Screening of Ty1-copia Retrotransposons in Water Onion (Crinum thaianum), an Endangered Species in Thailand

Abstract

Crinum thaianum, commonly known as water onion, is an endangered species which is primarily threatened by flood-control-related habitat destruction and illegal harvesting for export, resulting in a sharp population decline; its genetic data still remains poorly studied. Retrotransposon-based markers have received significant attention due to their higher potential informativeness compared to conventional marker methods in genetic diversity studies. This study focused on the screening of Ty1-copia retrotransposons, which have been widely studied and are commonly used as molecular markers in various plant species. Ty1-copia reverse transcriptase (rt) fragments were amplified using degenerate primers targeting conserved regions, followed by cloning and sequencing. Sequences were screened for rt gene homology and translated into amino acid sequences. Lineages were assigned by alignment, and phylogenetic analysis was performed for each isolated sequence with a set of well-classified rt genes. The p-distance values were calculated between the isolated sequences and their closest homologous sequences. A total of 123 isolated sequences were analyzed, representing conserved domains in the rt gene of Ty1-copia elements from C. thaianum and four other Crinum species. The results revealed sequence homology to the Ale, TAR, or Angela lineages, which showed the closest resemblance to 9, 4, and 110 isolated rt sequences, respectively. The conserved rt domain SIYGLKQA was mostly found in Angela (87.27%), while SLY/HGLKQS/L and SLYG/ELKQF/S were mostly found in Ale (66.67%) and TAR (75.00%), respectively. The p-distance values obtained from comparisons with Ty1-copia elements in other plants suggest that the Angela and TAR lineages are more evolutionarily conserved than the Ale lineage. Whilst our study sheds light on the variety of Ty1-copia retrotransposons in C. thaianum and other Crinum species, further research on additional Crinum species and other plants is required to enhance our understanding and facilitate future retrotransposon-based marker development.

1. Introduction

Crinum thaianum, commonly known as water onion, has a diploid chromosome number of 2n = 22 [1]. It is a native aquatic plant located in the southern areas of Thailand; the nomenclature ‘thaianum’ is attributed to its discovery exclusively in the Thai provinces of Ranong and Phang Nga [2]. Typically, this plant thrives along the banks of flowing streams, with its roots and bulbs submerged beneath the water. Its flowers form a prominent umbel structure above the water surface, and the plant can grow to an impressive height of 150 cm. Water onion, aside from its ecological role, offers various advantages. Its towering height, long leaves, and attractive foliage make it an ideal background ornamental plant for aquariums, contributing to its economic value. Moreover, it serves as an indicator of water quality in streams, as it can only grow in clear and clean water sources. Additionally, it holds potential for cosmetic and skincare product extraction [3]. In the past, the water onion blooming season in Ranong and Phang Nga provinces was a popular tourist attraction; today, however, the plant is categorized as an endangered species on the International Union for Conservation of Nature (IUCN) Red List due to both illegal harvesting for exportation and upstream destruction, generally for flood safety purposes.

Genetic diversity in plants can be explored using various DNA markers, including both dominant and co-dominant types. A specific investigation into the genetic diversity of water onion using the Polymerase Chain Reaction–Restriction Fragment Length Polymorphism (PCR-RFLP) marker technique was reported by Changcharoen et al. in 2014 [1]. Notably, there are currently no reports on Ty1-copia retrotransposons in the Crinum species. Long Terminal Repeat (LTR) retrotransposons, which are class I transposable elements (TEs), are widely distributed and abundant within plant genomes [4,5,6]. TEs, also known as mobile genetic elements or jumping genes, represent DNA sequences capable of relocating within a genome. This movement can induce the creation or reversal of mutations, thereby influencing the genetic identity and genome size of the cell. Such alterations contribute to the generation of diversity, rendering transposons an intriguing subject for research and exploration. Among the diverse types of TEs, the Ty1-copia family, a prominent subgroup of LTR-retrotransposons, has undergone extensive investigation in plants [4,5,6,7,8]. The crucial structures for transposition within Ty1-copia are the LTR elements, which are conserved in the flanking regions among Ty1-copia retrotransposons of the same family [9]. Ty1-copia elements form a diverse superfamily, with multiple families present across major plant lineages [10,11,12,13,14].

