Qianshi (Euryale ferox Salisb. ex Konig et Sims), also known as Qian and Jitou in Chinese, is an annual large herbaceous aquatic plant of Euryale genus in the family of Nymphaeaceae (Figure 1), distributed in lakes, ponds or low-lying wetlands (Zhao, 1999). Euryale ferox has a long history of cultivation. In recent years, the cultivation scale has gradually expanded, and the planting area in Jiangsu Province is more than 2×10⁴ hm² (Jiang et al., 2018). Euryale ferox can generally be divided into two types: Euryale ferox without thorns and Euryale ferox with thorns. Euryale ferox with thorns belongs to wild and semi-wild species. The leaves, stems and fruits of the plant are densely covered with thorns, while Euryale ferox without thorns is a cultivated variety. Only the veins on the back of the leaves and the edge of the leaves have thorns. Due to the large size, good quality and high economic efficiency of the seeds, it is gradually popularized and cultivated in many places in China (Jiang et al., 2018). Euryale ferox can be used as both medicine and food. It contains many effective ingredients such as sterols, flavonoids, cyclopeptides, sesquienolignans, cerebroside esters, phenols, etc. It has antioxidant, anti-fatigue, hypoglycemic, anti-aging, anti-cancer and other pharmacological effects. It is often used in clinical treatment of early diabetes, kidney disease, chyluria, sequela of apoplexy and chronic enteritis and other diseases (Liu et al., 2015). As one of the important aquatic vegetables in China, the seed kernel of Euryale ferox is rich in starch, protein, vitamins and other nutrients, and is deeply loved by people. Therefore, the breeding and development prospects of new varieties of Euryale ferox are increasingly broad.

At present, the germplasm improvement of Euryale ferox is often carried out by crossing between Euryale ferox without thorns and Euryale ferox with thorns. After years of screening seed kernel, shell and other characters, stable high-quality and high-yield offspring are finally obtained (Yin et al., 2015). With the deepening of the breeding work, it is difficult to distinguish the genetic relationship between the new varieties of Euryale ferox. However, using molecular biological technology to distinguish different varieties at the molecular level can make up for the lack of morphological identification to a certain extent.

Simple sequence repeat (SSR), also known as microsatellite DNA, is usually composed of 2 to 5 nucleotides (a few of them are 1 to 6) in series to form a repetitive DNA sequence (Luo et al., 2010). It has the characteristics of high polymorphism, good repeatability, simple operation and fast response. It has been widely used in the genetic diversity research of rice (Yu et al., 2004), wheat (Chen et al., 2011), pepper (Sheng et al., 2019), sweet potato (Dong et al., 2018) and other economic crops. However, there are relatively few studies on molecular markers of Euryale ferox. You et al. (2012) used AFLP marker and SRAP marker technology to study the genetic relationship between several Euryale ferox germplasm resources and Jiwanglian (Victoria cruziana×Euryale ferox) and Victoria cruziana respectively for the first time. Quan et al. (2009) screened 11 pairs of microsatellite marker primers from the Euryale ferox. Kumar et al. (2016) used RAPD and ISSR molecular markers to carry out DNA fingerprinting and diversity analysis on 16 cultivated varieties of Euryale ferox in India. Then Kumar et al. (2017) used SSR primers to carry out genetic analysis on the distribution of cultivated varieties of Euryale ferox in India in different regions. DNA molecular marker technology plays an important role in the genetic diversity analysis, variety classification and identification and breeding of various economic crops. In this study, transcriptome sequencing (RNA-Seq) was used to analyze the SSR site and distribution characteristics in the transcriptome of Euryale ferox, in order to provide effective theoretical support for the development of SSR markers and genetic diversity of Euryale ferox.

1 Results and Analysis

1.1 SSR site information in the transcriptome of Euryale ferox

After assembly and splicing, the total length of the transcriptome sequence of Euryale ferox was 73.78 Mb, including 86 829 non-redundant genes (Unigene), with an average length of 849.74 bp, N50 1 516 bp, N90 315 bp, and GC ratio of 43.96%. The search for the above Unigenes found that out of 8 437 Unigenes, there were 9 640 SSR sites, the frequency of occurrence was 9.72%, and the frequency of SSR sites was 11.10%, of which 1 026 Unigenes had more than one SSR site, accounting for 10.64% of the total SSR, and 424 Unigenes contained composite SSR sites, accounting for 4.4% of the total SSR (Table 1).

