Karyotype Analysis and Genome Size Estimation Using Flow Cytometry of the Genus Encyclia Hook. (Orchidaceae: Laeliinae)

Abstract

Encyclia Hook. is a tropical orchid known for its delightful fragrance and distinctive flower shapes, which have been widely used as superior parents for inter and intrageneric breeding. In this study, karyotype analysis shows that the number of chromosomes in diploid plants of Encyclia is 40, with all species exhibiting mesial and central filamentous chromosomes. The karyotype of diploid plants is primarily 2B. Among the three detected intergeneric hybrids, both Enanthleya Circus Lady ‘Coastal Star’ and Guaricyclia Kyoguchi ‘Fumi’ are triploid, while Robertsara ‘Green Wonder’ is tetraploid. By evaluating various lysates and tissues, a flow cytometry system (FCM) is developed with Galbraith’s buffer (GLB) and tender leaves. By using Dendrobium officinale as an external reference, the genome size of the Encyclia species and its intra and intergeneric hybrids are assessed, which ranges from 1.07 Gb to 3.23 Gb. These results will establish a foundation for high-throughput identification of germplasms, advance crossbreeding efforts, and enhance the understanding of the genome in Encyclia.

1. Introduction

Encyclia Hook. is a genus of Neotropical orchids found from Florida and Mexico to southeastern Brazil [1,2,3]. It currently includes 213 species, with 179 officially described and 25 likely of hybrid origin [4]. Within the Encyclia genus, there are several supposedly related species that exhibit distinct morphological traits, such as the shape of central lobe of the labellum, the presence of warts on the ovaries with peduncles, and the existence or absence of columnar processes on the column [5]. These plants typically grow in clusters, featuring short internodes that connect ovate and pear-shaped pseudobulbs. Each pseudobulb bears one to four jointed fleshy leaves and a single apical panicle [2].

Karyotype represents the initial phenotypic expression of a genotype [6] and can reflect chromosome number and structure, heterochromatic band diversity, and gene location. Changes in karyotypic symmetry typically involve changes in chromosome size and shape [6,7]. Detailed karyotyping is essential to identify chromosomal signatures and further determine differences and relationships between species and taxa [8,9,10]. Genome size (GS) reflects the ecological adaptability of species [11] and significantly affects certain epigenetic traits as well as the growth and development of plants [12]. Techniques for measuring genome size include genome sequencing [13], flow cytometry [14], and Feulgen densitometry [15].

The number and morphology of chromosomes in a few species of the genus Encyclia, such as E. flava (Lindl.) Porto & Brade, E. oncidioides (Lindl.) Schltr., E. seidelii Pabst, have been investigated and their genome size had been roughly estimated, with the smallest genome being E. seidelii with 2C = 2.87 pg and the largest being E. oncidioides and E. flava with 2C = 5.20 pg [16]. However, these species studied in the past are only a small part of the genus Encyclia and they are not common in practical production and ornamental applications. In addition, previous studies only focused on chromosome number and morphology but lacked detailed karyotyping and did not establish efficient systematic methods for assessing ploidy and genome size. This study aims to conduct karyotyping analysis of widely cultured Encyclia species and hybrids, establishing a high-throughput flow cytometry (FCM) analysis technique to assess ploidy and genome size, which will further facilitate species identification, crossbreeding and genomic research.

2. Materials and Methods

2.1. Plant Materials

The accessions include Encyclia species, as well as hybrids that are commonly grown (

Table 1. Species and hybrids of Encyclia.

Name	Types	Genus	Seed Parent	Pollen Parent
E. alata	Species	Encyclia	-	-
E. cordigera var. alba	Species	Encyclia	-	-
E. cordigera var. rosea	Species	Encyclia	-	-
E. granitica	Species	Encyclia	-	-
E. rufa	Species	Encyclia	-	-
E. tampensis var. alba	Species	Encyclia	-	-
E. tampensis ‘Gem’	Species	Encyclia	-	-
E. Orchid Jungle	Intrageneric hybrid	Encyclia	E. alata	E. phoenicea
E. Shinfong Smile	Intrageneric hybrid	Encyclia	E. Shinfong Thomas	E. cordigera
Eny. Circus Lady ‘Coastal Star’	Intergeneric hybrid	Enanthleya	Cattlianthe Trick or Treat	E. correllii
Gcy. Kyoguchi ‘Fumi’	Intergeneric hybrid	Guaricyclia	Guarianthe aurantiaca	E. incumbens
Robertsara Green Wonder	Intergeneric hybrid	Robertsara	Epicyclia Mabel Kanda	Cattlianthe Loog Tone

and Figure A1). All of them are cultivated in the germplasm nursery of the Tropical Crop Genetic Resource Institute, Chinese Academy of Tropical Agricultural Science (Danzhou, China).

