Tiger nut (Cyperus esculentus) is an annual plant of the genus Cyperus Linn., which belongs to the family Cyperaceae. There are about 380 species of Cyperus Linn., which are produced in temperate and tropical regions. There are about 30 species in China and distributed throughout the country. Common plants include nutgrass (Cyperus rotundus), ricefield flatsedge (Cyperus iria L.) and tiger nut (Cyperus esculentus) (Cui, 2012, China Agricultural Publishing House, pp. 287-288). At present, only one edible species of the genus Cyperus is known, The tiger nut originated from Africa in the late Cenozoic era. It may be the ancient Egyptians who planted the tiger nut first. There are pictures of the tiger nut found on the murals in ancient Egypt. Now it is cultivated in many countries, such as Egypt, Morocco, Nigeria, Congo, Spain, Italy, Bulgaria, Russia, China, etc. In 1952, the Beijing Botanical Garden of the Institute of Botany of the Chinese Academy of Sciences introduced tiger nuts back to China for trial planting in the former Soviet Union, and then introduced them from Bulgaria for cultivation in 1960. It has been nearly 70 years since the introduction of tiger nut into China. Due to the lack of harvesting machinery and insufficient understanding of its value, few people have planted it. In recent years, with the development of tiger nut harvesting machinery and the adjustment of the planting structure of the Ministry of Agriculture, the planting area of tiger nut has gradually expanded, but there is little research on the genetic diversity of tiger nut varieties and germplasm resources (Jing et al., 2015).

DNA molecular marker technology can not be affected by environmental conditions and temporal and spatial constraints of gene expression, reduce the blindness of material selection, and effectively improve the predictability of breeding work. Random amplified polymorphic DNA (RAPD) technology is a molecular marker method based on polymerase chain reaction (PCR) technology, which is widely used. It has the advantages of simplicity, speed, high accuracy and low test cost. It is widely used in variety classification, provenance relationship determination, plant germplasm resources identification and genetic diversity detection of germplasm resources, Many crops have been reported in this field (Hu et al., 2008; Gao et al., 2010; Zhao et al., 2013), but there is no report on RAPD technology in genetic diversity detection of tiger nuts germplasm resources in China. Therefore, this study tried to use RAPD technology to analyze 20 Selaginella materials, so as to clarify the genetic diversity and genetic relationship among materials, and provide reference for comprehensive utilization of test materials and selection of new varieties of tiger nuts.

1 Results and Analysis

1.1 Genomic DNA extraction

The DNA of the test materials was detected by 1% agarose gel electrophoresis, and the bands were clear and complete. Genomic DNA concentration determined by nucleic acid protein content analyzer is 30~70 ng/μL, OD_260/280 value is 1.8~2.0, which can be used for RAPD analysis.

1.2 Primer screening and RAPD amplification

24 primers with clear bands, high polymorphism and good repetition effect were screened from 96 random primers tested (Table 1), and 134 stable loci were obtained through twice repeated amplification, including 94 polymorphic loci with a polymorphism frequency of 70.15% (Figure 1).

1.3 Cluster analysis results

NTSYS-PC software was used to calculate the genetic similarity coefficient of the tested materials between tiger nut and nutgrass. The analysis results showed that the genetic similarity coefficient of the tested materials was between 0.41 and 0.46, the minimum genetic similarity coefficient between material 8 and aconite was 0.41, and the maximum genetic similarity coefficient between material 19 and aconite was 0.46; The genetic similarity coefficient between the tested materials was 0.91~1.00 (Table 2). The genetic similarity coefficient between material 1 and material 11 was the farthest, 0.91, and that between material 5 and material 20 was the closest, 1.00, which may be the same kind of tiger nut. According to the similarity coefficient value of the tested tiger nut materials, UPGMA method was used for cluster analysis and genetic map was constructed. When the similarity coefficient was 0.94, the tested materials were divided into four categories, in which material 1, material 10 and material 11 were grouped into one category, and the other 17 materials were grouped into a large category. Materials with the same grain size were not grouped into one category, and materials from the same region were not grouped into one category (Figure 2).

