Only 11 Simple Sequence Repeats Needed to Identify Chinese Cabbage (Brassica rapa L.) Cultivars

Abstract

Chinese cabbage is a popular leaf vegetable, and many cultivars have been developed for different regions and growing seasons. For the identification of the cultivars, many marker systems have been applied, but they usually require a large number of markers and expensive equipment. Therefore, it is necessary to develop an efficient and economical method for identifying Chinese cabbage cultivars. In this study, we aimed to develop a marker system with the minimum number of markers using simple PCR and gel electrophoresis. A total of 48 simple sequence repeats (SSRs) in a previous study were screened based on their chromosomal location and applied to 105 Chinese cabbage cultivars. The minimum number of markers was selected based on their genomic location, polymorphic information content, and allele frequency. To validate the cultivar identification capability of selected SSRs, they were applied to genetically similar cultivar pairs from a previous study. Eleven SSRs were finally selected, and they successfully identified cultivars with high genetic similarity, as well as all 105 Chinese cabbage cultivars tested. The proposed SSRs require only 11 primer sets, simple PCR, and gel electrophoresis, which need less time and resources compared to previous ones. These SSRs can be used not only in small seed companies and laboratories but also in large-scale seed companies.

1. Introduction

Chinese cabbage (Brassica rapa L., Brassicaceae, 2n = 20) is a popular leaf vegetable in Asia, and its cultivation has expanded to other regions in the world [1]. It plays a crucial role in traditional Korean cuisine, particularly for kimchi [2]. Chinese cabbage, is also used in various dishes in Asia; for example, it is often incorporated into traditional dishes or fermented for pickling in China [3]. In Thailand, it is commonly used as an ingredient in dishes, such as “Tom Yum soup” [4]. The majority of commercially available Chinese cabbage cultivars are F₁ hybrids, which are produced using self-incompatibility or male sterility [5]. Different types of Chinese cabbage cultivars have been developed for growing them in different seasons, such as spring, summer, autumn, and winter, for delayed bolting, enhanced heat tolerance, good taste and production, and adaptation to the southern coastal area in winter [6]. Utgari cultivars are semi-heading Chinese cabbage cultivars, grown in all seasons of Korea and harvested early compared to other cultivars. About 175 cultivars were registered in plant variety protection (PVP) systems of Korea (https://www.seed.go.kr, accessed on 4 September 2023) [7].

Cultivar identification is a critical aspect of the PVP system, but morphological characteristics alone pose challenges for accurate cultivar identification due to the large number of cultivars tested. Time and resources are also limited to evaluate these cultivars in field. In addition, the development of new cultivars in breeding programs involves the test of combining ability among many parental lines, necessitating time-consuming field evaluations of their F₁’s performance. Therefore, there is a need for an efficient and cost-effective method to identify cultivars and eliminate genetically similar or identical lines prior to conducting field observations.

Various marker systems have been employed to identify cultivars in B. rapa. Amplified fragment length polymorphism (AFLP) was first introduced to identify cultivars in B. rapa [8,9]. However, AFLP has several disadvantages, including the need for purified and high molecular weight DNA, non-homology of comigrated fragments, and the complexity of band interpretation. Moreover, it produces dominant markers [10]. Later, simple sequence repeats (SSR) [11] and single nucleotide polymorphism (SNP) [12] were used to identify commercial cultivars and to assay the genetic diversity of inbred lines and cultivars in B. rapa, respectively. SSRs and SNPs can have strengths in cultivar identification because of their codominance of alleles, high genomic abundance, and random distribution throughout the genome [13]. However, they need expensive equipment for genotyping and, therefore, cost-effective multiplexing during PCR or gel electrophoresis for the identification of PCR products is necessary to reduce genotyping cost [14].

