Establishment and Optimization of an Experiment System for Flow Cytometry in Oil-Seed Camellia
by
,
Ying Zhang
1,2,†,
Zhen Zhang
1,2,†,
Xiangnan Wang
1,2,
Rui Wang
1,2,*,
Zhilong He
1,2,
Gaohong Xiao
3,
Weiguo Li
3 and
Yongzhong Chen
1,2,* 1
Research Institute of Oil Tea Camellia, Hunan Academy of Forestry, Changsha 410004, China
2
National Engineering Research Center for Oil Tea Camellia, Changsha 410004, China
3
Huazhi Biotech Co., Ltd., Changsha 410125, China
*
Authors to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Horticulturae 2024, 10(7), 704; https://doi.org/10.3390/horticulturae10070704
Submission received: 6 May 2024
/
Revised: 1 July 2024
/
Accepted: 1 July 2024
/
Published: 3 July 2024
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
Abstract
:Ploidy identification is a basic method for determining germplasm resources and for breeding new varieties of oil-seed camellia. In this study, flow cytometry and K-mer analysis were used to identify the ploidy of oil-seed camellia germplasms. To determine the best tissue organ type, lysis time, and dyeing time, evaluation indices such as the presence of a clear main peak, the ease of sampling, and the coefficient of variation were used. A technique was established, and the ploidies of different oil-seed camellia germplasms were identified. The results showed that pollen was the best material and that using a 400 mL PI lysis solution for 10 min lysis, followed by dyeing with a 1600 mL DAPI dyeing solution for 10 min, was the most suitable technique. According to the peak value of the control diploid Camellia azalea, 15 samples were estimated to be diploid, 24 samples were tetraploid, 18 samples were hexaploid, and 13 samples were octoploid. In addition, the K-mer analysis results showed that among the five samples, CK, C051, and C047 were diploid, while C037 and C043 were tetraploid, results that are consistent with the results of the flow cytometry identification. This study is therefore valuable for the polyploid selection and use of different ploidy germplasm resources for the cross breeding of oil-seed camellia.
1. Introduction
Oil-seed camellia belongs to the genus Camellia in the family Theaceae and is an important woody, edible oil species with a history of more than 2000 years of cultivation and utilization. Together with oil palm, olive, and coconut, it is known as one of the world’s four major woody, edible oil plants [1]. Broadly speaking, oil-seed camellia refers to plants in the genus Camellia that have a high seed oil content and a certain level of economic cultivation value [2]. Currently, 238 species of the genus Camellia are known, of which more than 50 have a high oil content in their seeds. Oil-seed camellia has abundant germplasm resources and complex chromosome ploidies, with diploid, tetraploid, pentaploid, hexaploid, octaploid, and decaploid species existing in nature [3]. These rich ploidy levels provide high-quality resources for the genetic improvement of oil-seed camellia, laying the foundation for the breeding and development of new varieties and germplasm.
At present, research on the ploidy levels of oil-seed camellia mainly uses chromosome compression and flow cytometry. Chromosome compression is the most effective method of determining the ploidy of plants. Li et al. [4] determined the ploidy of tetraploid C. oleifera based on chromosome number, and similarly, Ye et al. [5] determined the ploidy of three C. oleifera varieties based on chromosome number. Chromosome compression can be used to directly observe chromosomes with high reliability, but it is time-consuming and not suitable for large-scale ploidy identification. In recent years, flow cytometry has been shown to have the advantages of fast detection and a large data collection and thus has been widely used in plant ploidy research. So far, it has played an important role in the ploidy identification of plants such as coffee [6], sunflowers [7], asparagus [8], torreya [9], pear [10], chrysanthemums [11], and birch [12]. Some researchers have used flow cytometry to identify the ploidy of oil-seed camellia. Yan et al. [13] determined the chromosome ploidy of 97 germplasms of C. oleifera using various methods such as flow cytometry, SSR-PAGE (polyacrylamide gel electrophoresis), and SSR sequencing. Ye et al. [5] determined the ploidy of 60 germplasms of C. hainanica using flow cytometry and chromosome counting. Jia [14], Xu et al. [15], and Wu et al. [16] also used flow cytometry to identify the ploidy of oil-seed camellia and confirmed its reliability in the identification of chromosome ploidy. Although flow cytometry can rapidly identify ploidy in plants, a standardized international reference for the nuclear DNA content in plants is lacking. Thus, discrepancies in the DNA content may arise from the utilization of different tissue organs and the application of difference lysis and dyeing time [16]. Therefore, each plant requires a different technique. Gong [17] conducted flow cytometry experiments on three different tissues of C. hainanica: the roots, leaves, and petals. Their results indicated that the petals were the best material, followed by young leaves, while the roots of cuttings were found to be unsuitable. Mature leaves showed severe browning after the addition of the staining solution, which led to an obstruction in the instrument and rendered the experiment infeasible. Among conventional solutions such as Galbraith, WPB, Tris-MgCl2, and OTTO, Yang et al. [18] found that a modified OTTO solution was the most suitable for flow cytometry in C. oleifera.
