Introduction

Wild Allium tuberosum from the Tibet is disturbed widely in Tibetan area [1]. Wild Allium tuberosum as a stylish food is attracting wide attention, especially in China, because of its rich and nutritional value, capability of removing free radicals and inhibiting the activity of pathogen [2-5]. In addition, for a long process of wild growth, wild Allium tuberosum has developed excellent characteristics including to strong resistance to root knot nematodes and easy cultivation [6]. Thus, wild Allium tuberosum can be used as the primary gene pool in modern cultivation and breeding programs for cultivated Allium tuberosum. Using these genes, they need genetic improvement for adaption to harsh environments and insect pest. Generally, the understanding of genetic information is the basic of genetic engineering. However, a wide range of genetic diversity on wild Allium tuberosum from Tibet has not been studied.

Here, we selected 11 wild Allium tuberosum samples germplasms. We examined the seeds morphology, seeds response to NaCl and PEG stress and analyzed genetic diversity by Sequence Related Amplified Polymorphism (SRAP) method. SRAP is cost-effective and reliable genetic marker to estimate genetic diversity and relationships in various plant species [7,8].

Materials and Methods

Plant Materials

All wild Allium tuberosum samples were collected from Langkazi Kare Country of SHANNAN site [(29^°13'48"-29^°14'05" (Longitude, E) and 90^°23'56"-90^°24'04" (Latitude, N)] from Tibet. All the detailed information of 11 samples was listed in (Table 1).

Seeds Morphology

Surface texture of seeds randomly collected from 30 individuals were observed and imaged using a digital camera (Olympus SZX7, lympus Corporation, Japan). Two exomorphic parameters including the length and width of seeds, the length and width of wings were measured (Figure1).

Measurement of Germination Percentage Under NaCl and PEG

Germination percentage was tested under NaCl and PEG. Five NaCl concentration (0 mM, 50 mM, 100 mM, 150 mM and 200 mM), five PEG concentrations (0%, 5%, 10%, 15% and 20%) and the combination of NaCl and PEG (NaCl (0 mM) + PEG (0%); NaCl (50 mM + PEG (5%); NaCl (100 mM + PEG (10%); NaCl (150 mM + PEG (15%);) was selected to calculate germination percentage. For all tests, three replicates of 30 seeds per treatment were sown on the surface of 2% agar in water in Petri dishes and incubated with complete darkness.

SRAP Analysis

DNA Extraction

Total genomic DNA of seeds was isolated according to the modified CTAB method of Doyle (1990). DNA concentration was measured by a spectrophotometer (Eppendorf, Eppendorf China Limited, China).

Ten SRAP primers were used for amplification. The information on all primers was listed in (Table 2). 
PCR condition was followed was carried out in 25 μl volume containing 50 ng DNA templates, 1U Taq polymerase (TakaRa, China) and 20 ng of forward and reverse primers and 2.5 μL 10×PCR buffer. PCR condition was followed: 95 ^°C denaturation for 5 min, 5 cycles for 1 min denaturation at 94 ^°C, 1 min at annealing at 35 ^°C, and then 72^°C elongation for 1 min, the next 30 cycles including 10 min annealing at 48 ^°C, 72 ^°C final extension. PCR products were detected on 6% polyacrylamide sequencing gel (Figure 2).

The distinct and reproducible SRAP bands were scored as absent (0) or present (1). The dendrogram was constructed according to the Jaccard's similarity coefficients by the NTSYS-pc Version 2.10e [9]. Some basic parameters including total number of fragments (TN), the Number of Polymorphic Fragments (NPF) and the percentage of polymorphic fragments (PPF, %) were calculated. Genetic diversity between samples was estimated based on the method of Analysis of Molecular Variance (AMOVA) Version 1.55 [10].

Results

Observation of Seeds

The parameters of seeds measurement are listed in (Table 1). The characteristics of seeds were showed in (Figure 1). Although there is little difference, the seed morphous of wild Allium tuberosum from four altitudes showed similar features. The seeds showed shield shape, had brown color. There is wrinkle or smooth on sexine ornamentation. The length of seeds ranges from 2287.75 µm to 3693.62 µm. The width of seeds ranges from 1654.75 µm to 1928.26 µm.

Under NaCl stress, there were significant treatment effects on germination percentage for cultivated and wild samples (Figure 3). NaCl stress resulted in the decrease of germination percentage for cultivated samples, while there were no treatment differences under 50 mM and 100 mM NaCl. 200 mM NaCl was found to be inhibitorier to germination percentage (0%) of cultivated samples, compared with wild samples (5%).