1.2 The type and distribution of SSR site in the transcriptome of Euryale ferox

A total of 6 repeat types were found in the transcriptome of Euryale ferox, including 5 965 dinucleotide repeat units, which were the main repeat types, accounting for 61.88% of the total number of SSRs, followed by trinucleotide and mononucleotide repeats, with 2 077 and 1 219 repeat units, respectively, accounting for 21.55% and 12.65% of the total number of SSRs. However, the number of repeats of tetra-, penta- and hexa-nucleotides is relatively small, with a total of 379, accounting for only 3.93% (Table 2). The average distribution distance of SSR was 7.65 kb in the transcripts of Euryale ferox. The distribution distance of SSR sites decreased with the increase of frequency.

1.3 Characteristics of SSR repeat elements in the transcriptome of Euryale ferox

There are 226 repeat primitives in the transcriptome of Euryale ferox. Among them, there were 21 kinds of single nucleotide repeats, and the frequency of A/T was the highest among them, accounting for 11.25% of the total number. There were 24 kinds of dinucleotide repeats, among which AG/CT had the highest frequency of 4 904, and the frequency of CG/CG was 50.87%, while CG/CG had a low frequency of only 15. There were 38 trinucleotide repeats, among which AAG/CTT had the largest number (628), and the occurrence frequency was 6.51%, followed by AGG/CCT, AGC/CTG and ACC/GGT, which accounted for 4.04%, 3.00% and 2.80% of the total number, respectively. There were 25, 45 and 73 types of tetra-, penta- and hexa-nucleotides, respectively, but the repetition frequency of each type was very low, all of which were below 0.3% (Figure 2).

The length of SSR motifs in Euryale ferox ranges from 12 to 60 bp, and most of them range from 12 to 20bp, with a total of 8 923 motifs, accounting for 92.56%, followed by 702 motifs ranging from 21 to 30 bp, accounting for 7.28%, and few with a length above 30 bp, accounting for 0.15% (Figure 3).

1.4 SSR primer screening

Primer 3.0 was used to select site with repeat units of 2, 3, 4 and 5 bases for primer design according to the obtained SSR site information. 200 pairs of primers were randomly selected for screening, and the detection results were analyzed according to the screening criteria. In the experiment, 11 pairs of primers with good amplification effect were selected. The amplified fragments ranged from 110 to 180 bp (Table 3).

2 Discussion

Microsatellite molecular markers are one of the effective tools for plant genetic analysis and genetic map construction, which are characterized by rapid and accurate discovery of functional genes. Currently, there are relatively few reports on the genetic diversity of Euryale ferox using microsatellite molecular markers. In this study, newborn leaves of Euryale ferox were adopted for transcriptome sequencing. By searching non-redundant genes in transcriptome, 9 640 SSR sites were found in 86 829 Unigene, with a frequency of 11.10%. This was consistent with the results reported earlier, which showed that the frequency of SSR site in dicotyledonous plants was 2% to 17% (Kumpatla and Mukhopadhyay, 2005). The occurrence frequency of SSR site in Euryale ferox was slightly higher than that of Nelumbo nucifera (9%), Eleocharis dulcis (5.8%) and Capsicum annuum (5.60%) (Zhang et al., 2014; Liu et al., 2015; Sheng et al., 2019), but lower than 14.97% of Luffa aegyptiaca (Zhu et al., 2016). The differences in the frequency of SSR site were influenced by various factors, such as different SSR search parameters, different measurement tools, and different genome size or structure. In addition, different search parameter settings and treatments also had an impact on SSR distribution density. The SSR site density of Euryale ferox was 7.65 kb, lower than that of Nelumbo nucifera (6.84 kb) and Colocasia esculenta (5.90 kb), but higher than that of Eleocharis dulcis (9.79 kb) (Zhang et al., 2014; Liu et al., 2015; You et al., 2015).

