与苦丁茶冬青雄性性状连锁的RAPD标记

RAPD Markers Linked to the Male Character of Ilex kudingcha C. J. Tseng

通过正交试验设计对影响苦丁茶冬青RAPD-PCR反应的5种因素4水平进行优化试验,最终确定苦丁茶冬青RAPD-PCR的最佳反应体系为:在25μL反应体系中,DNA模板20ng,Mg2+2.5mmol·L-1,引物浓度为0.3μmol·L-1,Taq聚合酶浓度为2.0U,dNTPs浓度为200μmol·L-1。最佳的RAPD-PCR扩增程序为:94℃预变性5min,然后94℃变性30s,36℃退火30s,72℃延伸120s,进行40个循环,最后72℃延伸10min;4℃保存。然后通过RAPD技术筛选了91条随机引物,共计有24条引物能在雌/雄DNA/样品池间显示多态性,其中引物S164和S191分别扩增得到2个雄性特异标记S164-900和S191-800。经多次重复实验,RAPD标记均能在雄性个体中稳定出现,故此标记可应用于苦丁茶冬青性别的早期鉴定。

An orthogonal design was made to optimize RAPD-PCR amplification system of llex kudingcha in 5 factors （ DNA template, Mg2 ＋ , primer, dNTPs, Taq polymerase） at 4 levels, respectively. Through comprehen- sive analysis, an optimized RAPD-PCR reaction system, was established： 10× buffer 2.5 μL, 20 ng DNA tem- plate, 2.5 mmol·L- 1 Mg： ＋ , 0.3 μmol·L - 1 primers, 2.0 U Taq polymerase, and 200 μmol· L-1 dNTPs in the 25 μL reaction system, and the optimized RAPD-PCR amplification program： predenaturing at 94 ℃ for 5 min, then denaturing at 94 ℃ for 30 s, elongation at 36 ℃ for 30 s and extension at 72 ℃ for 120 s, running for 40 cycles, and final extension at 72 ℃ for 10 min. The products were stored at 4 ℃. Furthermore, a total number of 91 random primers were screened in the RAPD-PCR. Polymorphic fragments were detected with 24 primers in female and male DNA samples pools. Two male-associated fragments （S164-900 and S191-800） were respectively generated with S164 primer and S191 primer. Repeated experiments indicated that these RAPD markers appeared stably in male individuals. So these RAPD markers can be applied to identify the sex of the young plantlets of Ilex kudingcha C. J. Tseng at the early stage.