2.2. Preparation and Staining of Chromosomes

The root tip was pretreated with saturated p-dichlorobenzene solution for 4 h at 4 °C. It was rinsed with ultrapure water and transferred to a Carnoy solution consisting of absolute ethanol and glacial acetic acid (3:1, v/v) for 24 h at 4 °C. After that, it was placed in 1 mol/L hydrochloric acid solution for 8 min at 60 °C. Finally, it was stained with modified carbolic acid magenta. The number of chromosomes was observed and counted under a microscope, and cells with well-defined and scattered chromosome morphology were selected for karyotyping photos. Chromosomes were measured using Image J v1.54g, and the measurement data were organized. Karyotyping was conducted according to the Stebbins classification criteria [17], and the karyotype asymmetry coefficient was calculated using Arano’s method [18]. Chromosomes were classified according to the method outlined by Levan [19].

2.3. Construction of Flow Cytometry System for Encyclia

Galbraith buffer (GLB), Woody Plant buffer (WPB), and Modified Gitschier buffer (mGb), as well as root tips, tender leaves and mature leaves of E. alata (Bateman) Schltr., were used to establish a systematic flow cytometry evaluation system. GLB buffer was chosen as the buffer for the experiment because of its single and sharp peak of flow in the cytometry histogram. The three plant tissues were placed in 1 mL of pre-chilled GLB lysate, quickly minced by a blade, and then stained for 1 min with 20 μL of DAPI fluorescent dye before being detected by the Sysmex CyFlow Ploidy Analyzer. Tender leaves were selected as the plant tissue material for experiments because of their flow cytometry histogram with a single and sharp peak.

2.4. Flow Cytometric Estimation of 2C DNA Content

The genome sizes of Encyclia species and hybrids were estimated according to the established flow cytometry system of Encyclia. Diploid Dendrobium officinale Kimura et Migo with a genome size of 1.1 Gb was used as a reference plant using the following formula:

$$\mathrm{Genome} \mathrm{size}=1.1 \mathrm{Gb}\times {\displaystyle \frac{\mathrm{Mean} \mathrm{florescence} \mathrm{value} \mathrm{of} \mathrm{the} \mathrm{tested} \mathrm{samples}}{\mathrm{Mean} \mathrm{florescence} \mathrm{value} \mathrm{of} D. \mathit{officinale}}}$$

2.5. Statistical Analysis

The number of replicates performed for the experiments on karyotyping and FCM was three. The FCM data collected were analyzed, and flow histograms of the test samples were plotted using FCS Express version 3. The data were summarized using Excel, and one-way ANOVA was performed using IBM SPSS Statistics 26 software.

3. Results

3.1. Karyotype Analysis

As shown in Figure 1, with a chromosome base of 20, six species were diploid (2n = 2x = 40), except for Robertsara Green Wonder, which was tetraploid (2n = 4x = 80) (Figure 1). The seven species studied included both metacentric and submetacentric chromosomes, with only E. cordigera var. alba having a pair of acrocentric chromosomes (Figure 2). Being tetraploid, Roertsara Green Wonder exhibited the greatest variation in relative lengths and the highest ratio of the longest to the shortest chromosome, as well as the highest nuclear asymmetric coefficient, categorized as karyotype type 2C. Among the diploid species, E. Shinfong Smile, an intrageneric hybrid, had the largest range of relative lengths and the highest ratio of the longest to the shortest chromosome, while the other diploid species were all unique. Both E. alata and E. cordigera var. alba had the same karyotype type, which was type 3B, while the other diploid species were classified as type 2B (

Table 2. Chromosome characteristics of Encyclia.

