Establishment of molecular identity of 22 pomelo varieties using fluorescent labeled SSR markers

Abstract:【Objective】Pomelo is a collection of perennial fruit trees that belong to one of the three basic species of citrus genus in Rutaceae. It is inferred that they are originated in Southeast Asia, or in South China where they have been cultivated for 3000 years. In China, there are a myriad of pomelo germplasm resources likely ascribed to the monembryony whereby pomelo plants are prone to genetically vary during long-term breeding, selection, and cultivation. Relatively low genetic diversity but high morphological variation makes traditional morphology identification among hundreds of pomelo germplasms and varieties difficult. The molecular technique at multiple levels can be applied to improve the accuracy of the identification. SSR is informative, codominantly inherited, highly polymorphic, easily utilized, but not affected by environments and phytomorphological characteristics. The identification of the size of target gene DNA fragment by capillary electrophoresis with primers of 5’-end fluorescence labeling (FAM, HEX, TAMRA) is accurate to even 1 bp, with reliable and stable detection performance. The molecular ID codes of 22 pomelo germplasm resources were established using SSR markers. 【Methods】The 22 germplasm resources were collected from the germplasm repository of GuangxiAcademy of Specialty Crops, and orchards of Quanzhou county, Guilin city, and Gaoming farm, Nanning city, Guangxi. According to literature reports, 21 pairs of primers with high polymorphism and good repeatability were synthesized, labeled with three kinds of fluorescence, and used for PCR amplification. The genomic DNA was extracted by magnetic bead method genomic DNA kit, and the purity, concentration and integrity of the extracts were assessed by NanoDrop and agarose gel electrophoresis. A 10 μL PCR system was adopted, including 5.0 μL of 2 × Taq PCR Master Mix, 0.5 μL of each of forward and reverse primers (10 mol·L^-1 ), 0.5 μL genomic DNA (about 20 ng · μL^-1 ), and 3.0 μL of ultrapure water. The PCR procedure was performed as follows: an initial predenaturation at 95 ℃ for 5 min, 35 cycles of denaturation at 95 ℃ for 30 s, annealing at appropriate temperature for 30 s, and extension at 72 ℃ for 30 s, a final extension at 72 ℃ for 20 min, and storage at 4 ℃. The PCR products of each primer pair with each of the 22 samples were subjected to fluorescence capillary electrophoresis. The GeneMarker analysis software was used to analyze the original data, and sizes of the amplified fragments were determined by comparing them with the internal standard of molecular mass, and the genotypes were further recognized. At the same time, the individual digits and lowercase English letters were used to mark each band to encode a fingerprint. In another word, each molecular ID card (the code) of the test samples was designed as concatenate markers in order from the largest band to the smallest.【Results】Four pairs of SSR primers that could amplify products with good polymorphism were screened and applied, which revealed a total of 49 polymorphic patterns and 41 polymorphic alleles, with an average of 12.25 patterns and 10.25 alleles, and the length of amplified fragments was 119-282 bp. Among the 22 pomelo germplasm samples, 12 had different molecular codes and could be distinguished. The same molecular identity code was found in Citrus grandis‘Shatianyou’, C. grandis ‘Guiyouyihao’, C. grandis‘Youxuan No. 1’and C. grandis‘Youxuan No. 2’Guangxi pomelo, Red pomelo and Guanxi Pomelo shared the same molecular identity code. The molecular ID code of pomelo Wuzheng and pomelo Cystis were also the same. These indicated that the pomelos with the same ID had similar SSR patterns, high genomic homologies, and close evolutionary relationships. This study showed that SSR fluorescence marker-based detection technology had the advantages of accurate, reliable, efficient, and high throughput. Only fewer markers needed the identity code of pomelo germplasm by double character coding method to facilitate differentiation between the homozygous and heterozygous genotypes.【Conclusion】Twenty-two molecular IDs of the pomelo germplasms including 12 specific samples were successfully constructed by the SSR pattern analysis, and the results from analyses in genetic distance clustering and the molecular ID were consistent. This study would not only provide a reference method for rapid, accurate and efficient identification of pomelo germplasm resources at molecular level, but also establish an important theoretical basis for identification and conservation of related pomelo varieties.