BIOCELL
DOI:10.32604/biocell.2022.020311
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Article

Development of polymorphic SSR markers and their applicability in genetic diversity evaluation in Euptelea pleiosperma

XIAOJUN ZHOU*, XIAOYU LU and XUBO WANG

College of Life Science, Luoyang Normal University, Luoyang, 471934, China
*Address correspondence to: Xiaojun Zhou, zhouxiaojun@lynu.edu.cn
Received: 16 November 2021; Accepted: 13 March 2022

Abstract: Euptelea pleiosperma is a characteristic species of East Asian flora with both ornamental and scientific values. Based on the reduced-representation sequencing (RRS) technology of RAD-Seq, this study conducted high-throughput Illumina paired-end sequencing to find SSR marker information in the genome of E. pleiosperma, and to screen and verify polymorphism of SSR markers. We obtained 5.5G of high-quality data using RAD-Seq. The total number of contigs of the RAD tags was 299,376, with the maximum contig length of 2,062 bp and the average length of 445 bp. From these sequences, we identified 20,718 SSR loci, with a distribution density of one SSR per 6.45 kb (1/6.45 kb). Among all SSRs, dinucleotides (52.00%) were the most detected SSRs, followed by mononucleotides (21.63%). AG/CT was the dominant motif in the SSR loci, accounting for 34.8%. Primers were successfully designed for 14,593 loci, and 100 pairs of these primers were randomly selected for chemical synthesis and validated by SSR-PCR amplification in 20 individuals of E. pleiosperma. Seventy-nine primers were able to amplify the target bands. Cervus 3.0 software was used to analyze the selected 20 SSR loci with good polymorphism. For the 20 SSR markers, the number of alleles ranged from 4 to 9, and the observed heterozygosity and expected heterozygosity were from 0.35 to 0.75 and 0.541 to 0.875, respectively. The information content of polymorphic loci ranged from 0.463 to 0.848, with an average value of 0.638. Among them, there were 18 highly polymorphic loci, and 20 SSR loci did not deviate from the Hardy-Weinberg equilibrium. Furthermore, the 20 pairs of SSR primers were used to conduct PCoA analysis based on Nei’s genetic distance of 51 individuals from three populations. The results showed that these SSR markers could distinguish genetic differences based on different geographical locations.

Keywords: Cluster analysis; E. pleiosperma; RAD-Seq; Rare species; SSR markers

Introduction

Euptelea pleiosperma J. D. Hooker & Thomson is a tertiary relict plant of the Eupteleaceae family, mainly distributed in Henan, Hebei, and Shanxi in China (Fu and Peter, 2001). This species forms deciduous shrubs or small arbors with flowers that open before the leaf flush, and clusters of red flowers and fruits, making it a beautiful ornamental tree. In addition, E. pleiosperma is a characteristic species of the East Asian flora, which has important academic value for the study of palaeoflora and paleoclimate (Wang et al., 2015). However, with the social development, the wild habitats have been disturbed or destroyed by human activities, affecting the growth and natural regeneration of E. pleiosperma (Chen et al., 2007). As a result, the number of E. pleiosperma has decreased dramatically and the distribution range has become increasingly smaller (Wang and Qin, 2011). Therefore, the species has been listed as a national third-class protected plant of China and the IUCN LC level (Wang and Xie, 2004; Sun, 2018). As a rare plant with both ornamental and scientific value, E. pleiosperma attracted the attention of many researchers. Much research has been conducted on habitats investigation of germplasm, seedling growth assays, or chemical composition analysis (Zhang et al., 2016; Yan et al., 2020).

At the molecular level, SCoT (start codon targeted polymorphism) and RAPD (random amplified polymorphic DNA) molecular markers were used to analyze the genetic diversity of related populations of E. pleiosperma (Wu et al., 2020; Wang et al., 2014; Zhang et al., 2016).

Simple sequence repeats (SSRs), are widely present in eukaryotes and consist of 1–6 nucleotides in tandem as repeating units (Varshney et al., 2005; Zhang et al., 2021). Compared with SCoT and RAPD molecular markers, SSRs have the characteristics of high polymorphism, co-dominant inheritance, and are suitable for high-throughput automated genotyping (Varshney et al., 2005; Victoria et al., 2011). SSRs have been widely used in genetic analysis and conservation of tobacco, soybean, and ginkgo (Liu et al., 2018; Qi et al., 2019). Reduced-representation sequencing (RRS) techniques reduce the complexity of the genome of a given species by sequencing only a subset of the genome (Choquet, 2021). Restriction-site associated DNA sequencing (RAD-Seq) is a simplified genome sequencing technique based on whole-genome restriction sites developed using next-generation sequencing (Baird et al., 2008). In the past few years, dozens of publications using RAD-Seq have been reported in molecular marker development, genetic diversity, and mapping studies (Etter et al., 2011; Gonen et al., 2014; Tsujimoto et al., 2020). Some of the main reasons are that RAD-Seq does not require a reference genome, it represents a cost-effective and high throughput method for generating comparative genomic information and it can be widely used in different species (Miller et al., 2007; Feng et al., 2020). The approaches are particularly useful for species that rather young plant groups (less than 50 million years), and across different plant systems (Eaton et al., 2017).

