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Global Research journal of Natural Science

& Technology (GRJNST)

Volume: 04 - Issue 2 (2026), 2050

ISSN P: 2790-7643 ISSN E: 2790-7651

www.grjnst.net

https://doi.org/10.53762/grjnst.04.02.0 2

Cross-Species PCR Amplification Performance of Wheat Stripe Rust

Resistance Gene-Specific Primers in Common Bean (Phaseolus Vulgaris L.)

Received: 31 December 2025. Accepted: 30 January 2026. Published: 31 March 2026

Muhammad Mehraj Riasat (Corresponding Author)

Department of Agriculture,

Hazara University, Mansehra 21300, Pakistan

Email: mehrajhum30278@gmail.com

Muhammad Waleed

Department of Agriculture,

Hazara University, Mansehra 21300, Pakistan

Uzair Ahmad

Department of Agriculture,

Hazara University, Mansehra 21300, Pakistan

Farhan Tahir

Department of Agriculture,

Hazara University, Mansehra 21300, Pakistan

Shafaq Munir

Department of Agriculture,

Hazara University, Mansehra 21300, Pakistan

Atika Liaqat

Department of Agriculture,

Hazara University, Mansehra 21300, Pakistan

Saqib Waqar Ahmed

Department of Agriculture,

Hazara University, Mansehra 21300, Pakista

GRJNST, Volume: 04 - Issue 2 (2026) / ISSN P: 2790-7643

Article ID: 2050

https://doi.org/10.53762/grjnst.04.02.02

Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use and distribution

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Abstract: PCR-based gene-specific primers are essential in crop genetics, but their cross-

genome effectiveness is unclear. This study tested whether wheat (Triticum aestivum L.)

stripe rust resistance markers Yr15 and Yr30 can be amplified in common bean (Phaseolus

vulgaris L.). Genomic DNA from 30 bean genotypes was amplified by PCR, with wheat

DNA and nuclease-free water serving as positive and negative controls. The PCR protocol

used an initial denaturation at 94°C for 5 minutes, 40 cycles (94°C for 60 seconds, 55.8°C

for Yr15 or 52.7°C for Yr30 for 60 seconds, 72°C for 45 seconds), and a final extension at

72°C for 10 minutes. PCR products were separated via agarose gel and visualized. Yr15 was

not detected in any bean genotype, while Yr30 showed the expected 120 bp band in some

genotypes with additional non-specific bands. These findings highlight that gene-specific

primers may fail or yield ambiguous results in unrelated species, emphasizing the need for

rigorous marker validation across species.

Keywords: PCR, Gene, Yr15, Yr30, Amplification, Triticum aestivum L., Phaseolus

Vulgaris L.

Introduction

Polymerase chain reaction (PCR) is a common lab method in crop breeding and genetics that helps

find, copy, and study specific pieces of DNA (Mullis & Faloona, 1987; Saiki et al., 1988). PCR

methods are important for selecting plants with desired traits, figuring out where genes are, and

studying useful traits in crops (Collard & Mackill, 2008; Varshney et al., 2005). The success of PCR

depends mostly on how well the starting pieces of DNA, called primers, match the DNA, how they

attach, and the conditions used, since primers decide which parts of DNA are copied in plants

(Dieffenbach et al., 1993; Kwok et al., 1990; Thornton & Basu, 2011).

Gene-specific primers amplify unique DNA regions linked to distinct genes or loci. These

primers are usually highly specific within their target species (Dieffenbach et al., 1993; Thornton &

Basu, 2011). However, sequence similarity, conserved motifs, primer–template mismatches, and

experimental conditions can decrease specificity. These issues may cause non-specific or cross-species

amplification (Kwok et al., 1990; Lefever et al., 2013). Undetected unintended amplification can lead

to inaccurate or incorrect experimental interpretations, especially when primer performance is not

thoroughly assessed or when primers are used outside their original genetic context (Thornton & Basu,

2011; Bustin et al., 2009).

Stripe rust (yellow rust), caused by Puccinia striiformis f. sp. tritici, is among the most

destructive fungal diseases affecting wheat (Triticum aestivum L.) globally (Chen, 2005; Wellings,

2011). To address this challenge, numerous stripe rust resistance (Yr) genes have been identified and

incorporated into breeding programs (McIntosh et al., 2010). Among these, Yr15 confers broad-

spectrum resistance, while Yr30 provides adult plant resistance. Accordingly, gene-specific or closely

linked primers for these loci are widely utilized for molecular screening and germplasm evaluation in

wheat (Klymiuk et al., 2018; Kokhmetova et al., 2021).

