Background

Tree peony (Paeonia suffruticosa) is one of the important ornamental plants in China, and Luoyang is famous for its various superior tree peony cultivars. In fact, tree peony and related industries account for large proportion in tourism income of Luoyang, Henan province. While most native tree peony cultivars flower in April, their ornamental period is very short, which limit the economic benefits increasing. For a long time, some introduced late-flowering cultivars from Japan, America etc. played an important role in delaying ornamental period. Hybridizing superior native cultivars with late-flowering introduced cultivars would be a valid method to breed late-flowering native tree peony cultivars to meet tourist who were free in May. In order to select appropriate parental combination, diversity analysis of the parent materials, revealing the relationship between the native cultivars and the introduced late-flowering cultivars is the first work. SSR, or Simple Sequence Repeat was proved to be the powerful tool for diversity analysis and genetic relationship identification in plants. Genomic derived SSRs were developed and used for population genetic studies in tree peony (Wang et al., 2009; Homolka et al., 2010; Hou et al., 2011a; Yuan et al., 2012). By now, two thousands and more EST sequences were deposited in NCBI dbEST database, which gave opportunity for EST-SSR markers develpoment. Some researchers reported no more than 10 polymorphic EST-SSRs development in tree peony (Homolka et al., 2010; Hou et al., 2011b). Here we reported the development of twenty novel polymorphic EST-SSR markers and diversity analysis of 41 tree peony cultivars by these EST-SSR markers, providing basis for parent selection in tree peony late-flowering breeding.

1 Materials and Methods

Forty-one tree peony cultivars (twenty-one central peony cultivars from Heze, Shangdong province and Luoyang, Henan province, nineteen foreign cultivars from Japan, one foreign cultivar from America) were kindly provided by International Tree peony Park, Luoyang. The forty-one cultivars and their three main phenotypic traits were listed in Table 1. Genomic DNA was extracted by CTAB method (Dellaporta et al., 1983) from leaves of six plants per cultivar on March 20, 2012.

EST-SSR primers design: Totally 2204 EST sequences from NCBI dbest were searched for SSR by Simple Sequence Repeats Identifed Tool (http://www.gramene.org/ db/searches/ssrtool) (Temnykh et al., 2001). Primers were designed flanking SSR with a minimum five repeats using DNAMAN 5.0 software.

PCR amplification:Polymerase chain reactions (PCRs) were performed in a 10μl reaction volume containing 30 ng of template DNA, 2.5 pmol of forward and reverse primers, 5μl of 2×Power Taq PCR Master Mix (BioTeke Biotechnology Co. Ltd., Beijing, China) and sterile distilled water. The conditions for amplification were 4 min at 94℃ followed by 34 cycles of 35s at 94℃, 60 s at 60℃~52℃, and 60s at 72℃, then with a final extension time of 5 min at 72℃. A total of 6μl PCR production was subjected to electrophoresis at 130V on 10% no-denaturing polyacrylamide gels for 3h and visualized by silver staining.

Analysis of genetic diversity: The parameters for SSR polymorphism evaluation, including observed number of alleles (Na), effective number of alleles (Ne), observed heterozygosity (Ho), expected heterozygosity (He) were calculated using POPGENE (version 1.31, Yeh et al., 1999). Cluster analyses were carried out on the matrix of GS using the Unweighted Pair Group Method using Arithmetic Averages (UPGMA) clustering algorithm. The similarity matrix and dendrogram were constructed with the program NTSYS-pc2.11 (Rohlf, 1995).

2 Results

From 2204 EST sequences, 48 SSR-containing uni-EST sequences were selected for primer designing. Among the 48 primer pairs, 21 gave no amplification or unclear bands, 7 gave monomorphic profiles, the remaining 20 primer pairs gave polymorphic profiles in the forty-one tree peony cultivars (Table 2). The observed number of alleles (Na) of the 20 SSR loci ranged from 2 to 10, with an average allele number of 4.3 per locus. The effective number of alleles (Ne) per locus ranged from 1.045 to 4.661, on average 2.466 alleles per locus. The observed heterozygosity (Ho) and expected heterozygosity (He) at each locus ranged from 0 to 0.857 and 0.044 to 0.795, respectively, on average 0.258 and 0.509 per locus (Table 3). So the 20 SSR loci showed a moderate polymorphic level (Figure 1).

