Construction And Analysis Of DNA Fingerprinting Database For Citrus Cultivars

Citrus is the most important fruit crop in the world. Its growing area reached about 6.7 million hectares all over the world, annual aggregate production exceeded 11 million tones, and annual trade value was about $23.4 billion. It has become the fourth lasted agricultural products, just followed about wheat, corn and soybean. China has the world widest citrus' growing area, annual aggregate yield climbed to 1.7 thousand tons, which was the second highest all over the world, just inferior to Brazil. Seedling is the headstream of citrus production, and be one of the key factors affecting continuable development of citrus industry. The quality of seedlings directly influences citrus' yield, quality, and planting value for planters. For recent thirty years, more than one hundred citrus cultivars have been obtained in other countries. And also in China, we have gained teens of excellent novel citrus cultivars by introducing, selecting and breeding. However, the manage system of citrus seedlings was incomplete in present. The phenomenons, like mixing up good and evil seedlings and using the false as the geniune often occurred, which has not only damaged croppers' advantage, but also brought huge threaten to the development of citrus industry. Along with China joined into WTO and Union for the Protection of New varieties of Plants (UPOV), and actualization of plants new cultivars protection statute which protecting breeding researchers' right, disputed examples relate to crop variety rights and researchers' benefits increased year after year. Therefore, it's increasingly important to establish a fast and exact technology system for detection of authenticity and purity among citrus seedings. It will be helpful in many areas, such as preventing the evil or false varieties get into market, protecting farmer's and researcher's advantage and so on.In this research, a simple and useful technology system, which suit for construction of citurs DNA fingerprinting database, has been established by optimization of PCR reaction system, primers and markers screening. A DNA fingerprinting database for 93 varieties have been constructed by analysis of 126 citrus cultivars, containing Clementina, sweet orange, hybrid, pomelo and lemon cultivars, using the technology system above. It will lay a foundation for detection of authenticity and purity among citrus seedings. The main results were as follows:Establishment of DNA fingerprinting detection systemFour molecular marker systems have been established by PCR reaction factors optimization.RAPD marker system: template DNA 50 ng, 10×PCR Buffer 2.5μL, 25mM Mg²⁺ 1.5μL, 2.5mM dNTP 1.5μL, Taq DNA polymerase 1.0 U, 20 ng/μL primer 1μL in 25μL system..SSR marker system: template DNA 50 ng, 10×PCR Buffer 2.5μL, 25mM Mg²⁺ 2.0μL, 2.5mM dNTP 2.0μL, Taq DNA polymerase 0.8 U, 20 ng/μL primers 1μL in 25μL system..ISSR marker system: template DNA 50 ng, 10×PCR Buffer 2.5μL, 25mM Mg²⁺ 1.6μL, 2.5mM dNTP 2.0μL, Taq DNA polymerase 1.0 U, 20 ng/μL primer 1μL in 25μL system..SRAP marker system: template DNA 50 ng, 10×PCR Buffer 2.5μL, 25mM Mg²⁺ 1.2μL, 2.5mM dNTP 1.2μL, Taq DNA polymerase 1.0 U, 20 ng/μL primers 1μL in 25μL system..Primers screeningBy screening primer pairs, 34 out of 105 SSR primer pairs, 6 out of 40 RAPD primers, 4 out of 20 SRAP pirmer pairs and3 out of 40 ISSR primers were obtained, which with high polymorphism and stability. Polymorphic primers accounted for 32.38%, 15%, 5% and 17.5% respectively. It's containing 14 SSR primers designed by ourselves. SSR marker was considered as the key method among citrus DNA fingerprinting, and ISSR marker as the accessorial method.Citrus DNA fingerprinting126 citrus cultivars were analyzed using SSR marker and ISSR marker. 24 lemon cultivars, 14 pomelo cultivars, 22 hybrid cultivars and 6 Clementina cultivars could be identified by SSR marker. ISSR marker could identified 5 unshui cultivars, 18 sweet orange cultivars and 4 ponkan cultivars which could not be identified by SSR marker. 93 out of 126 citrus cultivars were identified with SSR and ISSR together.Construction of DNA fingerprinting database in citrus cultvars12 SSR pimer pairs and 2 ISSR primers with high polymorphism and no linkage relationship were considered as the core primers for construction of citrus DNA fingerprinting database, including SS2, SS15, SS16, SS17, SS18, Cssr015, Cssr036, Cssr037, Cssr038, Cssr050, Cssr051, Cssr052, IS09 and IS22. A mode figerprint database contained 93 citrus cultivars has been constructed using Gel 2.0 written by Cao.Validatation of DNA fingerprinting databaseIt has been validated that the standard DNA fingerprinting database we constructed has high repetition. The results of cluster ananlysis and principal components analysis were consistent to traditional classification, which indicated that DNA fingerprinting database constructed in this research has well accuracy and reliability.