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. 2022 Mar 8;31(4):423–431. doi: 10.1007/s10068-022-01043-w

High-throughput SNP markers for authentication of Korean wheat cultivars based on seven complete plastomes and the nuclear genome

Woohyeon Cho 1, Jin-Kee Jung 2, Min-Young Kang 2, Yong Weon Seo 3, Jee Young Park 1,✉, Tae-Jin Yang 1,✉
PMCID: PMC8994797  PMID: 35464241

Abstract

Wheat (Triticum aestivum) has diverse uses in the food industry, and different cultivars have unique properties; therefore, it is important to select the optimal cultivar for the intended end use. Here, to establish an identification system for Korean wheat cultivars, we obtained the complete plastome sequences of seven major Korean cultivars. Additionally, the open access database CerealsDB was queried to discover single-copy genomic single-nucleotide polymorphisms (SNPs) in the hexaploid wheat genome. Ten SNPs were developed into allele-specific PCR (ASP) markers, and eight of the SNPs used for ASP markers were converted into TaqMan high-throughput genotyping markers. Phylogenetic analysis using SNP genotypes revealed the genetic diversity and relationships among 137 wheat lines from around the world, including 35 Korean cultivars. This research thus presents a high-throughput authentication system for Korean wheat cultivars that may promote food industry uses of Korean wheat.

Supplementary Information

The online version contains supplementary material available at 10.1007/s10068-022-01043-w.

Keywords: Wheat, Fluorescence, Plastome, Polyploidy, Authentication

Introduction

Common bread wheat (Triticum aestivum) is one of the three most widely grown crops in the world and is the second most consumed grain in Korea, with a daily consumption of 87.11 g per person (Ministry of Agriculture, Food and Rural Affairs, 2021). The Korean wheat industry relies on imports (98.9%) rather than domestic production (1.1%) because of unstable supply of ideal cultivar and lack of diverse wheat cultivars in Korea (Ministry of Agriculture, Food and Rural Affairs). Korean farmers sometimes cultivate wheat crops with self-produced mixed seeds, which result in low quality of wheat products. Therefore, a systematic study to improve the uniformity and quality of domestic wheat cultivars is needed to enhance consumption and establish a robust Korean wheat industry.

Presently, 52 wheat cultivars are registered by the Korea Seed & Variety Service (KSVS). Promoting food industry uses of Korean wheat, requires that each wheat production process, from farming to milling and processing into various food or industrial products, uses the ideal cultivar. Therefore, a wheat cultivar authentication system is necessary for the farm field as well as the food processing industry. High-throughput authentication systems can be used on plants, seeds, and even processed flours. Breeders can use these markers to maintain the purity of seeds for each cultivar and ensure that Korean farmers are growing genotypes with the right combinations of grower-preferred traits (such as disease resistance, yield) and consumer-preferred traits (such as gluten content). Moreover, producers, processors, and distributors can use these markers as quality control for wheat flours, enabling them to produce and sell high-value, locally grown wheat and wheat products, and thus boosting the Korean wheat industry.

In wheat, protein content determines the strength of the grain and in turn determines how the grain is used for bread, noodles, pastries, or other food products. Wheat cultivars with 13% or higher gluten content are used for ‘hard’ flour to make bread or pasta. ‘Strong’ flour with a gluten content of 10–12% is used for flour all-purpose, and ‘soft’ flour with a gluten content of 10% or less is used for cakes and cookies. Each of the Korean wheat cultivars has a distinct gluten content, which is an important characteristic for food processing. The cultivars ‘Baekkang’ and ‘Jokyoung’ are mainly used for bread; ‘Saekeumkang’, ‘Sooan’, ‘Baegjoong’, and ‘Keumkang’ are used for noodles; and ‘Goso’ is used for cookies (Rural Development Administration, 2021). Cultivar ‘Jokyoung’ was bred at the Rural Development Administration in 1995, and ‘Baekkang’ was bred at the National Institute of Crop Science in 2005. Presently, ‘Baekkang’ is often grown instead of ‘Jokyoung’ due to its higher resistance to Fusarium head blight, the most destructive disease of wheat, as well as its more stable yield (National Institute of Crop Science, 2021).

