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Article

Rapidly Evolved Genes in Three Reaumuria Transcriptomes and Potential Roles of Pentatricopeptide Repeat Superfamily Proteins in Endangerment of R. trigyna

by
Ruizhen Zhang
1,2,
Xiaoyun Cui
1,2,* and
Pengshan Zhao
1,2,*
1
Key Laboratory of Ecological Safety and Sustainable Development in Arid Lands, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
2
Key Laboratory of Stress Physiology and Ecology in Cold and Arid Regions, Gansu Province, Northwest Institute of Eco-Environment and Resources, Chinese Academy of Sciences, Lanzhou 730000, China
*
Authors to whom correspondence should be addressed.
Int. J. Mol. Sci. 2024, 25(20), 11065; https://doi.org/10.3390/ijms252011065
Submission received: 6 September 2024 / Revised: 4 October 2024 / Accepted: 8 October 2024 / Published: 15 October 2024
(This article belongs to the Section Molecular Biology)
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Versions Notes

Abstract

:
Reaumuria genus (Tamaricaceae) is widely distributed across the desert and semi-desert regions of Northern China, playing a crucial role in the restoration and protection of desert ecosystems. Previous studies mainly focused on the physiological responses to environmental stresses; however, due to the limited availability of genomic information, the underlying mechanism of morphological and ecological differences among the Reaumuria species remains poorly understood. In this study, we presented the first catalog of expressed transcripts for R. kaschgarica, a sympatric species of xerophyte R. soongorica. We further performed the pair-wise transcriptome comparison to determine the conserved and divergent genes among R. soongorica, R. kaschgarica, and the relict recretohalophyte R. trigyna. Annotation of the 600 relatively conserved genes revealed that some common genetic modules are employed by the Reaumuria species to confront with salt and drought stresses in arid environment. Among the 250 genes showing strong signs of positive selection, eight pentatricopeptide repeat (PPR) superfamily protein genes were specifically identified, including seven PPR genes in the R. soongorica vs. R. trigyna comparison and one PPR gene in the R. kaschgarica vs. R. trigyna comparison, while the cyclin D3 gene was found in the R. soongorica vs. R. trigyna comparison. These findings suggest that genetic variations in PPR genes may affect the fertility system or compromise the extent of organelle RNA editing in R. trigyna. The present study provides valuable genomic information for R. kaschgarica and preliminarily reveals the conserved genetic bases for the abiotic stress adaptation and interspecific divergent selection in the Reaumuria species. The rapidly evolved PPR and cyclin D3 genes provide new insights on the endangerment of R. trigyna and the leaf length difference among the Reaumuria species.