This study analyzed the SSR markers characteristics of E. pleiosperm based on RAD-Seq and explored the feasibility of developing polymorphic SSR markers. These SSR markers will provide opportunities for examining the genetic diversity and population structure of E. pleiosperma and contribute to the effective conservation of this species.

Materials and Methods

Research materials

The plant samples were collected in the Longyuwan National Forest Park (Luanchuan County, Luoyang City, China), the Daohuigou Park (Songxian County, Luoyang City, China), and the Xiaoqinling National Nature Reserve (Lingbao County, Sanmenxia City, China). Sampling information is shown in Table 1.

For each individual healthy young leaves were collected (different individuals should be more than 10 meters apart) and stored in the refrigerator at 4°C. DNA was extracted by modified CTAB (hexadecyl trimethyl ammonium bromide) method and detected by KAIAO ultra-micro spectrometer (K5500, Beijing, China).

Sequencing and data processing

Three qualified genomic DNA samples were mixed in equal amounts, treated with restriction enzymes, and P1 adaptors were added (the adaptors contain amplification primer sites, Illumina sequencing primers binding sites sequences, and short tag sequences to distinguish different samples). The DNA sample was interrupted into short sequences of 300–700 bp in length and added with P2 adaptors (which include the reverse complementary amplification primer sites), and then RAD tags were enriched by PCR amplification. The RAD libraries were sequenced by the Illumina NovaSeq 6000 platform. The raw reads obtained from sequencing were conducted quality control by twice data filtering, and then high-quality clean reads were used for subsequent analysis. High-quality Illumina sequencing reads were submitted to the NCBI (accession number: PRJNA749160). The specific sequencing and assembly procedures were described in references (Catchen et al., 2011; Willing et al., 2011).

Screening and validation of SSR

SSRs search was performed on the assembled sequences by the software MISA (Beier et al., 2017). The searching parameters of SSR loci were set to identify perfect mono-, di-, tri-, tetra-, penta-, and hexa-nucleotide motifs with a minimum of 15, 6, 5, 4, 4, and 4 repeats, respectively. Moreover, the distance between two SSRs must be at least 100 bp. If the distance between two SSRs is less than 100 bp, they are merged into one SSR marker. SSR primers were designed using Primer Premier 3.0 software in the flanking regions (Untergasser et al., 2012; Zhou et al., 2015). To validate the designed primers, a total of 100 pairs of primers were synthesized and PCR was carried out for amplification in 20 individuals of E. pleiosperma. The number of individuals from LY, XQ, and DH was 10, 5, and 5 respectively. The PCR procedure was carried out in 15 μl volume containing 7.5 μl of 2 × PCR Mixture (Tiangen, Beijing, China), 20 ng of genomic DNA, 0.25 μM of forward and reverse primers with conditions as follows: denaturation for 5 min at 94°C followed by 30 cycles of 50 s at 94°C, 45 s for annealing and 30 s min at 72°C. Amplification products were resolved by 10% polyacrylamide gel electrophoresis (PAGE) and visualized by silver staining. The size of each SSR-PCR product was determined in comparison with pBR322 DNA/MspI marker (Tiangen, Beijing, China).

Principal coordinate analysis

Twenty pairs of SSR primers with good polymorphism were selected to conduct the genetic analysis of the 51 individuals from three populations (LY, DH, and XQ). The amplified data were input into GenAlEx v6.5, and the principal coordinate analysis (PCoA) was performed based on Nei’s genetic distance of the individuals in different populations (see e.g., Karbstein et al., 2019).

Results

Sequencing quality assessment

The raw data obtained by sequencing and the clean data after filtering are shown in Table 2. It indicates that the obtained data are accurate and reliable and can be used for further analysis. The total number of contigs assembled from the RAD tags was 299,376 with a length average of 445 bp, a minimum of 159 bp, and a maximum of 2,062 bp.