Wheat has a hexaploid genome (2n = 6x = 42) about 17 Gbp in size, making it difficult to study (Brenchley et al., 2012; IWGSC, 2014). However, since wheat is an important grain crop worldwide, a high-quality genome has been published, with 21 paired chromosomes (Chapman et al., 2015; Marino et al., 1996; Zimin et al., 2017). Analysis based on the sequenced genome has revealed an abundance of variation, including single-nucleotide polymorphisms (SNPs), some of which have been developed into molecular markers for genotyping using diverse platforms. The online resource CerealsDB, an open access online database containing about 100,000 SNPs and expressed sequence tags (ESTs), was established in 2012 and is constantly updated (Wilkinson et al., 2012; 2020). CerealsDB contains SNP datasets validated by various marker platforms such as KASP, TaqMan, Axiom (Affymetrix), and iSelect (Illumina) (Allen et al., 2017; Burridge et al., 2018; Wilkinson et al., 2016; Winfield et al., 2016), and it is actively utilized by many research groups (Dong et al., 2014; Xue et al., 2015).

Plastid genomes (plastomes) are excellent targets for rapid and reliable marker development due to their unique characteristics. For example, plastomes range from 107 to 218 kb, which is significantly smaller than the nuclear genome (Daniell et al., 2016). Also, it is easy to amplify target sequences from plastomes, since each leaf cell has upwards of 10,000 copies of the chloroplast genome (Morley and Nielsen, 2016). Finally, the typically uniparental inheritance of this highly conserved genome facilitates reliable plant classification (Birky, 1995).

In 2013, a sequence characterized amplified region (SCAR) marker for domestic wheat cultivar identification was developed (Son et al., 2014). This marker can distinguish 13 domestic wheat cultivars, but the gel-based marker is costly and data interpretation is time consuming. Therefore, we attempted to establish a reliable and cost-effective high-throughput SNP genotyping platform. TaqMan is a commercial high-throughput tool to detect the accurate SNP genotype (Ayalew et al., 2019). In this study, we developed high-throughput SNP markers for the authentication of major domestic wheat cultivars in Korea compatible with the TaqMan system. We expect this marker set to find practical applications in improving wheat production in Korea.

Materials and methods

Plant materials and DNA extraction

Thirty-five Korean wheat cultivars registered in KSVS were used for this study (Table S1). Seeds provided by KSVS and Korea University (KU) were planted and grown in a greenhouse at Seoul National University. For each cultivar, seven individuals were randomly selected and pooled for DNA extraction. To study the heterogeneous genotype of ‘Jokyoung’, more than 200 seeds of ‘Jokyoung’ provided by KSVS and KU were analyzed using DNA from individual plants or pooled DNA. Fresh leaves were frozen in liquid nitrogen and ground with a mortar and pestle. DNA was extracted using the Plant SV mini kit (GeneAll Biotechnology, Seoul, Korea), and the amount and quality of DNA were measured using the NanoDrop ND-1000 (Thermo Fisher Scientific, Waltham, MA, USA).

Plastome assembly, annotation, and variant identification

Among the 35 Korean wheat cultivars, plastome assembly was conducted for seven major cultivars: ‘Sooan’, ‘Jokyoung’, ‘Baegjoong’, ‘Goso’, ‘Baekkang’, ‘Keumkang’, and ‘Saekeumkang’. Whole-genome shotgun sequencing was performed using the Illumina Miseq platform (Illumina, San Diego, CA, USA), producing 957 Mb to 1.37 Gb of raw data for each cultivar. The de novo plastome sequences of the seven major cultivars were assembled and manually corrected by following the de novo assembly of low-coverage whole-genome sequence (dnaLCW) method using the CLC genome assembler program (ver. 4.6 beta, CLC Inc., Aarhus, Denmark) (Kim et al., 2015).

Complete plastome sequences were annotated using GeSeq (https://chlorobox.mpimp-golm.mpg.de/geseq.html) (Tillich et al., 2017) and manually corrected using the Artemis program (Rutherford et al., 2000). Plastome sequences of seven major cultivars were compared to identify variants using the MAFFT 7.0 (https://mafft.cbrc.jp/alignment/server/) (Kuraku et al., 2013).

