Plant materials.

A total of 77 peach germplasms, including 54 P. mira Koehne ex Sargent from the Tibetan Plateau and 23 peach materials from Zhengzhou Fruit Research Institute (Zhengzhou, China), were examined (Table 1; Fig. 1).

Table 1.List and description of 77 individuals used in this study.

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DNA extraction.

The total genomic DNA was extracted using the modified CTAB method as described by Bouhadida et al. (2011). The DNA concentration and quality were estimated using a ultraviolet-VIS spectrophotometer (ultraviolet-1800; Shimadzu Corporation) and 0.8% agarose gel electrophoresis. The extracted DNA was then stored at –80 °C for polymerase chain reaction (PCR) amplification.

PCR amplification.

The AFLP reactions were performed according to the method described by Vos et al. (1995) with some modifications. All of the primers and adapters are listed in Table 2. The DNA (150 ng) was digested with 5 U of EcoRI and 5 U of MseI (Promega, Madison, WI) in a total volume of 40 μL at 37 °C for 3 h and then incubated at 75 °C for 15 min. The digested DNA products were then ligated with 1 μL of EcoRI adapter (50 μM) and 1 μL of MseI adapter (5 μM) at 16 °C for 16 h. After ligation, the mixture was diluted 10-fold with ddH₂O. The pre-amplified reaction was performed using pre-selective primers. The pre-amplified mixture consisted of 5 μL diluted solution, 10 × PCR buffer, 2 mm dNTPs, 20 mm MgCl₂, 20 mm pre-amplification primer, and 1 U of Taq DNA polymerase in a 20-μL volume. The pre-amplified reaction was performed under the following conditions: denaturation at 94 °C for 3 min, 30 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min and a final extension at 72 °C for 5 min. The amplified products were diluted 30-fold with ddH₂O and used as the template for selective amplification. The selective amplification reactions were performed in a 20-μL volume containing 5 μL of template, 10 × PCR buffer, 2 mm dNTPs, 25 mm MgCl₂, 20 mm selective amplification primer, and 1 U of Taq DNA polymerase. The PCR selective amplification was performed as follows: initial denaturation at 94 °C for 3 min, 13 cycles of 94 °C for 30 s, 65 °C for 30 s (–0.7 °C at each cycle) and 72 °C for 1 min, 23 cycles of 94 °C for 30 s, 56 °C for 30 s, and 72 °C for 1 min, and a final step at 72 °C for 5 min. The PCR products were separated on a 6% polyacrylamide gel for 2.5 h at 80 W and detected using silver staining as previously described by Bassam et al. (1991).

Table 2.Characteristics of the amplification products obtained using 13 amplified fragment length polymorphism primers.

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Data analysis.

Only reproducible, clear, and well-resolved AFLP fragments were scored as the presence (1) or absence (0) for each primer pair. The genetic parameters, including the total number of fragments, number of polymorphic fragments, and percentage of polymorphic fragments (%), were estimated. A dendrogram corresponding to the similarity coefficient and PCoA results was constructed using NTSYS-pc software (Version 2.10) (Rohlf, 2000). The resolving power (Rp) was used to assess the power of the primers to discriminate all individuals (Prevost and Wilkinson, 1999). The assessment of Rp was performed using the Gilbert formula: Rp = ∑Ib, where Ib = 1 – [2 × |0.5 –P|] and P is the frequency of the individuals containing the I band (Gilbert et al., 1999). In addition, the PIC was used to discriminate the ability of the primers. The formula was as follows:where k is the total number of alleles detected by a primer and Pi is the frequency of the allele in all individuals (Lynch and Ritland, 1999).

AFLP polymorphism.

Thirteen AFLP primer combinations generated a total of 377 scorable and clear fragments for 77 individuals, and all of these fragments (100%) were polymorphic (Table 1; Fig. 2). The total number of fragments of each primer pair varied from 15 for EcoRI-AC/MseI-AG to 42 for EcoRI-AG/MseI-AG with an average of 28.40. Moreover, the PIC values ranged from 0.91 to 0.96 with a mean of 0.95 (Table 2; Fig. 3). The mean of the Rp was 15.00 and ranged from 8.60 to 18.68 (Table 2).

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Genetic diversity analysis.