The variability in the condition of the test materials, the experimental platform settings, and the technical proficiency of the operators lead to differences in the determination results. Therefore, in order to ensure the accuracy of flow cytometry in measuring the DNA content of oil-seed camellia, a stable and suitable technique needs to be established. In this study, we also aimed to optimize a method for identifying oil-seed camellia using flow cytometry based on prior research that used flow cytometry for the identification of the ploidy of oil-seed camellia and other plants [5,14,15,16]. In this study, by comparing the effects of different tissue organ types, lysis times, and dyeing times on the ploidy identification of oil-seed camellia, a ploidy identification method based on flow cytometry is established, and the method is used to identify the ploidy of 70 oil-seed camellia germplasms. This not only provides a reliable new method for the rapid identification of ploidy in oil-seed camellia germplasms but also provides a theoretical basis for artificial hybrid pollination configurations, the hybrid breeding, the ploidy breeding, and other aspects of oil-seed camellia. Therefore, this study is of great significance for the ploidy identification and polyploid breeding of polyploid plants.
2. Materials and Methods
2.1. Experimental Materials
Seventy oil-seed camellia germplasm collections were used for this study (Table 1). Among them, 34 resources were selected as excellent trees from Hunan and Zhejiang provinces, China, including Changde (C001–C014), Xiangxi (C015–C030), Hengyang (C031), Changsha (C032), and Jinhua (C033 and C034), and 36 resources were selected from the National Camellia Oleifera Germplasm Resource Preservation Center (NCOGRPC), Changsha, China, including ancient C. oleifera trees that have lived for over three hundred years (C035 and C037), superior varieties of C. oleifera (C049 and C050), C. fraterna (C036 and C060), C. vietnamensis (C038 and C066), C. gauchowensis (C039, C061, C062, and C063), C. chekiangoleosa (C040), C. meiocarpa (C041–C046), C. furfuracea (C047 and C048), C. gigantocarpa (C051 and C052), C. sasanqua (C053 and C054), C. kissi var. megalantha (C056 and C058), C. pitardii (C064, C065, and C069), and C. semiserrata (C070). C. azalea, which is a known diploid species, was used as a control.
2.2. Reagents and Instrumentation
In this study, both the lysis solution and the staining solution were from a CyStain UV PreciseP Ploidy Test Kit, which was purchased from Kindi Future Biotechnology (Beijing, China). The ploidy identification was carried out using a Sysmex Cy Flow®Ploidy Analyser (Norderstedt, Germany); then, the data were exported; and the results were analyzed using Flomax2.81 software.
2.3. Experimental Method
2.3.1. Establishment of the Flow Cytometry Detection Method
(1) Sampling: In November 2022, young leaves and flowers were collected from the same tree and then brought back to the laboratory for further analysis.
(2) Sample preparation: Following the method of Yan et al. [13], 0.5 cm2 samples were added into a disposable culture dish; 400 mL of a PI lysis solution was added; then, the samples were quickly chopped with a single-sided blade. The material to be tested was completely submerged in the lysis solution during the whole process. Then, 1600 mL of a DAPI staining solution was added for dyeing, and the dyed sample was filtered through a microporous membrane into a sample tube.
(3) Determination of the tissue organs: The selection of materials is crucial during the process of ploidy identification using flow cytometry, as the ploidy identification outcomes can vary with different tissue types. In this study, the young leaves, pollen, petals, and filaments of oil-seed camellia were subjected to ploidy detection to ascertain the most suitable material for ploidy identification in oil-seed camellia.
(4) Determination of lysis and dyeing times: Lysis and dyeing times are principal factors affecting the efficacy of ploidy identification. The effects of ploidy identification vary with different lysis and dyeing times. In this study, we established gradients for lysis time (5, 10, 20, and 30 min) and dyeing time (5, 10, 20, and 30 min) to test the pollen of oil-seed camellia, with the aim of determining the optimal lysis and dyeing times for the detection of oil-seed camellia.
