Study On Molecular Markers Linked To Sex Of Salacca Zalacca

Salacca zalacca（namely salak） belongs to the genus of Salacca of the family of Palmaeand is endemic to the Malay peninsula to Java island. It was introduced into China20yearsago. Sexual reproduction is an important way of salak propogation, but it is difficult todistinguish male seedlings from female seedlings, and difficult to control sex ratio whenseedlings are planted. A sequence of genome DNA is screened out by RAPD for male salak,and it is considered to be used to identify sex of salak seedlings. The sequence is used toidentify sex of salak seedlings, as well as to proliferate female salak seedlings with embryo asexplant by tissue culture. It provides technical support to screen female seedlings from maleseedlings, and helps to fix sex ratio when planting. Therefore, this study is meaningfulsignificantly in improving China’s salak cultivation.1. Four methods were tested to extract genomic DNA from fresh leaves of salak, it isshowed that: the modified CTAB method removes such materials as phenolic substances,polysaccharides, and protein, it is proved to be an method to extract genomic DNA from salak.2. The different levels of concentration of Mg²⁺, dNTPs, Taq DNA polymerase andannealing temperature were tested for salak genomic DNA extracted by modified CTABmethod, the results show that: PCR amplification system for15μL:10×buffer1.5μL,0.12mmol/L dNTPs,0.12U/μL Taq DNA polymerase,1.4mmol/L Mg²⁺,1.0μmol/L RAPDprimers,12ng DNA template. The PCR amplification program:94°C pre-denaturation2min;then40cycles: denaturation at94°C for30s, annealing at39°C30s and72°C for2min;72°Cfor10min.3. Ten male plants and ten female plants of the ’Pondoh’ varieties were sampled and theirgenomic DNA were extracted and amplified by900random primers, a size of approximately1600bp is amplified by the primer S1431（5’-CCTGGGTCAG-3’）. Another10male and10female plants were sampled randomly and amplified to verify the amplication of male-specificsequence by the S1431primer. RAPD molecular is feasible to identify the gender of salak. 4. The male specific fragments of salak RAPD molecular markers was cloned andsequenced, the results show that: the specific fragment is a1640bp sequence.5. According to the sequence analysis, a pair of SCAR primers containing18bp, namelyLF （5’-AGCACAGCCTAGTTAGTT-3’） and LR （5’-TGACACCTCCTCCCATAT-3’） weredesigned. Twelve adult plants were amplified by the above primers at vegetative growth stage,and their sex were identified at reproductive growth stage in field. Male specific fragmentswere observed for male plants, while it is absent for the female plants. Twenty salak seedlingswere tested by the above primers, the results showed that: there are8samples with theamplification of the male fragment, and not any fragmen were observed for the other12samples. Three salak varieties including12tissue-cultured plantets from an explant of embryowere tested by the above primers, not any specific fragment were amplified.6. Leaves of seedlings were sampled and amplified by a pair of primer LF and LRcultured from fruit containing different seeds. Sex of seedling is determined by the presence orabsence of male-specific fragments, the results showed that: the ratio of male to female is1:1for seeds from fruits containing not less than two seeds; the frequency of male plants is80%for fruits containing one seed only; however, the ratio of male to female is1:1in general forseedlings from all fruits.7. Seed weight and both total leaf length and total root length of seedling were measured,seedlings of Salacca zalacca were grouped into male and female groups by presence ofmale-specific fragments, ANOVA analysis were applied to test difference between the twogroups by SPSS software, the results show that: the traits of male group is not significantdifferent from those of female group, which means these traits are not suitable to identify sexof seedlings.8. The costs for genomic DNA extraction, PCR reactions, electrophoresis, and labor werecalculated and analyzed for sex identification of Salacca zalacca at vegetative growth, theresults showed that: the identification cost is estimated at18.00yuan for a batch with only onesample, while the cost is150.00yuan for a batch with96samples, the later is affordable for most planters.It is concluded that it is feasible to apply the markers to screen male plants inpratically.