Under no PEG stress, we observed no obvious effects for cultivated and wild samples. However, there are strong effects of PEG on germination percentage on cultivated samples (Figure 4). In contrast, wild samples had significantly higher germination percentage value under PEG stress compared to cultivated samples (Figure 4).

To investigate the effects of different NaCl and PEG concentration on germination percentage, seeds sensitivity to exogenous NaCl and PEG was investigated. Stress (NaCl and PEG) lead to decrease of germination percentage, however, the decrease in wild samples under NaCl and PEG was less significant in Cultivated (Figure 5).

Genetic Diversity Analysis 

The genetic diversity on 11 samples was assessed using ten primer pairs (Table 2). 853 fragments were detected, of which 826 fragments were polymorphic. The percentage of polymorphic fragments was ranged from 79.59% (ME5-EM4) to 100% (ME4-EM4 and ME7-EM4) with an average of 96.83. In addition, the mean of similarity coefficients among all samples is found to be 0.463 (Table 3). The Min similarity coefficient (0.355) was found between A-2 and D-3 and the Max similarity (0.575) was found between D-2 and D-3, respectively. The SRAP profile amplified by the primer primers ME7-EM6 is shown in (Figure 2). Cluster analyses were carried out based on the UPGMA method. All samples were clustered into 3 groups (Cluster I, Cluster II and Cluster III) (Figure 6). Most of these samples clustered appeared scattered distribution.

Discussion

In our study, the color of the seeds of wild Allium tuberosum is brown. This would have indicated that, under special environmental conditions in Tibet, high seed germination could be environmental selection for conditional tolerance. A similar finding was reported for Sesamum indicum [11]. Their findings showed sesame genotypes characterized by brown seeds were more tolerant to PEG and NaCl stresses than sesame genotypes characterized by white seeds. Generally, the seed coat is a key tissue that serves as a conduit for nutrients and water [12,13]. It has been reported that environmental stress imposed during seed development can cause changes in seed coat morphology leading to negative effects on seed germination rate, seed quality and seedling vigor [14,15]. Therefore, shrinkle, twinkle and light seed coat of wild Allium tuberosum can facilitate the prevention of strong light, low temperature and drought from Tibetan environment.

In this study, we analyzed the seed germination traits of wild Allium tuberosum under NaCl and PEG. We found that wild Allium tuberosum belonging to alpine plant species generally showed higher germination percentage for NaCl and PEG stress, while cultivated species had lower germination percentage. These results confirmed the trend suggested by Wang (2017), who recorded a higher germination percentage under PEG (5%-15%) and NaCl (0.2%-0.4%). Indeed, these results were also consistent with the observations in other Tibetan plant species, such as Sophora moorcroftian, Hippophae rhamnoides and Avena fatua [16-18] which suggested that wild Allium tuberosum from The Tibet had fine regulation of tolerance under high altitudes conditions.

To improve the genetic characteristics, the understanding of genetic diversity on plant species is important. Additionally, the understanding of genetic diversity plays a key role for utilization new gene resources to enlarge genetic variation to plant breeding materials [19]. To data, few researches have used molecular markers technology to study the genetic diversity in wild Allium tuberosum [20]. In this study, SRAP proved to be a effective, useful and high-resolution technique to the detect the variation among all samples of wild Allium tuberosum. Taking no account of the relatively small sample sizes, 10 SRAP primers were sufficient to differentiate all wild Allium tuberosum samples from four altitudes.

Additionally, genetic cluster plot showed that all samples from different altitudes are randomly distributed. This scattered distribution suggested that there is high genetic diversity for wild Allium tuberosum from Tibet. [20] also reported that wild Allium tuberosum from Tibet maintained relatively more genetic diversity. Wild Allium tuberosum grows harsh environmental conditions including violent winds, low temperatures, drought, a low oxygen concentration, and strong Ultraviolet (UV) radiation [21]. Accordingly, wild Allium tuberosum develops many genetic variation characteristics to adapt to complicated environments. Therefore, these biological characteristics may contribute to maintain a high level of genetic variability of wild Allium tuberosum.

Acknowledgement

Figure 1: The morphous of seeds on Allium hookeri Thwaites from different altitude (A, 4057 m; B, 4022 m; C, 3929 m; D, 3900 m. Bar: 1cm).