(5) Detection method: The filtered samples were placed in an inlet, and the prepared samples were analyzed with a Sysmex Cy Flow®Ploidy Analyser. Then, the data were exported, and the results were analyzed using Flomax2.81 software. The peak value histogram and coefficient of variation (CV) in the DNA peaks of each sample were obtained, and the size of the CV values was used as a parameter for evaluating the detection effect.
2.3.2. Identification of Oil-Seed Camellia Ploidy by Flow Cytometry
Based on the flow cytometry ploidy detection technology system for oil-seed camellia, Cy Stain UV Precise P was used as the dissociation solution, with pollen as the sample, to identify the ploidy of 70 oil-seed camellia germplasms.
Following the method of Yan et al. [13], 0.5 cm2 samples were added into a disposable culture dish; 400 mL of a PI lysis solution was added; a single-sided blade was used to chop the samples in preparation for 10 min of lysing; 1600 mL of a DAPI staining solution was added for 10 min of dyeing; the dyed sample was filter through a microporous membrane into a sample tube; then, the sample was put on a machine for ploidy identification; and the peak value histogram and CV value were recorded for each sample.
The known diploid C. azalea was used as a control, and the samples to be tested were compared with it. The corresponding ploidy of oil-seed camellia was determined, and 3 sets of repetitions were set for each sample. The peak value of the reference sample was 25.82, and the ploidy of the test sample was calculated based on the following formula:
DNA ratio (ploidy ratio) of the test sample = the fluorescence intensity peak of the DNA content in the test sample/the fluorescence intensity peak of the DNA content in the reference sample.
2.3.3. K-Mer Analysis to Identify the Ploidy of Oil-Seed Camellia
A Beijing Tiangen Plant Genomic DNA Extraction Kit was used to extract the DNA. The DNA was detected using agarose gel electrophoresis and the Qubit fluorescent dye method. After the DNA sample passed the test, a small fragment library was constructed. After passing the library test, sequencing was performed using the BGI DNBSEQ-T7 platform (Shenzhen, China), which manufactured by MGI Tech. The obtained raw data were subjected to quality control filtering (removing adapters, duplicates, and low-quality reads) for the K-mer analysis.
3. Results
3.1. Influence of Different Tissue Organs on Detection Effect
C. vietnamensis was used as the research object. Young leaves, pollen, petals, and filaments were used for ploidy identification to determine the best test material. The results showed that the peak histograms of samples prepared from different tissue organs were significantly different (Figure 1). Young leaves (Figure 1A): The identification effect of flow cytometry for young leaves was the worst. There was no obvious main peak, and its ploidy could not be judged. Pollen (Figure 1B): Flow cytometry had the best identification effect for pollen, with a narrow main peak, a coefficient of variation less than 5%, a few stray peaks, more particles collected, and a fast speed. Petals (Figure 1C): The identification effect of flow cytometry was good for petals, with a wide main peak, a coefficient of variation above 10%, more particles collected, and a faster speed. Filaments (Figure 1D): The identification effect of flow cytometry was even better for filaments, with a wider main peak and a coefficient of variation around 5% to 10%, but few particles collected and a slow speed. Combining the results of the four tissue organs, we can see that there is no obvious main peak in the young leaves, but there are obvious main peaks in the other tissue organs; pollen has the clearest main peak, the narrowest main peak, the least miscellaneous peaks, many collected particles, a fast speed, an easy and short chopping process, and a simple operation, making it the best material for the ploidy identification of oil-seed camellia.
3.2. Influence of Different Lysis and Dyeing Times on Detection Effect
C. gauchowensis was used as the research object, and pollen was used as the research material. A PI lysis solution (400 mL) was used for lysis for 5 min, 10 min, 20 min, and 30 min and, then, a DAPI dye solution (1600 mL) was used for dyeing for 5 min, 10 min, 20 min, and 30 min in order to screen for the best lysis and dyeing times. The results showed that the peak histograms with different lysis and dyeing times were significantly different. Under conditions with 5 and 30 min of lysis, all dyeing times had poor identification effects and many impurity peaks. Under conditions with 10 min and 20 min of lysis, dyeing for 5 min had poor identification effects and many impurity peaks; the main peak was broad, few particles were collected, and the speed was slow. Dyeing for 10 min, 20 min, and 30 min had better identification effects and fewer impurity peaks; the main peak was narrow, many particles were collected, and the speed was fast.