1. Introduction

Reaumuria is a genus adapted to arid environment and wildly distributed across the arid regions of Central Asia [1]. Phylogenetic evidence has revealed that Reaumuria is monophyletic, with its estimated divergence from Tamarix dating back to the early Paleocene (69.99 million years ago; 66.36 Mya) [1,2]. The perennial xerophytic semi-shrub R. soongorica is a constructive and dominant species present in desert ecosystems, which is capable of surviving even after losing up to 95% of its water content [3,4]. Physiological studies have shown that sucrose, malate, and proline are important osmotic regulators and the antioxidative system can effectively remove superoxide anion and scavenge hydrogen peroxide during dehydration in R. soongorica [5,6]. Consistent with the findings, a set of genes relevant to aquaporins, proline transporters, flavonoid biosynthetic proteins, small heat shock proteins, and late embryogenesis abundant proteins is highly accumulated after drought stress treatment [3]. R. kaschgarica is a sympatric species of R. soongorica (Figure 1), and the divergence time between these two species could be dated to middle Miocene (13.13 Mya; [1]). It is characterized by flowers located at the apices of branchlets and longer leaves compared to those of R. soongorica.
In contrast with the broad distribution of R. soongorica and R. kaschgarica, the relict plant R. trigyna is restricted to a small region—the Alashan Left and Western Ordos of Inner Mongolia—which is recognized as one of the most noticeable endemic areas of China (Figure 1) [10]. Another characteristic feature of the Reaumuria habitat is high soil salinity. Both R. soongorica and R. trigyna can complete their life cycles in soils with sodium concentrations as high as 0.6 mg/g [8,11]. Thus, salt excretion is considered a crucial mechanism contributing to the salinity of resistance of Reaumuria [11,12,13,14]. Transcriptome analyses have revealed significant alterations in the expression levels of genes involved in ion transport and reactive oxygen species scavenging following salt stress treatment in R. trigyna [11].
Many specific traits, including succulent leaves, sunken stomata, and salt excretion glands, have evolved in Reaumuria species to enable it to survive in an arid environment [11,14,15]. While numerous physiological and molecular studies have elucidated the adaptation mechanisms of Reaumuria, the genetic mechanisms underlying the development of specific organs and phenotype diversity are poorly understood for the Reaumuria species. Previous study based on neutral markers, RAPD and ISSR, has revealed high genetic variations within R. trigyna populations [9]. However, the evolutionary constraints that result in the endangerment of R. trigyna remain understudied. In this study, we obtained the transcriptome of R. kaschgarica using the Illumina platform and characterized its transcriptome to expand the available information for the comparative transcriptomic analyses of the Reaumuria species. We also identified conserved genes and candidate genes, such as pentatricopeptide repeat (PPR) proteins and cyclin D3 genes, that have experienced adaptive evolution among R. soongorica, R. kaschgarica, and R. trigyna via comparative evolutionary analyses. PPRs are pivotal in regulating RNA processing within organelles, including the editing, splicing, and stability of mitochondrial and chloroplast transcripts. These processes are crucial for the proper functioning of the photosynthetic and respiratory machinery, which are essential for plant energy metabolism and growth [16,17,18]. Additionally, PPR proteins contribute to the plant’s ability to respond to abiotic stresses [19,20]. Understanding these evolution adaptations not only advances our knowledge of plant evolution but also informs conservation strategies for endangered taxa such as R. trigyna.

2. Results

2.1. Assembly and Annotation of R. kaschgarica Transcriptome

Total RNA was extracted from mixed materials including stems, leaves, and flowers. Paired-end sequencing of the library on a sequencer platform (HiSeq 4000, Illumina, San Diego, CA, USA) yielded 40 million clean reads, with 89.94% of bases having quality scores above Q30. These reads were assembled using Trinity (version r20140413p1) [21], resulting in the generation of 62,680 unigenes with a mean length of 744 bp and an N50 length of 1388 bp (Figure 2A). All unigenes were subsequently aligned against seven public databases, including the NCBI non-redundant nucleotide (Nt) and protein (Nr) database, Pfam, Clusters of Orthologous Groups of proteins (KOG/COG), the Swiss-Prot protein database, the KEGG Ortholog database (KO), and Gene Ontology (GO), using a threshold of less than 1 × 10−5. As shown in Table 1, 30,002 unigenes (47.86%) could be matched to protein sequences or domains in at least one of these databases. In addition, the taxonomic distribution based on Nr annotation revealed that 5357 (19.6%) unigenes had top hits to Vitis vinifera (Figure 2B). These results were highly consistent with the annotation of R. soongorica transcriptome [22].

2.2. Characterizing Transcriptomes of R. soongorica, R. kaschgarica, and R. trigyna

As a first step towards conducting a comparative evolutionary analysis, we evaluated the transcriptome of R. kaschgarica alongside previously published transcriptomes of R. soongorica and R. trigyna [11,22]. The average length of R. trigyna unigenes was 486 bp, which is shorter than those of R. soongorica (677 bp) and R. kaschgarica (Figure 3A). The GC content of R. soongorica (42.3%) and R. trigyna (42.9%) was higher than that of R. kaschgarica (40.5%; Figure 3B). We further examined the transcriptome quality using a Blastx comparison against the Arabidopsis TAIR10 protein database (comprising 35,386 peptides) as described by Dorn et al. (2013) [23]. The percentage of identity and coverage for each unigene versus the most similar Arabidopsis peptide were measured independently for three Reaumuria transcriptomes (Figure 3C,D). R. trigyna exhibited the highest identity value (63.5%) but the lowest coverage (33.9%), likely due to its shorter unigene length (Figure 3A). The high identity values and good coverage suggest that Reaumuria transcriptomes are viable genomic resources for further evolutionary analysis.

