Efficient Identification Techniques for 61 Oil Tea (Camellia Oleifera Abel) Cultivars Using the MCID Strategy and DNA Markers

Summary

This study employed a manual cultivar identification diagram (MCID) derived from Random Amplified Polymorphic DNA (RAPD) banding patterns to efficiently and reproducibly identify 61 Camellia oleifera Abel cultivars for germplasm resource utilization and oil tea cultivar identification.

Abstract

It is crucial to identify Camellia oleifera Abel [Oil tea (Ot)] germplasm materials and cultivars for evaluating germplasm resources and assessing mechanisms for protecting Ot varieties. This study employed an optimized RAPD technique by increasing the RAPD primer length and screening the PCR annealing temperature, which identified 61 Ot cultivars from various regions. Furthermore, a MCID was developed to design efficient RAPD markers for cultivar identification. From 80 RAPD arbitrary 11-nt primers, 10 primers producing clear polymorphic bands (PBs) were selected for PCR analysis of the 61 cultivars. Moreover, the MCID strategy was employed to manually distinguish 61 cultivars based on the PBs observed in DNA fingerprints from the 10 primers, which generated the Ot cultivar identification diagram (Ot-CID). This Ot-CID indicated each cultivar individually, utilizing PBs for cultivar identification, with the appropriate primers accurately positioned on the Ot-CID. The MCID strategy provides a simple and practical approach for plant identification using DNA markers. Furthermore, this approach provides a practical and efficient method for cultivar identification. The resulting Ot-CID of 61 Ot cultivars effectively distinguished Ot variants using RAPD markers, thereby facilitating cultivar differentiation for cultivar-right protection and early seedling identification in the nursery industry.

Introduction

Camellia oleifera Abel [Oil tea (Ot)] is among the most crucial woody edible oil tree species in the world¹. China is considered the origin and diversity hub for Ot, with abundant germplasm resources². The ancient tradition of Ot, vegetative propagation, and diverse global breeding programs has resulted in the emergence of numerous cultivars³. The widespread global cultivation of Ot culture has resulted in cultivars with homonyms (distinct cultivars with the same name) and synonyms (cultivars with more than 1 name)⁴. Therefore, it is crucial to develop techniques for managing a vast range of Ot germplasm and accurately distinguishing various cultivars^3,5.

Despite the application of DNA-based molecular markers in cultivar characterization, genetic research, and Ot identification, which can elucidate genetic diversity and differentiate plant individuals, no study has successfully identified a substantial number of Ot cultivars or provided a reliable basis for cultivar differentiation^6,7,8,9. The lack of reliable methods is the primary limitation for the application of DNA markers in plant crop identification. This might be because the analytical techniques used for DNA fingerprints cannot yield pertinent information that distinctly specifies primers and polymorphic markers for differentiating the cultivars that require identification. The analytic techniques for DNA banding patterns (called cluster analyses) are unsuitable for effectively distinguishing cultivars or species.

Although DNA molecular identification cards (IDs) have been developed for over 200 Ot cultivars utilizing SNP markers^2,3,10,11, there are still several challenges: (1) The Ot genome is complex, with the majority of cultivated camellias being hexaploid, while the reference genome utilized for constructing the DNA molecular IDs is triploid, which may result in certain inaccuracy³. (2) Whole-genome sequencing is expensive, specifically for laboratories with limited facilities, preventing its widespread application for large-scale variety analysis. Furthermore, PCR amplification products necessitate sequencing or fluorescence detection, further complicating process¹². (3) In the absence of distinguishing markers for new varieties, it is necessary to develop new marker primers, which will also require the re-coding of ID card codes for existing types. This results in an overwhelming quantity of code positions, diminishing the efficacy of variety differentiation. Therefore, it is essential to employ technique that provides easy, reliable, referable, and practical verification of Ot cultivars for the Ot nursery sector, agricultural business, plant patent protection, and the conservation and assessment of genetic resources⁵.

Traditional strategies for cultivar identification relied on morphological, physiological, and agronomic characteristics, but these characteristics have limits as they are susceptible to environmental influences and require a comprehensive evaluation of fully developed plants^13,14. Furthermore, molecular markers have the distinct benefit of being unaffected by environmental variables, thus serving as an efficient tool for the accurate characterization of cultivars^14,15. Recently, various DNA-based indices have been established for assessing fingerprinting, genetic diversity, cultivar, and purity^{16,17,18,19,20,21,22}. The RAPD marker is advantageous for cultivar analysis because of its simplicity, efficiency, and the non-requirement of previous sequence data^23,24. The RAPD approach can be enhanced by selecting 11-nucleotide (nt) primers along with their specified PCR annealing temperature for its application in plant fingerprinting, therefore establishing RAPD as a preferable method for plant variety identification and genetic diversity evaluation^25,26. These markers have been extensively utilized in crop differentiation and genetic correlation studies of various plant species, including apricot²⁷, Ornamental peach²⁸, pomegranate²⁹, landraces²⁰, and walnut³⁰(Çilesiz et al., 2025). Despite their widespread use, many DNA marker-based approaches remain insufficiently practical or streamlined for routine plant variety identification.

Therefore, this study utilized a novel analytical technique for the reproducible identification of various Ot cultivars. The MCID derived from RAPD banding patterns differentiated 61 Ot cultivars for germplasm resource utilization and cultivar identification.

The RAPD-MCID strategy described herein (Figure 1) is most appropriate for small- to medium-scale germplasm characterization in laboratories with standard PCR and gel electrophoresis infrastructure but limited access to sequencing facilities. It is also well-suited for rapid, cost-effective preliminary screening before more detailed genetic analysis. The method’s primary operational limits include its reliance on dominant markers and the need for careful standardization of PCR conditions to ensure across-laboratory reproducibility. For large-scale germplasm banks or legal variety registration, complementary use of co-dominant markers such as SSRs or SNPs is recommended.