Based on the results of the different time combinations of these two factors, combinations with 10 to 20 min of lysis and 10 min, 20 min, and 30 min dyeing were found to have better identification effects, with narrow main peaks, fewer stray peaks, a coefficient of variation less than 5%, and the collection of many particles at high speeds. Therefore, the optimal lysis time was 10 to 20 min, with identification following less than 10 min and more than 20 min being poor. Identification following dyeing for more than 10 min was better, while that following dyeing for less than 10 min was worse. Taking into account the test efficiency issue, a lysis time of 10 min + a dyeing time of 10 min was the best combination for effectively identifying the ploidy of oil-seed camellia within the shortest time (Figure 2).
3.3. Ploidy Identification of Oil-Seed Camellia Using Flow Cytometry
Based on the optimized ploidy identification method of oil-seed camellia, the ploidies of 70 oil-seed camellia germplasm resources were identified, and the known diploid C. azalea was used as a control. The results (Table 2) showed that the peak value of C. azalea was 25.82 while the peak values of the 70 oil-seed camellia germplasms ranged from 20.84 to 108.92. Among them, 15 samples had peak values between 20.84 and 30.97 and DNA content ratios ranging from 0.81 to 1.20, suggesting that they are diploid; 24 samples had peak ranges of 44.85~56.93 and DNA content ratios ranging from 1.74 to 2.20, suggesting that they are tetraploid; 18 samples had peak values between 68.69 and 83.53 and DNA content ratios ranging from 2.66 to 3.24, suggesting that they are hexaploid; and 13 samples had peak values ranging from 93.54 to 108.92 and DNA content ratios ranging from 3.62 to 4.22, suggesting that they are octoploid. Most of the CVs of the 70 oil-seed camellia samples were less than 5%, indicating that the test data were reliable and the identification results were relatively accurate. The results of the different ploidy flow cytometry measurements are shown in Figure 3.
3.4. K-Mer Analysis to Identify the Ploidy of Oil-Seed Camellia
Five oil-seed camellia resources were sequenced on the BGI DNBSEQ-T7 platform, and the sequencing data were analyzed using KMC for K-mer analysis (with a K value of 23). First, the original sequencing reads were analyzed by calculating the K-mer frequency in the sequencing data. K-mers are K-length DNA sequences. By counting the frequency of these K-mer sequences, we can obtain preliminary information about the genome size, heterozygosity, and reproducibility. Then, heterozygous K-mer pairs were extracted from the K-mer database using Smudgeplot (https://github.com/KamilSJaron/smudgeplot, accessed on 5 May 2024), with these heterozygous K-mer pairs referring to the presence of two or more different versions of K-mer in the genome; they may correspond to different haplotypes or alleles. By comparing the total coverage (Cova + CovB) and relative coverage (CovB/(Cova + CovB)) of heterozygous K-mers pairs, Smudgeplot was able to count the number of heterozygous K-mers pairs and to parse the genome structure accordingly. The heat map generated with Smudgeplot indicates that the genotype of C037 (Figure 4A) was AAAB (0.64), that of C051 (Figure 4B) was AB (0.80), that of C047 (Figure 4C) was AB (1.00), that of CK (Figure 4D) was AB (0.60), and that of C043 (Figure 4E) was AAAB (0.68). Therefore, C037 and C043 were inferred to be tetraploid, and CK, C047, and C051, diploid.
4. Discussion
The identification of ploidy is a technique of fundamental importance in genetic evolution studies, systematic classification, and hybrid breeding endeavors [19]. Currently, methods such as chromosome counting and flow cytometry are employed for ploidy identification. Chromosome counting is the most direct and reliable method of identifying the ploidy of plants, but it has high requirements for the test materials and operation techniques, and the process is cumbersome and inefficient. In contrast, flow cytometry is not restricted by the plant sampling site and the developmental period of the cells; the preparation of samples is simple; and it has high sensitivity, resolution, and accuracy, making it suitable for the ploidy detection analysis of a large number of samples within a short period of time [20]. The reliability of flow cytometry has been demonstrated by comparing the results of chromosome counting and flow cytometry for the identification of ploidy in mulberry [21], C. hainanica [5], and cherry rootstock [22]. Consequently, flow cytometry has emerged as the preferred method for ploidy identification.
However, the efficacy of flow cytometry depends on many factors, such as tissue organ type, lysis time, and dyeing time. For a specific plant species, determining the optimal method for flow cytometry detection is thus essential. Flow cytometry has been optimized in potato [23], strawberry [24], barley [25], and other plants. Although many studies have reported on the ploidy identification of oil-seed camellia, none have reported on a flow cytometry detection system for the ploidy identification of oil-seed camellia.
