We sampled six species, the broad-leaved trees Populous tomentosa Carr. (Y) and Salix babylonica L. (L), the needle-leaved trees Pinus koraiensis Sieb. (H) and Pinus sylvestris var. mongolica Litv. (S), and the succulent herb Hylotelephium spectabile (Bor.) H. Ohba (T) and Sedum sarmentosum Bunge (C), from the campus of Jilin University between June and July 2013. These leaves were at a similar growing status and free from fungus, other infections or dirt. We used three preservation methods to preserve the samples; they were divided into three groups and stored in cryogenic liquid nitrogen (N₂), preserved in a −20 °C ice chest (I), and rapidly dried in silica gel (D). In order to rule out the influence of individual genetic difference and experimental bias, each species was derived from the same individual, and 16 specimens were sampled. Eight samples from each group were preserved for three weeks, while the other 8 samples were preserved for three years.

We isolated genomic DNA of all the preserved samples and extracted fresh leaves for use as the control using the plant genomic DNA kit (Tiangen Biotech. Beijing Co. Ltd., China), following the manufacturer's instructions. Each preservation method for all species was repeated 8 times in case of random errors. DNA quality and quantity were estimated both spectrophotometrically, as well as visually, by ethidium bromide staining after electrophoresis on 0.8% agarose gels. The DNA samples were diluted to the concentration of 50 ng/μL and stored at −20 °C for use. A set of 30 anchored microsatellite primers and 20 decamer arbitrary primers were synthesized from Sangon Biotech. Beijing Co. Ltd., China, and at least 8 primers were selected for each species (see Tables S1–S4). ISSR–PCR was performed in 25-μL PCR reaction tubes containing 2 μL of genomic DNA, 1.0 μL of primer (20 μM stock, 0.8 μM final concentration), 3.0 μL of dNTP mix (2.5 mM stock, 0.3 mM final concentration), 2.5 μL of standard Taq reaction buffer (10×), and 0.2 μL of Taq DNA polymerase enzyme (5000 unit/mL; TransGen Biotech, China). Thermocycling proceeded as follows: 94 °C for 5 min followed by 40 cycles at 94 °C for 45 s, 49–59.8 °C annealing for 45 s, 72 °C for 1 min; completion of these cycles was followed by a final extension at 72 °C for 10 min. The annealing temperature was usually adjusted according to the T_m of the primer used in the reaction. RAPD-PCR was performed in a 25-μL volume having 1× PCR buffer, dNTP (0.2 mM), primer (10 pmol), 5 ng template and Taq DNA polymerase (1 U) (TransGen Biotech, China). RAPD amplification was performed with initial denaturation at 94 °C for 5 min, followed by 45 cycles of 1 min denaturation at 94 °C, 1 min annealing at 37 °C, 1 min extension at 72 °C and final extension at 72 °C for 10 min. The amplified products were mixed with bromophenol blue gel loading dye and were analyzed by electrophoresis on a 1.2% agarose gel using a 0.5× Tris Acetate EDTA buffer pH 8.0 at room temperature. In general, the quality of the patterns generated differed from primer to primer (Fig. S1).

The amplified fragments, with the same mobility according to their molecular weight (bp), were scored according to whether a binary code was present (1) or absent (0). Only the consistently reproducible bands were scored, and smeared and weak bands were excluded. The number of bands from each group was manually counted and least significant difference (LSD) multiple comparisons were performed with SPSS (version 19). In order to detect the influence of different preservation methods for plant materials on ISSR and RAPD analysis, we calculated the genetic index used for each molecular marker. The statistical analysis of ISSR and RAPD markers were analyzed using POPGENE software (version 1.31) [20]. An analysis of molecular variance (AMOVA) was used to examine population genetic differentiation within and among the seven groups preserved by different methods during different times using AMOVAPREP and AMOVA 1.55 [21]. The dendrograms were constructed using an unweighted paired group method of cluster analysis with arithmetic averages (UPGMA) on NTSYS-pc program (version 2.10e) [22].