Single-copy SNP detection in the nuclear genome

For identification of single-copy SNPs, nuclear genome–derived SNPs and their ESTs were obtained from the open access online resource CerealsDB (https://www.cerealsdb.uk.net/cerealgenomics/Cereals-DB/) (Wilkinson et al., 2012). Additional genotyping-by-sequencing (GBS) data for Korean wheat cultivars were provided by KSVS. The SNPs existing alone in each EST were selected, and paralogous sequences were removed by identifying the 150-bp flanking sequence of each SNP using BLAST (Altschul et al., 1990). Sequences were aligned to the reference whole-genome sequence of wheat cultivar Chinese Spring with the maximum E-value set to 10. Candidate SNPs matching a single region of the whole-genome sequence of wheat cultivar Chinese Spring were selected for further analysis.

SNP validation with allele-specific PCR markers

Detected candidate SNPs from plastid and nuclear genomes were validated with allele-specific PCR (ASP) markers. One set of ASP markers consists of two allele-specific primers and one common primer. To amplify the target region more specifically, a mismatch was inserted one or two base pairs away from the SNP towards the 5′ end. To design markers specifically amplifying only the target region, BLAST in CerealsDB and the PrimerBlast program (Ye et al., 2012) were used with maximum E-values of 10. SNPs detected based on plastome sequences were developed into ASP markers using the PrimerBlast program.

PCR amplification was conducted in 25-µL reactions containing 1 unit of Taq polymerase (Inclone Biotech, Seongnam, Korea), 2.5 µL of 10 × reaction buffer, 0.2 mM dNTPs, 20 ng genomic DNA, and 10 pM of each primer. The thermal cycling conditions were as follows: 95 °C for 5 min, 38 cycles of 95 °C for 15 s, 62 °C for 15 s, 72 °C for 15 s, and one cycle of 72 °C for 7 min. The PCR products were loaded into 1% agarose gels containing safe gel stain (Inclone Biotech) with a 100-bp ladder. Electrophoresis was conducted for 30 min at 100 V, and bands were visualized under UV light. Verification of designed ASP markers was performed by testing all seven cultivars. Valuable markers that revealed genotype variation between the seven major cultivars and only amplified the target locus were selected.

Development and application of markers for high-throughput genotyping

TaqMan markers were developed with the SNPs validated by ASP markers, and 35 Korean wheat cultivars were tested. PCR amplification was performed in 20-µL reactions containing 10 µL of 2 × TaqMan Fast Advanced Master Mix (Applied Biosystems, Waltham, MA, USA), 1 µL of 20 × TaqMan marker, 20 ng genomic DNA, and 7 µL of nuclease-free water. The thermal cycling and plate reading were performed using a Roche LC480 (Roche Diagnostics, Penzberg, Germany). The thermal cycling conditions were as follows: 50 °C for 2 min, 95 °C for 20 s, 40 cycles of 95 °C for 3 s, and 60 °C for 30 s.

Genetic diversity analysis

Major allele frequency (MAF), gene diversity (GD), observed heterozygosity (Ho), and polymorphism information content (PIC) were calculated with genotyping results using the PowerMarker v3.25 program (Liu and Muse, 2005). The dendrogram was drawn using genotypes of 112 foreign wheat germplasm lines and 35 Korean wheat cultivars (Table S1 and S2). Genotypes of foreign wheat lines at each SNP position were obtained from CerealsDB. The phylogenetic analysis was performed based on the unweighted pair group method with arithmetic mean (UPGMA) algorithm using the PowerMarker program. The dendrogram was visualized with genetic distance statistics using the MEGA v6.0 program (Tamura et al., 2013).