Therefore, in this study, ploidy was identified in different tissue organs of oil-seed camellia. The results indicated that young leaves exhibited the least effective results, compared with the results of previous studies, possibly due to the leaves having more secondary metabolites such as polyphenols and their tendency to produce sticky residues when chopped, leading to the adsorption of sexual substances onto the cell nuclei and resulting in a low nuclear yield. Secondly, the leaves are prone to browning and thus clog the instrument. More background debris, interference from stray peaks, and scattered main peaks also appeared more easily [26]. Therefore, young leaves were the least effective. Conversely, pollen was the best material, with fewer fragments and clearer peak histograms, and no clogging or oxidation occurring during the experiment, probably because pollen grains are small and easy to cut; therefore, pollen is more suitable for the large-scale ploidy identification of oil-seed camellia.
Moreover, the lysis and dyeing times directly influenced the identification effect. Excessive lysis and dyeing times could lead to an abundance of background fragments [27]. In this study, the most suitable lysis and dyeing times were screened by comparing different lysis and dyeing times; the results of the study determined that lysis times of 10 to 20 min and dyeing times of 10 to 30 min were optimal. The optimal lysis time, 10 min, and dyeing time, 10 min, were screened by taking into account the efficiency issues of the test.
In this study, 70 oil-seed camellia germplasms were identified using the optimized identification system. The results showed various ploidy types, including diploid, tetraploid, hexaploid, and octoploid, a result that is consistent with previous research results [28,29,30]. C. meiocarpa were tetraploid. However, C. gauchowensis, C. sasanqua, C. vietnamensis, C. brevistyla, and C. kissi var. megalantha showed ploidy diversity. These results further verified the results of Zhuang [3] and Zhang [31], and the reliability of the methodology and results of this study.
In addition, the CV analysis in this study further validated the reliability of the results. CV is a crucial parameter in flow cytometry analysis, indicating the reliability of the data. Generally speaking, a CV of less than 5% is deemed reliable [32], while for polyphenolic plants, generally, less than 8% is considered credible [33]. In this study, the CV ranged from 2.15% to 7.75%, and most were below 5%, indicating that the data were reliable. The wider the main peak of the histogram due to cell debris and adhesions in the sample, the greater the CV will be, indicating potential challenges in data interpretation.
This study used a method that combines flow cytometry and K-mer analysis to identify oil-seed camellia ploidy, which can improve the accuracy of ploidy estimation and provide a reference for whole-genome sequencing research on oil-seed camellia. Based on the research results and the current trend of sequencing development, for testing the oil-seed camellia genome, the use of a complementary sequencing strategy with two or more sequencing methods is recommended when conducting oil-seed camellia genome sequencing and further promoting molecular biology research and the molecular breeding of oil-seed camellia.
5. Conclusions
In this study, by comparing the effects of different tissue organ types, lysis times, and dyeing times on the ploidy identification of oil-seed camellia, a technique for identifying ploidy in oil-seed camellia using flow cytometry was established. Compared with other tissue organs, pollen was the optimal material for preparing nuclei suspensions of oil-seed camellia, and the use of 400 mL of a PI lysis solution for 10 min of lysing, followed by dyeing with 1600 mL of a DAPI dyeing solution for 10 min, was the most suitable technique.
The ploidy levels of 70 oil-seed camellia resources were identified using an optimized method. The results showed various ploidy types, including diploid (15 samples), tetraploid (24 samples), hexaploid (18 samples), and octoploid (13 samples). The results of the K-mer analysis were consistent with those of the flow cytometry identification, indicating that the identification results of flow cytometry are reliable. This finding will provide a new and reliable method for quickly identifying the ploidy of oil-seed camellia and will lay a foundation for germplasm innovation and the ploidy breeding of oil-seed camellia.
Author Contributions
The contributions of the respective authors are as follows: Conceptualization, Y.Z., R.W. and Y.C.; methodology, Y.Z. and R.W.; software, Z.Z.; validation, Z.Z.; formal analysis, Y.Z. and Z.Z.; investigation, Y.Z., Z.Z., X.W., Z.H., G.X. and W.L.; resources, Y.C.; data curation, Y.Z., Z.Z., X.W. and Z.H.; writing—original draft preparation, Y.Z., Z.Z., X.W. and Z.H.; writing—review and editing, R.W. and Y.C.; visualization, Y.Z.; supervision, R.W. and Y.C.; project administration, Y.Z.; funding acquisition, Y.Z. All authors have read and agreed to the published version of the manuscript.
Funding
This research was funded by the Key Research and Development Program of Hunan (2023NK2005), the Forestry Scientific and Innovative Program of Hunan (XLKY202206) and the Major Special Project of Changsha Science and Technology Bureau (KQ2102007).
