Species-specific Identification Of Larix Gmeline, L.olgensis And L.principis-rupprechtii By RAPD And ISSR Marker

The needles and seed embryos of the main local Larch species (Larix gmeline, Larix olgensis and Larix principis-rupprechtii) from the northern area of China are taken as the materials, RAPD and ISSR and other molecular techniques are used to identify three larch species in this study. Three species-specific bands which are amplified by RAPD reactions are reclaimed and sequenced, and then transformed into SCAR primers for species-specific identification of these larch species as a new molecular marker system.The genomic DNA is extracted from the needles and seed embryos of the Larch by improved CTAB method after the extraction methods are confirmed. At the same time, the optimal RAPD-PCR and ISSR-PCR reaction system and procedure are determined by gradient comparison of dNTP, Mg²⁺ and primer in concentration as control factors, that RAPD reaction system in 20uL includes 1.5uL of template DNA(30ng), 1.5uL of dNTP(2mM), 2.0uL of Taq polymerase, 10×buffer(500mM KC1, 100mM Tris-HCl), 1.6ul of Mg²⁺(25mM), 2uL of primer DNA(4uM), and PCR procedure consists four steps running in 40 cycles: pre-denaturation in 3min at 93℃, denaturation inlmin at 93℃, annealing in 1min at 37℃, extension in 2min at 72℃, following by final extension in 10min at 72℃. ISSR reaction in 20ul contains 1.5uL of template DNA(30ng), 2.0uL of dNTP(2.0mM), 2.0uL of Taq polymerase, 10×buffer(500mM KC1, 100mM Tris-HCl), 1.6uL of Mg²⁺(25mM), 2uL of primer DNA(4uM), PCR procedure also consists four steps in 35 cycles: pre-denaturation in 3min at 93℃, denaturation in 30s at 93℃, annealing in 45s at 55/57/60/63℃ (changing with the primers at Tm+1 as usual), extension in 90s at 72℃, following by final extension in 7min at 72℃.The primers are selected from 47 RAPD and 20 ISSR primers according to the needle and seed embryo samples. 3 species-specific RAPD primers are obtained to identify Larch seedlings, which are OPB-11, OPN-07 and OPX-14. And 3 species-specific RAPD primers and 2 species-specific ISSR primers are obtained to identify Larch seeds which are OPB-11, OPN-17, OPX-14, ISSR-44 and ISSR-46.Three species-specific bands from the seed embryo samples amplified by RAPD reactions are reclaimed, cloned and sequenced. 3 pairs of species-specific SCAR primers are designed based on the DNA sequences, which are SCB₁/SCB₂, SCN₁/SCN₂, SCX₁/SCX₂. All samples of three Larch species are re-tested by these SCAR primers and good result is obtained, even though the percentage of the band's appearance is not in 100% that probably causes by the mutations in primer site among individuals of a species. Therefore, the detected samples must be in enough numbers, randomization and species-specific. This study provides an effective and reliable method for identification of different larch species.