Analysis Of Genetic Diversity And Organic Acids Of Prunus Mume In Fujian Province

Fujian province is an important productive area of Prunus mume in China. Wild germplasm resources of Prunus mume sieb.et zucc have, however, became less and less. As one of traditional Chinese medicinal materials, Prunus mume is mainly depended on artificial cultivation. Yet, classification, identification and medical quality evaluation of Prunus mume cultivars/varieties have not been examined systematically. The aim of this study is to investigate the genetic diversity of 18 Prunus mume cultivars collected from Fujian province by analyzing nuclear 18S-26S rDNA internal transcribed spacer (ITS) region sequences, and to characterize their medical value by detecting the contents of organic acids with reverse-phase HPLC, so that to develop a scientific basis for evaluating germplasm resources and medicial quality of Prunus mume collections and for its breeding in practice.The length of the ITS1, 5.8S and ITS2 regions of nuclear ribosomal DNA from 18 Prunus mume cultivars collected ranged from 628 to 634 bases, with an ITS1 of 223-224 bp, a 5.8S of 164bp, and an ITS2 of 241-246 bp. The content of G+C of ITS1 region is from 57.9 % to 62.7%, ITS2 region is from 64.4 % to 66.4%. All the sequences tested were uploaded into Genbank, and the accession numbers are EF523482-EF523493, EF529435, EF529436 and EU334746.There are 83 variable sites in ITS region, 41 variable sites in ITS1,10 in 5.8S rDNA and 32 in ITS2 by using DNAMAN software. The samples have individually specific single nucleotide variation sites and sequence homology among Prunus mume samples collected from different geographical regions is more than 94.7%. The Kimura two-parameter distances ranged from 0.000-0.058 among the samples. Based on ITS1, ITS2 and 5.8S sequences, a phylogenetic tree was reconstructed using software MEGA3.1. It is indicated that the samples from the same cultivation area were not always clustered to a same clade, and the samples from different regions might be grouped in a branch. The results have led to a suggestion that ITS sequences would be used as suitable markers for identification of Prunus mume and the molecular technique is feasible for its breeding in practice as well.A method for simultaneous and rapid determination within Prunus mume with reverse-phase high performance liquid chromatography (HPLC) was also developed. HPLC was performed on a Hypersil ODS C₁₈ column (250mm×4.6mm i.d., 5μm) at 21℃, using KH₂PO₄ 0.01mol·L^-1 (pH2.8) as mobile phase at the flow rate of 0.8mL·min^-1 and the detection wavelength of 215nm. The recoveries of the organic acids ranged from 75.4% to 121.0% with relative standard deviations of 0.78%-2.12% and the limits of detection were 0.022-1.290μg·mL^-1. Using the developed reverse-phase HPLC, which is powerful, convenient and sensitive tool, 7 organic acids, including oxalic, tartaric, malic, lactic, acetic, citric and succinic acid within 4 Prunus mume were detected.