Molecular Markers Of Sexuality For Trichosanthes Kirilowii Maxim

1. In order to isolar high quality DNA from Trichosanthes kirilowii Maxim leaves enriching polysaccharides and polyphenols, the effects of DNA extraction were studied with four different methods including CTAB regular method, CTAB subarea method, SDS regular method, Low pH extraction medium with high salts.Moreover, the CTAB subarea method was used for the first time for genomic DNA isolation from Trichosanthes kirilowii Maxim. The results showed that the CTAB subarea method was optimal, the value of OD₂₆₀/OD₂₈₀ was over 1.8 and the extracted DNA by this method was appeared as a clear band when electrophoresed in 1.5% agrarose gel, and produced clear polymorphic patterns amplified by PCR.2. L₁₆(4⁵) orthogonal experiment was made to investigate the amplification efficiency of different amplification conditions, optimum combination of amplification conditions(genomic DNA, dNTP density, primer density, Mg²⁺ density, Taq polymerase density)were identified rapidly and effectively. The result shows that the optimal concentration was 1 ng/μL, dNTP 0.2 mM, primer 0.04 μM, Mg²⁺2.0 mM, Taq polymerase 1.0 U and the most suitable program was one cycle at 94℃ pre-denaturation for 2 min, 36cycles of 94 ℃ for 30s, 37 ℃ for 40s, 72 ℃ for 1.5 min, and 10 min 72 ℃ extension.3. In this study, the random amplified polymorphic DNA technique was employed with the objective of finding the specific sex-associated DNA markers for the further research on early sexual identification of Trichosanthes kirilowii Maxim. The reaction systems and cycling parameters of RAPD for Trichosanthes kirilowii Maxim were confirmed after the reduplicate experiments of genomic DNA abstraction and RAPD analysis. A total of 80 10-mer primers was tested. A male-associated fragment with a length of about 0.6kb was generated with AN 18 primer. The fragment was cloned and sequenced. In order to convert the RAPD marker into SCAR marker, the length of primers about 20-mer were constructed and used for PCR amplifying. TKM 647 was false positive marker and yielded band in the female. This research was an attempt to identify the six in early stage of the Trichosanthes kirilowii Maxim on molecular level in China.4. Because of its hypervariability, neutrality, co-dominance and ubiquity in eukaryoticgenomes, microsatellite DNA is becoming preferred molecular marker for population genetic studies, as well as for strain identification, kinship analysis, and mapping purpose. In this test, some microsatellite sequence for Trichosanthes kirilowii Maxim were cloned. The genome DNA was first restricted into fragments of varied length with Hinalll, BamU I and ligated with synthetic adapters, then enriched microsatellite sequence with magnetic beads which was linked with biotinylated (GA)10 probes. About 71 clones ware selected and screened, and 49 clones were positive. From those positive clones, 12 clones were sequenced, and 1 microsatellite sequence were gotten. This result showed that the magnetic beads enriched method could be a high efficiency and low cost method and can be employed as a reliable option for any molecular laboratory to develop SSR markers.