Data Availability Statement
The original contributions presented in the study are included in the article, further inquiries can be directed to the corresponding authors.
Acknowledgments
We thank three reviewers for their helpful suggestions for this manuscript.
Conflicts of Interest
Authors Gaohong Xiao and Weiguo Li were employed by the company Huazhi Biotech Co., Ltd. The remaining authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.
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Figure 1.
Peak value histograms of different tissue organs: (A): young leaves; (B): pollen; (C): petals; (D): filaments.
Figure 1.
Peak value histograms of different tissue organs: (A): young leaves; (B): pollen; (C): petals; (D): filaments.
Figure 2.
Peak value histograms of different lysis and dyeing times: (A): lysis 5 min + dyeing 5 min; (B): lysis 5 min + dyeing 10 min; (C): lysis 5 min + dyeing 20 min; (D): lysis 5 min + dyeing 30 min; (E): lysis 10 min + dyeing 5 min; (F): lysis 10 min + dyeing 10 min; (G): lysis 10 min + dyeing 20 min; (H): lysis 10 min + dyeing 30 min; (I): lysis 20 min + dyeing 5 min; (J): lysis 20 min + dyeing 10 min; (K): lysis 20 min + dyeing 20 min; (L): lysis 20 min + dyeing 30 min; (M): lysis 30 min + dyeing 5 min; (N): lysis 30 min + dyeing 10 min; (O): lysis 30 min + dyeing 20 min; (P): lysis 30 min + dyeing 30 min.
Figure 2.
Peak value histograms of different lysis and dyeing times: (A): lysis 5 min + dyeing 5 min; (B): lysis 5 min + dyeing 10 min; (C): lysis 5 min + dyeing 20 min; (D): lysis 5 min + dyeing 30 min; (E): lysis 10 min + dyeing 5 min; (F): lysis 10 min + dyeing 10 min; (G): lysis 10 min + dyeing 20 min; (H): lysis 10 min + dyeing 30 min; (I): lysis 20 min + dyeing 5 min; (J): lysis 20 min + dyeing 10 min; (K): lysis 20 min + dyeing 20 min; (L): lysis 20 min + dyeing 30 min; (M): lysis 30 min + dyeing 5 min; (N): lysis 30 min + dyeing 10 min; (O): lysis 30 min + dyeing 20 min; (P): lysis 30 min + dyeing 30 min.
Figure 3.
Peak value histograms of different ploidies: (A): C. azalea (diploid, used as a control); (B): C. gigantocarpa (diploid); (C,D): C. meiocarpa (tetraploid); (E,F): C. oleifera (hexaploid); (G): C. pitardii (octoploid); (H): C. vietnamensis (octoploid).
Figure 3.
Peak value histograms of different ploidies: (A): C. azalea (diploid, used as a control); (B): C. gigantocarpa (diploid); (C,D): C. meiocarpa (tetraploid); (E,F): C. oleifera (hexaploid); (G): C. pitardii (octoploid); (H): C. vietnamensis (octoploid).
Figure 4.
Heat map of a ploidy analysis for five oil-seed camellia: (A): C037; (B): C051; (C): C047; (D): CK; (E): C043.
Figure 4.
Heat map of a ploidy analysis for five oil-seed camellia: (A): C037; (B): C051; (C): C047; (D): CK; (E): C043.
Table 1.
List of oil-seed camellia germplasms in this study.
