Cost-effective duplex Kompetitive Allele Specific PCR markers for homologous genes facilitating wheat breeding
BMC Plant Biology volume 23, Article number: 119 (2023)
Owing to successful cloning of wheat functional genes in recent years, more traits can be selected by diagnostic markers, and consequently, effective molecular markers will be powerful tools in wheat breeding programs.
The present study proposed a cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system that combined multiplex PCR and KASP™ technology to yield twice the efficiency at half the cost compared with the common KASP™ markers and provide great assistance in breeding selection. Three dKASP markers for the major genes controlling plant height (Rht-B1/Rht-D1), grain hardness (Pina-D1/Pinb-D1), and high-molecular-weight glutenin subunits (Glu-A1/Glu-D1) were successfully developed and applied in approved wheat varieties growing in the middle and lower reaches of the Yangtze River and advanced lines from our breeding program. Three markers were used to test six loci with high efficiency. In the approved wheat varieties, Rht-B1b was the most important dwarfing allele, and the number of accessions carrying Pinb-D1b was much greater than that of the accessions carrying Pina-D1b. Moreover, the number of accessions carrying favorable alleles for weak-gluten wheat (Null/Dx2) was much greater than that of the accessions carrying favorable alleles for strong-gluten wheat (Ax1 or Ax2*/Dx5). In the advanced lines, Rht-B1b and Pinb-D1b showed a significant increase compared with the approved varieties, and the strong-gluten (Ax1 or Ax2*/Dx5) and weak-gluten (Null/Dx2) types also increased.
A cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed, which would greatly improve the efficiency of marker-assisted selection of wheat.
A cost-effective duplex Kompetitive Allele Specific PCR (dKASP) marker system was proposed to achieve double efficiency at half the cost, and three dKASP markers were successfully developed and applied.
Numerous essential traits of wheat, including disease resistance, grain quality, and grain yield, are typical quantitative traits whose significant genotype-by-environment interactions hinder the accuracy of phenotypic evaluation [1,2,3]. Conversely, molecular markers reflecting differences in genome sequences are independent from environmental influences, and hence, have emerged as a powerful tool in wheat breeding programs [4, 5]. Fhb1 to Fusarium head blight (FHB) and Sr2 to stem rust are valuable resistance genes in wheat; however, these genes are linked in the repulsion phase. Zhang  successfully developed elite lines with two genes using diagnostic markers, remarkably promoting wheat resistance breeding. Pina and Pinb are the major genes controlling grain hardness, and when foreground and background selections were performed simultaneously using molecular markers, dozens of BC2F2 soft lines were obtained . In recent years, many functional genes have been successfully cloned [8,9,10], thereby enabling the selection of more traits by diagnostic markers and ensuring the cost-efficiency of marker-assisted selection (MAS).
In the past two decades, substantial progress has been achieved in the field of molecular marker technology, which has considerably contributed to genetic research and breeding application. From hybridization-, PCR-, and to the latest sequencing-based markers [11,12,13], the quantity, capacity, and operability were greatly improved. Recently, single nucleotide polymorphism (SNP) markers have been receiving more attention owing to their wide distribution and excellent stability , and several identification technologies corresponding to different situations have also been developed, such as SNP array , genotyping by target sequencing (GBTS) , cleaved amplified polymorphic sequence (CAPS) , and Kompetitive Allele Specific PCR (KASP™) . SNP array and GBTS, which integrate thousands of SNPs, have widely been used in genetic mapping and genetic diversity analysis; however, their high cost limited the sample size. CAPS markers were based on enzyme digestion and are suitable for a small sample. KASP™ is a homologous, fluorescence-based technology that is particularly suitable for large samples with few markers, and therefore, prefect for the MAS in large-scale breeding.
In recent years, some databases including numerous wheat KASP™ markers have been established, which will assist wheat breeders and scientists to select the most appropriate markers for MAS breeding [5, 18]. CerealsDB version 3.0 is an online resource that contains a wide range of genomic datasets for wheat , and provides a total of 4992 mapped KASP™ markers, which can be used in germplasm resources evaluation and MAS. Another marker database containing 122 functional KASP™ markers is proposed by Chinese Academy of Agricultural Sciences , and markers covering many key wheat traits including yield, disease resistance, and grain quality, have been widely used in many breeding institutions [19, 20].
