Genetic relationship analysis among Nanfeng citrus using SSR markers

Abstract:【Objective】Nanfeng tangerines (Citrus reticulate Blanco‘Kinokuni') are one of the most famous local cultivars of China. It has more than 1 300 years of cultivation history. Due to the high frequency of natural variation and natural hybridization, the Nanfeng tangerine gradually formed the Nanfeng tangerine group (such as small-fruit Nanfeng tangerines, large-fruit Nanfeng tangerines, early-ripe Nanfeng tangerines, ‘Guihuadi' Nanfeng tangerines, etc.) and the Nanfeng guangju group (such as‘Hongguang' ‘Miguang' etc.) . Based on the comprehensive collection of Nanfeng citrus germplasms, this study utilized SSR markers to explore their genetic relationships from the molecular level, in order to provide a theoretical basis for the Nanfeng citrus germplasm resources preservation and utilization.【Methods】Twenty-eight germplasm accessions of Nanfeng citrus were used as materials for analyzing their genome polymorphism, including 15 small-fruit Nanfeng tangerine cultivars, two large-fruit Nanfeng tangerine cultivars, two earlyripe Nanfeng tangerine cultivars, ‘Guihuadi'Nanfeng tangerine, ‘Hongguang'‘Miguang'‘Xiaoyeguang'‘Huoguang'‘Red tangerine'‘Huoju'‘Bendizaoju'and kumquat all collected from the Nanfeng Tangerine Germplasm Repository in Nanfeng county, Shuinan and Dabao village in Qincheng town, Nanfeng county, and the Nanfeng Tangerine Germplasm Repository in Horticultural Sciences Institute of Jiangxi Agricultural Sciences Academy. The collected fresh leaves were brought back to the laboratory iniced cassettes. After their surfaces were cleaned by clear water, they were rapidly frozen in liquid nitrogen and stored at-80 ℃ until required for testing. The improved CTAB method was used to extract the total DNA genome, 91 pairs of SSR primers were used from previous reports. The total PCR reaction volume was 25 μL: 2×Taq PCR Master Mix 12.5 μL, 10 μmol·L^-1 forward and reverse primers each 0.5 μL, 50 mg·L^-1 template DNA 0.5 μL, dd H2 O 11 μL. The cycles were programmed as follows: one initial denaturing cycle at 94 ℃ for 4 min, 32 cycles of 30 s denaturing at 94 ℃, 45 s annealing at 45-60 ℃, 45 s elongation at 72 ℃ and one final cycle of 5 min at 72 ℃, stored at 4 ℃. The products were electrophoresised on 6% (ρ) denaturing polyacrylamide gel and visualized using silver staining. The polymorphism was determined according to the presence (scored as“1”) or absence (scored as“0”) of the SSR band. The similarity coefficient was calculated using NTSYS-pc2.1 software. UPGMA (unweighted pair group method arithmetic averages) were determined by using clustering analysis. The polymorphic information content (PIC) values for each marker were calculated according to the following formula: PIC=1-ΣPij2, where Pijis the frequency of the jth pattern for SSR marker i.【Results】Thirteen out of the 91 SSR loci were used for genotpying and they were able to distinguish all accessions. Sixty-four alleles were identified across all loci, with an average of 4.92 alleles per locus. UPGMA analysis showed that the kumquat (Fortunella crassifolia Swing.) was separated from the other 27 accessions of Citrus at the similarity coefficient of 0.18. At the coefficient of 0.59, the remaining 27 accessions of Citrus were divided into five major groups. All the 20 Nanfeng tangerine cultivars and Miguang could be clustered into one group (Ⅰ) , and‘Xiaoyeguang'‘Huoguang'and‘Huoju'were clustered into another group (Ⅱ) .‘Hongguang'‘Red tangerine'and‘Bendizao tangerine'were distributed in group Ⅲ, Ⅳ and Ⅴ, respectively. When the similarity coefficient was 0.70, group Ⅰwas divided into 2 subgroups.‘Guihuadi'Nanfeng tangerine, early-ripe Nanfeng tangerine‘LS-1'and14 small-fruit Nanfeng tangerine cultivars were distributed in the Ⅰ-1 subgroup. The biological characteristics and economic traits of‘Guihuadi'Nanfeng tangerine and early-ripe Nanfeng tangerine‘LS-1'were fundamentally the same as the small-fruit Nanfeng tangerine except for sepals like osmanthus flower shape and an early mature period of 7-10 d, respectively. Small-fruit Nanfeng tangerine‘97-2', earlyripe Nanfeng tangerine‘97-1', large-fruit Nanfeng tangerine‘LS-1', large-fruit Nanfeng tangerine‘97-1'and‘Miguang'were distributed in subgroupⅠ-2. Each of the Nanfeng tangerine strains did not separate during clustering. However, the small-fruit Nanfeng tangerine was primarily concentrated in subgroup Ⅰ-1.【Conclusion】The genetic relationships between the‘Miguang'and Nanfeng tangerine were closer than that between the ‘Hongguang' and Nanfeng tangerine. In addition, ‘Xiaoyeguang'‘Huoguang'and‘Huoju'were clustered together, indicating they have close genetic relationships.