ISSR Analysis Of The Citrus Germplasm Resources In Fujian

The citrus was a fruit tree belonged to the family of Rutacease. It was one of the most important fruit tree in south china. The citrus had six Genuses. Thereinto, Poncirus, Fortunella and Citrus had important economic value. They were used as fruit cultivation and rootstocks.In south china, the nature climatic condition was superior, abundant citrus germplasm resources were holed in store. It was one of the major production areas of world citrus. Fujian was located the coast of southeast China, belonged to the subtropical maritime monsoon climate zone, was one of the major citrus production areas in China.In 1958~1959, investigation showed that the Fujian citrus germplasm resources had 3 Genuses, 13 species, and about 400 breeds. In recent 50 years, abundant new citrus germplasm resources were introduced from elsewhere, enriching the breed type of Fujian citrus germplasm. However, the citrus had particular characteristics; it made a variety of citrus appear. As had been noted, Fujian citrus germplasm resources were mistiness for us; it impeded breeding process of the citrus.ISSR(Inter-Simple Sequence Repeats)synthesize various kinds advantage of other molecular marker, was appropriate for analyzing Fujian citrus germplasm resources.The purpose of this study was to analyze the genetic background of 75 citrus germplasm resources in Fujian. The results could provide scientific basis for making right protection strategy and help to foster new cultivars. The main results were as follow:Firstly, faced the fact that it was difficulty to extract DNA from the Citrus which contained phenolic compound and quite a lot of sugar,the researcher used improved CTAB-mini method to extract DNA of Citrus,which meet ISSR requirements in quantity and purity. So far it was quite a proper way to extract DNA from Citrus. Secondly, the technological system of ISSR-PCR reaction of the Citrus had been built up. In the 20μL reaction system,there was 1ng/μL template DNA,0.35 mmol·L-1 dNTPs,0.15μmol·L-1 Primer,1 U Taq DNA enzyme and 3μL 10×Buffer(Contain Mg2+). According to the system,the experimental steps were as follows. 5 min predenaturation at 94℃,30 seconds denaturation at 94℃,60 seconds annealing,90 seconds extension at 72℃. The cycle repeats forty times and ends in 7 min extention at 72℃.Thirdly, a total of 14 ISSR primers were employed on 75 individuals of cultivated Citrus from 95 primers and 219 scorable amplified polymorphic were detected,of which 206(94.06%) were polymorphic loci. The similarity coefficient of 75 citrus germplasm resources between 0.443～1.000.Finally, according to the clustering analysis of ISSR results,the genetic diversity , similarity and genetic relationships of 75 citrus germplasm resources were revealed. In addition, we analyzed the biological evolution of Fujian special citrus germplasm. All of these data laid a theory foundation for the citrus Introduction and breed.