After UPGMA analysis based on 20 SSR amplification data, the dendrogram with forty-one tree peony cultivars was constructed (Figure 2). Two clusters could be unambiguously formed at the genetic similarity coefficient of 0.64. One cluster included two cultivars “Luoyanghong” and “Shin Shichifukujin”, The other cluster included the remaining 39 tree peony cultivars. When the genetic similarity coefficient was at about 0.72, seven clusters could be formed. ClusterⅠincluded fifteen cultivars, except for two Japanese cultivars “Togawakan” and “Kanzakura Jishi”, the remaining thirteen were all native cultivars from Luoyang, China. ClusterⅡincluded fifteen cultivars, too, they were all from Japanese. In this cluster, “Taiyoh” and “Shima Nishiki” showed very close relativeness, their genetic similarity coefficient reached 0.93, which was in accord with the fact that “Shima Nishiki” was a sport from “Taiyoh”. Cluster Ⅲ included seven cultivars, they were all native cultivars from Luoyang, China. The remaining four clusters each included one cultivar. Cluster Ⅳ included “Okina Jishi”, a Japanese cultivar, clusterⅤ included “High Noon”, an American cultivar, cluster Ⅵ included “Shin Shichifukujin”, a Japanese cultivar and clusterⅦ included “Luoyanghong”, a native cultivar from Luoyang.

3 Discussion

In this study, twenty novel EST-SSR markers were developed from tree peony. The average number of alleles per locus was 4.3, which was equal to Hou’s report (Hou et al., 2011a), lower than 5.5 (Wang et al., 2009), but higher than 3.9, 2.6 and 3.5 (Homolka et al., 2010; Hou et al., 2011b; Yu 2013). The average value of observed heterozygosity was 0.258, which was lower than 0.41 (Wang et al., 2009), 0.37 (Homolka et al., 2010) and 0.43 (Hou et al., 2011a). The average value of expected heterozygosity was 0.509, which was lower than 0.67 (Wang et al., 2009), 0.60 (Hou et al., 2011a), but higher than 0.410 (Hou et al., 2011b). The twenty novel EST-SSR markers developed in this study were informative and suitable for diversity study of tree peony germplasm resources.Based on the twenty EST-SSR data obtained, forty-one tree peony cultivars were grouped into seven clusters. For most of the tree peony cultivars studied, the clustering groups based on EST-SSR markers were largely consistent with the geographic origin of these cultivars. For example, clusterⅠand cluster Ⅲ mainly consisted of native cultivars, clusterⅡconsisted of Japanese cultivars, cluster Ⅴ consisted of one American cultivar. Zhou et al. (2011) studied 89 tree peony cultivars by morphological traits, clustering analysis based on morphological traits showed that all the 89 tree peony cultivars could be grouped into two clusters: one cluster mainly consisted of foreign cultivars, the other consisted of native cultivars and foreign cultivars. Their studies indicated that most foreign cultivars showed great genetic differences with native cultivars. In the present study, except two Japanese cultivars “Togawakan” and “Kanzakura Jishi”, which grouped into clusterⅠ, showing a relatively close kinship with native cultivars, the remaining foreign cultivars showed great genetic differences with native cultivars. Zhou et al., (2012) studied the diversity of the same 89 tree peony cultivars by ISSR markers, UPGMA clustering grouped the 89 cultivars into two clusters, which was largely accorded with their clustering result based on morphological traits (Zhou et al., 2011). Some inconsistencies existed between our study and Zhou’s reports. For example, two Japanese cultivars, “Okina Jishi” and “Shin Shichifukujin”, formed two separate clusters (Ⅳ, Ⅵ) in our study, while grouped into one cluster with other Japanese cultivars in Zhou’s reports. This may be attributed to the difference in sample size. Sixty Japanese cultivars in Zhou’s studies was much more than nineteen Japanese cultivars in our study. The difference in molecular markers used (EST-SSRs in our study and ISSRs in Zhou’s study) may be another reason for the inconsistencies. Conclusively, our study and Zhou’s reports both supported that genetic distance between foreign tree peony cultivars and native tree peony cultivars was larger than genetic distance among native cultivars, indicating the use value of foreign cultivars as parents to hybridize with native cultivars to produce novel tree peony varieties. For example, the genetic distance between American cultivar “High Noon” and native cultivars was very far in our study. With the strong disease- resistance, vigorous growth, pure yellow flower and late-flowering feature, “High Noon” would be an ideal parent to hybridize with native cultivars for late-flowering, superior tree peony variety breeding.

Authors' contribution

Jia Xiaoping carried out the molecular genetic studies, participated in the sequence alignment and drafted the manuscript. Fan Bingyou carried out the immunoassays. Hou Dianyun participated in the sequence alignment. Shi Guo’an participated in the design of the study and performed the statistical analysis. Dai Lingfeng conceived of the study, and participated in its design and coordination. All authors read and approved the final manuscript.

Acknowledgements

I thank Zhang Shu-Ling for providing tree peony plant leaves for DNA isolation, this work was supported by The Youth Science Foundation of Henan University of Science and Technology (grant no. 2009QN001).       

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