The results showed that the genetic similarity coefficient between nutgrass and the tested tiger nut was small, and the genetic diversity was rich. The RAPD analysis method could be used to analyze and identify the genetic differences among tiger nut germplasm interspecies. The similarity between the tested tiger nuts materials is very high, the genetic basis is similar, and there is no obvious regional characteristics.

2 Discussion

Most researchers at home and abroad have studied tiger nuts as a kind of malignant weed (Hill et al., 1963). There are few reports on the genetic basis of tiger nuts, and the conclusions are not uniform. Zhao and Wei (2011) used SRAP markers to analyze the materials from different sources of tiger nuts, and the average genetic distance between the tested materials was 0.37, which concluded that the genetic basis of tiger nuts was rich; Okoli et al (1997) used the RAPD molecular marker technique to analyze the genetic diversity of the tiger nut and nutgrass. The results showed that there were significant genetic differences between the two materials, and there were some differences between the two materials, while there were very small differences between the two materials; But Horak and Holt (1986) used isozyme method to study the materials from Florida, California and other different regions of the United States, and reached the conclusion that the genetic basis of tiger nut is narrow.

In this experiment, 20 materials were tested, one of which was nutgrass, and 19 were tiger nut material. RAPD analysis showed that there was a large genetic difference between nutgrass and tiger nut material, which was consistent with the results of Okoli et al. (1997), and the genetic relationship was far away; RAPD analysis method can be used to identify genetic diversity among Cyperus species; The RAPD analysis results of the tested tiger nut materials showed that the genetic similarity was high and the genetic basis was similar, which was consistent with the research results of Horak and Holt (1986). There may be the following reasons for the conclusions drawn from this experiment. Firstly, the source of tiger nut varieties is single. The planting of tiger nuts has been recognized for a short time in China. At present, there is only one variety registered. It is the "Zhongyou Sha No. 1" (Zhao and Wei, 2019), which was cultivated by the Institute of Oil Crops of the Chinese Academy of Agricultural Sciences in 2018. The new variety has just started to be popularized. At this stage, the seed sources of tiger nuts are basically the same everywhere. Although tiger nuts are used earlier in foreign countries, scientific research is mostly focused on food processing, and there is little research on breeding of tiger nuts. Secondly, currently, tuber asexual reproduction is the main way to produce tiger nut, with little genetic variation. At present, there is no report on the identification of new hybrid varieties of tiger nut. The oil crop variety "Zhongyousha No. 1" approved by the Chinese Academy of Agricultural Sciences was selected by mutation (Zhao and Wei, 2019). Thirdly, it is possible that the primers selected in this experiment are universal primers, and the specific region of tiger nut could not be amplified. The RAPD analysis method uses universal primers with low specificity. If specific primers are designed for the genome of tiger nut, the results may be different, and this subject will continue to do further research.

The genetic basis of germplasm resources of tiger nut is narrow, and long-term planting will lead to its gene degradation. The self reseeding of tiger nut and the imperfect breeding system are also unavoidable factors that cause the degradation, and asexual reproduction is the main way, with virus accumulation, which seriously restricts the development of tiger nut industry. At present, cultivating high-quality, virus free tiger nut varieties is the primary task of breeding workers. tiger nut mainly reproduce asexually, generally do not blossom, and rarely blossom and bear fruit (Wu, 2009, Lanzhou University, pp. 1). Therefore, conventional cross breeding techniques have limitations in the improvement of genetic traits and the cultivation of new varieties of tiger nut. The use of physical and chemical mutation techniques and genetic engineering techniques to innovate germplasm resources and cultivate high-quality, high-yield, pest resistant tiger nut has broad prospects (Li, 2010, Southwest University, pp. 1-2); Stem tip tissue culture may solve the problem of virus accumulation caused by long-term asexual reproduction of tiger nut, establish an efficient regeneration system and explore limited gene transformation paths (Li, 2010, Southwest University, pp. 1-2), which will be one of the focuses of genetic improvement research of tiger nut in the future.