2.3. Comparative Evolutionary Analysis of Reaumuria Transcriptomes

A total of 13,334 orthologous groups were isolated using the OrthoMCL program [24], in which 5182 groups contained three unigenes, each originating from R. soongorica, R. kaschgarica, and R. trigyna transcriptomes (Figure 4A). The non-synonymous (Ka) and synonymous (Ks) nucleotide substitution values were calculated for each orthologous group from the three pair-wise comparisons (Figure 4E–G). The number of orthologous pairs with a Ka/Ks ratio of less than 0.1 was 1920 for R. kaschgarica vs. R. soongorica, 2067 for R. kaschgarica vs. R. trigyna, and 2149 for R. soongorica vs. R. trigyna (Figure 4B). Six hundred orthologous pairs were shared among the three comparisons (Supplementary Table S1), indicating that the sequences of these genes have been highly conserved throughout the evolutionary history of the Reaumuria species.
To gain more insights into the functions of highly conserved genes, Arabidopsis genes with the highest similarity to each orthologous group were subjected to GO enrichment analysis using the AgriGO program v2.0 [25]. The enriched GO terms were mainly related to basic biology processes, including ‘vesicle-mediated transport’, ‘post-embryonic development’, ‘aromatic compound biosynthetic process’, and ‘protein folding’ (Supplementary Figure S1). Interestingly, the GO term ‘response to radiation’ was also overrepresented, which is consistent with the strong light selection pressure in the habitats of the Reaumuria species. Moreover, at least 21 transcription factors, including AP2, bHLH, C2H2, TCP, and NAC, were identified among the conserved Reaumuria genes (Supplementary Table S1).
The numbers of orthologous pairs with Ka/Ks ratios above one ranged from 71 to 116 across three comparisons (Figure 4C and Supplementary Table S2). There were eleven groups in which Ka/Ks ratios of one Reaumuria gene compared to two other genes were both over one. Only one orthologous group (OG03284), which encoded a heat shock protein (HSP) 20-like chaperone protein, showed rapid evolutionary patterns in all three species. Although none of the GO categories were overrepresented according to the GO enrichment analysis, a number of genes coding pentatricopeptide repeat (PPR) superfamily proteins were identified as positively selected genes (Figure 4D and Table 2). For example, eight PPR genes were detected in the R. soongorica vs. R. trigyna comparison and one in the R. kaschgarica vs. R. trigyna comparison.