| Germplasm ID | Species | Source | Germplasm ID | Species | Source |
|---|---|---|---|---|---|
| CK | C. azalea | NCOGRPC | C036 | C. fraterna | NCOGRPC |
| C001 | C. oleifera | Changde, Hunan | C037 | C. oleifera | NCOGRPC |
| C002 | C. oleifera | Changde, Hunan | C038 | C. vietnamensis | NCOGRPC |
| C003 | C. oleifera | Changde, Hunan | C039 | C. gauchowensis | NCOGRPC |
| C004 | C. oleifera | Changde, Hunan | C040 | C. chekiangoleosa | NCOGRPC |
| C005 | C. oleifera | Changde, Hunan | C041 | C. meiocarpa | NCOGRPC |
| C006 | C. oleifera | Changde, Hunan | C042 | C. meiocarpa | NCOGRPC |
| C007 | C. oleifera | Changde, Hunan | C043 | C. meiocarpa | NCOGRPC |
| C008 | C. oleifera | Changde, Hunan | C044 | C. meiocarpa | NCOGRPC |
| C009 | C. oleifera | Changde, Hunan | C045 | C. meiocarpa | NCOGRPC |
| C010 | C. oleifera | Changde, Hunan | C046 | C. meiocarpa | NCOGRPC |
| C011 | C. oleifera | Changde, Hunan | C047 | C. furfuracea | NCOGRPC |
| C012 | C. oleifera | Changde, Hunan | C048 | C. furfuracea | NCOGRPC |
| C013 | C. oleifera | Changde, Hunan | C049 | C. oleifera | NCOGRPC |
| C014 | C. oleifera | Changde, Hunan | C050 | C. oleifera | NCOGRPC |
| C015 | C. oleifera | Xiangxi, Hunan | C051 | C. gigantocarpa | NCOGRPC |
| C016 | C. oleifera | Xiangxi, Hunan | C052 | C. gigantocarpa | NCOGRPC |
| C017 | C. oleifera | Xiangxi, Hunan | C053 | C. sasanqua | NCOGRPC |
| C018 | C. oleifera | Xiangxi, Hunan | C054 | C. sasanqua | NCOGRPC |
| C019 | C. oleifera | Xiangxi, Hunan | C055 | C. brevistyla | NCOGRPC |
| C020 | C. oleifera | Xiangxi, Hunan | C056 | C. kissi var. megalantha | NCOGRPC |
| C021 | C. oleifera | Xiangxi, Hunan | C057 | C. brevistyla | NCOGRPC |
| C022 | C. oleifera | Xiangxi, Hunan | C058 | C. kissi var. megalantha | NCOGRPC |
| C023 | C. oleifera | Xiangxi, Hunan | C059 | C. brevistyla | NCOGRPC |
| C024 | C. oleifera | Xiangxi, Hunan | C060 | C. fraterna | NCOGRPC |
| C025 | C. oleifera | Xiangxi, Hunan | C061 | C. gauchowensis | NCOGRPC |
| C026 | C. oleifera | Xiangxi, Hunan | C062 | C. gauchowensis | NCOGRPC |
| C027 | C. oleifera | Xiangxi, Hunan | C063 | C. gauchowensis | NCOGRPC |
| C028 | C. oleifera | Xiangxi, Hunan | C064 | C. pitardii | NCOGRPC |
| C029 | C. pitardii | Xiangxi, Hunan | C065 | C. pitardii | NCOGRPC |
| C030 | C. pitardii | Xiangxi, Hunan | C066 | C. vietnamensis | NCOGRPC |
| C031 | C. yuhsienensis | Hengyang, Hunan | C067 | C. tunganica | NCOGRPC |
| C032 | C. handelii | Changsha, Hunan | C068 | C. tunganica | NCOGRPC |
| C033 | C. nitidissima | Jinhua, Zhejiang | C069 | C. pitardii | NCOGRPC |
| C034 | C. nitidissima | Jinhua, Zhejiang | C070 | C. semiserrata | NCOGRPC |
| C035 | C. oleifera | NCOGRPC |
Table 2.
Nuclear DNA contents and ploidy of 70 oil-seed camellia germplasm resources.
| Germplasm ID | Peak Value | CV (%) | Ratio of DNA Content | Ploidy Estimation |
|---|---|---|---|---|
| CK | 25.82 | 4.37 | 1.00 | 2 |
| C001 | 69.14 | 4.75 | 2.68 | 6 |
| C002 | 50.02 | 4.19 | 1.94 | 4 |
| C003 | 56.93 | 2.15 | 2.20 | 4 |
| C004 | 45.76 | 5.85 | 1.77 | 4 |
| C005 | 52.28 | 6.90 | 2.02 | 4 |
| C006 | 74.95 | 3.54 | 2.90 | 6 |
