The Establishment Of AFLP Silver-staining Analysis System And Fingerprinting Of Walnut

AFLP(amplified fragment length polymorphism) is a new DNA molecular marker technique with high efficiency, promptitude, stability and accuracy. It has been widely used in classification, cultivar identification, genetic mapping, breeding, location of target gene in animals and plants.Walnut originated in China and has been cultivated for a long time in China. It has rich germplasm and widely distributed in China. Understanding the relationship of different walnut cultivars and their genetic diversity will be benefit to the utilization of the germplasm and improve the breeding work. At recent years, biochemical markers and RAPD marker were used in research of the germplasm. But all these methods have limitations.In order to provide scientific basis for the identification,conservation and utilization of walnut germplasm,AFLP silver-staining technique system suitable for walnut was established.The fingerprinting of 58 walnut materials were constructed,and the genetic diversity of these materials was analysed.1. The genomic DNA of walnut was extracted by a modified CTAB method.The modified isolation buffer was: 100 mmol/LTris-HCl (pH 8.0), 20 mmol/LEDTA, 1.4 mol/LNaCl, 2% CTAB, 3% PVP40, 50 mmol/L beta -Mercaptoethanal and 10 mmol/L Na2S2O5.The RNA was eliminated from the solution with RNase,and the DNA was purified by phenol/chloroform (one),chloroform/isoamyl alcohol (twice).The purified walnut genomic DNA was suitable for AFLP analysis.2.After systematically study on the main factors involved,an AFLP silver-staining analysis system suitable for walnut genomic DNA was established. The purified walnut genomic DNA450 ng, NEB buffer2 2 μL, BSA(10 mg/mL) 0.2μL, EcoR I 3 U, Mse I 3 U and ddH2O were in a reaction volume of 20μL, and incubated at 37 ℃ for 4 h. 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 pre-amplifiedproduce was also diluted 5 time with TE buffer and used for selective amplification.At the end of the selective amplification,the walnut samples were denatured by adding 10 μL Loading buffer and heated up at 95 癈 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.Ten EcoRI/Msel primer pairs showing high level of polymorphism and scorable strong band were selected out from 64 primer combinations and used for further practical AFLP analysis.560 bands were scored,94.11% of which (527 ) were polymorphic.The rates of polymorphic band varied among 46 walnut cultivars(Juglans regia L.), 8 Hebei walnuts(J. hopeiensis Hu) and 4 walnut Chinese catalpas(J. mandshurica Max.).4.The fingerprinting of 58 germplasm of walnut materials were established based on 560 AFLP bands using 10 primer combinations.These germplasm could be separated into 2 groups by Ward's method. Their genetic diversity was discussed.