3. Discussion

Physiological studies have demonstrated that the Reaumuria species are xerophyte and recretohalophyte plants [4,5,6,8,12,14]. In the present study, 600 relatively conserved genes were identified according to their Ka/Ks ratios (Supplementary Table S1). Highly conserved genes are typically associated with fundamental cellular functions and organismal survival, remaining unchanged throughout evolution because they are crucial for the organism’s survival. For example, the transcriptome analysis of R. soongorica and sand rice revealed that the post-embryonic development genes are conserved [26]. The analysis of drought-treated transcriptomes revealed that the expression levels of 18 out of these 600 R. soongorica unigenes decreased, while 3 unigenes showed increased expression [3]. Moreover, several ion transporter-related genes, such as cation/H+ exchanger 20 (OG06920), sodium hydrogen exchanger 3 (NHX3; OG08509), and NHX6 (OG08209), which are involved in the salt response of R. trigyna [11], were identified as conserved genes in the Reaumuria transcriptomes. The conservativeness of these genes indicates that they are crucial for maintaining ion balance and adapting to high-salinity environments, suggesting that the same genetic modules are employed by three Reaumuria species to survive in drought and salinity environments.
Salt excretion is an important characteristic of the Reaumuria species [12,13,14]. A recent comparative transcriptome analysis of Limonium bicolor has revealed that genes controlling salt gland development may also be involved in trichome formation [27]. Eight genes, such as GLABRUS 2 (GL2), GL3, and TRANSPARENT TESTA GLABRA 1 (TTG1), are suggested to be involved in salt gland initiation, and eighteen genes related to plasmodesmata, vesicle transport, cuticular wax, and lignin participate in the ultrastructure differentiation of salt glands [27]. In this study, one orthologous group (OG05769) encoding a TTG1 protein was categorized to be under purifying selection. In addition, the GO term ‘vesicle-mediated transport’ was enriched among the 600 conserved genes. This evidence implies that the mechanism of salt gland development in Reaumuria species is similar to that in L. bicolor and underscores the key role of vesicle transport in salt secretion [27].
A total of 250 orthologous pairs exhibited rapid evolution with signs of strong selection (Figure 4C), and their orthologous genes in Arabidopsis were isolated from the Blastx comparison results to identify the possible functions (Table 2 and Supplementary Table S2). The diversification of these genes may be driven by environmental pressures or ecological interactions, thereby enhancing the organism’s ability to adapt to specific ecosystems. For example, the HSP20-like chaperone showed rapid evolution in all three comparisons and the corresponding Arabidopsis protein targets to the matrix of peroxisome, which is an important organ to scavenge the reactive oxygen species (ROS) [28]. The RING E3 ubiquitin ligase ATL78 (homologoue of OG13726), which exhibited Ka/Ks ratios greater than one in the R. kaschgarica vs. R. trigyna and R. soongorica vs. R. trigyna comparisons, plays a role in the ROS-mediated abscisic acid (ABA) signaling pathway, conferring drought tolerance [29,30]. Therefore, the variation in these genes suggests that different Reaumuria species may have developed unique adaptations to varying local environmental conditions, such as drought stress.
The PPR family is known as one of the largest gene families in terrestrial plants, with Arabidopsis alone containing around 450 members. The expansion of this family is likely attributed to the retrotransposition events [31,32]. PPR proteins are sequence-specific RNA-binding proteins with universal influence on the RNA processing within organelles [31]. Organelle RNA metabolism significantly influences plant photosynthesis, respiration, and environmental responses [16,17,18,19,20]. Large variations in the RNA editing extent and products have been reported among different Arabidopsis ecotypes, which can be explained by the ecotype-specific variations in PPR protein sequences [33,34,35,36,37]. Previous molecular evolution studies have shown that some PPR genes are maintained under strong negative selection, whereas the tandem repeated genes, such as the restorer of fertility (Rf) and Rf-like (RFL) genes, are subjected to positive selection in Arabidopsis [32,33,38]. In this study, we focused on potential functions of the 8 rapidly evolved PPR genes that were specifically identified in R. soongorica vs. R. trigyna or R. kaschgarica vs. R. trigyna comparisons (Figure 4D). One gene of interest, RFL9 (homologue of OG14530), acts as a mitochondria RNA editor limited to Arabidopsis accessions genetically close to Columbia-0 and is associated with the suppression of a cytoplasmic male sterility in Arabidopsis (CMS; [33]). It is possible that some variations fixed in R. trigyna RFL9 gene could affect the fertility system leading to the endangerment of R. trigyna. PPR proteins are also involved in other biological processes, and mutations of PPRs can result in diverse phenotypes, such as aberrant leaf development, slow growth, embryo abortion, and hypersensitivity to environmental stresses or ABA [31,39]. Among the rapidly evolved PPR genes, BIGYIN/FISSION1A (homologue of OG10059) encodes a protein that is triple-targeted to peroxisomes, mitochondria, and chloroplasts, and the disruption of its expression leads to growth inhibition and abnormal fission of peroxisomes and mitochondria in Arabidopsis [40,41,42,43]. Numerous studies have demonstrated that both biotic and abiotic stresses can alter the expression patterns of PPR genes. Research has identified various PPR genes that are linked to responses to salt stress, drought stress, cold stress, and defense mechanisms [44,45]. For instance, Luo et al. (2022) conducted an analysis of PPR gene expression in rice under different stress conditions [46]. Their findings revealed that out of the total PPR genes studied, 16 out of 81, 15 out of 127, and 27 out of 35 were either upregulated or downregulated in reaction to osmotic, salt, and oxidative stress, respectively [46]. For the Arabidopsis homologs of the remaining six orthologous groups, various expression patterns have been shown in Arabidopsis seedlings subjected to different abiotic stresses (Supplementary Figure S2; [47,48,49]). The possibility that variations in these genes could compromise the adaptation ability of R. trigyna to the arid regions cannot be ruled out.
Leaf length difference is one of significant phenotype variations among R. soongorica, R. trigyna, and R. kaschgarica (Figure 1), and we are still far from knowing how this variation happened during the evolution of the Reaumuria species. Cyclin D3 is known to play an important role in the control of cell number in developing leaves in Arabidopsis [50]. For example, the overexpression of AtCYCD3;1 resulted in significant alterations in leaf architecture, including the loss of distinct spongy and palisade mesophyll layers and the presence of an epidermis made up of numerous poorly differentiated polygonal cells [51]. Furthermore, the overexpression of AtCYCD3;2 led to the formation of rosettes resembling propellers, featuring narrow, dome-shaped leaves [52]. The finding that cyclin D3 (OG19460) has been under positive selection in the R. soongorica vs. R. trigyna comparison implies a possible reason for the shorter leaves observed in R. soongorica. Moreover, there were eight rapidly evolved PPR genes in the R. kaschgarica vs. R. soongorica comparison. Mitochondrial editing factor 29 (homologue of OG14044) dually targets mitochondria and chloroplasts in Arabidopsis, and mutation of its ortholog PPR2263 in maize results in narrow and short leaves [53]. However, the R. trigyna unigene was absent in this orthologous group (OG14044), indicating that further experiments are necessary to elucidate the role of this gene in leaf development.
Overall, these genetic differences could potentially contribute to R. trigyna’s endangerment by affecting its fertility, growth, stress tolerance, and overall adaptation to its environment. This means that the species’ endangerment could be exacerbated if these genetic factors negatively impact its ability to thrive and reproduce in its natural habitat. However, a further study is needed to confirm the roles of the identified PPR proteins and cyclin D3 genes in stress tolerance and reproductive success. This can be achieved through techniques such as gene editing and expression analysis. Additionally, it is also necessary to understand how these genetic variations interplay with environmental factors like drought and salinity. Comparative genomic analyses with other arid-adapted species and population genetics studies of R. trigyna will further elucidate the mechanisms underlying its adaptability and inform conservation strategies.

