| Using the technique of random amplyfication polymophism DNA(RAPD) markers, genitic alynasis of 19 hybrid citrus varieties in Chendu and Pujiang of Sichuan Province were conducted with 9 additional varieties of Citrus L.(Rutaceae, Aurantinoideae) or related Genera from Chongqing Citrus Research Institute used as control.We hope the results can provide information on methods and theory for collecting,preserving,identificating,evaluating,classifying hybrid citrus germplasm resources and study their relationships.Meanwhile,genetic backgrounds and genetic differences were analyzed to provide scientific basis for identifying or preventing variety degration of"Shiranui tangerine".The results are as follows:1,Comparing 5 differient DNA extration methods by observing the nucleic acid precipitation appearance,UV spectrophotometric,gel electrophoresis detection and RAPD-PCR amplification,it was indicated that methods such as SDS,nuclear DNA method,CTAB and microwave method which was used in microbial DNA extraction were all efficient except the modified miacrowave method.Specifically,micarowave method was the most efficient with its high-quality extracted DNA and least time cost.2,Some essential factors that might affect the results of RAPD assay were tested and compared,optimization for miscellaneous kom RAPD amplification system was: template DNA 25ng,10×buffer 2μl,Mg2+2.0 mmol/L,primers 0.5μmol/L, dNTPs 0.22mmol/L,TaqDNA polymerase 1.4 U,total volume 20μl.PCR conditions were as the follows:94℃/1min,94℃/1mim,36℃/1min,72℃/2mim,total 43 cycles, then 72℃/10min and hold at 4℃.3,With differient fruit characters,5 materals " Shiranui tangerine ","Star Ruby", "Pengna","A late ripen white yuzu" and "Trifoliate orange" were used for RAPD analysis.Among 100 random primers tested,16 primers gave amplified band patterns. Each primer revealed differient amounts of polymorphic RAPD locis,ranging from 7 to 22.A total of 270 PAPD bands were generated,among which 254(94.07%) bands were polymophic.This indicated that hybrid citrus were so rich in DNA polymophism that the technique PAPD markers can be competent for checking out these discrepancies.4,19 hybrid citrus cultivars fell into 2 clusters.'amakus','shiranui tangerine', 'kiyomine tangerine','tunohide tangerine','yang xiang','mo kete','nuo wa','ri hui', 'chime 14 tangerine' fell into the cluster of looseskinned orange and 'ariage tangerine', 'ming ne','xing lubi','gape-citrus aurantium','red flesh citrus aurantium','huang guogan','katuyamayokan','migautiiyokan tangerine'and 'ka lizuo' fell into another but not in tdfoliate orange.5,These highly-polymiphic bands indicated the wide range of genitic diversities among the cultured vadeties.A characteristic band was abserved by comparing all bands between 28 hybrid citrus material and the control:trifoliate orange.This band was amplyfied by oligonucleotide primers "GTGACGTAGG" and had 1519bp,we speculated that it might be related to trifoliate orange's resistance.6,The amplified results also indicated that "Shiranui tangerine" in Sichuan and Chongqing could be devided into 2 groups.One was introduced from Chongqing Citrus Research Institute,the other from Central China Agricultural University. |