Recently, a simple and economical method was reported to identify cultivars with a minimum number of markers using simple PCR and gel electrophoresis in radish (Raphanus sativus L. Brassicaceae) [15], a closely related species of B. rapa. The method to develop markers is so simple that can be easily adopted in other crops [15]. Moreover, SSRs can be a good candidate for cultivar identification if their polymorphisms can be visualized in gel electrophoresis since they can produce many amplicons per primer pair and are located throughout the genome. SSRs incorporated into the linkage map [16] can be a good choice since the information on their genomic locations is also available. Therefore, it is necessary to develop an improved marker system using SSRs for identifying Chinese cabbage cultivars with a smaller number of markers than the previous studies [8,9,11,12] and no expensive equipment for genotyping, using simple PCR and gel electrophoresis as in the previous study [15].

In this study, we present a rapid and efficient marker system for identifying Chinese cabbage cultivars using SSRs. The SSRs require only 11 primer pairs, conventional PCR, and gel electrophoresis systems for identifying cultivars so that it is suitable for small seed companies and independent breeders that cannot afford expensive genotyping equipment.

2. Materials and Methods

The method to select the minimum number of markers for Chinese cabbage cultivar identification was based on Hong et al. (2023) [15] with minor modifications, such as using different primer pairs and cultivars.

2.1. Selection of Molecular Markers Showing Polymorphism among Chinese Cabbage Cultivars

2.1.1. Plant Materials and DNA Preparation

For initial polymorphism detection, 12 Chinese cabbage cultivars were chosen such as ‘Marathonwoldong’ (Nongwoo Bio Co., Ltd., Suwon, Republic of Korea), ‘Chukwang’, ‘Chungwang’, ‘Hwiparamgold’ (Sakatakorea Co., Ltd., Seoul, Republic of Korea), ‘Golaengjiyeoleum’, ‘Norangbom’, ‘Bulamplus’, ‘Taekwang’, ‘High star’ (Farm Hannong Co., Ltd., Seoul, Republic of Korea), ‘Urideul’ (Hyundae Seed Co., Ltd., Icheon, Republic of Korea), ‘Bughaedo2’ (Chocheonsingwa Co., Ltd., Wuhan, China), and ‘Geugpumhanggeun’ (Fusida Co., Ltd., Kumming, China). DNA extraction was performed using the modified CTAB DNA extraction method [17] using chloroform and 100% EtOH instead of chloroform-isoamyl alcohol (24:1) and isopropanol, respectively. Leaves of approximately 4 cm in length were used for DNA extraction, which were obtained from greenhouse-grown plants three weeks after sowing. The quantity and quality of DNA were assessed using a UV spectrophotometer (DS-11, DeNOVIX, Wilmington, DE, USA).

2.1.2. Amplification of Genome-Anchored Markers

Forty-eight markers distributed in all ten chromosomes were initially screened from a B. rapa linkage map [16] and applied to the 12 Chinese cabbage cultivars listed in 2.1.1. PCR reactions were carried out in a 20 μL volume containing 10 ng of genomic DNA, 0.5 μM of forward and reverse primers, and 5.0 μL of AccuPower^® Taq PCR PreMix (K–2601, Bioneer, Daejeon, Republic of Korea). The PCR conditions consisted of an initial denaturation at 95 °C for 5 min, followed by 35 cycles of denaturation at 95 °C for 30 s, annealing at 52–60 °C (depending on annealing temperatures of primers) for 30 s, extension at 72 °C for 30 s, and a final extension at 72 °C for 5 min. The amplicons were separated on 1.5% agarose gels in 0.5× TBE buffer and visualized using Loading Star (Dyne bio, Seongnam, Republic of Korea). Gel electrophoresis was conducted at 200 V for two hours. All PCR and gel electrophoresis were replicated at least two times.