4. Materials and Methods

4.1. Plant Materials, RNA Extraction, Transcriptome Sequencing, and Analyses

Fresh shoots of R. kaschgarica, including stems, leaves, and flowers, were sampled in Dulan County, Qinghai Province, China. The average annual precipitation of Dulan County is 179.1 mm. The distribution areas of R. kaschgarica have an average altitude of 2613.9 m (ranging from 1050 to 4931 m), an average annual temperature of 2.4 °C (ranging from −7.8 to 13.2 °C), and an average annual precipitation of 192.5 mm (with a range from 27 to 545 mm). Solar radiation varies from 0.17 to 0.78 kJ m−2 day−1, with an average of 0.43 kJ m−2 day−1. The wind speed ranges from 13,751 to 16,953 m s−1, with an average of 15,888 m s−1. Water vapor pressure varies from 1.3 to 4.1 kPa, with an average of 2.7 kPa (Supplementary Figure S3). All materials were immediately frozen in liquid nitrogen and brought back to our laboratory for analysis. The stems, leaves, and flowers were pooled, and total RNA was extracted using a Plant Total RNA Kit (#DP432, TIANGEN, Beijing, China). The RNA purity was measured using spectrophotometric A260/A280 and A260/A230 ratios, ensuring that the ratios were greater than 2. RNA integrity was assessed using agarose gel electrophoresis and automated systems, such as the Agilent Bioanalyzer 2100 (Agilent Technologies, Santa Clara, CA, USA), ensuring a RIN value above 7 and checking for DNA contamination. To create the library, 25 µL of RNA at a concentration of 470 µg/µL was used. The cDNA library was constructed following the description by Zhao et al. [54] and sequenced using the Illumina Hiseq 4000 platform and 150 bp paired-end reads at Novogene (Beijing, China). A total of 6 Gbp of clean bases were generated, with 89.94% of the bases having a quality score of Q30 or higher.
Clean reads were assembled using Trinity (r20140413p1; [21]) with default parameters. The assembled unigenes were annotated by aligning using Blast against several public databases, including Nr, Nt, Pfam, KOG/COG, Swiss-Prot, KO, and GO, using an e-value cutoff of 1 × 10−5. Gene frequency units can be expressed as FPKM (number of Fragments Per Kilobase of transcript sequence per Millions base pairs sequenced), which normalizes for gene length and sequencing depth.