From the perspective of SSR structure, there are 5 965 dinucleotide repeats in the transcriptome of Euryale ferox, accounting for the highest proportion of 61.88%, which is close to the results of most crops such as lotus (64.5%), taro (64.95%), sweet potato (43.32%), pepper (46%) and pumpkin (49.82%) (Wang et al., 2010; Zheng et al., 2015; You et al., 2015; Wang et al., 2016; Sheng et al., 2019), followed by trinucleotide and mononucleotide repeats, which accounted for 21.55% and 12.65% of SSR total, respectively. The number of repeats of tetra-, penta-, hexa-nucleotide is relatively small. There are 226 kinds of repeated motifs in Euryale ferox, and AG/CT is the most repeated motifs, accounting for 50.87%, which is most similar to Nelumbo nucifera 51.12% (Zhang et al., 2014). The occurrence frequency of the trinucleotide motif AAG/CTT was the highest in Euryale ferox, accounting for 6.51%, which was also reported in lotus, sweet potato, pecan and other plants (Wang et al., 2010; Zheng et al., 2015; Jia et al., 2019). However, the contents of dinucleotide motif CG/CG and trinucleotide motif CCG/CGG in Euryale ferox are low, which is consistent with the results of previous studies on dicotyledon plants (Zeng et al., 2010).

Both the number and length of repeats of SSR motifs are important factors to evaluate their polymorphism. The longer the repeats, the higher the polymorphism (Lin et al., 2019), and the number of repeats affects the length of SSR motifs. Temnykh et al. (2001) divided rice SSR elements into two categories according to the length of nucleotides. When the length of microsatellite was 20 bp, it was highly polymorphic, while when the length of microsatellite was 12~20 bp, it tended to be conservative, and representative site occasionally occurred, which might be caused by short SSR mismatch. The length of SSR motifs in Euryale ferox was 12~20 bp, with a total of 8 923, accounting for 92.56%, while the length was 21~30 bp, with a total of 702, accounting for 7.28%. Therefore, it is predicted that the SSR site discovered by the transcriptome sequencing of Euryale ferox are polymorphic and can be used for subsequent primer screening experiments.

Euryale ferox is used as both medicine and food, which has a wide application prospect. Molecular marker technology is adopted to evaluate the genetic relationship and genetic structure of Euryale ferox germplasm resources, which can better protect and develop the germplasm resources. However, the traditional hybrid breeding of Euryale ferox is very complicated, which is one of the important reasons restricting the development of Euryale ferox. Therefore, it is very important to carry out relevant research at the molecular level. This study analyzed the distribution type and structural characteristics of SSR in the transcriptome of Euryale ferox, and integrated the occurrence frequency, repetition type, length of motif and other characteristics of SSR in the transcriptome, which is helpful for genetic diversity analysis and molecular marker-assisted breeding of Euryale ferox.

3 Materials and Methods

3.1 Experimental material

The experimental materials were fresh leaves of purple flower Euryale ferox without thorn, collected from Caohu Resource Nursery Conservation Base of Suzhou Academy of Agricultural Sciences. The newly grown leaves of the plants were taken, washed and quickly placed in liquid nitrogen and stored at -80℃. The transcriptome sequencing was commissioned by Tsingke Biotechnology (Nanjing) Co., Ltd.

3.2 Experimental method

Total RNA from Euryale ferox leaves was extracted, the sequencing library was prepared after the samples were tested qualified, and sequencing was completed by Illumina platform. Use the De novo assembly software Trinity for assembly, and then use TGICL to remove redundancy and splicing to obtain non-redundant Unigene, and make further statistics and quality control on Unigene. Use MicroSAtellite (MISA) recognition tool to analyze the SSR of the transcriptome of Euryale ferox and remove the long and short sequences in the EST sequence. The search criteria were set as follows: the minimum number of repeats of mono-, di-, tri-, tetra-, penta-, hexa-nucleotide repeats was 10, 6, 5, 5, 4 and 4, respectively.