2.2. Development of a Minimum Number of Markers for Identifying Cultivars

2.2.1. Plant Material, DNA Preparation, and Genotyping

A total of 105 Chinese cabbage cultivars (Supplementary Table S1) and two radish cultivars as out-groups were utilized for selecting the minimum number of markers to identify cultivars. The cultivars were composed of 74, 26, 4, and 1 cultivars for Korea, China, Thailand, and Indonesia, respectively, and they were used for cultivation in spring, summer, autumn, and winter. In addition, four Utgari cultivars were also included. The method for genotyping was the same as those described in Section 2.1.2. All PCR and gel electrophoresis were replicated at least two times, and if there were gel distortions, the gel electrophoresis was repeated until at least two same results without gel distortion were obtained.

2.2.2. Cluster Analysis

Cluster analysis was performed to evaluate the genetic relationships among the 105 Chinese cabbage cultivars based on the SSRs showing polymorphisms. A binary matrix from coding 1 and 0 for their presence and absence of amplicons, respectively, was used to generate a distance matrix using Jaccard’s similarity coefficient [18]. The unweighted pair-group method with arithmetic averages (UPGMA) protocol using NTSYS-pc version 2.21p software [19] was employed to construct dendrograms.

2.2.3. Procedure for Selecting the Minimum Number of Markers

First, 26 SSRs (Supplementary Table S2) showing polymorphism among 12 cultivars were applied to 105 Chinese cabbage cultivars (Supplementary Table S1). The informativeness of the markers was determined using the polymorphic information content (PIC) by the following equation:

$${\mathrm{P}\mathrm{I}\mathrm{C}}_i=1-\sum _{i=1}^n{P_i}^2$$

where n represents the sum of the number of different alleles observed at a specific locus, and P_i represents the frequency of the ith allele at the locus [20]. Second, based on the markers with the highest PIC values or the highest number of alleles on each linkage group, ten SSRs were initially selected, and the remaining markers with the highest PIC values were sequentially added until all 105 cultivars could be distinguished in the UPGMA dendrogram. Third, the population structure of the cultivars was analyzed using the STRUCTURE program (Pritchard Lab, Stanford University, Stanford, CA, USA), and the optimal K value was determined using STRUCTURE HARVESTER [21]. Lastly, the cultivar identification capability of the selected SSRs was tested using pairs of genetically similar Chinese cabbage cultivars from a previous study [11]. The cultivar names and their genetic similarities are presented in

Table 1. The pairs of genetically similar Chinese cabbage cultivars and their genetic similarity identified in a previous study using simple sequence repeats [11].

Cultivars		Genetic Similarity z
Goranengjiyeoreum	Yeoreimsingwan	0.97
Victory	CR-chunhailpoom	0.97
CR-saesinrokutgari	Chamsin	0.97
Chunhanoran	Norangbom	0.95
CR-ok	Lipoomyeoreum	0.93

^z Jaccard’s coefficient of similarity.

.

2.2.4. Phenotypic Observation of Chinese Cabbage Cultivars

The phenotypic variations of Chinese cabbage cultivars were investigated according to the definition of the Chinese cabbage test guideline prescribed by the Korea Seed and Variety Service [7] in the experimental plot of Farm Hannong Breeding Research Institute, Anseong, Korea (37°04′75″ N, 127°19′95″ E) for two years. Seeds of 105 Chinese cabbage cultivars were sown in plug cell trays and grown in a greenhouse for 25 days in two autumn seasons. Young seedlings were transplanted to open field with a spacing of 40 × 40 cm between and within rows, respectively. The field was prepared with preplant broadcast manure (80 kg/10a) and basal fertilizer containing 10.4 kg/10a N, 6.4 kg/10a H₃PO₄, 6.4 kg/10a K₂CO₃, 1.6 kg/10a Mg₂SiO₄, and 0.16 kg/10a B and mulched with black polyethylene film. Additional fertilization was applied twice, on the 30th and 45th days after planting, with a dose of 10.4 kg/10a N, 1.6 kg/10a H₃PO₄, 9.6 kg/10a K₂CO₃, 1.6 kg/10a Mg₂SiO₄, and 0.16 kg/10a B. Experiments were carried out using a randomized complete block design with three replications. Each replication contained 14 plants and was harvested 70 days after transplanting. Plant morphologies were investigated for 4–6 plants per cultivar based on four categories: inside color, heading type, width, and length.