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Primers designed for wheat Yr genes are generally validated exclusively within wheat genetic

backgrounds, and there is limited information regarding their amplification performance in non-target

plant species. Therefore, assessing primer specificity beyond the target genome is crucial for identifying

the limitations of PCR-based assays and for preventing misinterpretation due to non-target

amplification (Bustin et al., 2009; Lefever et al., 2013).

The objective of this study was to evaluate the PCR amplification behavior of wheat stripe rust

resistance gene-specific primers, Yr15 and Yr30, both recognized for their roles in conferring resistance

to wheat stripe rust when applied to common bean (Phaseolus vulgaris L.) genomic DNA. To clarify

the scope, this research focuses solely on amplification patterns and primer specificity, and does not

address gene function, phenotypic traits, or disease resistance.

Material and Methods

Thirty (30) common bean genotypes were collected from diverse agroecological locations in the

Mansehra region of Pakistan. Genomic DNA from 30 bean genotypes was amplified by PCR, with

wheat DNA and nuclease-free water serving as positive and negative controls, respectively. A standard

buffer-based protocol was followed, including purification and precipitation. DNA integrity and

quality were verified by agarose gel electrophoresis prior to PCR amplification. Gene-specific primers

linked to wheat stripe rust resistance genes, Yr15 and Yr30, were used to examine cross-species

amplification behavior. PCR reactions were performed in a 10 µL volume using a commercial master

mix, genomic DNA template, and primer pairs. Amplification was conducted under optimized cycling

conditions. These included denaturation at 94°C for 5 minutes; 40 cycles of 94°C for 60 seconds;

annealing at 55.8°C for Yr15 and 52.7°C for Yr30 for 60 seconds; 72°C for 45 seconds; and final

extension at 72°C for 10 minutes. PCR products were resolved on 1.5% agarose gels alongside a DNA

ladder for fragment size estimation. Gel electrophoresis was performed under standard conditions.

Banding patterns were visualized under ultraviolet illumination and documented using a gel imaging

system. Interpretation was based on direct visualization of amplification profiles in the gel images. For

Yr15, the expected diagnostic fragment size was 390 bp. For Yr30, the expected fragment size was 120

bp. Only bands corresponding to these expected sizes were considered relevant for interpretation. Non-

specific fragments, including lower molecular weight bands and primer dimers, were disregarded. The

results are presented as gel images, which allow clear visual assessment of amplification patterns across

genotypes.

Results and Discussion

Yr15 amplification profile

PCR amplification with the Yr15 gene-specific primer failed to generate the expected 390bp

diagnostic fragment in any of the tested common bean genotypes (Figure 1). No bands corresponding

to the target size were detected in any sample. This lack of amplification aligns with the established

species specificity of Yr15, which encodes the tandem kinase protein WTK1 and has been functionally

characterized in wheat and closely related species (Klymiuk et al., 2018). The absence of cross-

amplification in common bean, a phylogenetically distant legume, likely results from the lack of

GRJNST, Volume: 04 - Issue 2 (2026) / ISSN P: 2790-7643

Article ID: 2050

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conserved primer-binding regions necessary for specific amplification. Comparable findings have been

reported for wheat rust resistance markers, which generally do not amplify in non-cereal species due to

genomic divergence (Lagudah et al., 2009; McIntosh et al., 2010). The consistent absence of the

390bp fragment, therefore, constitutes a biologically meaningful result, underscoring the high

specificity of the Yr15 marker within its native genomic context.

Figure 1. PCR amplification profile of Yr15 in common bean genotypes.

Yr30 amplification profile

PCR amplification with the Yr30 gene-specific primer produced heterogeneous banding patterns

among the analysed common bean genotypes (Figure 2). Some samples had fragments close to the

expected size of 120bp, while others showed bands with slightly lower molecular weight (about 90–

110bp) or no amplification at all. To ensure consistency, only clearly resolved bands near 120bp were

interpreted, with lower molecular weight fragments excluded from analysis. This observed variability in

amplification profiles likely results from partial or non-specific primer binding, a phenomenon

common when gene-specific markers are used outside their native genomic context. Such size

differences have been well documented and are attributed to sequence divergence and primer template

mismatches in non-target species (Kwok et al., 1990; Dieffenbach et al., 1993; Smith et al., 2007).

Importantly, the presence of120bp fragments in some genotypes does not confirm that the Yr30

resistance gene is present, but rather reflects amplification behavior under cross-species conditions. The

absence of amplification for Yr15 contrasts with these results, further highlighting differences in

primer specificity and binding efficiency.

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Figure 2. PCR amplification profile of Yr30 in common bean genotypes.

Conclusion

This study shows that wheat gene-specific primers vary in amplification with non-target species. The

Yr15 primer failed to amplify target-size fragments in any common bean genotype, while the Yr30

primer showed variable results, sometimes producing expected-size fragments. These results suggest

primer specificity, not biological resistance, caused the differences. This highlights the limitation of

using crop-specific molecular markers in different plant genomes and stresses the need for careful

validation before applying PCR-based primers in broader molecular studies.