Name	Karyotype Formula	Relative Length Range (%)	Average Arm Ratio	Asymmetry Index (%)	Longest/ Shortest	Scale of Arm Ratio > 2 (%)	Karyotype
E. alata	2n = 2x = 40 = 8 m + 32 sm	0.27~0.54	2.11	66.71	2.33	0.53	3B
E. tampensis var. alba	2n = 2x = 40 = 24 m + 16 sm	0.43~0.88	1.77	63.33	2.11	0.20	2B
E. tampensis ‘Gem’	2n = 2x = 40 = 8 m + 32 sm	0.40~0.81	1.90	64.57	2.13	0.38	2B
E. cordigera var. alba	2n = 2x = 40 = 4 m + 34 sm + 2 st	0.32~0.72	2.19	67.91	2.42	0.58	3B
E. rufa	2n = 2x = 40 = 26 m + 14 sm	0.31~0.66	1.55	60.43	2.20	0.08	2B
E. Shinfong Smile	2n = 2x = 40 = 24 m + 16 sm	0.70~2.00	1.76	62.24	3.03	0.25	2B
Robertsara Green Wonder	2n = 4x = 80 = 60 m + 20 sm	1.01~2.50	1.62	80.00	5.16	0.14	2C

).

3.2. Flow Cytometry Analysis of Encyclia

Flow cytometry histograms indicated that all three lysates produced peaks, but there were variations in fluorescence intensity and CV values, with the WPB sample exhibiting notably higher CV values compared to the other two samples (

Table 3. Flow cytometry results for three different lysates.

Lysis Solution	Fluorescence Intensity	Coefficient of Variation (%)
GLB	6881.17 ± 138.32 c	4.11 ± 0.11 ab
WPB	7796.33 ± 5.51 a	4.37 ± 0.03 a
mGb	7571.50 ± 75.75 b	3.84 ± 0.35 b

Note: Lowercase letters indicate significant differences among treatments of different lysates at the 0.05 level using a Tukey test.

). The flow cytometry histograms for WPB and mGb displayed multiple and prominent impurity peaks, whereas GLB showed a single and sharp peak (Figure 3). Consequently, GLB was selected as the lysate for further experiments. Tender leaves were chosen as the experimental material due to their flow cytometry histogram with a single and sharp peak.

3.3. Genome Size Estimation of Encyclia

Using GLB as the lysate and the tender leaves from each sample as the material, the genome size was estimated through flow cytometry, maintaining the CV value within 5%. The flow histograms of the samples were obtained (Figure 4). The genome size of the sample being measured was calculated using a formula with diploid Dendrobium officinale as the external reference sample (

Table 4. Genome size estimation of native species and hybrids of Encyclia.

Name	Fluorescence Intensity	Ratio	Genome Size (Gb)	Coefficient of Variation (%)	Ploidy Level
D. officinale	6313.33 ± 119.26	1.00	1.11	4.53 ± 0.37	Diploidy
E. alata	6395.33 ± 17.39	1.01 ± 0.02	1.12 ± 0.02	4.56 ± 0.02	Diploidy
E. cordigera var. rosea	6320.17 ± 62.12	1.00 ± 0.02	1.11 ± 0.03	4.53 ± 0.27	Mixoploidy
E. tampensis ‘Gem’	6061.50 ± 165.40	0.96 ± 0.04	1.07 ± 0.04	4.64 ± 0.14	Diploidy
E. granitica	10,194.17 ± 102.38	1.62 ± 0.05	1.79 ± 0.05	4.44 ± 0.06	Triploidy
E. Orchid Jungle	6421.67 ± 94.03	1.02 ± 0.03	1.13 ± 0.03	4.15 ± 0.59	Diploidy
E. Shinfong Smile	6181.83 ± 156.86	0.98 ± 0.04	1.09 ± 0.04	4.95 ± 0.03	Diploidy
Eny. Circus Lady ‘Coastal Star’	9708.17 ± 122.68	1.54 ± 0.04	1.71 ± 0.04	4.59 ± 0.05	Triploidy
Gcy. Kyoguchi ‘Fumi’	11,008.67 ± 157.33	1.74 ± 0.01	1.94 ± 0.01	4.63 ± 0.18	Triploidy
Robertsara Green Wonder	18,351.50 ± 421.00	2.91 ± 0.07	3.23 ± 0.08	3.31 ± 0.12	Tetraploidy

Note: Ratio means fluorescence intensity of the sample to be tested/fluorescence intensity of the external reference sample.