3. Results

3.1. Selection of Markers Showing Polymorphism among Chinese Cabbage Cultivars

Out of the 48 SSRs screened from a Chinese cabbage genetic map [17], 26 SSRs exhibited polymorphism among the 12 cultivars (Supplementary Table S2), and their polymorphisms can be detected in agarose gel electrophoresis. These polymorphic SSRs were distributed across all ten chromosomes, with 2.6 SSRs per chromosome on average (Supplementary Table S2).

3.2. Development of the Minimum Number of Markers for Cultivar Identification

Eleven SSRs were finally selected from the 26 SSRs for identifying Chinese cabbage cultivars (

Table 2. Linkage groups, the numbers of alleles, allele frequencies, and polymorphic information content values of 11 SSRs selected for cultivar identification among 105 cultivars in Chinese cabbage.

Primers	Linkage Group	NA z	Allele Frequency				PIC y
			Allele 1	Allele 2	Allele 3	Allele 4
Cnu_m474a	1	3	0.439	0.140	0.037	–	0.786
Cnu_m046a	2	4	0.523	0.355	0.224	0.019	0.549
Cnu_m241a	3	4	0.551	0.280	0.150	0.140	0.575
Cnu_m256a	4	3	0.486	0.262	0.178	–	0.664
Cnu_m471a	5	3	0.617	0.308	0.187	–	0.489
Cnu_m257a	5	3	0.869	0.664	0.299	–	–
Cnu_m049a	6	3	0.411	0.383	0.112	–	0.671
Cnu_m308a	7	3	0.402	0.243	0.121	–	0.757
Nia_m095a	8	4	0.570	0.159	0.140	0.019	0.630
Cnu_m008a	9	3	0.542	0.449	0.075	–	0.499
Nia_m034a	10	3	0.729	0.355	0.103	–	0.332

^z The number of alleles. ^y Polymorphism Information Content.

). Polymorphisms of the amplicons were visually detected through agarose gel electrophoresis (Supplementary Figure S1). Each SSR resided in each linkage group, except for Cnu_m471a and Cnu_m257a, both of which were located in the same linkage group 5 (Table 2). Eight SSRs produced three amplicons, and Cnu_m046a, Cnu_m241a, and Nia_m095a generated four amplicons. The allele frequencies of the SSRs ranged from 0.019 to 0.869, with all SSR having at least one allele with a frequency greater than 0.402 (Table 2). PIC values of SSRs ranged from 0.332 to 0.786, with an average of 0.595 (Table 2).

The 11 SSRs successfully distinguished all 105 Chinese cabbage cultivars (Figure 1). The genetically closest cultivars exhibited a genetic similarity of approximately 0.88, differing by four amplicons between the two cultivars. The two radish out-groups were clearly separated out and the Chinese cabbage cultivars were organized into five small groups and a large group with six subgroups. However, the groups did not match with either cultivar types or countries (Figure 1, Supplementary Table S1). For example, group one (G1) was comprised of a summer, unidentified and Utgari cultivars derived from China, Thailand, and Korea, respectively. Group two (G2) contained a spring, an autumn, and two winter cultivars, and all cultivars in this group were developed for China. Group three (G3) was composed of two spring and one summer, and all these cultivars were from Korea. Group four was divided into six subgroups. Subgroup 4-1 (G4-1) was comprised of seven spring, three autumn, a winter, an Utgari, and three unidentified cultivars. The subgroup included six Korean, seven Chinese, and two Thai cultivars. Subgroup 4-2 (G4-2) was consisted of a spring, a summer, four autumn, and three winter cultivars, and divided into six Korean and three Chinese cultivars. Subgroup 4-3 (G4-3) included three spring, one summer, five autumn, and one winter cultivars, and seven and three of which were developed for Korea and China, respectively. Subgroup 4-4 (G4-4) included three spring, seven summer, two autumn and an unidentified cultivar, and the subgroup consisted of 12 Korean and one Indonesian cultivars. Subgroup 4-5 (G4-5) consisted of nine spring, one summer, eight autumn, three winter, one Utgari, and one unidentified cultivar. The subgroup included 18 Korean, four Chinese, and one Thailand cultivars. Subgroup 4-6 (G4-6) included ten spring, 11 autumn, one winter, and one Utgari cultivars and was divided into 18 Korean and five Chinese cultivars. Group five (G5) was composed of two spring cultivars, all of which were developed for Korea.

