Studies On Molecular Makers For Sexual Identification Of Female And Male Plants In Ginkgo Biloba L.

Ginkgo (Ginkgo biloba L.), a plant of dioecism, has important economic, ecological, aesthetic and research values. Its seedling trees show a series of characteristics such as a longer junior period, sexual differentiation relatively slow, flowering and seed late and so on. The different characters of growth and development between female and male plant of Ginkgo make their resources utilization significantly different. It's difficult to accurately identify the sex type of seeding plants in Ginkgo before its flowering based on morphological features,In order to meet the need of early sexual identification on the seedling plants of Ginkgo for its optimization of resources allocation, in this study, the male and female plants from the germplasm resources garden of Ginkgo of Yangzhou University were used as experimental materials to determine their sex type by ISSR, SRAP and SCAR markers respectively. The main results are as follows:1. The DNA of high quality from the young leaves of Ginkgo, was successfully extracted by method of modified CTAB, the band of which is bright without any smearing or diffusion through agarose gel electrophoresis, its value of A260/A280 detected by UV is within 1.8-2.0.2. 25 ISSR primers were used to be amplified in male and female DNA pools of Ginkgo respectively, out of which only 21 primers could obtain stable amplification products with clear background , each primer could amplify 6-12 bands, and a male-specific band about 200bp long was got by primer AW988.3. The four factors (Taq enzyme,Mg2+,dNTP, primer) which could impact the SRAP-PCR were optimized by a method of single-factor gradient, and the optimal SRAP-PCR system obtained was as follows: template DNA 30-60ng, 0.75 U Taq enzyme, 0.18 mmol. L-1 dNTP, 1.8 mmol. L-1 Mg2+, 1.0 umol ? L-1 primers, the rest was filled with ddH2O.4. The optimum annealing temperatures of 51°C -55°C were definite after selecting and optimization of the primer combinations for SRAP with the DNA pools from male and female plants of Ginkgo as a template in the stable reaction system obtained.5. Out of 66 pairs of SRAP primer combinations, 46 pairs can get stable and efficient amplification products on Ginkgo genomic DNA, each produces 4-13 polymorphic bands about 100bp-2000bp in size. A female-specific band with about 600bp was generated by primer combination me5-em2 and another female-specific band 250bp length was amplified by primer combination me6-em3.6. The male-specific ISSR marker and female-specific SRAP markers received in Ginkgo were cloned and sequenced, the SCAR primers were screened by the sequence information of specific bands.7. The specific ISSR marker was transformed into SCAR marker, the gender of Ginkgo male and female trees each 20 whose sex type already known were detected using the SCAR marker, and the accuracy rate of identification is 85%.8. The method of specific SRAP marker transform into SCAR marker needs further study.The above results have great theoretical significance and application value for the early selection, optimal allocation and rational development of germplasm resource in male and female plants of Ginkgo.