Results and discussion

Assembly of complete plastomes for seven major Korean wheat cultivars

The plastome is a major molecular target for plant taxonomy, comparative analysis, and resource identification because of its comparatively conserved sequence than the nuclear genome. For Korean wheat cultivars, this study presents the first complete plastome sequencing effort and development of DNA markers based on the plastome. We completed the plastome sequences for seven major Korean wheat cultivars. The total length of the plastome was 135,909 bp for ‘Sooan’, ‘Baegjoong’, ‘Goso’, ‘Keumkang’, and ‘Saekeumkang’, and 135,900 bp for ‘Jokyoung’ and ‘Baekkang’. The lengths of large single copy (LSC), small single copy (SSC), and inverted repeat (IR) regions were 80,005–80,014 bp, 12,791 bp, and 21,552 bp, respectively (Table 1). The average sequence read coverage ranges from 90.4 × to 231 × for plastome assembly of each cultivar. All seven cultivars’ plastomes have 131 functional genes, which includes 85 protein-coding genes, 38 tRNA genes, and 8 rRNA genes (Fig. 1).

Table 1.

Sequencing and de novo assembly of the plastomes of seven major Korean wheat cultivars

Cultivar NGS information Average coverage (×) Plastome (bp) GenBank Acc. No
Total read bases (bp) Total reads Total LSC SSC IR
Baegjoong 957,761,332 3,241,732 90.4 135,909 80,014 12,791 21,552 MW889054
Baekkang 1,139,847,268 3,786,868 152.3 135,900 80,005 12,791 21,552 MW889055
Goso 1,315,569,262 4,370,662 231.0 135,909 80,014 12,791 21,552 MW889056
Jokyoung 1,134,534,618 3,769,218 204.1 135,900 80,005 12,791 21,552 MW889057
Keumkang 1,371,467,972 4,556,372 133.5 135,909 80,014 12,791 21,552 MW889058
Saekeumkang 1,191,377,866 3,958,066 125.1 135,909 80,014 12,791 21,552 MW889059
Sooan 1,298,377,948 4,313,548 103.8 135,909 80,014 12,791 21,552 MW889060
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LSC, large single copy; SSC, small single copy; IR, inverted repeat

Fig. 1.

Fig. 1

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Complete plastome map of seven Korean wheat cultivars. Genes shown on the outside of the outer circle are transcribed clockwise and genes inside the circle are transcribed counter-clockwise. The grey area in the inner circle indicates the GC content, and the color of the gene boxes represents the classified function. Red and blue arrows indicate SNP and InDel variations identified between the seven major Korean wheat cultivars

Plastome sequences of ‘Sooan’, ‘Baegjoong’, ‘Goso’, ‘Keumkang’, and ‘Saekeumkang’ were identical, differing from ‘Jokyoung’ and ‘Baekkang’, which were identical to each other. There were four SNPs and four insertion and deletions (InDels) in two types of plastome sequences (Table 2). Among the variants, four SNPs were developed into ASP markers and validated in all seven wheat cultivars (Fig. 2A and Table S3). Markers were designed from unique sequences in the plastome while avoiding the mitochondrial plastid DNA (MTPT), which occurred via intracellular transfer of plastome sequence into the mitochondrial genome and can cause ambiguity in interpreting genotypes (Park et al., 2020).

Table 2.

Genotypes of seven major Korean wheat cultivars on variants identified from the plastome

Variant type Positiona Major Korean wheat cultivar ASP marker ID TaqMan marker ID
SA BJ GS KK SKK JK BK
SNP 29,539 T T T T T C C whcpASP001 –
46,215 A A A A A T T whcpASP002 –
74,336 A A A A A G G whcpASP003 whTM004
77,287 T T T T T C C whcpASP004 whTM005
InDel 60,785 (T)16 (T)16 (T)16 (T)16 (T)16 (T)17 (T)17 – –
62,666 (T)11 (T)11 (T)11 (T)11 (T)11 (T)12 (T)12 – –
65,666 5 × 2 5 × 2 5 × 2 5 × 2 5 × 2 5 × 1 5 × 1 – –
78,601 (A)14 (A)14 (A)14 (A)14 (A)14 (A)8 (A)8 – –
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aPosition information is based on the ‘Baekkang’ sequence

SA, ‘Sooan’; BJ, ‘Baegjoong’; GS, ‘Goso’; KK, ‘Keumkang’; SKK, ‘Saekeumkang’; JK, ‘Jokyoung’; BK, ‘Baekkang’

Fig. 2.