All 377 AFLP fragments scored were used for genetic diversity studies. A high level of similarity coefficients was observed. The average of the similarity coefficients values was 0.56. The lowest similarity (0.23) occurred between Dejichun and Dianzhan2 and the combination of Youpantao 36-3 and Zhongyou 10 showed the highest similarity (0.76) (data not shown).

Based on Nei’s genetic distance, an unweighted pair group method with arithmetic average (UPGMA) dendrogram was constructed (Fig. 4). In the dendrogram, all of the materials were divided into three main groups at the similarity level of 0.31 (Fig. 3). Group I consisted of most of the materials from the Tibetan Plateau. Group II included some of the materials from the Tibetan Plateau and all of the individuals from the center of the peach resource. Only the Dejichun individual belonged to Group III. In addition, Group II contained two groups (Subgroups I and II). There were 10 individuals from the Tibetan Plateau in Subgroup I, and all of the materials from the Zhengzhou Fruit Research Institute were in Subgroup II.

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PCoA analysis.

The relationship among all of the individuals was also estimated using PCoA. The results of the PCoA analysis largely corresponded to those obtained using cluster analysis. The three principal axes accounted for 2.6%, 5.79%, and 25.26% of the total variation, respectively. The first two axes separated all of the individuals into two groups. One group included all of the individuals from the center of the peach resource. The remaining individuals (from the Zhengzhou Fruit Research Institute) with the exception of Dejichun were clustered together to form one group (Fig. 5).

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AFLP polymorphism.

AFLP has been extensively used in previous studies as a technique to detect molecular polymorphisms in cultivars and wild peach germplasms (Aranzana et al., 2003; Hu et al., 2005; Wang et al., 2008). Using AFLP, Wang et al. (2006) found a polymorphic percentage of 29.5% for nine primer combination pairs among 94 wild peach germplasms. Sixteen AFLP primer combinations produced a total of 837 fragments and 146 polymorphic bands with a polymorphism percentage of 17.5%, as reported by Xu et al. (2006). These results suggested that a low level of genetic diversity is present among peach germplasms based on the AFLP technique. Compared with previous studies, high levels of polymorphism (100%) were found in this study through the evaluation of the genetic diversity in Prunus mira (P. mira Koehne ex Sargent) germplasms based on AFLP. This result suggested that P. mira and other peach species have a broad relationship. However, some potential reasons for these different levels of polymorphism may be correlated with the material size and the level of polymorphism of the primer combinations.

Although the AFLP technique may be useful for detecting genetic diversity in different peach germplasms, none of the results can be simply compared with those of other studies as a result of different genotypes and primer pairs. Thus, different marker techniques should be used to estimate DNA polymorphisms in peach germplasms (Testolin et al., 2000). For instance, Bouhadida et al. (2011) analyzed 94 peach cultivars using six simple sequence repeat (SSR) markers and found that SSR is very useful for future peach identification studies. Cao et al. (2012) detected highly polymorphic SSR markers among 104 peach landrace accessions using 53 SSR markers. Despite having a high variability for the identification of all peach cultivars, SSR was found to be relatively less variable compared with other species (Aranzana et al., 2012). Thus, considering the limitation of different marker techniques, the combination of two or more molecular marker systems is most likely efficient for the conservation and management of peach germplasms. Furthermore, the effects of factors on morphological traits and fruit development should be taken into account when elucidating the genetic structure in peach germplasm resources (Cao et al., 2012).

Genetic diversity analysis.

In China, peach, as a cultivated plant, has existed for thousands of years (Everett, 1982). As a self-pollinating plant, peach has very low genetic variation (Scorza et al., 1985). To enrich the genetic diversity of peach, we need to identify more wild peach resources. The rich genetic basis of wild peach germplasms is well known and has been demonstrated; however, the relationships among these germplasms remain unclear (Wang et al., 2008).

