The screening and identification of DNA barcode sequences for Rehmannia.

In this study, ITS, ITS2, matK, rbcL and psbA-trnH in Rehmannia were successfully amplified and sequenced, but some ITS sequences need to be proofread according to ITS2 sequences. Compared with rbcL, matK and psbA-trnH, ITS and ITS2 had higher mutation rate and more information sites, and ITS2 had higher interspecific diversity and lower intraspecific variation in Rehmannia, but the interspecific genetic variation of rbcL and matK was lower. Furthermore, the obvious barcoding gap was found in psbA-trnH or ITS2 + psbA-trnH, and the overlap between interspecific and intraspecific variation of ITS, ITS2 or matK was less. In addition, the phylogenetic tree based on ITS or ITS2 indicated that R. glutinosa, R. chingii or R. henryi with obvious monophyly could be successfully identified, but R. piasezkii and R. elata were clustered into one branch, R. solanifolia could not be distinguished from R. glutinosa, and R. chingii was closer to R. henryi. In phylogenetic tree based on psbA-trnH or ITS2 + psbA-trnH, cultivars and wild varieties of R. glutinosa could be distinguished, were clearly separated from other Rehmannia species, and cultivars or wild varieties of R. glutinosa could be also distinguished by matK. Taken together, ITS2 has great potential in systematic study and species identification of Rehmannia, the combination of ITS2 and psbA-trnH might be the most suitable DNA barcode for Rehmannia species.

[Molecular cloning and expression analysis of an Aux/IAA gene (RgIAA1) from Rehmannia glutinosa].

To clone and analyze a member of the Auxin/indole-3-acetic acid (Aux/IAA) gene family, RgIAA1, from Rehmannia glutinosa. The transcriptional EST database of R. glutinosa was used to clone the new Aux/IAA gene by cDNA probe of AtIAA14. Bioinformatics was applied to analyze the sequence characteristics of RgIAA1 protein and construct phylogenetiC trees. Quantitative RT-PCR has been applied to detect the transcription level of RgIAA1 in seven tissues as well as in leaves under three stresses. The results showed that, the cDNA sequence of RgIAA1 contains 903 bp was obtained. The open reading frame (ORF) of RgIAA1 was 681 bp encoding 226 amino acids, which has typical structural domains and characteristic sequence of Aux/IAA family proteins. RgIAA1 showed the highest expression level in unfolded leaf, followed by the stem. And the expression of RglAA1 was quickly decreased with leaf growing up. The transcription level increased under continuous cropping conditions while it reduced both in salinity and waterlogging stresses. RgIAA1, an Aux/IAA gene from R. glutinosa has been obtained for the first time, which can lay the foundation for further studies about its molecular function in development and responses to stress.

[Genetic diversity of Rehmannia glutinosa germplasms].

OBJECTIVE: To provide molecular evidences for phylogenetic analysis by studying ITS sequences of Rehmannia glutinosa from different areas. METHOD: The DNAs were extracted from leaves of R. glutinosa by means of CTAB method. The products of PCR amplification were cloned . The data were analyzed by MEGA4.0 software. RESULT: The results showed that the size of the ITS of R. glutionsa tested was from 613 to 614 bp and the length variation was only 1 bp. The sequence of ITS1 was 224-225 bp, and G + C content varied from 60.4% to 63%. The sequence of ITS2 was 224-225 bp and G + C content varied from 57.1% to 65.3%. The sequence of 5. 8S rDNA was 164 bp, it's very conservative in these species. Phylogram tree based on ITS sequence data indicated that the kinship between Bejing No. 2 R. glutinosa and the others were far. There was obvious diversity within wild R. glutinosa varieties, while there was no different among cultivated R. glutinosa varieties. In cultivated R. glutinosa varieties, there was no diversity between R. glutinosa varieties from Henan and those from others provinces. In wild varieties, R. glutinosa from Shengnongshan and Qingtianhe of Henan province showed a closer systematic relationship with cultivated R. glutinosa from Shandong province, while there was no difference between wild R. glutinosa varieties and cultivated varieties from Henan and Shanxi provinces. CONCLUSION: The genetic relationship among R. glutinosa varieties was very close, there was no distinct systematic differentiation.

Genetic relationships among Rehmannia glutinosa cultivars and varieties.

Many cultivars of Rehmannia glutinosa are grown in China for medicinal uses, but detailed agronomic and morphological descriptions are available for only a few. Knowledge of genetic relationships among most of the cultivars is also scanty and poorly documented. Here, cultivars, varieties and some sexually produced seeds of R. GLUTINOSA were raised in the field and studied for morphological diversity including shape, color, edges of leaves, color of anther, cornal and root, as well as yield of the medicinal part of the roots. Random amplified polymorphism DNA (RAPD) and amplified fragment length polymorphism (AFLP) were used to determine genetic relationships and ribosome DNA internal transcribed spacer (ITS) sequences were used for analyzing sequence variations and phylogenetic history. The 118 and 1019 polymorphic markers produced by 10 RAPD and 8 AFLP primers discriminated cultivars and varieties satisfactorily. Sixty-eight accessions were clustered in three main groups at 0.69 similarity levels by unweighted pair-group method arithmetic average (UPGMA) cluster analysis using RAPD in combination with AFLP markers. The average polymorphism information content (PIC) and Shannon index were 0.438 and 2.19 in RAPD and 0.476 and 26.68 in AFLP primers, respectively. This indicates that AFLP markers would be more efficient than RAPD for screening large numbers of R. GLUTINOSA accessions. The analysis of ITS sequences indicated that ITS1 - 5.8S-ITS2 of R. GLUTINOSA was informative in its 611 - 614-bp-long sequence and had 106 variable sites. Phylogenetic trees generated based on ITS sequences as well as the dendrogram obtained from two molecular markers identified four accessions: BY3, BY5, BY6 and Wildness6, with great genetic divergence.