Profile of the SSR loci of E. pleiosperma

After searching 299,376 contigs by MISA software (http://pgrc.ipk-gatersleben.de/misa/), a total of 20,718 SSR loci were detected. Of these, 19,034 (91.87%) were complete SSRs and 1,684 (8.13%) were complex SSRs. The 20,718 SSR loci were distributed in 18,135 contig sequences, of which 2,171 sequences contained two or more SSR loci. The distribution density of the SSR was 0.155 SSR/kb, with an average of one SSR locus per 6.45 kb (1/6.45 kb). The sequences containing SSR loci accounted for 6.06% of the total number of sequences, and detected SSR loci accounted for 6.92% of the total number of sequences.

SSRs motif types

In the present study, dinucleotide was the most abundant SSR marker, accounting for 52.00% (10,773) of all SSRs, followed by mono- (21.63%, 4,482), tri- (16.19%, 3,355), tetra- (6.21%, 1,287), hexa- (2.11%, 437) and pentanucleotide (1.85%, 384). The A/T motif accounted for 96.3% of the mononucleotide repeat motifs and 20.8% of all SSR loci. AG/CT was the most common dinucleotide motif, accounting for 67.1% of all dinucleotide repeat motifs and 34.8% of all SSR loci. AAG/CTT was the dominant trinucleotide motif, accounting for 40.1% of all trinucleotide repeat motifs and 6.3% of all SSR loci. For tetranucleotide repeats, the most frequent motif was AAAT/ATTT, which accounted for 63.2% of all tetranucleotide repeat motifs and 2.4% of all SSR loci. The total number of pentanucleotides and hexanucleotides accounted for 7.1% of all SSR loci (Fig. 1).

Figure 1: Statistics of E. pleiosperma SSR motifs. The x-coordinate is the SSR motif type, and the y-coordinate is the proportion of different motif types among all SSRs. The others represent pentanucleotides and hexanucleotides motif types.

Length distribution of SSRs motifs

The SSR motifs length of the E. pleiosperma genome was in the range of 12–142 bp, and there were 17,862 SSRs in the range of 12–20 bp, accounting for 86.0% of all SSRs. There were 2,919 SSR motif sequences longer than 20 bp, accounting for 14.0% of all sequences. There were 16,403 SSR loci between 12 and 19 bp and 4,378 ≥ 20 bp in the genomes of E. pleiosperma. Therefore, in the genome of E. pleiosperma, most SSR loci (78.9%) showed moderate polymorphism, and the loci with high polymorphism account for 21.1%. The number of SSRs with motif length of 16 bp was the highest (3,542), representing 17.0% of all SSRs (Fig. 2).

Figure 2: Length Distribution of SSRs motifs. The x-coordinate is the SSR motif length, and the y-coordinate is the number of SSRs with different motif lengths.

SSRs validation and principal coordinates analysis

A total of 14,593 primers were successfully designed for all these SSR loci. One hundred primers were randomly selected to chemically synthesize and conducted SSR-PCR amplification in 20 E. pleiosperma DNA samples, of which 79 could amplify the target bands.

To verify the polymorphism of these primers, 20 highly polymorphic loci were selected and analyzed by CERVUS 3.0 software. The detailed information of the 20 primers is shown in Table 3. Results showed the average allele of 20 SSR loci was 5.25, the observed heterozygosity ranged from 0.350 to 0.740, and the expected heterozygosity ranged from 0.541 to 0.875. In this study, the polymorphic information content (PIC) of the 20 polymorphic loci ranged from 0.463 to 0.848, with an average of 0.638 (Table 3). Among them, there were 18 highly polymorphic loci (PIC > 0.5), two moderately polymorphic loci (0.25 < PIC < 0.5), and the 20 SSR loci did not deviate from the Hardy-Weinberg equilibrium. In addition, the 20 polymorphic SSR loci were used to conduct PCoA analysis of 51 individuals of E. pleiosperma. The results showed that LY and DH populations tend to be clustered into one group because the distance between LY and DH populations is even closer (Fig. 3). Two coordinates explain 24.1% and 18.97% of the overall genetic variation, respectively. The results showed that the SSR markers could distinguish genetic differences among populations based on different geographical locations.

Figure 3: PCoA of genetic distance for the 51 individuals from the three populations sampled. Coordinate axis 1 explains 24.1% of the variation, coordinate axis 2 explains 18.97% of the variation.

Discussion

RRS technology can obtain many tag sequences representing the genome of a species through high-throughput sequencing of part of the genome, which has the advantages of short experimental periods, high accuracy, and reliable results. RAD-Seq is one of the most used sequencing technologies. It has many advantages, such as simple operation, low experimental cost with high throughput, and has been widely used in many fields such as genome comparison, genetic analysis, and germplasm conservation (Basak et al., 2019). Due to the above advantages, RAD-Seq technology is an ideal method for SSR development and analysis for non-model plant E. pleiosperma.