Protocol

1. Plant Material

1. Collect young leaves from healthy, actively growing shoots of each oil tea cultivar from the National Camellia Germplasm Repository, Shucheng County, Anhui Province.
2. Refer to Table 1 for the names and origins of these cultivars.

2. Genomic DNA extraction

1. Extract total genomic DNA using a plant DNA extraction kit according to the manufacturer’s instructions.
2. The DNA was eluted with 1× TE buffer. After elution, confirm that the extracted DNA solution appears clear and colorless. Measure the DNA concentration using a microvolume spectrophotometer.
3. Adjust the DNA concentration to 30 ng·µL^-1 with 1× TE buffer and store the adjusted DNA solution at -20 °C.

3. RAPD primer selection

1. Develop 80 random primers for RAPD analysis and analyze them using a few genotypes. Select 11-nt RAPD primers that produce clear and unequivocal banding patterns across all tested genotypes.
2. Use the selected primers for genotyping to increase the credibility of the amplified fragments. Select 10 well-resolved primers with reproducible bands for further analysis, as listed in Table 2.

4. Random Amplified Polymorphic DNA (RAPD) PCR

1. Prepare the amplification reactions by slightly modifying the previously described method²³. Prepare the reaction solutions according to the composition listed in Table S1.
2. Perform PCR using a thermal cycler with the following conditions: pre-denaturation at 94 °C for 5 min; 42 cycles of denaturation at 94 °C for 30 s, annealing for 1 min at the optimized temperature listed in Table 2, and extension at 72 °C for 2 min; and a final extension at 72 °C for 10 min.

5. Detection and analysis of PCR products

1. Separate the amplified DNA fragments by gel electrophoresis at 100 V in 1.3% agarose (w/v) prepared in 1× TAE buffer containing 0.001 M EDTA and 0.04 M Tris-acetate at pH 8.0. Stain the gels with ethidium bromide at 0.5 µg·µL^-1.
   CAUTION: Ethidium bromide is a potent mutagen. Wear appropriate personal protective equipment when handling ethidium bromide solutions or stained gels.
2. Run the gel at a constant voltage of 100 V for 45–60 min, or until the bromophenol blue dye front has migrated approximately two-thirds of the gel length. After staining and destaining, observe the gel under UV transillumination.
   NOTE: Confirm that distinct DNA bands are clearly visible under UV transillumination. Ensure the DNA ladder exhibits sharp, well-separated bands ranging from 100 bp-2,000 bp.
3. Record the polymorphic bands (PBs) of the cultivars based on their presence or absence under ultraviolet light.
4. Perform each PCR amplification in triplicate to ensure reproducibility. Score only bands that are consistently present or absent across all three independent replicates for downstream analysis. Confirm that the concordance rate of band scoring among replicates exceeds 95%.

6. Data analysis

1. Score the bands for cultivar identification based on their presence or absence by assigning “+ o” to clearly visible bands and “− ” to absent bands at the corresponding molecular size position across all cultivars. Include only bands that are consistently observed across three independent replicates.
2. Classify cultivars with distinct bands in the fingerprint produced by a single primer independently. Categorize cultivars with identical banding patterns into the same subgroup. Differentiate all oil tea cultivars progressively and completely based on these parameters.

7. Diagram’s application and efficiency in cultivar identification

1. Randomly select two oil tea cultivar groups from the intra- and inter-groups to verify the applicability and functionality of the diagram illustrating 61 oil tea cultivars. Perform the selection manually by referring to different branches of the Ot-CID diagram, without the use of any software.
   NOTE: 2 Ot cultivar groups were randomly selected to verify the applicability and functionality of the diagram illustrating 61 Ot cultivars.
2. Designate the two selected groups as “A” and “B”. Screen the relevant primers for each group from the Ot-CID.

8. Hazardous-waste disposal guidance

1. Prepare and handle all ethidium bromide solutions in a designated area. Collect all ethidium bromide-contaminated solid waste and liquid waste separately for disposal according to institutional hazardous-waste guidelines.
2. Dispose of all ethidium bromide-contaminated materials in designated hazardous waste containers. Contact the institutional environmental health and safety office for approved disposal procedures if uncertain.

Results

The Representative Results presented here illustrate the complete workflow of the RAPD-MCID strategy, from initial primer screening through hierarchical cultivar discrimination to final validation. The stepwise discrimination process is documented in Figure 2, Figure 3, Figure 4, Figure 5, Figure 6<...

Discussion

DNA markers are powerful tools that are used for identifying plant cultivars and species²⁴. These markers have been developed and employed in thousands of studies on genetic analysis, cultivar identification, etc.; however, they are neither easy nor widely utilized in genotyping^24,31. This is because of the lack of efficient and easy methods that use DNA markers for cultivar identification, phylogenetic trees, or fingerprints. Alt...

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Centrifuge	Eppendorf,Germany	5427R	Used for genomic DNA extraction from oil tea leaves
Hi-DNAsecure Plant Kit	Tiangen, China	DP350	Used for genomic DNA extraction from oil tea leaves
PCR gene amplification instrument	Monad, China	MP60901	Used for PCR amplification
SolarRed Agarose LE	Solarbio, China	A8202	Used for PCR fragment electrophoresis
TAE	Tsingke, China	TSG001	Used for PCR fragment electrophoresis
TaKaRa Ex Taq	Takara, Japan	RR001A	Used for PCR amplification
Thermo Scientific NanoDrop 2000C spectrophotometer	ABI,USA		Used to determine RNA concentration