The Ty1-copia retrotransposons in various plants have been predominantly analyzed using PCR methods, particularly with degenerate primers designed for amplifying copia-like reverse transcriptase (rt) gene fragments [4]. This technique facilitates the exploration of retrotransposon variety across numerous plants, leading to phylogenetic comparisons amongst copia element populations. Recently, retrotransposon-based markers have gained popularity for their use in analyzing the abundant LTR-retrotransposons found in plant genomes. The markers provide great benefits, serving as useful tools for diverse genomic applications, including polymorphism screening, genome mapping, and marker-assisted breeding, as well as diversity evaluation and evolutionary studies [15,16].

There is limited genetic data available for Crinum species, particularly regarding retrotransposon activity and evolution; no existing information on the diversity of retrotransposons in C. thaianum has been reported. In this study, we aimed to isolate and classify rt fragments of Ty1-copia retrotransposons from the genomes of C. thaianum and selected Crinum species to provide the first insights into their diversity. We employed degenerate primers to evaluate the heterogeneity and phylogenetic relationships among these sequences, particularly between C. thaianum, the endangered native plant of Thailand, and other Crinum species. This study is expected to offer valuable insights into the diversity of Ty1-copia retrotransposons in Crinum species, supporting the future development of retrotransposon-based molecular markers.

2. Materials and Methods

2.1. Plant Materials and DNA Extraction

Six water onion (C. thaianum) samples were sourced from Ranong and Phang Nga provinces, as well as from the White Cane Company and Suan Luang Rama IX in Bangkok, Thailand (Figure 1A). Four other Crinum species were also included in the research (Figure 1B–E). The genomic DNA of Crinum plants was obtained from leaves using a modification of the protocol outlined by Doyle and Doyle, 1990 [17]. The quality of the genomic DNA was observed using 0.8% agarose gel electrophoresis stained with ethidium bromide to visualize DNA bands under UV light, and its concentration was determined through spectrophotometry. All the genomic DNA samples were stored at −20 °C, and the extracted DNA was appropriately diluted for PCRs.

2.2. Isolation of Ty1-copia rt Fragments

PCRs were performed using two primer pairs, F: 5′-ACNGCNTTYYTNCAYGG-3′/R: 5′-ARCATRTCRTCNACRTA-3′ [5] and F: 5′-CARATGGAYGTNAARAC-3′/R: 5′-CATRTCRTCNACRTA-3′ [18], where N represents A, G, C, or T; Y represents T or C; and R represents G or A. These primer pairs were specifically designed to target and amplify a conserved area within the rt gene of Ty1-copia retrotransposons, facilitating phylogenetic analyses [19,20,21]. The two primer pairs were selected based on their success in the PCR-based approach with the Crinum species. The PCRs were carried out in 25 µL mixtures consisting of 100 ng DNA, 0.5 µM of every primer, 2.0 mM of MgCl₂, 1.25 µM of dNTP, and 2 units of Taq DNA polymerase (Invitrogen, Carlsbad, CA, USA) in 1× PCR buffer. The thermal cycling profile started with an initial denaturation at 94 °C for 3 min, then 35 cycles of 94 °C for 30 s, 55 °C for 1 min, and 72 °C for 1 min, ending with a final extension at 72 °C for 10 min. The PCR products, approximately 300 bps in size, were stored at 4 °C for subsequent analysis. Subsequently, the purified PCR products were ligated into pGEM^®-T easy vectors (Promega, Madison, WI, USA), and then the vectors were transformed into Escherichia coli (E. coli) strain JM 109. Approximately 10 colonies with the recombinant plasmids were chosen and cultured for every plant sample studied. The plasmids from these cultures were then purified and sent for sequencing using a BigDye Terminator v3.1 Cycle Sequencing kit in an ABI Prism 3730xl Genetic Analyzer (Applied Biosystems, Carlsbad, CA, USA).

