| Ilex kudingcha C. J. Tseng is a species of dioecious, perennial arbor. Its adult leaves and young buds can be used both for beverage and for medicine. It has lots of health protection functions and medicinal effect. Moreover, it is also a good species for ornamental. The excellent female plants can be used for enjoying fruits, excellent male plants can be used to enjoy flowers. It is a very time-consuming task to determine the sexuality of the breeding materials in the cross breeding work of Ilex kudingcha C. J. Tseng because its physical period for sexuality-mature is very long (for5-6years after setting-up), a young plant, or an adult plants in the season without flower and fruit, has not any morphological characteristic indicating its sexuality.In this study, a dual molecular marker technique (RAPD-SCAR) was used to identify the sexuality of I, kudingcha in the early stages. That is, at first RAPD molecular marker technique was used to obtain the specific RAPD marker (specific RAPD fragment) closely linked the sexuality. Then this specific fragment was retrieved, cloned, and sequenced. In order to improve the annealing temperature, enhance preciseness and repeatability of the reaction, a pair of longer SCAR primer was designed based on the sequencing results, which be extended for10-17bp at both ends of the original RAPD primer. And then, the newly-designed specific SCAR primer was used for PCR amplification to convert the specific RAPD marker into specific SCAR marker which only detects the single band. With simple operation and low testing cost, the SCAR marker technology was suitable for the identification of the sexuality of large quantity of samples of I, kudingcha in the early stage, so it could be applied in the production in future. The experimental results of these were as follows:1. The orthogonal design was used to optimize RAPD-PCR amplification system of I. kudingcha in5factors (DNA template, Mg2+, primer, dNTPs, Taq polymerase,) at4levels, respectively. Through comprehensive analysis, the optimized RAPD-PCR reaction system was established as follows:10×buffer2.5μL,20ng DNA template,2.5mmol/L Mg2+,0.3μmol/L primers,2.0U Taq polymerase,200μmol/L dNTPs were contained in25μL reaction system, and the optimized RAPD-PCR amplification program was that predenaturing at94℃for5min, then denaturing at94℃for30s, elongation at36℃for30s and extension at72℃for120s, for40cycles, final extension at72℃for10min. The productions were stored at4℃.2. Using the optimized reaction system and RAPD-PCR amplification program, a total number of91random primers were screened in the RAPD-PCR. Polymorphic fragments are detected with24primer in female and male DNA samples pools.2male-associated fragments were respectively generated with S164primer and S191primer, for the time being they were named for S164-900and S191-800.3. The S164-900and S191-800fragments were recovered from electrophoresis gel and ligated into the pMD(?)18-T vector system for being sequenced. The results showed that the2fragment respectively consisted of928bp and824bp with specific nucleotide sequence.2pairs of primers will be respectively designed on based of the2fragments sequencing results. The S164-900marker was successfully converted into a SCAR marker, but S191-800marker was fail.4. A total number of90I. kudingcha (their gender was known) was actual tested for their sexuality with this SCAR marker, the result indicated that the SK-928specific band associated with the male traits appeared in all of50male germplasm materials, but only3germplasm materials can amplified SK-928band in a total number of40female germplasm materials. It can be proved that this SCAR marker(SK-928) can multitudinous be use for the identification of the sex in the early stage of I, kudingcha... |
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