KASP™ primers comprise two allele-specific primers (F1, F2) carrying fluorescence tails and one common primer (C) . Generally, the two allele-specific primers correspond to a favorable and unfavorable allele. The ultimate goal of breeding selection is to obtain lines carrying favorable alleles, which enables further improvement in the cost and efficiency by optimizing the present marker system. Here a duplex KASP (dKASP) marker system was designed for certain homologous genes with the aim of providing a more efficient method for conducting MAS.
Development of the dKASP markers
The two allele-specific primers of the KASP™ markers usually correspond to the different alleles of one functional gene, and hence, the genotyping result included four types of alleles: AA, BB, AB, and no template control (NTC). In this study, we developed the dKASP markers whose allele-specific primers corresponded to the different alleles of the two homologous genes (Fig. 1), which also showed four types: allele A for gene 1 + allele A for gene 2, allele A for gene 1 + allele B for gene 2, allele B for gene 1 + allele A for gene 2, and allele B for gene 1 + allele B for gene 2. In this manner, two genes were identified in one reaction, and the detection efficiency was doubled while halving the cost.
In the present study, the dKASP markers for the major genes controlling plant height (PH) (Rht-B1 and Rht-D1), grain hardness (GH) (Pina-D1 and Pinb-D1), and high-molecular-weight glutenin subunit (HMW-GS) (Glu-A1 and Glu-D1) were developed (Fig. 2 and Table 1), and the detection results of the dKASP markers were consistent with those of the diagnostic markers (some accessions were repeated twice) (Fig. 3 and Supplementary Table S1) and previous studies [19, 21,22,23]. Pina-D1b was produced by the loss of Pina, and a specific primer based on the Pina-D1a sequence was designed. Three allelic variations were present at Glu-A1, and the materials carrying Ax1 and Ax2* exhibited better gluten strength than those carrying Null. In the present study, Ax1 and Ax2* shared the same allele and were categorized into one group, whereas the marker, Glu-AD, was used to differentiate Null from Ax1 and Ax2*.
Distribution of the major genes in the approved wheat varieties in the middle and lower reaches of the Yangtze River
Three dKASP markers were used to screen 212 wheat accessions, including the approved varieties and elite lines, in a regional test from 1966 to 2021 (Fig. 4 and Supplementary Table S2). Rht-B1b was found to be the most important dwarfing allele, accounting for over three fourths of the tested accessions, and the accessions carrying Rht-D1b were mainly from institutions in Hubei province, while only three accessions had both Rht-B1b and Rht-D1b. Meanwhile, 11 accessions showed no dwarfing alleles at the two loci. More than half the accessions were soft type (Pina-D1a/Pinb-D1a), and the number of accessions carrying Pinb-D1b (39.34%) was much greater than that of the accessions carrying Pina-D1b (8.06%), while no Pina-D1b/Pinb-D1b type was detected. Types A (Ax1 or Ax2*/Dx5) and B (Null/Dx2) at Glu-A1/Glu-D1 were the favorable alleles for strong- and weak-gluten wheat, respectively, while the number of type B accessions was much greater than that of type A accessions, which was consistent with the classification of Chinese wheat regions based on quality .
More accessions carrying Rht-B1b were identified over time, and those without dwarfing alleles decreased significantly. Moreover, there were no accessions carrying both Rht-B1b and Rht-D1b after 2010. A substantial growth from 17.86 to 51.11% was observed for type A at Pina-D1/Pinb-D1, while an opposite trend was observed for types B and C. Both types A and B of Glu-A1/Glu-D1 presented a growing trend, indicating a directional selection of wheat quality.
Distribution of the major genes in the advanced lines
In total, 518 advanced lines from the breeding program were screened using the three developed dKASP markers (Fig. 5 and Supplementary Table S3). The distribution was in accordance with that of the 212 wheat accessions over time. Types B, C, and D of Rht-B1/Rht-D1 accounted for only less than 10% in the advanced lines, while Rht-B1b was the dominant dwarfing allele. Type A of Pina-D1/Pinb-D1 was the most common, while type B decreased to below 1%. The proportions of types A and D of Glu-A1/Glu-D1 significantly increased compared with those in the above 212 wheat accessions.