2.3. Ty1-copia rt Sequence Screening

The isolated nucleotide sequences were analyzed for Ty1-copia rt gene homology by cross-referencing with the National Center for Biotechnology Information (NCBI) database. Finally, these sequences were translated into amino acid sequences. In cases where frameshift mutations disrupted the conserved domain inside the amino acid sequences, gaps were introduced into the nucleotide sequences to restore the correct reading frames. Sequences displaying uncertainty, such as those lacking rt gene homology or containing frameshift mutations, were excluded from the study.

2.4. Classification of Isolated Retrotransposons

Six major evolutionary lineages of Ty1-copia retrotransposons, namely Ale, Angela, Bianca, Ivana, Maximus, and TAR, were divided in accordance with an extensively studied and widely accepted category diagram proposed by Wicker and Keller, 2007 [22]. A set of 105 well-classified rt gene Ty1-copia elements from previous research served as references [20,22,23,24]. Individual ClustalW alignments were conducted for every amino acid sequence and a phylogenetic tree was constructed based on the Maximum Likelihood method with the Jones–Taylor–Thornton (JTT) model and a thousand bootstrap replications using the Molecular Evolutionary Genetic evaluation (MEGA) software program (version 10). The ensuing phylogenetic tree was constructed to categorize the isolated rt sequences. Reference rt sequences, demonstrating the very best homology to the isolated sequences, were diagnosed and utilized to determine the lineages to which the isolated rt sequences should be assigned.

2.5. Comparison with Ty1-copia Elements in Other Plants

In an initial analysis, the isolated amino acid sequences were identified against the NCBI database using the Standard Protein Basic Local Alignment Search Tool or BLASTp (BLAST+ version 2.16.0). The isolated rt sequences and their closest homologous sequences from the database were aligned, and then the p-distance values and average values were calculated for every group and lineage.

3. Results

3.1. Ty1-copia rt Fragments of Crinum Species

A total of 153 DNA fragments representing Ty1-copia rt sequences were isolated through PCR amplification, cloning, and sequencing. Of these, 30 sequences either lacking homology to rt genes or exhibiting unidentifiable frameshift mutations were eliminated from the study. The remaining 123 sequences were analyzed and then submitted to the GenBank database (PP134481-PP134603). Upon translation to amino acid sequences, it was found that 100 sequences (81.3%) maintained intact reading frames, whereas the remaining 23 sequences (18.7%) displayed disrupted reading frames and/or premature stop codons (

Table 1. Crinum Ty1-copia rt sequences sorted by presence of deleterious mutations and lineage characterization.

Sample	Total rt Sequences	Translated Sequences		Classification
		Function	Non-Function	Ale-Like	TAR-Like	Angela-Like
C. thaianum_1	13	11	2	1	1	11
C. thaianum_2	11	7	4	0	0	11
C. thaianum_3	13	12	1	0	0	13
C. thaianum_4	12	12	0	0	0	12
C. thaianum_5	16	13	3	1	1	14
C. thaianum_6	11	9	2	2	0	9
C. natans	14	10	4	1	2	11
C. asiaticum L.	12	9	3	3	0	9
C. latifolium L.	9	6	3	1	0	8
C. erubescens L.f. ex Aiton	12	11	1	0	0	12
All samples	123	100	23	9	4	110

).

In total, 110 isolated rt sequences exhibited the highest homology with Angela-like references, whereas only 9 and 4 isolated rt sequences were found to cluster with Ale-like and TAR-like references, respectively (Table 1). Out of the 123 isolated rt sequences, 49 sequences were derived using the primer pair designed by Flavell et al., 1992 [5] and were clustered with the Angela, TAR, and Ale lineage reference sequences; meanwhile, the remaining 74 sequences, obtained using the primer pair designed by Voytas et al., 1992 [18], were grouped with only Angela and TAR lineage reference sequences.