Authors’ contributions

XBW was the experimental designer and executor of this study, responsible for the writing of the first draft of the thesis; ZRH and XJ participated in data sorting and writing the first draft of the thesis; SFF participated in some experiments; YYL was the project leader, guiding experimental design, data statistics, thesis writing and revision. All authors read and approved the final manuscript.

Acknowledgments

This research was funded by "Breeding of High-quality New Varieties of Characteristic Aquatic Vegetables" (20035).

References

Chen X.D., Sun D.F., Rong D.F., Peng J.H., and Li C.D., 2011, A recessive gene controlling male sterility sensitive to short day length/low temperature in wheat (Triticum aestivum L.), J. Zhejiang Univ. Sci. B., 12(11): 943-950.

https://doi.org/10.1631/jzus.B1000371

Dong L.X., Su Y.J., Dai X.B., Wang J., Tang J., Zhao D.L., and Cao Q.H., 2018, Genetic diversity analysis of overground sweetpotato special-purpose varie-ties based on simple sequence repeats (SSR) markers, Jiangsu Nongye Xuebao (Journal of Southern Agriculture), 34(4): 741-746.

Jia Z.Y., Xuan J.P., Zhang J.Y., Wang G., Jia X.D., and Guo Z.R., 2019, Analysis of SSR information in Carya illinoinensis transcriptome, Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 17(10): 3305-3311.

Jiang J.Z., Zou F.G., Diao W.P., Jiang J., and Yang Y.C., 2018, Analysis of Euryale ferox industry development in jiangsu province, Changjiang Shucai(Journal of Changjiang Vegetables), (20): 24-25.

Kumar H., Priya P., Singh N., Kumar M., Choudhary B.K., and Kumar L., 2016, RAPD and ISSR marker-based comparative evaluation of genetic diversity among Indian Germplasms of Euryale ferox: an aquatic food plant, Applied Biochemistry and Biotechnology, 180(7): 1345-1360.

https://doi.org/10.1007/s12010-016-2171-z

Kumar N., Shikha D., Kumari S., Choudhary B.K., Kumar L., and Singh I.S., 2017, SSR-Based DNA fingerprinting and diversity assessment among Indian Germplasm of Euryale ferox: an aquatic underutilized and neglected food crop, Applied Biochemistryand Biotechnology, 185(1): 34-41.

https://doi.org/10.1007/s12010-017-2643-9

Kumpatla S.P., and Mukhopadhyay S., 2005, Mining and survey of simple sequencerepeats in expressed sequence tags of dicotyledonous species, Genome 48(6): 985-998.

https://doi.org/10.1139/g05-060

Liu L., Liu Y.Y., Zhan Y., Fan B.T., Wei Q., Xu D.H., and Liu T.H., 2015, Research progress in chemical components, pharmacological action and clinical application of Semen Euryales, Zhonghua Zhongyiyao Zazhi (China Journal of Traditional Chinese Medicine and Pharmacy), (2): 477-479.

Luo R., Wu W.L., Zhang Y., and Li Y.H., 2010, SSR marker and its application to crop genetics and breeding, Jiyinzuxue Yu Yingyong Shengwuxue (Genomics and Applied Biology), 29(1): 137-143.

Lin H., Li Y.P., Xue Z.Z., Chen M.D., Zhu H.S., and Wen Q.F., 2019, Analysis of SSR loci in transcriptome and development of molecular markers in Brassica oleracea L.var.botrytis L., Xibei Nonglinkeji Daxue Xuebao (Journal of Northwest A&F University (Nat.Sci.Ed.)), 47(3): 85-93.

Liu H.B., You Y.N., Zhu Z.X., Zheng X.F., Huang J.B., Hu Z.L., and Diao Y., 2015, Leaf transcriptome analysis and development of SSR markers in water chestnut (Eleocharis dulcis), Genet. Mol. Res., 14(3): 8314-8325.

https://doi.org/10.4238/2015.July.27.20

Quan Z.W., Pan L., Ke W.D., and Ding Y., 2009, Polymorphic microsatellite markers in Euryale ferox Salisb. (Nymphaeaceae), Mol. Ecol. Resour., 9(1): 330-332.

https://doi.org/10.1111/j.1755-0998.2008.02402.x

Sheng W.T., Deng J.L., Rao Y.S., Chai X.W., and Yao Y.P., 2019, Analysis of SSR information of EST resource in Capsicum annuum L., Fenzi Zhiwu Yuzhong (Molecular Plant Breeding), 17(14): 4698-4703.