3 Materials and Methods

3.1 Test materials

The 20 materials for the test were collected in 8 provinces and cities across the country. Because the test materials are unregistered varieties, they are only represented by serial numbers. The specific regional sources and seed shape information are in the table below. The 10th material is the wild nutgrass plant from Sanya, Hainan. The test tiger nut material was planted in the greenhouse of Economic Plant Research Institute of Jilin Academy of Agricultural Sciences on January 20, 2019, and the sampling time was March 5, 2019.

3.2 DNA extraction and detection

Take the fresh and tender leaves from the top of tiger nut in the field, put them in a heat preservation box with ice bags, and store them in a -20 ℃ refrigerator after returning to the laboratory. The improved CTAB method was adopted to extract genomic DNA from the leaves of tiger nut (Zhang, 2016, Xinjiang Agricultural University, tutor; Li, pp. 41), prepare 1% agarose gel, detect the DNA quality of tiger nut samples by electrophoresis, measure the DNA concentration of samples by nucleic acid protein content analyzer, dilute the samples with ddH₂O to reach 50 ng/ μ 50. Store at - 20 ℃ for standby.

3.3 RAPD primer synthesis and screening

Through literature review, 92 RAPD primer sequences with high polymorphism were selected from other crops (Li et al., 2016). The primers were synthesized by Shanghai Sangong. The primers were diluted for use according to instructions. The mixed sample DNA and representative sample DNA were used as templates for amplification. The primers were screened according to the amplification results, repeated three times, and finally the amplification bands were selected to be clear and recognizable The primers with more specific bands were used for population amplification (Wei et al., 2011a).

3.4 RAPD amplification system and procedure

The total reaction volume is 25 μL. Amplification condition: template DNA 1 μL; H₂O₈ μ L; Primer 1 μL (10 ng/ μL)；Mix 15 μL (2Taq PCR Star Mix With Loading Dye, Beijing Kangrun Chengye Biotechnology Co., Ltd.).

The amplification procedure was: 94 ℃ pre denaturation for 3 min; Denaturation at 94 ℃ for 1 min, renaturation at 36 ℃ for 1 min, extension at 72 ℃ for 2 min, 43 cycles (Zhao, 2020); Extension at 72 ℃ for 10 min; Store at 4 ℃. PCR products were detected by 1% agarose gel electrophoresis, the electrophoresis buffer was 1xTAE, 120 V voltage, about 40 min, observed and photographed under the ultraviolet imaging system.

3.5 Data analysis

After electrophoretic detection of PCR amplification products, take pictures and keep pictures. According to the pictures, count the diversity bands. If a band appears at the same position, it will be marked as "1". Otherwise, if there is no band, it will be marked as "0". Use Excel to record the statistical results. Finally, generate a matrix consisting of "1" and "0". Use NTSYS software to analyze the sample. First, calculate the similarity coefficient between different materials of tiger nut, and then use the class average method to cluster analysis, Generate similarity coefficient matrix and tree diagram (Wei et al., 2011b).

Authors’ contributions

WZM was the designer and research executor of this experiment, who completed the data analysis of the experiment and article writing; WZH, YXD and NL completed the data analysis and the writing of the first draft of the paper; CY and LJY participated in the experimental design and analysis of the experimental results; MZS was the designer and director of the project, guiding experimental design, data analysis, article writing and revision. All authors read and approved the final manuscript.

Acknowledgements

This research was jointly funded by the National Key R&D Program of China (2019YFD1002603), Major Science and Technology Project of Jilin Province (192480NY010166906) and Agricultural Science and Technology Innovation Project of Jilin Province (CXGC2019DC007).

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