AFLP-based Sex Identification In Torreya Grandis Fort.

AFLP (amplified fragment length polymorphism) has been applied to the differentiation of the male and female in Torreya grandis Fort. The following have been obtained:1. It is the first time to successfully isolate high quality DNA from leaves and endosperm of Torreya grandis Fort, by a CTAB-silicon-based method. The DNA extracted had an OD260/OD280 value between 1.8-2.0 and was bright and clear when electrophoresized in agarose gel.2. It is also the first time to establish a silver staining-based AFLP system suitable for AFLP analysis with either leaves or endosperm of Torreya grandis Fort. This result would lay a foundation for further studies of molecular biology and genetic map construction inTorreya grandis Fort. The AFLP system is as follows:Double enzyme digestion of extracted DNA and adaptor ligation of digested DNA fragments were realized in one step, which was as follows: a total volume of 25μl composed of DNA, Mse I, EcoR I, T4 ligase, M-adaptor, E-adaptor, 100×BSA, 10×NEBuffer 2 and T4 ligation buffer. Complete digestion and ligation were achieved overnight (about 12 hr.) at 37℃.Preamplification was conducted in a volume of 20ul, in which there were DNA, dNTP, Mg +, Taq polymerase, Mse I preamplification primer, EcoR I preamplification primer and buffer. PCR was conducted at 25 cycles, each of which consisted of denaturing at 94℃for 30s, annealing at 56℃for 60s and extension at 72℃for 60s, and extension at 72℃for 5 min.An optimized reaction system for selective amplification was as such: a total volume of 20μl that was composed of DNA, dNTP, Mg2+, Taq polymerase, Mse I amplification primer, EcoR I amplification primer and buffer. PCR was done following the program of 13 cycles of denaturing at 95℃for 30s, annealing at 65 for 40s, and extension at 72℃for 60s, each of which had an annealing temperature 0.7℃lower than that in the previous cycle, 25 cycles of denaturing at 95℃for 30s, annealing at 56℃for 40s and extension at 72℃for 60s, and extension at 72℃for 5min.The selectively amplified products were electrophoresized in denatured PAGE gel and silver stained.3. 20 pairs of primers have been screened from 64 pairs of primers, out of which 20 pairs were suitable for endosperm, 15 pairs suitable for leaves, and 7 pairs suitable for both.4. A band specific for the female of Torreya grandis Fort using the primer pair of E-AGC/M-CAT had been got.5. This female-specific band can only be used to differentiate the male and female of Torreya grandis Fort. It can't be used in identification between the female and monoecia as well as among the female of Torreya grandis Fort, ex Lindl. cv. Merrillii Hu and the other types of Torray grandis Fort. This result can be used in production to identify the male and female seedlings of Torreya grandis Fort.