Screening And Identification Of AFLP Maker Linked To The Fertility Gene In Watermelon

Fl generation of watermelon generally shows obvious heterosis, it can greatly improve yield of watermelon and increase farmers' income, so nowadays the production of watermelon all use the F1 generation in our country. Using the male sterile line to product hybrids, it can greatly economizes man-power and thing-power, reduces the effective cost. Through studying the molecular mechanism of genic-male-sterility in watermelon, we can obtain makers and gene fragment, and provide basis to the early identification of male-sterile plant and the cultivation of good sterile line. AFLP(amplified fragment length polymorphism) is a DNA molecular marker technique with high efficiency, promptitude, stability and accuracy. It has been widely used in molecular marker-assisted breeding. The experiment is to construct AFLP reaction system for watermelon, and to obtian the molecular marker which link to fertility gene by using AFLP to sduty G17AB. It can provide a rapid and simple way for early identification of male-sterile plant. The main results are as follows:1. The genomic DNA of watermelon was extracted by a modified CTAB method. The modified isolation buffer was: 100 mmol/L Tris-HCl (pH 8.0), 20 mmol/L EDTA(pH 8.0), 1.4mol/L NaCl, 2% CTAB, 1% PVP40, 0.2%β-Mercatoethanal. The RNA was eliminated from the solution with RNase, and the DNA was purified by chloroform/isoamyl alcohol (twice). The purified watermelon genomic DNA was suitable for AFLP analysis.2. After systematically studied on the main factors involved, an AFLP silver-staining- analysis system suitable for watmelon genomic DNA was established. The purified watermelon genomic DNA 500ng, Tango buffer 2μl, EcoR I 3U, Mse I 3U and ddH20 were in a reaction volume of 20μl, and incubated at 37℃for 4h and 65℃15min. Double-stranded adaptors were added to the restriction fragments at 37℃for more than 10 h(or overnight). The linked produce was diluted 5 times with TE buffer and used as templates for. pre-amplification The system of pre-amplification was: template DNA 5.0μl, 10×Buffer 2.0μl, dNTP(2.5mM) 1.6μl, Moo(50ng/μl) 0.6μl, Eoo(50ng/μl) 0.6μl, Taq polymerase(5U/μl) 0.2μl, ddH2O 10.0μl. The pre-amplified produce was also diluted 5 time with TE buffer and used for selective amplification. The system of selective amplification was: template DNA 5.0μl, 10×Buffer 2.0μl, dNTP(2.5mM) 1.8μl, Moo(50ng/μl) 0.8μl, Eoo(50ng/μl) 0.8μl, Taq polymerase(5U/μl) 0.2μl, ddH2O 9.4μl. At the end of the selective amplification, the watmelon samples were denatured by adding 10μl Loading buffer and heated up at 95℃for 10 min, then cooled on ice immediately. The PCR reaction products were analysed on 6% denatural acrylamide/bisacrylimade gels and then stained by silver.3. 238 EcoRI/MseI primer pairs showing high level of polymorphism and scorable strong band were selected out from 256 primer combinations and used for further practical AFLP analysis. The rate of effective primer is up to 92.97%. 4427 bands were scored, the average is 18.6 bands, the most is 45 bands, the least is 10 bands.4. The genic male-sterile and male-fertile lines were compared by using 256 primers with the AFLP techniques. A specific amplified DNA fragment, E31M50, that is linked to the pollen fertility was identified from watermelon. The genetic distance between E31M50 and the fertility gene is 5.0cM. The 337 bp E31M50 segment was recovered and sequenced. The marker could potentially be used for breeding and gene identification.