| Rehmannia is one of the most valuable Chinese herbal medicines in China. With the modernizationand internationalization of Chinese medicine, studies on rehmannia at home and abroad has becomeincreasingly prevalent and far-reaching,the demand for rehmannia has been increased. The requirementsfor rehmannia yield and quality becomes higher and stricter. However, the phenomena that variousdisordered varieties and random names appear in the production of rehmannia are not beneficial to thescientific selection of new high yield and good quality rehmannia varieties. In this study, the geneticdiversity of rehmannia varieties grown in the germplasm garden of the Agriculture Research Institute ofWen county was analysed by RAPD markers,and specific RAPD markers were transformed intoRAPD-SCARs to establish a quick and simple method for Rehmannia varieties identification andMarker-assisted selection (MAS). The main results are as follows:1. The single factor method is adopted in the experiment to optimize RAPD-PCR elements such as theamount of DNA template, Mg2+concentration, dNTPs concentration, primer concentration, and TaqDNApolymerase dosage.As a result, one optimal reaction system of25μl total volume, containing10X PCRbuffer2.5μl,dNTP (10mM)0.5μl,Taq enzyme (2U/μl)1μl,primer(10μmol/L)2μl,DNA(20ng/μl)2μl and double distilled water17μl, was established. PCR amplification procedure:94°C for5min;94°Cfor1min,36°C for1min,72℃for2min, altogether40cycles;72°C for10min and stored at4℃.2.6RAPD primers with good polymorphism and reproduciability were screened from100ones usingPCR with three DNA templates (Mixian County's wild rehmannia, xiao hei ying, Beijing1#). the speciesare studied on their hereditary diversity and classification. A total of36DNA bands were amplified from21Huaiqing rehmannia varieties genomic DNAs using PCR with the6RAPD primers, of which27bandsare polymorphic. Statistic analyses revealed that polymorphic loci rate is75%, the observed allelic genenumber1.7500, valid allelic gene number1.3074, Nei's gene diversity degree0.1907, Shannon' diversityrate0.3021,showing higher genetic diversity of rehmannia varieties. Clustering dendrogram withSoftware MEGA4divided21rehmannia varieties into five classes: Class A:0821,03-2, Xiaohiying,Jinzhuangyuan,08-13, Jinsanhuang, Shanxi Beixiang,85-5, Beijing1#, Beijing3#,9302, Hongshuwang, 08-6; Class B: Jiyuan' and Xinmi' wild rehmannia; Class C:08-08and Fangzhuang of Xiuwu; Class D:Guolimao,03-18,0802; class E: Shengjin1#.3. A specific band was amplified by RAPD primer s135from Jiyuan wild rehmannia genomic DNA,which can not be done from that of other21rehmannia varieties. The band is eluted from agarose gel.Nucleotide sequencing indicated that it is composed of three different fragments:360bp,433bp and355bpin length,named as F1,F2and F3,respectively. Three pairs of specific primers such as ZX1F/ZX1R,ZX3F/ZX3R, ZX5F/ZX5R were designed according to the three RAPD fragment sequences,andsuccessfully used in their transformation into SCAR markers such as SCAR1(340bp), SCAR2(413bp) andSCAR3(315bp). They can accurately distinguish Jiyuan wild rehmannia from other21rehmannia varieties.4Their nucleotide sequence analyses for sequence uniqueness by comparing with the known DNAsequences available at NCBI database, using BLAST showed that they are rich in AT: F1's AT content is75.2%, F2's AT content is65.8%, and F3's AT content is65.6%. Besides, the15-143area of F1is an OpenReading Frame (ORF) encoding a protein consisting of42amino acid residues, whose en-codable proteinand the guanine nucleotide exchange factor VAV3(AAL06249.1) have a high similarity of76%;there is also an ORF in area1-153of F2, encoding a protein containing51amino acid residues,whosesimilarity with a known protein T-cell receptor beta(AAB488882.1)in NCBI is82%; The27-108area ofsequence V also is an ORF, encoding a protein of26amino acid residues, whose similarity with a knownprotein Putative aminopeptidase(zp09127781.1)is88%. |
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