The Complete Plastid Genome of Magnolia zenii and Genetic Comparison to Magnoliaceae species.

Magnolia zenii is a critically endangered species known from only 18 trees that survive on Baohua Mountain in Jiangsu province, China. Little information is available regarding its molecular biology, with no genomic study performed on M. zenii until now. We determined the complete plastid genome of M. zenii and identified microsatellites. Whole sequence alignment and phylogenetic analysis using BI and ML methods were also conducted. The plastome of M. zenii was 160,048 bp long with 39.2% GC content and included a pair of inverted repeats (IRs) of 26,596 bp that separated a large single-copy (LSC) region of 88,098 bp and a small single-copy (SSC) region of 18,757 bp. One hundred thirty genes were identified, of which 79 were protein-coding genes, 37 were transfer RNAs, and eight were ribosomal RNAs. Thirty seven simple sequence repeats (SSRs) were also identified. Comparative analyses of genome structure and sequence data of closely-related species revealed five mutation hotspots, useful for future phylogenetic research. Magnolia zenii was placed as sister to M. biondii with strong support in all analyses. Overall, this study providing M. zenii genomic resources will be beneficial for the evolutionary study and phylogenetic reconstruction of Magnoliaceae.

Reduced incompatibility in the production of second generation hybrids between two Magnolia species revealed by Bayesian gene dispersal modeling.

PREMISE OF THE STUDY: Hybrid zones are areas where gene flow between related species is currently occurring, so information on the compatibility between related species and their hybrids is essential for predicting the dynamics of such zones generated by introgressive hybridization. In this study, we quantified the compatibility among Magnolia stellata, M. salicifolia, and their hybrids in a hybrid zone using gene dispersal modeling. METHODS: After determining the genealogical classes of adult trees in the hybrid zone, the paternity of 574 open-pollinated seeds from 37 known maternal trees was analyzed with microsatellite markers. A neighborhood-based Bayesian gene dispersal model developed by us for estimating compatibility was then applied to the paternity data. KEY RESULTS: When M. stellata or M. salicifolia were mothers, interspecific mating to produce F1 hybrids yielded significant incompatibility, but backcrossing with F1 hybrids did not. Furthermore, when F1 hybrids became mothers, no significant incompatibility resulted from backcrossing to parental species or intra-F1 mating to produce F2 hybrids. The estimated proportion of F1 hybrids in the outcrossed seeds (1.7%) in the hybrid zone was much lower than that in the adult trees (14.0%). CONCLUSIONS: While it is difficult to obtain F1 hybrids, their low incompatibility makes it easy to produce advanced generation hybrids, once they have been successfully obtained. Although the production of F1 seeds is rare, heterosis and/or weak selection pressure in an empty niche between the parental species' niches may have contributed to the increased proportion of adult F1 hybrids in the hybrid zone.

[Screening target genes for bimolecular marking methods of Magnolia quality].

The study used use bimolecular marking methods to evaluate the lignans of Magnolia officinalis and M. officinalis var. biloba. First, we compare the chemical constituents between M. officinalis and M. officinalis var.biloba. There were significant differences in concentration of magnolignan I between leaves of these two varieties. Then we further select the p-hydroxyphenyl lignin to mining the key enzyme genes of biosynthesis from Magnolia transcriptome, and screened an encoding cinnamyl alcohol dehydrogease gene as the candidate marker of bimolecular marking methods of Magnolia quality by comparing of the expression level and structure variation in homologous gene between M. officinalis and M. officinalis var.biloba. The established method provides the technical support for bimolecular marking methods of Magnolia quality evaluation.

[Identification and bioinformatics analysis of genes associated with MVA pathway in Magnolia officinalis].