All the materials preserved in the seven groups resulted in sufficient levels of genomic DNA for use in ISSR and RAPD analysis. However, there were some differences in the number of bands (Figs. 1 and 2), except for P. koraiensis analyzed by RAPD, which resulted in 67 bands in all groups (Fig. 2c). In the ISSR analysis, the fresh samples of salicaceae and pinaceae showed the more bands. In particular, there were significantly more bands in the fresh pinaceae group than in other groups. Crassulaceae preserved in liquid nitrogen showed the more bands; the range of bands between the highest and lowest groups was higher than that in salicaceae and pinaceae. Significant differences were observed between short-time and long-time preservation, storage in liquid nitrogen of Se. sarmentosum and storage in −20 °C ice chest of Po. tomentosa, S. babylonica, and P. sylvestris var. mongolica. However, no significant differences were evident using other methods. In RAPD analysis, fresh samples of Po. tomentosa showed the more bands, but the number of bands was not significantly different from that of samples preserved in liquid nitrogen (Fig. 2a); S. babylonica preserved in liquid nitrogen and dried in silica showed more bands; P. sylvestris var mongolica preserved in liquid nitrogen showed significantly more bands than other groups; with regards to crassulaceae, the fresh samples showed the least number of bands and no significant difference between other H. spectabile samples was observed. The results show that different preservation methods have wide-ranging effects on the number of bands of different plant types, in both ISSR and RAPD analyses.

The genetic diversity detected by ISSR and RAPD is shown in Table 1. In the ISSR analysis, Po. tomentosa and H. spectabile, preserved in a −20 °C ice chest, showed the highest percentage of polymorphic loci (PPL) and the highest Shannon information index (i), whereas S. babylonica, P. koraiensis, and Se. sarmentosum, which were preserved by drying in silica gel, and P. sylvestris var mongolica, which were preserved in liquid nitrogen and dried in silica gel, showed a higher PPL. In the RAPD analysis, Po. tomentosa, which was preserved in liquid nitrogen, showed the most PPL and for S. babylonica was preserved in −20 °C ice chest; the fresh and dried in silica groups of P. koraiensis and H. spectabile got the higher PPL; the fresh groups of Se. sarmentosum, preserved in liquid nitrogen got higher PPL values; the groups of P. sylvestris var mongolica preserved in liquid nitrogen groups and dried in silica got higher PPL values.

The genetic variation attributed among the groups deviated from 50%, in particular P. sylvestris var mongolica, Se. sarmentosum, and S. babylonica with both ISSR and RAPD markers (Table 2). Only H. spectabile samples had variation of nearly 50% among the groups. However, the similar genetic variation between groups and within groups does not indicate that the different methods had no effect on ISSR or RAPD analysis.

The UPGMA cluster analysis data showed that most of the individuals preserved by the same methods clustered together regardless of how long they were stored (Figs. 3 and 4). In the ISSR analysis of Po. tomentosa, the groups clustered into three clades with a high similarity coefficient. The first clade was formed by YI and YN, and was clustered with YD, which also clustered with YF (Fig. 3a). For S. babylonica, all individuals of each group were not able to be separated (Fig. 3b). For P. koraiensis, HF clustered with HI, which were then clustered with other groups, which were not well separated. For P. sylvestris var mongolica, the groups clustered into three clades that were made up by SF, SD and the remaining individuals. For Se. sarmentosum and H. spectabile, the groups clustered according to the different preservation methods. In RAPD analysis, for Po. tomentosa, YF and YD clustered in the first clade and YI and YN clustered in the second clade which were connected together. For S. babylonica, LF and LI formed one clade and then clustered with other individuals. For P. koraiensis, HI and HN formed one clade and clustered with HD, which then clustered with HF. For P. sylvestris var mongolica, SI and SN formed the first clade and clustered with SF, and SD clustered with the whole clade. For Se. sarmentosum, CN and CD formed the first clade and clustered with CI, which formed a sister clade with CF. For H. spectabile, the groups clustered into two clades where TF formed the first clade and other individuals formed the remaining one. In both analyses, all the fresh samples clustered together.

In order to compare the differences between groups, we computed the genetic identity (GI) using Nei's unbiased measures based on ISSR and RAPD data (Tables 3–8). GI values between groups preserved by the same method, for different time periods ranging from 0.913 to 0.999, suggest that the same preservation methods for different storage periods had no effect on GI, both in RAPD and ISSR analyses. However, the GI values between some groups that were preserved by different methods were lower. For Po. tomentosa, the GI value between groups preserved in a −20 °C ice chest and fresh samples was 0.845 using ISSR; between groups preserved in a −20 °C ice chest and dried in silica, the result was 0.769 by RAPD. This result is lower than that observed in other groups, indicating that Po. tomentosa is not ideally suited for preservation in a −20 °C ice chest compared to other methods. Similarly, we found that drying in silica gel was not a suitable method for preserving P. koraiensis, P. sylvestris var mongolica, and H. spectabile; and storage in liquid nitrogen was not a suitable method for Se. sarmentosum. These results were consistent with cluster analysis. For S. babylonica, the GI value was more than 0.9 between all groups by ISSR analysis, and very high in RAPD analysis, indicating that the different preservation methods have little effect on the analysis.