Population structure showed a somewhat different pattern compared to UPGMA dendrogram and had three distinct populations (K = 3) (Figure 2). In the first population (P1), there were 16 cultivars for spring, 16 for autumn, one for winter, and two Utgari. This population also included 29 Korean and six Chinese cultivars, with the predominant cultivars for spring and autumn in Korea. The second population (P2) was comprised of ten spring, two summer, five autumn, four winter, two Utgari, and four other cultivars. This population included 12 Korean, 12 Chinese, and three Thailand cultivars. The third population (P3) contained 12 spring, nine summer, 14 autumn, six winter, and two unidentified cultivars. It is divided into 33 Korean, eight Chinese, one Thailand, and one Indonesian cultivars. Most summer cultivars fall within this population (Figure 2). However, these populations also did not represent either country for cultivation or cultivated seasons of cultivars, similar to the UPGMA analysis (Figure 1).

The morphology of Korean, Chinese, Indonesian, Thailand, and Utgari cultivars in field is shown in Figure 3 and Figure 4. The first three cultivars, ‘Tongkeunchuseok’ (Figure 3A), ‘Chunmyeong’ (Figure 3B), and ‘Golaengjiyeoleum’ (Figure 3C), were similar to each other in morphology, and Thailand cultivar (Figure 3D) was smaller than those three cultivars. Utgari cultivar was clearly different from other cultivars, with semi-heading morphology (Figure 3E). In cross-sections, Korean, Chinese, Indonesian, and Thailand cultivars were similar to each other except for the smaller size of a Thailand cultivar (Figure 4A–D). However, Utgari cultivar did not form head (Figure 4E), which was different from other cultivars.

To confirm the cultivar identification capability of the selected 11 SSRs, various pairs of genetically similar Chinese cabbage cultivars from a previous study (Table 1) [11] were tested. All pairs of genetically similar cultivars were successfully distinguished by at least one SSR (Figure 5). For example, the pair ‘Goranengjiyeoreum’ and ‘Yeoreimsingwan’ (Figure 5A) and the pair ‘Chunhanoran’ and ‘Norangbom’ (Figure 5B) were differentiated by an SSR, Cnu_m471a. Cnu_m474a also successfully identified two cultivars, ‘CR-saesinrokutgari’ and ‘Chamsin’ (Figure 5C). Cnu_m241a differentiated the two cultivars ‘Victory’ and ‘CR-chunhailpoom’ (Figure 5D). Cnu_m008a also distinguished two genetically similar cultivars, ‘CR-ok’ and ‘Lipoomyeoreum’ (Figure 5E).

4. Discussion

Previous marker systems used for identifying germplasm, cultivars, and breeding lines in Chinese cabbage did not prioritize efficiency or cost-effectiveness in genotyping [8,9,11,12]. Molecular markers for cultivar identification should be not only inexpensive and easy to use [9] but require a smaller number of markers [15]. Recently, Hong et al. (2023) [15] proposed a method to select the minimum number of markers for identifying radish cultivars. Different from other studies that applied markers to as many cultivars as possible, they first selected markers among about 100 cultivars and applied them to genetically similar cultivars determined by previous research in order to test the cultivar identification capability of the selected markers. Finally, they could identify the smallest number of markers for cultivar identification, which were easily detected through simple PCR and gel electrophoresis [15].