Methyl valerate (MVA) pathway is one of the important ways for synthesis of terpenoids. This study was based on data of the transcriptome sequencing of Magnolia officinalis, the associated genes MoACOT, MoHMGS, MoHMGR, MoMK in methyl valerate (MVA) pathway, were completed in detail by using bioinformatics methods. The results of analysis showed that MoACOT and MoMK were stable hydrophobic proteins, MoHMGS and MoHMGR were unstable hydrophobic protein. The secondary structures of all proteins were hybrid architecture,and alpha helical were the major motifs. There were no clear transmembrane domains in MoACOT, MoHMGS and MoMK, but two transmembrane domains were founded in MoHMGR which were from 39-61 aa and 82-104 aa resepectively. The results of evolutionary relationship analysis showed that MoACOT, MoHMGS, MoHMGR and MoMK had relative close relationship to angiosperm or dicotyledonous plants, and accorded with genetic evolution rule. From transcriptome data, transcripted level of MoACOT, MoHMGS, MoHMGR, MoMK in M. officinalis and M. officinalis var. biloba was not significantly different. The result provided theoretical reference for study on Methyl valerate (MVA) pathway of terpenoid of M. officinalis.

[High-throughput pyrosequencing of the complete chloroplast genome of Magnolia officinalis and its application in species identification].

Chloroplast genome sequences have comprehensive application prospects in DNA barcoding and chloroplast engineering in traditional Chinese medicine. The complete chloroplast genome of Magnolia officinalis sequenced by high-throughput pyrosequencing and a sequencing procedure was established. Fourteen contigs were obtained after de nove assembly. The sequencing percent of coverage was 99.99%. The chloroplast genome is 160 183 bp in size, and has a typical quadripartite structure with the large (LSC, 88 210 bp) and small copy (SSC, 18 843 bp) regions separated by two copies of an inverted repeat (IRs, 26 565 bp each). chloroplast genes were successfully annotated, of which 17 genes located in each IR region. The chloroplast genome features in Magnolia officinalis are nearly identical to those from other Magnoliid chloroplast genomes. Phylogenetic analyses were performed based on 81 shared coding-genes for a total of 9 Magnolia samples of 5 closely related species. Results showed that distinguishing among species was generally straightforward at the species and population level. This study confirmed the effectiveness of our chloroplast genome sequencing procedure. The chloroplast genome can provide distinguishing differences to help identify Magnolia officinalis and its closely related plants.

[Population genetic study of Magnolia officinalis and Magnolia officinalis var. biloba].

OBJECTIVE: population genetic study of Magnolia officinalis and M. officinalis var. biloba was performed in this paper. The objectives of this study are to provide baseline data of molecular pharmaceutics for quality control of Cortex Magnoliae Officinalis. METHOD: A total of 15 populations of M. officinalis and M. officinalis var. biloba were collected. PCR amplification and sequencing were performed with two chloroplast intergenic spacers psbA-trnH and trnL-trnF. Chloroplast haplotype frequencies were calculated, genetic diversity and genetic structure were estimated by using the program HAPLONST, and a haplotype network depicting the mutational relationships among distinct haplotypes was drawn following the principle of parsimony by TCS version 1.13. RESULT: The differentiation of haplotype frequencies between M. officinalis and M. officiunalis var. biloba was significant although there was no private haplotypes of themselves. CONCLUSION: There was certain genetic divergence between M. officinalis and M. offcinalis var. biloba although the monophyletic clade of themselves was not still formed.

[Construction on ISSR fingerprint of Magnolia officinalis from Enshi in Hubei province].

OBJECTIVE: To establish the ISSR fingerprint of the cortex of Magnolia officinalis produced from Enshi. METHODS: Cultivar and habit identification was based on Inter-simple sequence repeats (ISSR). RESULTS: Two ISSR primes could be used to construct the ISSR fingerprint of M. officinalis from Enshi. CONCLUSION: The ISSR marker is an effective method for the cultivar and habit identification of the cortex of M. officinralis.

Population structure and genetic diversity distribution in wild and cultivated populations of the traditional Chinese medicinal plant Magnolia officinalis subsp. biloba (Magnoliaceae).

