Identification Fokienia Hodginsii Clonal Genetic Relationship By The Molecular Marker

In order to understand the resource characters of Fokienia hodginsii in different regions and promote the discovery and application of high-quality resources in Fokienia hodginsii, this study analyzed the genetic differences of Fokienia hodginsii clones in the major growing areas of Fujian Province using ISSR (Inter-simple sequence repeat)and RAPD (Random amplification polymorphic DNA) markers and investigated the phylogenetic relationship of Fokienia hodginsii clones. These results can provide a reference for further discovery and use of quality resources of Fokienia hodginsii. The main results are as follows:1. PCR conditions were optimized by Changing single factor and a PCR reaction system and procedure was established for ISSR and RAPD analysis in Fokienia hodginsii clones.As lacking information for the study of molecular biology in Fokienia hodginsii, we analyzed effect of the different components changes, which included formamide, Taq enzyme, primer and template concentration, and the number of cycles and annealing temperature, on the PCR reaction results. The PCR conditions were established for ISSR and RAPD analysis in Fokienia hodginsii, That is:1) The ISSR-PCR reaction was performed in a25μ1volume containing the followingcomponents:2x PCR Reaction Mix containing0.1mM of each dNTP (12.5u L),0.75unit of Taq DNA polymerase(Golden DNA Polymerase),0.4μM of a single primer,35ng of genomic DNA,2%formamide and add ddH2O to25μ L. PCR amplification was performed in the following procedure:after initial denaturation for3min at94°, then35cycles were performed with30s at94°,30s at48.5°,1min at72°, and a final extension of5min at72°.2) The RAPD-PCR reaction was performed in a25μ1volume containing the followingcomponents:2x PCR Reaction Mix containing0.1mM of each dNTP (12.5u L),0.75unit of TaqDNA polymerase(Golden DNA Polymerase),0.12μM of a single primer,35ng of genomic DNA and add ddH2O to25μ L. PCR amplification was performed in the following procedure:after initial denaturation for2min at94°, then35cycles were performed with45s at94°,45s at36°,1min at72°, and a final extension of5min at72°.2. Some ISSR and RAPD primers were screened as DNA fingerprint analysis of Fokienia hodginsii clones.Based on Fokienia hodginsii clones, Polymorphism of ISSR and RAPD markers was analyzed. A total of100ISSR primers and88RAPD primers were used in the survey of ISSR-PCR or RAPD-PCR among the clones.41of the primers (20ISSR primers and21RAPD primers) produced distinguishable, reproducible and polymorphic bands which can be applied in DNA fingerprint analysis of Fokienia hodginsii clones. In the twenty ISSR primers, most of the primers (17primers out of20) that produced distinct bands were comprised of dinucleotide repeats. There were only two primers with trinucleotide motif, one primers with penta-nucleotide motif also yielding discrete bands. Primers based on (AG/GA)n and (AC/CA)n repeats gave the most polymorphic bands.3. The establishment of ISSR and RAPD fingerprinting of115Fokienia hodginsii clones.Using the polymorphic ISSR and RAPD primers, the DNA fingerprinting of115Fokienia hodginsii clones were analyzed. The20ISSR primers produced115amplified bands in the clones, fragment length300-1500bp, of which91were polymorphic. On the average, one primer could generate5.7bands and the percentage of polymorphic band was79.1%. The21RAPD primers screened could produce131amplified bands including90polymorphic locus, fragment length300-2000bp. On the average, each primer amplified approximately6.2bands with polymorphic rate of about68.7%. Relatively, ISSR-PCR could generate more amplified polymorphic bands than RAPD-PCR for systematic classification study on Fokienia hodginsii.4. Preliminary drawing phylogenetic relationship diagrams of the115Fokienia hodginsii clones by the ISSR and RAPD data.An analysis software NTSYS-pc version2.10e was used for cluster analysis of the ISSR and RAPD data. The results showed that genetic similarity of the115 Fokienia hodginsii clones was0.2241-0.9483based on the ISSR data, the average genetic similarity was only0.5862, which suggested that there was greater genetic distances among some of Fokienia hodginsii clones. The genetic similarity was0.7978-0.9727based on the RAPD data, with an average of genetic similarity by0.8852. Relatively, the genetic similarity revealed by the ISSR data was lower than that of the RAPD data. This suggested that ISSR-PCR could generate more genetic polymorphisms, and more suitable for molecular identification and relationship study of Fokienia hodginsii clones.