关键词: 柑橘, SSR, 毛细管电泳, 品种鉴定, DNA指纹图谱库

Abstract: 【Objective】The purpose of the present work aims at the construction of DNA fingerprint library of citrus cultivar by using a series of SSR core primers in order to provide references for citrus nursery plant identification, cultivars registration and plant variety protection. 【Method】SSR primers were primarily screened by analyzing 8 mainly cultivated species belonging to Citrus while the PCR products were separated by agarose gel electrophoresis, the primers that generated high polymorphic and stable PCR products were chosen for further PCR amplification with more citrus cultivars, and the PCR products were separated by polyacrylamide gel electrophoresis. the further screened primers were labeled with fluorescent and used for amplification of the all tested cultivars. The PCR products were detected by fluorescence capillary electrophoresis and the primers suitable for fluorescent capillary electrophoresis were screened according to their PIC, number of alleles identified, and employed to construct the citrus cultivar DNA fingerprinting library, and the selected primers were used to identify the tested cultivars.【Result】Firstly, 80 pairs of polymorphic and stable primers were chosen from 362 SSR primers by 4% agarose gel electrophoresis with representative cultivars of 8 species of Citrus genus(including Ota ponkan, Shatian pummelo, Yuanjiang soar orange, Dahong sweet orange, Duncan grapefruit, citron, Femminello lemon, Mexico lime); Secondly, 60 pairs of SSR primers with high polymorphism, stability and clear amplification products were further selected from the above 80 pairs of primers by 6% polyacrylamide gel electrophoresis with 64 citrus cultivars and labeled with fluorescent dyes; Thirdly, 21 pairs of SSR core primers were verified for the construction of fingerprint library from above 60 pairs of primers by capillary electrophoresis with 168 citrus cultivars on the basis of PIC, the allele number, peak reading difficulty, amplification stability and chromosome location. The corresponding reference cultivars were selected for 21 pairs of primers to eliminate the errors caused by different instruments and experimental batches. Through the multiple electrophoresis with the 5 combination groups of 21 primers according to the color and size of amplified fragment, we established 500 citrus cultivars fingerprinting library. Among the tested cultivars, 111 were identified by using only one pair of primers, 86 with the combination of two pairs of primers; and 24 with the combination of 3 pairs of primers, the remaining 279 cultivars were not able to be identified individually, they could, however, be divided into 87 groups, between which it was clearly distinguished but within which the cultivars were indistinguishable.【Conclusion】The DNA fingerprint library containing 500 citrus cultivars was constructed by using 21 pairs of screened SSR core primers, the specific markers for some cultivars were selected; 221 cultivars and 87 cultivar groups were identified; and a citrus cultivar identification system based on SSR fluorescence labeling was constructed.

Key words: Citrus, SSR, capillary electrophoresis, variety identification, DNA fingerprints