Magnolia officinalis subsp. biloba, a traditional Chinese medicinal plant, experienced severe declines in the number of populations and the number of individuals in the late 20th century due to the widespread harvest of the subspecies. A large-scale cultivation program was initiated and cultivated populations rapidly recovered the loss in individual plant numbers, but wild populations remained small as a consequence of cutting. In this study, the levels of genetic variation and genetic structure of seven wild populations and five domestic populations of M. officinalis subsp. biloba were estimated employing an AFLP methodology. The plant exhibited a relatively high level of intra-population genetic diversity (h = 0.208 and H(j) = 0.268). The cultivated populations maintained approximately 95% of the variation exhibited in wild populations, indicating a slight genetic bottleneck in the cultivated populations. The analysis of genetic differentiation revealed that most of the AFLP diversity resided within populations both for the wild group (78.22%) and the cultivated group (85.92%). Genetic differentiation among populations in the wild group was significant (F(ST) = 0.1092, P < 0.005), suggesting wild population level genetic structure. Principal coordinates analysis (PCO) did not discern among wild and cultivated populations, indicating that alleles from the wild population were maintained in the cultivated gene pool. Results from the present study provide important baseline data for effectively conserving the genetic resources of this medicinal subspecies.

[Studies on random amplified polymorphic DNA fingerprinting of Cortex Magnoliae Officinalis].

OBJECTIVE: To identify Chinese traditional medicine (CTM) "Hou-pu" (Cortex Magnoliae Officinalis), its counterfeits and substitues. METHODS: Total genomic DNA samples of ten plant species were amplified by randomly amplified polymorphic DNA (RAPD). RESULTS: Ten samples were able to be distinguished through their amplified DNA banding patterns on the agarose gels after electrophoresis. CONCLUSION: RAPD is able to identify "Hou-pu", its counterfeits and substitutes quickly and truly, which is also quite valuable for correctly introducing plant.

[Quality evaluation and utilization of germplasm resources of Magnolia officinalis].

OBJECTIVES: To lay a theoretical foundation for studies on strategies for improvement of Magnolia officinalis and select superior gemplasm resources to meet the demand for modernization, industrialization and internationalization of Chinese medicine. METHODS: Seeds of Magnolia officinalis from 13 main habitats of 7 provinces were collected and strewn in a place of Jingning County, Zhejiang. At the age of seven, 195 samples were collected from the same height of the trunk of 15 individual trees of each provenance, and assayed for effective ingredients with HPLC. RESULTS: Differences in the content of phenols were significant among the seed sources and even greater among individuals within a seed source. CONCLUSIONS: 3 seed sources viz. Wufeng, Hefeng and Enshi of Hubei were obviously superior to other seed sources on account of high contents of magnolol, honokoiol and total phenols. Extension and application of these 3 seed sources is an effective path leading to quality improvement of Magnolia officinalis. With great differences in the content of phenols existing among individuals within each source, there is a big gap between production of medicinal materials by merely using superior seed sources of Magnolia officinalis and the demand of stable and controllable quality for modernization and internationalization of Chinese medicine. But the great difference has laid a material foundation and brought about a great potential for genetic improvement of Magnolia officinalis. Therefore, the superior individuals within a superior seed source are an excellent material for the breeding of Magnolia officinalis.

[Research on DNA molecular marker of Magnolia officinalis Rehd. et Wils.--RAPD study on certified species].

AIM: To discuss the intraspecific relationship in Magnolia officinalis and the genuineness of Cortex Magnoliae officinalis, and to find some DNA characters of certified "Houpo". METHODS: Thirty-three samples from eleven locations, which can represent most of the distribution of M. officinalis, were selected. The total DNA was extracted. Severty-four random primers were tried to get good amplification. RESULTS: One hundred and sixteen bands amplified from seventeen primers, were clustered by NTSYS-pc software. Three branches were obtained. Some distinctive primers and bands, which represent certified species or fine breed, were obtained also. CONCLUSION: 1) M. officinalis should be divided into three geographic clans instead of two subspecies or varieties, they are, a) typical officinalis, b) typical biloba and c) Middle type. This conclusion agrees with the leaf form and other characters. 2) The genetic difference between "Chuanpo" and "Wenpo" is evident and the difference is in correspondence with the quantities of their chemical constituents. So, the genetic difference is the main reason of the genuineness of Cortex Magnoliae officinalis. 3) These results may be used to establish DNA database for identification of Cortex Magnoliae officinalis.