SSRs are located throughout the genome and have many alleles per primer pair. Therefore, they can be the marker of choice for the cultivar identification of not only Chinese cabbage but also other crops. A number of SSRs have been developed for B. rapa [22,23,24,25,26]. In a previous study [22], a size-fractionated genomic library was used to develop 36 SSR, and they detected a total of 232 alleles in 19 cultivars of B. rapa. Lowe et al. (2004) [23] developed expressed sequence tag (EST)-derived SSRs (EST-SSRs) using 182,703 EST sequences, and they were used to construct a high-density integrated map [24]. The 234 mapped EST-SSR markers also applied to 24 B. rapa. accessions and showed variable PIC values, with an average of 0.4 [24]. Song et al. (2015) [25] identified a total of 20,836 genome-wide SSRs in non-heading Chinese cabbage, and 5435 SSRs (26.08%) were derived from 4569 genes. Among 74 EST-SSRs randomly selected for validation, 63 EST-SSRs produced amplicons of the expected sizes [25]. Next-generation sequencing of the transcriptome in a Chinese cabbage accession ‘FushanBaoTou’ detected 10,420 EST-SSRs from 51,694 non-redundant unigenes [26]. These SSRs from previous studies can be useful resources for B. rapa. research and breeding programs. For example, 21 SSRs were selected for cultivar identification in Chinese cabbages [11] using SSRs developed in previous studies [22,23]. However, the polymorphism of SSRs was usually detected using expensive fluorescence-labeled primers followed by fragment analysis that needed expensive equipment. They should be easily detected through simple PCR and gel electrophoresis [14] for cost reduction for genotyping.

In the present study, we first selected 48 SSRs from a genetic linkage map of Chinese cabbage [17] and applied them to 12 cultivars to detect polymorphism among cultivars. Eleven SSRs, which were distributed across all Chinese cabbage chromosomes, were finally selected and successfully identified all 105 Chinese cabbage cultivars. Additionally, these SSRs effectively distinguished genetically similar cultivars that were reported in a previous study [8]. This confirms that the capability of the 11 SSRs for cultivar identification is comparable to previous studies in Chinese cabbage, although our SSRs require a significantly smaller number of markers than previous studies [8,9,11,12]. However, the UPGMA dendrogram using the 11 SSRs failed to group the cultivars based on their countries or cultivation seasons, in contrast to previous studies where Chinese cabbage cultivars were grouped according to their morphologies [8,9,11,12]. This might be due to the smaller number of markers in the present study than in previous studies [8,9,11,12]. It indicates that the 11 SSRs can be used for cultivar identification but not for the assessment of genetic relationship or diversity among Chinese cabbage cultivars or germplasm.

Previous studies have utilized AFLP [8,9] for Chinese cabbage cultivar identification, which suffers from a time-consuming process, high molecular weight DNA, and the complexity of band interpretation. The identification of Chinese cabbage cultivars with SSR [11] or SNPs [12] requires a lot of markers and expensive equipment. In addition, these previous marker systems did not have any information on their chromosomal locations. Previous studies with SSRs and SNPs used 21 and 60 markers, respectively [11,12], which required a significantly higher number of markers than the present study, which only required 11 SSRs to identify Chinese cabbage cultivars. Consequently, the present SSRs significantly reduce the time and effort for genotyping cultivars. Furthermore, the 11 SSRs rely on simple PCR and gel electrophoresis for detecting polymorphisms, eliminating the need for expensive equipment and high genotyping costs.

5. Conclusions

The SSRs developed in this study offer a highly efficient and economical approach to cultivar identification since they require only 11 primer pairs, simple PCR, and gel electrophoresis for genotyping. It enables widespread application in both large-scale seed companies and small-scale laboratories with relatively shorter time and lower costs. This study suggests that the method to develop the minimum number of markers in radish by Hong et al. (2023) [15] can also be applicable to Chinese cabbage cultivar identification. This method may offer a practical and accessible tool for cultivar identification in other crops.