| C007 | 68.91 | 6.36 | 2.67 | 6 |
| C008 | 71.14 | 3.74 | 2.76 | 6 |
| C009 | 69.21 | 4.99 | 2.68 | 6 |
| C010 | 55.76 | 3.45 | 2.16 | 4 |
| C011 | 54.86 | 3.63 | 2.12 | 4 |
| C012 | 54.27 | 4.92 | 2.10 | 4 |
| C013 | 54.44 | 4.04 | 2.11 | 4 |
| C014 | 54.57 | 6.43 | 2.11 | 4 |
| C015 | 107.03 | 2.45 | 4.15 | 8 |
| C016 | 101.83 | 4.51 | 3.94 | 8 |
| C017 | 101.92 | 2.64 | 3.95 | 8 |
| C018 | 93.77 | 3.68 | 3.63 | 8 |
| C019 | 69.05 | 4.77 | 2.67 | 6 |
| C020 | 108.92 | 2.88 | 4.22 | 8 |
| C021 | 101.80 | 4.29 | 3.94 | 8 |
| C022 | 82.19 | 3.03 | 3.18 | 6 |
| C023 | 76.62 | 4.66 | 2.97 | 6 |
| C024 | 93.54 | 3.00 | 3.62 | 8 |
| C025 | 100.93 | 3.34 | 3.91 | 8 |
| C026 | 103.13 | 3.54 | 3.99 | 8 |
| C027 | 82.12 | 5.95 | 3.18 | 6 |
| C028 | 55.80 | 7.51 | 2.16 | 4 |
| C029 | 100.17 | 3.29 | 3.88 | 8 |
| C030 | 98.45 | 3.73 | 3.81 | 8 |
| C031 | 47.56 | 7.18 | 1.84 | 4 |
| C032 | 30.84 | 7.59 | 1.19 | 2 |
| C033 | 25.11 | 7.57 | 0.97 | 2 |
| C034 | 20.91 | 6.90 | 0.81 | 2 |
| C035 | 70.79 | 3.71 | 2.74 | 6 |
| C036 | 27.21 | 5.29 | 1.05 | 2 |
| C037 | 44.96 | 4.41 | 1.74 | 4 |
| C038 | 95.46 | 2.90 | 3.70 | 8 |
| C039 | 53.53 | 3.85 | 2.07 | 4 |
| C040 | 22.45 | 4.57 | 0.87 | 2 |
| C041 | 44.85 | 4.53 | 1.74 | 4 |
| C042 | 49.30 | 4.88 | 1.91 | 4 |
| C043 | 50.42 | 4.29 | 1.95 | 4 |
| C044 | 44.85 | 5.29 | 1.74 | 4 |
| C045 | 47.10 | 4.61 | 1.82 | 4 |
| C046 | 48.95 | 4.20 | 1.90 | 4 |
| C047 | 20.84 | 4.76 | 0.81 | 2 |
| C048 | 22.42 | 3.75 | 0.87 | 2 |
| C049 | 70.34 | 5.18 | 2.72 | 6 |
| C050 | 70.51 | 4.58 | 2.73 | 6 |
| C051 | 24.22 | 4.27 | 0.94 | 2 |
| C052 | 30.97 | 4.63 | 1.20 | 2 |
| C053 | 55.51 | 7.35 | 2.15 | 4 |
| C054 | 68.69 | 4.28 | 2.66 | 6 |
| C055 | 22.86 | 6.10 | 0.89 | 2 |
| C056 | 24.91 | 6.41 | 0.96 | 2 |
| C057 | 28.28 | 6.35 | 1.10 | 2 |
| C058 | 45.26 | 6.02 | 1.75 | 4 |
| C059 | 48.02 | 3.01 | 1.86 | 4 |
| C060 | 72.99 | 6.51 | 2.83 | 6 |
| C061 | 71.36 | 3.49 | 2.76 | 6 |
| C062 | 83.53 | 3.74 | 3.24 | 6 |
| C063 | 80.84 | 7.58 | 3.13 | 6 |
| C064 | 81.53 | 5.04 | 3.16 | 6 |
| C065 | 104.49 | 4.96 | 4.05 | 8 |
| C066 | 55.66 | 7.75 | 2.16 | 4 |
| C067 | 47.79 | 4.95 | 1.85 | 4 |
| C068 | 29.71 | 6.17 | 1.15 | 2 |
| C069 | 20.90 | 5.28 | 0.81 | 2 |
| C070 | 22.38 | 4.69 | 0.87 | 2 |
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Zhang, Y.; Zhang, Z.; Wang, X.; Wang, R.; He, Z.; Xiao, G.; Li, W.; Chen, Y. Establishment and Optimization of an Experiment System for Flow Cytometry in Oil-Seed Camellia. Horticulturae 2024, 10, 704. https://doi.org/10.3390/horticulturae10070704
AMA Style
Zhang Y, Zhang Z, Wang X, Wang R, He Z, Xiao G, Li W, Chen Y. Establishment and Optimization of an Experiment System for Flow Cytometry in Oil-Seed Camellia. Horticulturae. 2024; 10(7):704. https://doi.org/10.3390/horticulturae10070704
Chicago/Turabian StyleZhang, Ying, Zhen Zhang, Xiangnan Wang, Rui Wang, Zhilong He, Gaohong Xiao, Weiguo Li, and Yongzhong Chen. 2024. "Establishment and Optimization of an Experiment System for Flow Cytometry in Oil-Seed Camellia" Horticulturae 10, no. 7: 704. https://doi.org/10.3390/horticulturae10070704
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