In the present study, the distribution density of SSR in the genome of E. pleiosperma was similar to that of Camellia sinensis (1/3.55 kb), Piper nigrum (1/6.3 kb), Hibiscus esculentus (1/7.81 kb), and Chimonanthus praecox (1/5.00 kb) (Sharma et al., 2009; Kumari et al., 2019; Li et al., 2018; Li et al., 2013). Compared with cotton (1/20.8 kb), Sorghum bicolor (1/220 kb), and wheat (1/578 kb), the distribution frequency of SSR loci in E. pleiosperma was significantly higher (Liu et al., 2019; Yonemaru et al., 2009; Morgante et al., 2002). These differences may be due to the use of different sequencing methods, but they also reflect differences in the genomic characteristics of these species.

Among these E. pleiosperma SSR loci, the dinucleotide motif was the main repeat type, followed by the mononucleotide motif. This is like the situation for both genomes of palm trees and Dimocarpus longan, which are dominated by mono- and dinucleotide motifs (Manee et al., 2020; Hu et al., 2019). However, it differs from strawberry, Cicer arietinum, and Corchorus capsularis, which are dominated by di- and trinucleotide motifs repetition (Zorrilla-Fontanesi et al., 2011; Asadi et al., 2020; Yao et al., 2019). The AG/CT repeat motif is the most abundant one detected in the genome of E. pleiosperma, which is the same as Jatropha curcas, Toona sinensis, and Paeonia lactiflora (Yadav et al., 2011; Yu et al., 2019; Mercati and Sunseri, 2020).

Polymorphism of SSR loci is mainly caused by changes in the number of repeats of the motif, and the higher the number of repeats, the higher the potential for polymorphism (Marshall et al., 2002). In addition, SSRs with low polymorphism are usually less than 12 bp in length, SSRs greater than 12 bp and less than 20 bp in length are often moderate polymorphic, while SSRs with higher polymorphism are usually greater than 20 bp in length (Gao et al., 2003; Temnykh et al., 2001). The results indicate that the development of SSR markers for E. pleiosperma is feasible using the RAD-Seq approach, and all of the SSR loci developed in this study had above moderate polymorphism.

Takezaki and Nei (1996) suggested that the range of heterozygosity calculated from SSR was 0.3–0.8. Heterozygosity indicates the degree of individual genetic variation within a population, and high values indicate large variation. The expected heterozygosity (He) and observed heterozygosity (Ho) of the 20 polymorphic SSR loci in this study were generally consistent with the criteria proposed by Takezaki, except for some loci (EP16, EP37, and EP91). According to the polymorphic information content index proposed by Bostein et al. (1980) to measure the degree of gene variation, a locus is considered low polymorphic when PIC < 0.25, moderately polymorphic when 0.25 < PIC < 0.5, and highly polymorphic when PIC > 0.5. In this study, two loci (EP88 and EP99) have PIC values between 0.25 and 0.5, indicating that they are moderately polymorphic, whereas the other 18 loci have PIC values greater than 0.5, indicating that they are highly polymorphic.

Principal coordinates analysis (PCoA) is a powerful tool for assessing the genetic structure of a population. The results demonstrate that the polymorphic SSR markers developed in this study are effective in the genetic analysis of E. pleiosperma and lay the foundation for the effective conservation of this species.

Conclusions

This study used RAD-Seq to investigate the characteristics of E. pleiosperm SSR markers and the potential of developing polymorphic SSR markers. Analysis of SSR loci in the genome of E. pleiosperma indicated that most of these SSR loci have polymorphism potential. Primers developed from the important genetic resources were successfully utilized for genetic analysis in E. pleiosperma and revealed good heterozygosity and PIC values. Furthermore, 14,593 SSR primers that were designed in the present study will provide opportunities for examining the population structure of E. pleiosperma and contribute to the effective conservation of this species. The results obtained in this study demonstrate that RAD-seq can be used as an efficient method for E. pleiosperma SSR markers development and genetic research.

Authors Contribution: The authors confirm contribution to the paper as follows: study conception and design: X.J.Z.; analysis and interpretation of results: X.J.Z. and X.B.W.; draft manuscript preparation: X.J.Z. and X.Y.L. All authors reviewed the results and approved the final version of the manuscript.

Funding Statement: This work was supported by the National Natural Science Foundation of China (31870697).

Conflicts of Interest: The authors declare that they have no conflicts of interest to report regarding the present study.

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