Figure 2: Fingerprint patterns generated by primers ME7-EM6 from the genomic DNA of the 11 genotypes of Allium hookeri Thwaites.

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Figure 3: Effects of different NaCl concentration on the germination percentage of Allium hookeri Thwaites.

Figure 4: Effects of different PEG concentration on germination percentage of Allium hookeri Thwaites.

Figure 5: Effects of different NaCl and PEG concentration on germination percentage of Allium hookeri Thwaites.

Figure 6: Dendrogram of 11 samples resulting from the UPGMA cluster analysis based on Jaccard’s similarity coefficients obtained from SRAP.

Table 1: The information and parameters of seeds of Allium hookeri Thwaites.

Table 2: Information on each primer pairs among all samples in this study.

Table 3: The similarity coefficients among all samples based on UPGMA dendrogram.

1. Wu Z (1987) Flora of Tibet. 5 Beijing Science Press: 556.
2. Hu G，Lu Y, Wei D (2006) Chemical characterization of Chinese chive seed（Allium tuberosum Rottl). Food Chemistry 99: 693-697.
3. Liu J, Zhao L，Su W (2006) Research progress of bioactive constituent and molecular biology in Allium tuberosum. Food Science and Technology 8: 67-70.
4. Hong J, Chen T, Hu P (2014) Purification and characterization of an antioxidant peptide（GSQ）from Chinese leek (Allium tuberosum Rottler) seeds. Journal of Functional Foods 10: 144-153.
5. Fang Y，Cai L，Li Y, Wang JP, Xiao H, et al. (2015) Spirostanol steroids from the roots of Allium tuberosum. Steroids 100: 1-4.
6. Li G, Quiros CF (2001) Sequence-Related Amplified Polymorphism (SRAP), a new marker system based on a simple PCR reaction: its application to mapping and gene tagging in brassica. Theor Appl Genet 103: 455-461.
7. Peng X, Ji Q, Fan S, Zhang Y, Zhang J (2015) Genetic diversity in populations of the endangered medicinal plant Tetrastigma hemsleyanum revealed by ISSR and SRAP markers: implications for conservation. Genet Resour Crop Ev 62: 1069-1078.
8. Rohlf F (2000) NTSYS-PC, numerical taxonomy and multivariate analysis system for the PC Exeter Software, Version 2.1. Applied Biostatistics Inc Setauket NY 2000.
9. Harfi ME, Hanine H, Rizki H (2016) Effect of Drought and Salt Stresses on Germination and Early Seedling Growth of Different Color-seeds of Sesame (Sesamum indicum). Int J Agric Biol 18: 1088-1094.
10. Ranathunge K, Shao SQ, Qutob D, Gijzen M, Peterson CA, et al. (2010) Properties of the seed coat cuticle change during development. Planta 231: 1171-1188.
11. Weber H, Borisjuk L, Wobus U (2005) Molecular physiology of legume seed development. Annu Rev Plant Biol 56: 253-279.
12. Dornbos DL, Mullen RE (1991) Influence of stress during soybean seed fill on seed weight, germination, and seedling growth-rate. Can J Plant Sci 71: 373-383.
13. Egli DB, TeKrony DM, Heitholt JJ, Rupe J (2005) Air temperature during seed filling and soybean seed germination and vigor. Crop Sci 45:1329-1335.
14. Ren ZJ (2013) Effects of PEG on seed germination of Hippophae rhamnoides. Agricultural science and technology communication 7: 101-103.
15. Zhang YF, Yao W, Guo Q (2015) Effcet of drought stress on seed germination and seedling growth of Sophora moorcrofiana. Journal of Northwest A&F University 43: 45-56.
16. Huo YL, Chai SH, Li T (2016) Effects of saline-alkali stress on seed germination and seedlings of three Tibetan herbage. Science and technology in Tibet 8: 68-71.
17. Meng F, Peng M, Wang R, Guan F (2014) Analysis of genetic diversity in Aconitum kongboense L. revealed by AFLP markers. Biochemical Systematics and Ecology 57: 388-394.
18. Wang Z, Lang J, Sheng G (2017) Evaluation of flavor and main nutritional quality of three wild populations of Allium tuberosum in Tibet. Acta Horticulturae Sinica 44：1189-1197.
19. Shen Q, Fu L, Dai F, Jiang L, Zhang G, et al. (2016) Multi-omics analysis reveals molecular mechanisms of shoot adaption to salt stress in Tibetan wild barley. BMC Genomics 17: 889.