Fig. 2

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Genotyping with 10 ASP markers for seven wheat cultivars. (A-B) Agarose gel image of four plastome (A) and six nuclear genome-derived (B) markers. Rows indicate markers, and columns indicate each of the seven major Korean cultivars. The left and right bands represent genotypes of each cultivar in each marker. Major and minor alleles are indicated by uppercase and lowercase, respectively, and cultivars having minor alleles are marked with blue asterisks for each marker. Heterogeneous genotypes are marked by red asterisks for the two markers whASP018 and 026. For the whASP010 marker, an off-target band smaller than 100 bp, which was presumed to be a primer-dimer, was ignored when interpreting the genotype results. L, ladder; SA, ‘Sooan’; BJ, ‘Baegjoong’; GS, ‘Goso’; KK, ‘Keumkang’; SKK, ‘Saekeumkang’; JK, ‘Jokyoung’; BK, ‘Baekkang’

Effective marker development from a single-copy region of the hexaploid wheat genome

Multiple rounds of genome duplication and hybridization generated the modern allohexaploid bread wheat genome (Marcussen et al., 2014), in which the paralogous sequence or repetitive sequence accounted for over 70% of the genome (Brenchley et al., 2012; Wanjugi et al., 2009; Zimin et al., 2017). Various markers have been developed for wheat (Allen et al., 2017; Burridge et al., 2018; Wilkinson et al., 2012; Winfield et al., 2016), and genotyping using these many markers are always confused because of the interference of homoeologous sequences (Makhoul et al., 2020; Wen et al., 2017). Therefore, we tried to develop SNP markers from single-copy regions and avoid homoeologous regions.

A total 93,363 SNPs and corresponding ESTs were obtained from CerealsDB. From these SNPs, 907 single-locus unique SNPs were detected through BLAST analysis by removing paralogous sequences. From these 907 SNPs, 55 were randomly selected and developed into ASP markers, with 37 being validated as target-specific markers (Table S4). The use of ASP markers is an efficient method to allow genotype confirmation of SNPs using a single standard PCR reaction followed by agarose gel electrophoresis. Among the 37 markers, five informative markers showed genotype diversity between the seven major cultivars. The whASP010, whASP015, whASP019, and whASP034 markers have minor alleles for ‘Goso’, ‘Sooan’, ‘Keumkang’, and ‘Baegjoong’, respectively (Fig. 2B). Two markers, whASP018 and whASP026, are unique in ‘Baekkang’. Meanwhile, pooled DNA from ‘Jokyoung’ showed heterogeneous genotypes for two markers, whASP018 and whASP026, indicating a mixture of two homozygous genotypes in the ‘Jokyoung’ population.

Development of high-throughput TaqMan markers

Eight SNPs, two from the plastome and six from the nuclear genome, were converted into TaqMan markers (Table S5). The eight TaqMan markers were validated using 35 Korean wheat cultivars. Six of the eight markers showed two distinctive clusters of fluorescence signal that represent the respective homozygous genotypes (Fig. 3 and S1). Meanwhile, two markers, whTM001 and whTM003, showed a heterozygous genotype for the ‘Jokyoung’ DNA pool, and ‘Anbaek’ was not amplified with the whTM001 marker.

Fig. 3.

Fig. 3

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Endpoint fluorescence scatter plots of eight TaqMan markers applied to 35 Korean wheat cultivars. The X and Y axes of the endpoint fluorescence scatter plot represent the FAM (483–533 nm) and VIC (523–568 nm) values, respectively, which represent the genotype of the SNPs. The two black dots on the lower left indicate the no-template control

