Analysis of genetic relationship of pummelo germplasms by SSR markers in Zhejiang province

 Abstract: 【Objective】Zhejiang province is one of the main distribution area of pummelo genetic germ⁃plasms. According to researches, the local pummelo germplasms were mainly originated from seedlingvariation of‘Fujian-wendan’, such as‘Zaoxiangyou’‘Foxiangyou’and‘Gaoxie-xiangyou’. Whereas,the origin and genetic relationship of‘Gusangyou’‘Mulanyou’‘Guannan-hongyou’and other local pummelo cultivars have remained unclear up to now. With the wide spread of newly selected cultivars, the production area of local pummelo cultivars has decreased and some pummelo germplasms with low cultiva⁃tion value has even disappeared. Therefore, it is necessary to analyze the genetic relationship of the localgermplasms and to get enough information for protecting and utilizing them in the future. In this study,twenty-three simple sequence repeat (SSR) primers mined in Citrus were used to detect the genetic diversity of 18 local pummelo germplasms collected in Zhejiang province, and to construct germplasm charac⁃terization map.【Methods】A total of eighteen local pummelo germplasms were collected from the ten citiesof Zhejiang province according to the Citrus germplasm resource description specifications, the eighteenlocal pummelo germplasms were divided into four groups based on the pulp colour. Six germplasms includ⁃ing‘Yuhuanyou’‘Zaoxiangyou’and‘Gaoxie-xiangyou’belonged to the pale yellow group. The pulp color of‘Mulanyou’‘Sijiyou’‘Mabu-wendan’and other seven germplasms was pink. Four germplasms in⁃cluding‘Gusangyou’‘Guannan-hongyou’‘Chuhongyou’and‘Yongjiahongxinyou 1’belonged to thered group and the pulp color of‘Changshan-huyou’was orange. Young leaves were sampled for DNA extraction and twenty-three citrus SSR primers with abundant polymorphisms were employed for genetic diversity analysis. The amplified products were sequenced on MegaBACE 1000 DNA Analysis System. Theobserved number of alleles (Ao), effective number of alleles (Ae), observed heterozygosity (Ho), expected heterozygosity (He) and polymorphism information content (PIC) were calculated with Popgene 1.31 software.The genetic similarity coefficients were calculated with NTSYS-pc 2.1 software and phylogenetic tree wasconstructed by the weighted average method.【Results】A total of 91 alleles were detected by twenty-threeSSR primers, ranged from two to seven and with an average of 4.0 alleles per locus. The mean observedand expected heterozygosity values were 0.51 and 0.54, respectively. The average polymorphism information content of 23 SSR loci was 0.47. The genetic similarity coefficients between genotypes ranged from0.55 to 0.94 and with an average of 0.71. The above results showed a rich genetic polymorphisms and com⁃plicated genetic background among pummelo germplasms in Zhejiang province. The UPGMA dedrogrambased on the genetic similarity coefficients grouped 18 pummelo accessions to six major groups at the coefficient of 0.71 as follows: seven germplasm including‘Sijiyou’‘Hongrou-Sijiyou’‘Foxiangyou’‘Mabuwendan’‘Zaoxiangyou’‘Yuhuanyou’and‘Gaoxie-xiangyou’were clustered to A1 group; the three pinkgermplasm including‘Yongjia-hongxinyou 2’‘Qingtian-hongxinyou’and‘Mulanyou’were clustered toA2 group; the four red germplasm including‘Chuhongyou’‘Yongjia-hongxinyou 1’‘Gusangyou’and‘Guannan-hongyou’were clustered to A3 group;‘Guannan-tuyou’and‘Wencheng-hongxinyou’wereseparately clustered to B1 and C1; and the two hybrids from the cross of pummelo and other citrus wereclustered to D1 group. The results showed that most local pummelo cultivars were clustered preferentiallyto their pulp colors except for a few germplasms, indicating that the local red pummelo germplasm couldbe originated from a common ancestor. However, the germplasms between pale yellow and pink were nottotally distinguished. Moreover, four SSR primers AG14, CAG01, CMS-16 and CMS-26 could rapidlyand accurately identify all the tested materials from each other, and a corresponding germplasm characterization map of Zhejiang province was constructed, which could be applied to seedling authenticity characterization.【Conclusion】There was a rich genetic diversity of pummelo germplasm resources in Zhejiangprovince, and four SSR primers could be used for rapid and accurate identification of local pummelo germplasms.