The predicted amino acid sequences were aligned, showing that the conserved domain SIYGLKQA (Figure 2) of the consensus region was mostly found in the Angela lineage (87.27%), while the domains SLY/HGLKQS/L and SLYG/ELKQF/S were mostly found in Ale (66.67%) and TAR (75.00%), respectively. When considering only 110 amino acid sequences of the Angela lineage, 90 patterns of amino acid sequences with at least one amino acid difference were found (Figure 2). In addition to the most commonly found conserved rt domain SIYGLKQA (87.27%) in the amino acid sequences, ten other amino acid motifs were identified in the Angela lineage: SIYGLNQA (one sequence), SIYGFKQA (one sequence), SIYELKQA (one sequence), SIYRLKQA (three sequences), SIYRLKQV (one sequence), SIYGLKQV (two sequences), STYGLKQA (one sequence), FIYGLKQA (two sequences), LIYGLKQA (one sequence), and PIYGLKQA (one sequence) (Figure 2).

3.2. Classification of Isolated rt Sequences

The classification of isolated rt sequences, shown in Figure 3, revealed that 36 out of the 123 sequences were clustered with bootstrap values exceeding 50, and were aligned with at least one of the reference sequences. Of these 36 rt sequences, 23, 4, and 9 sequences were identified as similar to the Angela, TAR, and Ale lineages, respectively. Considering the p-distance values, 87 out of the 123 isolated rt sequences demonstrated the highest similarity to the Angela lineage. In summary, 110, 4, and 9 isolated rt sequences were identified as being maximally similar to rt sequences from the Angela, TAR, and Ale lineages, respectively (Table 1).

Ty1-copia retrotransposons based on an isolated rt sequence from our Crinum plants were identified within the Ale, Angela, and TAR lineages, suggesting that these three lineages may be the representatives of Ty1-copia retrotransposons in C. thaianum and four other Crinum species: C. asiaticum, C. natans, C. latifolium, and C. erubescens.

3.3. Comparative Analysis of Ty1-copia Factors

A comparative analysis of Ty1-copia factors in numerous florae was conducted. The obtained reverse transcriptase (rt) sequences were compared with reference sequences for characterization. The outcomes of these rt sequences, when compared to their closest counterparts in the NCBI protein database, exhibited a consistent pattern. p-distance values were then subsequently computed between the isolated rt sequences and their closest counterparts, resulting in values ranging from 0.148 to 0.452.

The p-distance values for Ale-like, Angela-like, and TAR-like sequences ranged from 0.291 to 0.452, 0.161 to 0.368, and 0.148 to 0.368, respectively, with average values of 0.395, 0.309, and 0.299 (Figure 4). In all cases, the p-distance values between the isolated sequences and their most similar counterparts from the database were consistently low for Angela- and TAR-like sequences in comparison to Ale-like sequences. The results indicate that Ale-like sequences generally exhibited lower homology to any of the reference sequences than Angela- and TAR-like sequences.

Ty1-copia elements found in C. thaianum, C. asiaticum, C. natans, C. latifolium, and C. erubescens were most similar to the Angela and TAR lineages, exhibiting greater homology to their reference sequences in other plants than those most similar to the Ale lineage. This observation suggests that Ty1-copia elements in the Angela and TAR lineages are more conserved across the evolutionary lineages of these species than those in the Ale lineage. Varying degrees of conservation are conceivable among distinctive Ty1-copia retrotransposon families.

4. Discussion

This study focuses on screening Ty1-copia retrotransposons, which are widely studied and used as markers in various plants. In total, 153 putative Ty1-copia rt sequences were collected from water onion samples and other Crinum plants. After analysis, 30 sequences were eliminated due to either lacking homology to rt genes or exhibiting unidentifiable frameshift mutations. Of the remaining sequences, 81.3% (100 out of 123) maintained intact reading frames, while 18.7% (23 out of 123) had disrupted reading frames and/or premature stop codons, suggesting potential interference with gene function [9]. Considering all the amino acid sequences obtained, the consensus domain SIYGLKQA was mostly found to be highly conserved (Figure 2) in Crinum plants (78.86%), and most sequences in the Ty1-copia superfamily of angiosperms were also found to be SLYGLKQA [25,26].