To date, peach is widely cultivated in China as an important fruit crop. In particular, many wild peach germplasms have attracted much attention as a result of their specific characteristics (Aradhya et al., 2004; Guan et al., 2014; Wang et al., 2006). This may be helpful for breeding peach cultivars with potentially new biotic and abiotic stresses (Cao et al., 2012). However, their genetic relationships are controversial and unclear. For example, Gansutao (P. kansuensis Rehd) and Prunus mira (P. mira Koehne ex Sargent) have a relatively close relationship, as reported by Zong and Duan (1987). However, this result was inconsistent with our findings. In the present study, Gansutao was clustered with Zhongyou6 (P. persica var. necturina), which is consistent with the results obtained by Yu et al. (2004). In addition, Youpantao36-3 (P. persica var. platycarpa) was closely clustered with Zhongyou10 (P. persica var. necturina), which is consistent with the findings reported by Cheng et al. (2001). In addition, Chunmitao (P. persica L. Batsch) and Huangpantao (P. persica var. platycarpa) were clustered together in Subgroup I, most likely as a result of their evolutionary relationship (Cheng et al., 2001, 2002; Yang et al., 2001). It is common that Shouxingtao (P. persica var. densa) is derived from Maotao (P. persica var. aganopersica Reich.) (Yang et al., 2001). However, Maotao was clustered not with Shouxingtao but with Fenchuizhi (P. persica var. pendula). We hypothesized that Maotao may be closely related to Chuizhitao (P. persica var. pendula). Guo et al. (1986) reported that a close relationship existed among Gansutao, Shantao (P. davidiana Francht), and cultivated P. persica L., which is consistent with our findings.

The dendrogram showed that all of the individuals from the Zhengzhou Fruit Research Institute were localized in Group II, possibly as a result of the existence of a common genetic basis within this group. Moreover, the high genetic distances (data not shown) detected between these materials confirmed the higher diversity of individuals from the Zhengzhou Fruit Research Institute in this study. Importantly, the Zhengzhou Fruit Research Institute of the Chinese Academy of Agricultural Sciences, which was founded in 1960, is a national professional research organization of deciduous fruit that studies peach germplasms and develops new cultivars. Thus, we should enrich the peach gene pool to conserve them for the breeding of new varieties and to avoid genetic erosion.

In the present study, the relationship between P. mira and other peach germplasms was remote (Fig. 4). This finding indicated that P. mira demonstrates a genetic difference with other P. persica germplasms. Wang and Zhou (1990) confirmed that P. mira is the ancestor of cultivated peach. In addition, at the level of 0.31, Dejichun formed a distinct group (Group III), indicating its clear differentiation from the other materials. This result is most likely because of the adaptation to a socioeconomic factor or geographical isolation. Indeed, the Tibetan Plateau is a unique geographic area with many geographic barriers such as the Tanggula Mountains, Lanchang River, Kunlun Mountains, and Nujiang River (Zhang and Jiang, 2006). Thus, many specific germplasms may be formed in the Tibetan Plateau.

Based on the genetic similarity matrices, the UPGMA dendrogram indicated that most of the individuals from Bomi City in the Tibetan Plateau were clustered to form one group (Subgroup I). This result indicated that most of the individuals belonging to the Bomi group had close genetic relationships. This low diversity may be the result of a self-mating system, environmental similarities, and a small population size, which would not facilitate genetic drift and cause a low level of genetic variation.

Conservation of wild peach germplasms.

China is the native homeland of the peach tree and has abundant peach germplasms. However, the genetic narrowness of modern peach cultivars is a bottleneck to the breeding of new peach varieties. Wild peach germplasms have a high level of genetic diversity and are known to have many valuable genes that have not been used, e.g., resistance to disease and environmental stress (Cho et al., 2012; Sauge et al., 1998). However, there is an extremely dispersed distribution of wild peach germplasms. Moreover, some wild peach germplasms have been in danger of extinction as a result of human activities such as agriculture and pasture needs (Li et al., 2013; Wang et al., 2010; Wu et al., 2003). Thus, these germplasms should receive special attention to ensure their conservation, which would necessitate a variety of conservation approaches depending on the specific biological factors. These approaches should include the conservation of ecosystems/agro-ecosystems, naturally occurring gene pools, and specific genetic stocks such as the tubers and roots used by communities (Altieri et al., 1987; Brush, 1991). Furthermore, more national research institutes have been founded as priority conservation sites, and areas where the wild peach germplasms are found within the protected areas should be demarcated to not allow any activity.

Overall, our results indicated that AFLP is a powerful and effective tool for the analysis of the genetic diversity of wild and cultivated peach germplasms. Furthermore, further analyses using several molecular markers are required to confirm our conclusions, and more potential peach germplasms should be collected for the breeding of new varieties.