According to the technique used, plant breeding can be split into four major stages . Whereas the international commercial seed industries of developed countries are shifting to breeding 4.0, known as molecular design breeding, most institutions in China remain in a stage of breeding from 2.0 to 3.0, which involves selection by phenotype statistics and molecular MAS, respectively . Breeding 3.0 and 4.0 involves the necessary screening of the major genes that control key traits, and hence, in these breeding techniques, a molecular marker platform plays a crucial role. Presently, electrophoretic markers, such as simple sequence repeat and sequence tagged site markers, remain the most commonly used markers in numerous breeding institutions ; however, their detection efficiency is low. KASP™ genotyping assay, proposed by the Laboratory of the Government Chemist in the United Kingdom, is a homogeneous fluorescence-based assay that enables the accurate bi-allelic discrimination of known SNPs and InDels. KASP™ has become popular owing to its high detection efficiency and acceptable cost . In wheat breeding, numerous KASP™ markers for functional genes have been developed and widely applied [4, 5, 28]. In the present study, we proposed a cost-effective method dKASP that combines KASP™ technology and multiplex PCR to test two functional genes simultaneously to achieve twice the efficiency at half the cost.
The dKASP markers were developed based on the KASP™ technology, which involves two allele-specific primers and one common primer. Unlike the usual KASP™ markers, the two allele-specific primers of the dKASP markers are based on the sequence of two homogeneous genes. To date, many homogeneous wheat genes have been identified, here we successfully developed three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) [29,30,31,32]. These markers were applied in the approved wheat varieties and advanced lines as well. Sequence homology limited the development of dKASP markers for numerous non-homologous genes. We attempted to apply the common multiplex PCR system involving two groups of specific primers for two non-homologous genes, such as Fhb1/Fhb7 for Fusarium head blight (FHB) resistance and Fhb1/Pm21 for resistance to FHB and Powdery mildew [8, 10, 33]. Four reaction systems with different ratios of the primers and two programs with different touchdown temperatures (61–55 °C and 65–59 °C) were tested, but unfortunately the results were not stable at present, and the system required further optimization. Other solutions need to be proposed to enhance the development of dKASP markers. Additionally, although the dKASP markers are dominant, both homozygous and heterozygous lines with favorable genotypes are usually retained to obtain more useful and noteworthy progenies during practical breeding; hence, dominant markers also engender a suitable effect. Moreover, the dKASP markers could be applied to other crops, including rice and maize, thereby improving the efficiency of MAS.
In this study, three dKASP markers were used to test six loci of the approved wheat varieties and advanced lines; this efficiently clarified the historical and present distribution of the major genes in the middle and lower reaches of the Yangtze River. Artificial selection is critical for local breeding and retains more favorable alleles, such as type A of Rht-B1/Rht-D1, type A of Pina-D1/Pinb-D1, and types A and D of Glu-A1/Glu-D1. Rht-B1b was the major dwarfing allele in the tested lines, which was consistent with the result of previous studies, while Rht-D1b was mainly distributed in the Huanghuai wheat region [34, 35]. As a serious disease in the middle and lower reaches of the Yangtze River, FHB resistance received heavy attention in local breeding, and Rht-D1b presented a greater negative effect on FHB resistance than Rht-B1b . This might be due to its linkage drag with resistance QTL and its positive effects on spikelet density [35, 37]. The frequent use of elite parents, such as Yangmai 158, Ningmai 8, Ningmai 9, and Emai 12, might be another reason for the dominant proportion of Rht-B1b [19, 38, 39]. Moreover, decreased genetic diversity is a critical problem in modern breeding, and hence, the exploration and application of genetic resources have been receiving more attention recently. With the application of resistance genes and prevention technology to FHB, we could attempt to introduce Rht-D1b into the present breeding program to improve the genetic diversity and agronomic traits of wheat [35, 40].
With the development of society, wheat quality has been receiving increasing attention. GH is an important index for evaluating the quality and certifying the variety, and HMW-GS has proved to be significantly correlated with the processing quality of wheat [41, 42]. Pina-D1a/Pinb-D1b (type A) had significantly softer grain, higher break flour yield, flour yield, milling score, and loaf volume than Pina-D1b/Pinb-D1a (type B) [43, 44]. Ma et al. detected Pina-D1/Pinb-D1 in 1787 accessions, and the number of accessions carrying Pina-D1a/Pinb-D1b was much greater than that of accessions carrying Pina-D1b/Pinb-D1a . Similar results were observed in numerous other studies [46, 47]. In the present study, both the favorable types of strong-gluten wheat varieties, i.e., those with type A of Pina-D1/Pinb-D1 and type A of Glu-A1/Glu-D1, exhibited a significant increase in the advanced lines, thereby meeting the demand for strong-gluten wheat varieties in the market. Overall, 118 lines (22.78%) with favorable types for strong-gluten wheat varieties were selected in addition to 37 lines (7.14%) with favorable types for weak-gluten wheat varieties, including those with type C of Pina-D1/Pinb-D1 and type B of Glu-A1/Glu-D1. This provided abundant candidate lines for special usage wheat breeding. Meanwhile, Ax2* has rarely been identified in Chinese wheat accessions [48,49,50]; hence, the favorable type of strong-gluten at Glu-A1 might mostly be Ax1. Therefore, the dKASP markers of Pin-ab and Glu-AD can be useful for the directional selection of wheat quality breeding.