4.2. Transcriptome Characterization Using Blastx Program Versus Arabidopsis Protein Database

The Arabidopsis protein database (containing 35,386 peptides) was downloaded from the TAIR10 release (www.arabidopsis.org, accessed on 17 October 2015). Unigenes from R. kaschgarica in this study, as well as those from R. soongorica [22] and R. trigyna [11], were compared with the Arabidopsis peptides using the Blastx program (v2.2.18), employing a threshold of less than 1 × 10−5. The best Arabidopsis hit for each unigene was isolated based on sequence identity and e-value [26]. Statistical analyses were conducted using the R software (v4.4.1), as described by Dorn et al. [23].

4.3. Ortholog Grouping and Ka/Ks Calculation

The Reaumuria orthologous genes were identified using the OrthoMCL method [24]. To analyze the evolutionary pressures on these genes, we calculated the non-synonymous (Ka) and synonymous (Ks) nucleotide substitutions in three transcriptome comparisons (R. kaschgarica vs. R. soongorica, R. kaschgarica vs. R. trigyna, and R. soongorica vs. R. trigyna) using the Codeml program of Phylogenetic Analysis by Maximum Likelihood (PAML) package (version 4.9a) with the basic model [55]. By comparing the ratio of Ka to Ks, we can infer the type of selection pressure acting on the gene. If Ka/Ks > 1, it indicates positive selection; if Ka/Ks = 1, it suggests neutral evolution; and if Ka/Ks < 1, it implies purifying or negative selection. In this study, genes with 3 > Ka/Ks ratios > 1 were considered to be under positive selection pressures, indicating adaptive changes in their sequences [56], whereas genes with Ka/Ks ratios < 0.1 were selected as under purifying selection, which acts to maintain the function of genes [57].

4.4. GO Enrichment Analysis

The orthologous Arabidopsis genes for each Reaumuria group were subjected to GO annotation using the AgriGO program with all parameters set as default [25]. Enriched GO terms were analyzed using the hypergeometric test and corrected by Yekutieli (FDR under dependency). The cutoff of FDR was set at 0.01, with 46 biological processes, 59 cellular components, and 21 molecular function-related terms enriched for the 600 relatively conserved Reaumuria genes.

5. Conclusions

The first catalog of expressed transcripts of R. kaschgarica was presented, and the pair-wise comparisons of R. soongorica, R. trigyna, and R. kaschgarica identified 600 relatively conserved genes and 250 rapidly evolved genes among the Reaumuria species. Gene annotation suggests that the same genetic elements underlie the salt and drought stress adaptations of the Reaumuria in extreme habitats, with some equivalent components being potentially involved in salt gland development. The driving forces behind the endangerment of R. trigyna remain uncertain. The study of the rapidly evolved PPR genes provides novel insights on this evolutionary issue. Genetic variations in PPR genes could result in abnormal fertility systems or compromise the organelle RNA editing extent of R. trigyna. Experimental analysis of the rapidly evolved genes provided in this study will enhance our understanding of the morphology and evolution of the Reaumuria species.

Supplementary Materials

The supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms252011065/s1.

Author Contributions

R.Z. was responsible for investigation and formal analysis. X.C. was responsible for writing—original draft preparation and writing—review and editing. P.Z. was responsible for conceptualization, writing—review and editing, and funding acquisition. All authors have read and agreed to the published version of the manuscript.

Funding

This study was supported by the Western Light Project of CAS (xbzg-zdsys-202204), National Natural Science Foundation of China (No. 32271760), Pioneer Hundred Talents Program (Category B) of CAS (E4290501), Gansu Provincial Science and Technology Planning Project (No. 23ZDFA018), and Excellent Member of Youth Innovation Promotion Association, CAS (Y2022104).

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

Raw data are available in the NCBI Sequence Read Archive (SRA) under accession number PRJNA1138088.

