},\"publishedOn\":\"2025-12-19 15:58:20\",\"publishedOnDateReadable\":\"December 19th, 2025\"},\"versionCreatedAt\":\"2025-10-08 18:42:23\",\"video\":\"\",\"vorDoi\":\"10.1186/s12859-025-06322-x\",\"vorDoiUrl\":\"https://doi.org/10.1186/s12859-025-06322-x\",\"workflowStages\":[]},\"version\":\"v1\",\"identity\":\"rs-7229783\",\"journalConfig\":\"researchsquare\"},\"__N_SSP\":true},\"page\":\"/article/[identity]/[[...version]]\",\"query\":{\"redirect\":\"/article/rs-7229783\",\"identity\":\"rs-7229783\",\"version\":[\"v1\"]},\"buildId\":\"8U1c8b4HqxoKbykW_rLl7\",\"isFallback\":false,\"isExperimentalCompile\":false,\"dynamicIds\":[84888],\"gssp\":true,\"scriptLoader\":[]}","source_license":"CC-BY-4.0","license_restricted":false}