In the present study, a cost-effective dKASP marker system that combined multiplex PCR and KASP™ technology was proposed to achieve double the efficiency at half the cost compared with the common KASP™ markers. Three dKASP markers for the major genes controlling PH (Rht-B1/Rht-D1), GH (Pina-D1/Pinb-D1), and HMW-GS (Glu-A1/Glu-D1) were successfully developed and applied in the approved wheat varieties and advanced lines, and indicated a dominant proportion of Rht-B1b and directional selection of wheat quality in the present breeding program.
Materials and methods
This study used three panels of wheat lines. The first included 21 Chinese wheat varieties or accessions and one introduced accession that were used for marker development (Table S1). The second included 212 wheat varieties approved from 1966 to 2021 growing in the middle and lower reaches of the Yangtze River, which represented the distribution of the tested genes in modern wheat varieties (Table S2). The third included 518 F5 lines developed by our breeding program showing the application of favorable alleles (Table S3). The total genomic DNA of the materials was isolated from young leaf tissues using the cetyltrimethylammonium bromide (CTAB) method.
Development and application of the dKASP markers
Three homologous gene groups were selected for marker development (Table 2). Diagnostic markers were synthesized according to Rasheed . A function module of AlignX in software Vector NTI was used to search for sequence differences. Specific primers were artificially designed based on the differences, evaluated by Primer 6.0, and synthesized by Shanghai Sangon Biotech Co., Ltd. Standard FAM (5′GAAGGTGACCAAGTTCATGCT 3′) and HEX (5′ GAAGGTCGGAGTCAACGGATT 3′) tails were added to the front of the allele-specific primers F1 and F2, respectively.
KASP™ assays were performed in 384-well PCR plates with 2.5 μL of KASP™ V4.0 2X Master mix standard ROX (LCG Genomics, Beverly, USA), 0.07 μL of assay mix (12 μL of each allele-specific forward primer [100 μM], 30 μL of reverse primer [100 μM], and 46 μL of TRIS [10 mM, pH 8.3]), and 2.43 μL of 30 ng μL− 1 genomic DNA. The PCR program was as follows: 94 °C for 15 min; 10 touchdown cycles of 94 °C for 20 s and 61 °C–55 °C for 60 s (decreasing by 0.6 °C per cycle); and 26 cycles of 94 °C for 20 s and 55 °C for 60 s. The PCR reactions were conducted in Hydrocycler 16 water instrument (LGC Genomics, Beverly, USA), and PCR fluorescence detection was performed using PHERAstar (BMG LABTECH, Germany) microplate reader. KlusterCaller software (LGC Genomics, Beverly, USA) was used for data analysis.
Availability of data and materials
The data that support the findings of this study are available in the supplementary material of this article.
Duplex Kompetitive Allele Specific PCR
Fusarium head blight
Single nucleotide polymorphism
Genotyping by target sequencing
Cleaved amplified polymorphic sequence
No template control
High-molecular-weight glutenin subunit
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This work was financially supported by Jiangsu Provincial Key Research and Development Program (BE2022346 and BE2022337) and Seed industry revitalization project of Jiangsu Province (JBGS(2021)012 and JBGS(2021)006). The funding bodies played no role in the design of the study and collection, analysis, and interpretation of data and in writing the manuscript.
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Genotyping of the diagnostic markers and dKASP markers for plant height, grain hardness, and high-molecular-weight glutenin in 22 wheat varieties and germplasm resources. A, B, C, and D correspond to the genotypes in Table 1.
Genotyping of the dKASP markers for plant height, grain hardness, and high-molecular-weight glutenin in 212 wheat varieties approved from 1966 to 2021 growing in the middle and lower reaches of the Yangtze River. A, B, C, and D correspond to the genotypes in Table 1.
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Jiang, P., Fan, X., Zhang, G. et al. Cost-effective duplex Kompetitive Allele Specific PCR markers for homologous genes facilitating wheat breeding. BMC Plant Biol 23, 119 (2023). https://doi.org/10.1186/s12870-023-04116-y