Studies On Identification And Classification Of Ziziphus Jujuba Germsplasm Resources Using AFLP Molecular Markers

As a new DNA marker, AFLP (amplified fragment length polymorphism) has been widely used in cultivars identification, genetic diversity, and genetic linkage mapping in fruit trees. As one of the important fruit trees, Ziziphus jujuba originated from China. It has very long culture practice history, a large number of cultivars, and very wide distribution. The classification evolved from morphological diversity to genetic diversity. The studies on Ziziphus jujuba classification involved the fields of morphology, palynology, biochemistry and molecular biochemistry. Because of the different standard adopted and each kind of classification's limitation, both synonym and homonym are presented very seriously. In addition, the genetic relationships among cultivars were not very clear. AFLP silver-staining technique system suitable for Ziziphus jujuba was established in this study. The fingerprints of 82 materials were constructed, and the genetic diversity was analysed. The aim was to set up a classification system on Ziziphus jujuba and supply theoretical basis for conservation and utilization as well as technological foundation for cultivars registration, identification and protection. The main results were briefly summarized as follows:1. The genomic DNA of Ziziphus jujuba was extracted by a modified CTAB method. The modified extraction buffer contained 100 mmol/L Tris-HCl (pH 8.0) ; 20 mmol/L EDTA, 1.4mol/L NaCl, 2% CTAB, 2% PVP₄0, and 50 mmol/L P -Mercaptoethanal (added before isolation). RNA was removed from the solution with RNase, and DNA was purified by phenol / chloroform, chloroform / isoamyl alcohol. The purified genomic DNA was suitable for AFLP analysis.2. AFLP silver-staining analysis system suitable for Ziziphus jujuba genomic DNA was established. 450ng DNA was digested with 3 units of EcoR I and 3 units of Mse I enzymes in a reaction volume of 20 U L and incubated at 37°C for 3h.. Double-stranded adaptors were added to the restriction fragments at 37°C for more than 10 h(or overnight). The linked products was diluted 10 times with TE buffer and used as templates for pre-amplification. The pre-amplification was carried out using an EcoRI and a Msel site primer, both without selective bases. The pre-amplification products were diluted 10 times...