Acknowledgments

We thank Yubin Liu for her generous offer of the photograph of the R. trigyna and the drought transcriptome DEG dataset of R. soongorica, and Xingke Fan for the photograph of R. kaschgarica, we acknowledge Yingchun Wang from Inner Mongolia University for the supply of the R. trigyna transcriptome, and we thank the natural field survey on the R. trigyna by Haikui Chen from Beifang University of Nationalities and the kind gift of the dry leaf sample.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

RkR. kaschgarica
RtR. trigyna
RsR. soongorica
PPRpentatricopeptide repeat
HSPheat shock protein
Kanon-synonymous nucleotide substitution
Kssynonymous nucleotide substitution
NHX3sodium hydrogen exchanger 3
TTG1transparent testa glabra 1
ROSreactive oxygen species
RFL9restorer of fertility like 9
CMScytoplasmic male sterility

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Figure 1. Distribution and morphology of three Reaumuria species. (A) Distribution of three Reaumuria species. The background imagery is from GEBCO_2014 Grid, version 20150318 (http://www.gebco.net), and the desert dataset (in orange) is provided by National Cryosphere Desert Data Center (NCDC, http://www.ncdc.ac.cn/). The map was generated based on the latitude and longitude of the location sites of each species. The information for each red dot of R. soongorica was from the natural field survey and previous report [2]. The distribution information of R. kaschgarica (green dots) was modified according to the description by Hao et al. [7]. Dark gray dots represent the location of R. trigyna, and the information was based on the natural field survey and previous publications [7,8,9]. (B) Morphology of R. soongorica, R. trigyna, and R. kaschgarica in their habitat. (C) Dry leaves and leaf length of R. soongorica, R. trigyna, and R. kaschgarica (n = 20).
Figure 1. Distribution and morphology of three Reaumuria species. (A) Distribution of three Reaumuria species. The background imagery is from GEBCO_2014 Grid, version 20150318 (http://www.gebco.net), and the desert dataset (in orange) is provided by National Cryosphere Desert Data Center (NCDC, http://www.ncdc.ac.cn/). The map was generated based on the latitude and longitude of the location sites of each species. The information for each red dot of R. soongorica was from the natural field survey and previous report [2]. The distribution information of R. kaschgarica (green dots) was modified according to the description by Hao et al. [7]. Dark gray dots represent the location of R. trigyna, and the information was based on the natural field survey and previous publications [7,8,9]. (B) Morphology of R. soongorica, R. trigyna, and R. kaschgarica in their habitat. (C) Dry leaves and leaf length of R. soongorica, R. trigyna, and R. kaschgarica (n = 20).
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Figure 2. Analysis of the R. kaschgarica transcriptome. (A) Length distribution of assembled unigenes. (B) Taxonomic distribution of the top Blast hits in Nr database for each unigene.
Figure 2. Analysis of the R. kaschgarica transcriptome. (A) Length distribution of assembled unigenes. (B) Taxonomic distribution of the top Blast hits in Nr database for each unigene.
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Figure 3. Characteristics of three Reaumuria transcriptomes. (A,B) Boxplots of unigene length and GC content of assembled unigenes. (C,D) Boxplots of percentage identity and percentage coverage of each Reaumuria unigene versus an Arabidopsis peptide. The percentage coverage is the longest positive hit/protein length ratio [23]. Rk, R. kaschgarica; Rt, R. trigyna; Rs, R. soongorica.
Figure 3. Characteristics of three Reaumuria transcriptomes. (A,B) Boxplots of unigene length and GC content of assembled unigenes. (C,D) Boxplots of percentage identity and percentage coverage of each Reaumuria unigene versus an Arabidopsis peptide. The percentage coverage is the longest positive hit/protein length ratio [23]. Rk, R. kaschgarica; Rt, R. trigyna; Rs, R. soongorica.