Genetic diversity analysis

Statistics for the eight TaqMan markers were calculated using genotype data from the 35 Korean wheat cultivars (Table S6). Major allele frequency ranged from 0.6286 to 0.9143 and gene diversity ranged from 0.1567 to 0.4669. The observed heterozygosity was 0 for all the markers, which indicates that all the cultivars are genetically fixed by recurrent self-hybridization. As a powerful index for evaluating the degree of polymorphism, PIC values ranged from 0.1445 to 0.3579 with an average of 0.2713. A circular dendrogram was drawn with genotype information from 147 different wheat germplasm lines using five nuclear genome–derived SNPs obtained from CerealsDB (Fig. 4A). The 112 foreign wheat germplasm lines are widely distributed throughout the tree, but the 35 Korean wheat cultivars showed limited divergence and fit into only two groups. The cultivars ‘Baekkang’ and ‘Jojoong’ grouped together and separated from the other Korean cultivars. A dendrogram was generated using the eight TaqMan markers in the seven major cultivars (Fig. 4B). The seven cultivars are split into two groups, with ‘Baekkang’ and ‘Jokyoung’ forming one group and the other five cultivars forming the other. All seven major cultivars can be differentiated from each other by the genotypes of the eight high-throughput TaqMan markers.

Fig. 4.

Fig. 4

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Genetic diversity of wheat germplasm and barcoding method for seven major Korean wheat cultivars. (A) Genetic relationship of 112 foreign wheat germplasm lines and 35 Korean wheat cultivars. UPGMA analysis was conducted with genotype data of five SNPs obtained from CerealsDB and were used to develop the TaqMan assay. (B) Dendrogram of seven major Korean wheat cultivars based on eight SNP genotypes. The color code on the right represents allele combinations for each TaqMan marker. X, homozygous X; Y, homozygous Y; H, heterozygous

Authentication system for major wheat cultivars in Korea

Heterogeneity was observed in ‘Jokyoung’ for whASP018 and whASP026 markers (Figs. 2B and 3). After observing heterogeneity within the ‘Jokyoung’ DNA pool, we validated the genotypes by applying the whASP018 and whASP026 markers to eight ‘Jokyoung’ individuals (Fig. S2A). The eight individuals had two different homozygous genotypes in a ratio of 1:7, which indicates a mixture of seeds having different genotypes at the whASP018 and whASP026 markers. Heterogeneity in ‘Jokyoung’ was also observed in DNA pooled from 13 ‘Jokyoung’ individuals provided by KU (Fig. S2B). Because of this, the whASP018 and whASP026 markers represent useful tools for controlling ‘Jokyoung’ seed purity. Previous studies have developed gel-based markers for Korean wheat cultivars using AFLP and ISSR techniques (Son et al., 2013; 2014). In contrast to those gel-based markers, here we present fluorescence-based markers that enable accurate genotyping and can be used for precise and objective purity tests (Fig. 3). These high-throughput markers may be useful for seed purity tests to maintain uniformity and consistency of each Korean wheat cultivar.

In this study, we developed efficient, high-throughput markers for authentication of Korean wheat cultivars by obtaining seven complete plastome sequences and developing single-copy SNPs using a nuclear genome database. The eight high-throughput TaqMan markers can authenticate each wheat cultivar, and combinations of the eight markers can be used to authenticate the seven major cultivars. Each marker can be applied at any developmental stage of wheat, including harvested seeds, plants, and processed wheat flours. These markers can be broadly applied to maintain seed purity for each cultivar and ensure the right genotypes are being grown in Korean wheat fields. Moreover, these markers can be applied as quality control assurance for wheat flours in the processing industry.

Supplementary Information

Below is the link to the electronic supplementary material.

Supplementary file 1 (DOCX 236 kb) (236.7KB, docx)
Supplementary file 2 (XLSX 26 kb) (26.5KB, xlsx)

Acknowledgements

This work was supported by Korea Institute of Planning and Evaluation for Technology in Food, Agriculture and Forestry (IPET) through Innovative Food Product and Natural Food Materials Development Program funded by Ministry of Agriculture, Food and Rural Affairs (MAFRA) (320010-02-1-HD030).

Declarations

Conflict of interest

The researchers claim no conflicts of interest.

Footnotes

Publisher's Note

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

Contributor Information

Woohyeon Cho, Email: whyeonc@snu.ac.kr.

Jin-Kee Jung, Email: jinkeejung@korea.kr.

Min-Young Kang, Email: kmyjj3802@korea.kr.

Yong Weon Seo, Email: seoag@korea.ac.kr.

Jee Young Park, Email: jypark74@snu.ac.kr.

Tae-Jin Yang, Email: tjyang@snu.ac.kr.

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