). Among the species, E. alata, E. cordigera var. rosea, and E. tampensis ‘Gem’ were found to be diploid, with genome sizes of (1.12 ± 0.02) Gb, (1.11 ± 0.03) Gb, and (1.07 ± 0.04) Gb, respectively. E. granitica was identified as triploid, with a genome size of (1.79 ± 0.05) Gb. E. Orchid Jungle and E. Shinfong Smile, both intrageneric hybrids of Encyclia, were diploid with genome sizes of (1.13 ± 0.03) Gb and (1.09 ± 0.04) Gb, respectively. As intergeneric hybrids, Eny. Circus Lady ‘Coastal Star’ and Gcy. Kyoguchi ‘Fumi’ were triploid, while Robertsara Green Wonder was tetraploid, with genome sizes of (1.71 ± 0.04) Gb, (1.94 ± 0.01) Gb, and (3.23 ± 0.08) Gb, respectively.

4. Discussion

The research revealed that six species exhibited somatic chromosomes with a configuration of 2n = 2x = 40, consistent with findings from other species within the same genus, such as E. flava, E. oncidioides and E. seidelii, in earlier studies [16]. In contrast, Robertsara Green Wonder displayed a chromosome count of 2n = 4x = 80, indicating it is tetraploid. In this study, the tetraploid Robertsara Green Wonder showed a higher asymmetry coefficient. Chromosomal morphological diversity may result from changes in chromosomal symmetry due to intragenomic translocations, interarm inversions, fusions, or divisions, which may or may not influence genome size [6,20].

The fusion of 2n gametes is one of the mechanisms of polyploidy formation, and it has been observed that distant hybridization has a higher probability of producing 2n gametes compared to species [21,22]. Through intrageneric, and even intergeneric hybridization with more distant relatives, a 2n gamete could be obtained with a high probability compared with the species, resulting in polyploidy [21,23]. As a triploid, the male parent of Eny. Circus Lady ‘Coastal Star’ is an intergeneric hybrid, and the female parent is a species. Robertsara Green Wonder is a tetraploid intergeneric hybrid, and its parents are both intergeneric hybrids. Most of the other species are diploids, which shows that polyploidy can be formed through distant hybridization.

In previous experiments, plant leaves were commonly used as material for flow cytometry [24,25]. However, the presence of high concentrations of starch, polysaccharides or other cellular metabolites in mature leaves have a certain impact on the results of flow cytometry analysis. Containing the appropriate concentration of TritonX-100, GLB is used to remove sugars, phenols and other viscous substances from cells, resulting in a uniform and sharp peak. Therefore, GLB and young leaves were used as lysates and tissue materials for flow cytometry detection in this experiment. Intranuclear replication was prevalent in Orchidaceae, which showed two peaks on the flow cytometry histogram. In this study, E. cordigera var. rosea is judged to be mixoploid because it produces a signal peak lower than the G0/G1 period in the G2/M range of histogram.

As a genus of the subtribe Laeliinae (Orchidaceae), the genome size ranges from 1.07 to 1.79 Gb. Eny. Circus Lady ‘Coastal Star’, Gcy. Kyoguchi ‘Fumi’ and Robertsara Green Wonder were obtained by crossing a member of the genus Encyclia with a related genus, with genome sizes ranging from 1.71 to 3.23 Gb. The DNA content varied in the range of 2C = 2.85 pg (Prosthechea fragrans) ~ 2C = 5.76 pg (Laelia gouldiana), and the minimum diploid genome in the genus was 2C = 2.93 pg (E. ionosma), while the maximum diploid genome in this study was 2C = 1.13 Gb (1.15 pg) [16]. Flow cytometry of 60 Phalaenopsis varieties showed that the genome size was 1.93~7.78 Gb, and there was a significant difference between varieties [26]. Therefore, there are some differences in the genome size of different species within the same genus, which may be related to parentage.

5. Conclusions

This research utilized karyotype analysis to examine the chromosomal features of diploid plants belonging to the genus Encyclia. Additionally, a flow cytometry system for Encyclia was developed to assess the genome size of four native species, two hybrids within the genus, and three hybrids between different genera, providing valuable insights for future genomic studies.