As shown in Figure 3, the isolated rt sequences were classified into three clusters of Ty1-copia retrotransposon lineages: Angela, TAR, and Ale. In contrast, Wicker and Keller, 2007 [22] reported a significant presence of Ty1-copia retrotransposon elements from the Ivana and Maximus lineages in rice and triticeae, both of which are monocots, as with Crinum plants. However, Alipour et al., 2013 [19] and Kolano et al., 2013 [20] studied different plants with the same isolation method used herein and discovered the selective isolation of Ty1-copia retrotransposons closely related to the Ale, Angela, and TAR lineages. This suggests that the choice of primers used in various studies could possibly introduce a bias towards the specific lineages. In addition, a study using lily—from the same family as Crinum—constructed a fosmid library from its genomic DNA, and screened and selected clones using a hybridization technique with a reverse transcriptase (rt) domain-specific probe [23]. This approach identified only the Ale lineage of Ty1-copia elements, suggesting that using different screening methods or species sampled can lead to varying results.

In addition, the Ty1-copia elements of C. thaianum, C. asiaticum, C. natans, C. latifolium, and C. erubescens were more similar to the Angela and TAR lineages than the Ale lineage, as shown by high homology to the reference sequences from other plants. This suggests that the Ty1-copia elements in the Angela and TAR lineages were more conserved than those in the Ale lineage across the Crinum plants in this study. These findings imply varying degrees of conservation among different Ty1-copia retrotransposon families. However, LTR-retrotransposon lineages have been reported to be considerably conserved across a wide variety of plant species in various studies [25,27,28,29].

DNA markers such as Amplified Fragment Length Polymorphism (AFLP), Inter Simple Sequence Repeat (ISSR), and Simple Sequence Repeat (SSR) have been widely used for genetic variation studies in plants. Retrotransposons provide an alternative way to observe genetic variation, as they can occasionally induce or reverse mutations, thereby influencing the cell’s genetic identity and genome size [30,31,32]. These effects contribute to genetic variation in organisms. Considering the limited research on retrotransposons in C. thaianum and other Crinum species, further retrotransposon studies are essential to gain a more comprehensive understanding.

Several studies have utilized retrotransposon elements to identify varieties and explore genetic diversity [33,34,35,36]. Sequence-specific Amplified Polymorphism (SSAP) and Inter-Retrotransposon Amplified Polymorphism (IRAP), PCR, and retrotransposon-based markers generate DNA fragments from more than one site in genomes through conserved domain-specific primers within LTR-retrotransposons [15]. These primers need to be designed to ensure specificity to the conserved regions of LTR-retrotransposon families across the studied samples to produce informative fingerprints. This will allow these techniques to be universally applied across all samples, providing substantial genomic information. In our study, the isolated rt sequences, especially the Angela-like sequences which accounted for a large majority, were aligned (Figure 2). Many regions could be used to design specific primers for testing across multiple individuals/populations/species for studying the population genetics, conservation genetics, genetic diversity, species identification, and retrotransposon tracking of related species in future work.

5. Conclusions

Our study confirmed the existence of LTR-retrotransposon families, providing valuable insights into the Ty1-copia diversity of C. thaianum and four other Crinum species. Out of 123 isolated rt sequences, 9, 4, and 110 isolated sequences revealed homology to the Ale, TAR, and Angela lineages. The conserved rt domain SIYGLKQA was mostly found in the Angela lineage, while SLY/HGLKQS/L and SLYG/ELKQF/S were mostly found in the Ale and TAR lineages, respectively. Further investigation into additional Crinum species is necessary to enhance our understanding of LTR-retrotransposons. This is particularly beneficial information for developing retrotransposon-based markers for endangered native species in Thailand, enabling the study of genetic diversity and genetic identity evaluation for future C. thaianum conservation practices and sustainable usage.