[Analysis of genetic diversity of wild Rehmannia glutinosa by using RAPD and ISSR markers].

OBJECTIVE: To analyze the genetic diversity of wild Rehmannia glutinosa and evaluate and compare random amplified polymorphic DNA (RAPD) and inter sample sequence repeat (ISSR) for analysis of R. glutinosa accessions. METHOD: Two molecular markers, RAPD and ISSR were used for analyzing 55 wild R. glutinosa accessions. RESULT: Average 16.00 and 19.08 bands were amplified by RAPD primers and ISSR primers respectively, and the percentage of polymorphic bands were 89.58% and 94.32% respectively; Fifty-five R. glutinosa accessions categorized into 7 clusters were identified by unweighted pair-group method, arithmetic average (UPGMA) method. CONCLUSION: A high level of genetic diversity of wild Rehmannia glutinosa was displayed at DNA level, and genetic diversity coefficient of R. glutinosa from different production areas was 0.63-0.98, and ISSR marker can detect higher genetic diversity of R. glutinosa germplasms than RAPD marker.

[RAPD analysis on genetic relationship among varieties of Rehmannia glutinosa].

OBJECTIVE: To define the genetic relationship and other genetic characters of different varieties of Rehmannia glutinosa. METHOD: RAPD reactions were conducted on total 54 samples from 16 varieties and 2 populations of R. glutinosa. The data were analyzed by Phylips, Popgene and Arlequin software. RESULT: The majority rule consensus tree defined the 54 samples to 3 groups. Population genetics analysis and AMOVA showed that the genetic diversity among varieties/populations much greater than that within varieties/populations, but within group II the genetic diversity among varieties was similar to that within varieties. CONCLUSION: Due to the long-time asexual propagation of R. glutinosa, some genetic differentiation has been accumulated and fixed within the species. It was shown that the genetic distance between wild population and cultivated varieties was greater than the genetic distance among cultivated varieties. The wild resource of the plant should be paid more attention and studies.

[Identification of categories of Radix Rehmannia by IR spectroscopy and compare software].

OBJECTIVE: To identify the categories of Radix Rehmannia between the wild and the planting, different stored time, (gained in 2000,2002) , trueborn place (Henan) and no-trueborn places. METHODS: FTIR combined compare software, and the data was dealt with the means of normalization in computer. RESULT: The wild and the planting have evident difference. They can be identified for the different stored-time completely. The identification percentage between trueborn place and no-truedborn places is more than 90%. CONCLUSION: The method is fast, accurate.

[Assessment of genetic diversity of Rehmannia glutinosa germplasm detected by RAPDs and ISSRs].

RAPD and ISSR markers were used to assess the germplasm genetic diversity among 10 individuals of Rehmannia glutinosa, including 8 cultivars and 2 virus-free lines micropropagated by tip tissue culture. 17 RAPD primers and 10 ISSR primers, with polymorphic and informative patterns, were selected from a total of 80 RAPD ones and 44 ISSR ones to determine these individuals' genetic diversity. The 17RAPD primers and 10 ISSR primers generated 177RAPDfragments and 110 fragments, respectively. The number of effective loci, the percentage of polymorphic loci, Shannon's Information index (I) and effective number of alleles (Ne) is in turn109, 61.58%, 0.3135, 1.3641 for RAPD makers, and 79, 71.82 %, 0.3577 and 1.4037 for ISSR markers; Jaccard's genetic similarity matrice and dendrograms for the 10 individuals were formed based on RAPD and ISSR-generated polymorphic bands. In dendrograms, they could be divided into two groups: one group containing six individuals such as Zupei 85.5, Datian 85.5, jinzhuangyuan, Jinbai, Zupei 9302 and Datian9302; the other composed of 4 ones such as Beijing No.1, Dahongpao, Dihuang 9104 and wild dihuang; the correlation coefficient of 0.649 between RAPD and ISSR markers GSs indicated that these two markers were significantly correlated. The results revealed that RAPD and ISSR markers were suitable for assessment of germplasm genetic diversity of Rehmannia glutinosa, and ISSR marker was superior to RAPD marker.

[Comparison and correlative analysis on characters of Rehmannia glutinosa Libosch. Varieties].

OBJECTIVE: To compare the economical characters, yield characters and content of catalpol on Rehmannia glutinosa Libosch. varieties. METHODS: To study characters by field randomized block test and analysis of variance. To analyse the content of catalpol by HPLC. RESULTS: The results by analysis of variance were that the F value in plant width was 15.4 (F0.01 = 5.54), the F value in length of leaves was 12.2, the F value in width of leaves was 13.35, the F value in yield of single plant was 55.7 and the F value in content of catalpol was 8.03. The results by correlative analysis were that the linear correlation coefficient of signal plant yield with length of leaves was 0.9639, with width of leaves was 0.9073, with amount of earthnuts was 0.7060 and with plant fresh weight was 0.9950. The linear correlation coefficient of content of catalpol with plant width was 0.9169, with length of leaves was 0.7046 and with width of leaves was 0.7159. CONCLUSION: There were significant differences in plant width, length of leaves width of leaves, and yield of single plant and content of catalpol of Rehmannia glutinosa Libnosch. varieties. There were significantly positive correlations in signal plant yield with plant fresh weight, length of leaves, width of leaves and amount of earthnuts. There were significantly positive correlations in content of catalpol with plant width, length of leaves and width of leaves.