Author Contribution

Muhammad Mehraj Riasat: Supervise the research, Muhammad Waleed and Uzair Ahmad: Lab work,

Farhan Tahir: laboratory assistance, Shafaq Munir and Atika Liaqat: Write original manuscript, Saqib

Waqar Ahmed: Sample collection. All authors contributed to the review and editing of the manuscript.

Declaration of Funding

This research was supported by the Department of Agriculture, Hazara University, Mansehra, Pakistan.

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References

Bustin, S. A., Benes, V., Garson, J. A., Hellemans, J., Huggett, J., Kubista, M., & Wittwer, C. T.

(2009). The MIQE Guidelines: Minimum Information for Publication of Quantitative Real-

Time PCR Experiments.

Collard, B. C., & Mackill, D. J. (2008). Marker-assisted selection: an approach for precision plant

breeding in the twenty-first century. Philosophical Transactions of the Royal Society B:

Biological Sciences, 363(1491), 557-572.

Chen, X. M. (2005). Epidemiology and control of stripe rust [Puccinia striiformis f. sp. tritici] on

wheat. Canadian journal of plant pathology, 27(3), 314-337.

Dieffenbach, C. W., Lowe, T. M., & Dveksler, G. S. (1993). General concepts for PCR primer

design. PCR methods appl, 3(3), S30-S37.

Kwok, E. S., Kellogg, D. E., McKinney, N., Spasic, D., Goda, L., Levenson, C., & Sninsky, J. J. (1990).

Effects of primer-template mismatches on the polymerase chain reaction: human

immunodeficiency virus type 1 model studies. Nucleic acids research, 18(4), 999-1005.

Klymiuk, V., Yaniv, E., Huang, L., Raats, D., Fatiukha, A., Chen, S., & Fahima, T. (2018). Cloning of

the wheat Yr15 resistance gene sheds light on the plant tandem kinase-pseudokinase

family. Nature communications, 9(1), 3735.

Kokhmetova, A., Rsaliyev, A., Malysheva, A., Atishova, M., Kumarbayeva, M., & Keishilov, Z. (2021).

Identification of stripe rust resistance genes in common wheat cultivars and breeding lines from

Kazakhstan. Plants, 10(11), 2303.

Lefever, S., Pattyn, F., Hellemans, J., & Vandesompele, J. (2013). Single-nucleotide polymorphisms

and other mismatches reduce performance of quantitative PCR assays. Clinical

chemistry, 59(10), 1470-1480.

Lagudah, E. S., Krattinger, S. G., Herrera-Foessel, S., Singh, R. P., Huerta-Espino, J., Spielmeyer, W.,

& Keller, B. (2009). Gene-specific markers for the wheat gene Lr34/Yr18/Pm38 which

confers resistance to multiple fungal pathogens. Theoretical and Applied Genetics, 119(5),

889-898.

Mullis, K. B., & Faloona, F. A. (1987). [21] Specific synthesis of DNA in vitro via a polymerase-

catalyzed chain reaction. In Methods in enzymology (Vol. 155, pp. 335-350). Academic Press.

McIntosh, R. A., Dubcovsky, J., Rogers, W. J., Morris, C., Appels, R., & Xia, X. C. (2010). Catalogue

of gene symbols for wheat: 2010 supplement. Annual wheat newsletter, 56, 273-282.

Saiki, R. K., Gelfand, D. H., Stoffel, S., Scharf, S. J., Higuchi, R., Horn, G. T., & Erlich, H. A. (1988).

Primer-directed

enzymatic

amplification

of

DNA

with

a

thermostable

DNA

polymerase. Science, 239(4839), 487-491.

GRJNST, Volume: 04 - Issue 2 (2026) / ISSN P: 2790-7643

Article ID: 2050

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G. 2050

Page 7

Smith, A. G., Croft, M. T., Moulin, M., & Webb, M. E. (2007). Plants need their vitamins

too. Current opinion in plant biology, 10(3), 266-275.

Thornton, B., & Basu, C. (2011). Real‐time PCR (qPCR) primer design using free online

software. Biochemistry and molecular biology education, 39(2), 145-154.

Varshney, R. K., Graner, A., & Sorrells, M. E. (2005). Genomics-assisted breeding for crop

improvement. Trends in plant science, 10(12), 621-630.

Wellings, C. R. (2011). Global status of stripe rust: a review of historical and current

threats. Euphytica, 179(1), 129-141.

GRJNST, Volume: 04 - Issue 2 (2026) / ISSN P: 2790-7643

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