Temnykh S., DeClerck G., Lukashova A., Lipovich L., Cartinhour S., and McCouch S., 2001, Computational and experimental analysis of microsatellites in rice (Oryza sativa L.): frequency, length variation, transposon associations, and genetic marker potential, Genome Research, 11(8): 1441-1452.

https://doi.org/10.1101/gr.184001

Wang Y.Y., Shan W.Q., Xu W.L., Cui C.S., and Qu S.P., 2016, Analysis on SSR information in transcriptome and the polymorphism of Cucurbita maxima, Yuanyi Xuebao (Acta Horticulturae Sinica), 43(3): 578-586.

Wang Z.Y., Fang B.P., Chen J.Y., Zhang X.J., Huang L.F., and Chen X.L., 2010, De novo assembly and characterization of root transcriptome using Illumina paired-end sequencing and development of cSSR markers in sweetpotato (Ipomoea batatas), BMC Genomics, 11(1): 726.

https://doi.org/10.1186/1471-2164-11-726

Yin Y.L., Sun F.F., and Sun H.Y., 2015, Breeding of stingless and high yield varieties of Gusuqian No.5, Changjiang Shucai (Journal of Changjiang Vegetables), 396(22): 58-60.

You Y.N., Liu D.C., Liu H.B., Zheng X.F., Diao Y., Huang X.F., and Hu Z.L., 2015, Development and characterisation of EST-SSR markers by transcriptome sequencing in taro (Colocasia esculenta (L.) Schoot), Molecular Breeding, 35(6): 134.

https://doi.org/10.1007/s11032-015-0307-4

You Y.N., Yin Y.L. Diao Y., Sun F.F. Zheng X.F., Zhou M.Q., Bao Z.Z., and Hu Z.L., 2012, Genetic analysis and evaluation of Euryale ferox germplasm resources and their hybrid progenies, Changjiang Shucai (Journal of Changjiang Vegetables), (16): 34-38.

Yu J.K., Rota M.L., Kantety R.V., and Sorrells M.E., 2004, EST derived SSR markers for comparative mapping in wheat and rice, Mol. Genet. Genomics, 271(6): 742-751.

https://doi.org/10.1007/s00438-004-1027-3

Zeng S.H., Xiao G., Guo J., Fei Z.J., and Xu Y.Q., Bruce A Roe., and Wang Y., 2010, Development of a EST dataset and characterization of EST-SSRs in a traditional Chinese medicinal plant, Epimedium sagittatum(Sieb. Et Zucc.) maxim, BMC Genomics 11(1): 94.

https://doi.org/10.1186/1471-2164-11-94

Zhang W. W., Tian D.K., Huang X., Xu Y.X., Mo H.B., Liu Y.B., Meng J., and Zhang D.S., 2014, Characterization of flower-bud transcriptome and development of genic SSR markers in Asian lotus (Nelumbo nucifera Gaertn.), Plos One, 9(11): e112223.

https://doi.org/10.1371/journal.pone.0112223

Zhao Y.W., 1999, Chinese aquatic vegetable, Beijing: China Agriculture Press, pp: 55-56.

Zheng X.F., You Y.N., Diao Y., Zheng X.W., Xie K.Q., Zhou M.Q., Hu Z.L., and Wang Y.W., 2015, Development and characterization of genic-SSR markers from different Asia lotus (Nelumbo nucifera) types by RNA-seq, Genetics and Molecular Research, 14(3): 11171-11184.

https://doi.org/10.4238/2015.September.22.11

Zhu H.S., Zhang Q.R., Liu J.T., Chen M.D., Wang B., and Xue Z.Z., 2016, Analysis on SSR Loci in transcriptome and development of molecular markers in Luffa cylindrical, Zhongguo Xibao Shengwuxue Xuebao (Chinese Journal of Cell Biology), 38(9): 1118-1127.