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Figure 4. Venn diagram of orthologous genes and scatter diagram of Ka and Ks values for three Reaumuria species. (A) Orthologous genes of three Reaumuria species. There are 5182 orthologous groups, each containing three genes from R. kaschgarica, R. trigyna, and R. soongorica. (B) The number of genes under purifying selection in each pair-wise transcriptome comparison. Six hundred relatively conserved genes are identified in three Reaumuria species. (C) Positively selected genes in three pair-wise comparisons. Only one highly divergent gene is found among three Reaumuria species. (D) Rapidly evolved PPR genes in Reaumuria. (E–G) Ka and Ks values of three comparisons were estimated. Orthologous genes with Ka > 1 are excluded. Green dots represent divergent ortholog genes with Ka/Ks > 1, red dots indicate conserved ortholog genes with Ka/Ks < 0.1, and blue dots represent no positive or negative selections were detected.
Figure 4. Venn diagram of orthologous genes and scatter diagram of Ka and Ks values for three Reaumuria species. (A) Orthologous genes of three Reaumuria species. There are 5182 orthologous groups, each containing three genes from R. kaschgarica, R. trigyna, and R. soongorica. (B) The number of genes under purifying selection in each pair-wise transcriptome comparison. Six hundred relatively conserved genes are identified in three Reaumuria species. (C) Positively selected genes in three pair-wise comparisons. Only one highly divergent gene is found among three Reaumuria species. (D) Rapidly evolved PPR genes in Reaumuria. (E–G) Ka and Ks values of three comparisons were estimated. Orthologous genes with Ka > 1 are excluded. Green dots represent divergent ortholog genes with Ka/Ks > 1, red dots indicate conserved ortholog genes with Ka/Ks < 0.1, and blue dots represent no positive or negative selections were detected.
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Table 1. Summary of the annotation of R. kaschgarica assembled unigenes.
Table 1. Summary of the annotation of R. kaschgarica assembled unigenes.
Number of UnigenesPercentage (%)
Nr27,32543.59
Nt13,14220.96
KO933014.88
Swiss-Prot19,69631.42
Pfam18,95930.24
GO19,41430.97
KOG10,19916.27
Annotated in all databases41316.59
Annotated in at least one database30,00247.86
Total Unigenes62,680100
Table 2. Partial list of genes with positive selection. Note: n.d. means no data available.
Table 2. Partial list of genes with positive selection. Note: n.d. means no data available.
Orthologous GroupsGenesGene SymbolsKa/Ks
Rk vs. RsRk vs. RtRs vs. Rt
OG03284Rk|c19117_g1 Rs|Unigene52639_A_Rs
Rt|Unigene3477_Rt		HSP20-like1.521.3291.20
OG14217Rk|c41245_g1 Rs|Unigene22663_A_Rs
Rt|Unigene66872_Rt		PPR1.020.691.20
OG13726Rk|c17221_g1 Rs|CL3395.Contig2_A_Rs
Rt|Unigene50964_Rt		ATL780.972.091.07
OG10059Rs|Unigene46659_A_Rs Rk|c6241_g1
Rt|Unigene62804_Rt		BIGYIN/FIS1A0.220.00011.69
OG14530Rs|CL10303.Contig1_A_Rs, Rt|Unigene64640_RtRFL9n.d.n.d.1.62
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Zhang, R.; Cui, X.; Zhao, P. Rapidly Evolved Genes in Three Reaumuria Transcriptomes and Potential Roles of Pentatricopeptide Repeat Superfamily Proteins in Endangerment of R. trigyna. Int. J. Mol. Sci. 2024, 25, 11065. https://doi.org/10.3390/ijms252011065

AMA Style

Zhang R, Cui X, Zhao P. Rapidly Evolved Genes in Three Reaumuria Transcriptomes and Potential Roles of Pentatricopeptide Repeat Superfamily Proteins in Endangerment of R. trigyna. International Journal of Molecular Sciences. 2024; 25(20):11065. https://doi.org/10.3390/ijms252011065

Chicago/Turabian Style

Zhang, Ruizhen, Xiaoyun Cui, and Pengshan Zhao. 2024. "Rapidly Evolved Genes in Three Reaumuria Transcriptomes and Potential Roles of Pentatricopeptide Repeat Superfamily Proteins in Endangerment of R. trigyna" International Journal of Molecular Sciences 25, no. 20: 11065. https://doi.org/10.3390/ijms252011065

APA Style

Zhang, R., Cui, X., & Zhao, P. (2024). Rapidly Evolved Genes in Three Reaumuria Transcriptomes and Potential Roles of Pentatricopeptide Repeat Superfamily Proteins in Endangerment of R. trigyna. International Journal of Molecular Sciences, 25(20), 11065. https://doi.org/10.3390/ijms252011065

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