Identification of white flesh loquat germplasms of Zhejiang province with MCID strategy using genic-SSR markers

Abstract：【Objective】Zhejiang province is one of the most important original area of white flesh loquat germplasms in China. There are still many white flesh germplasms not recorded in the annals of the Chinese loquat. These different germplasms are usually called‘Baisha'by the local grower. Phenomenon of homonym or synonym often occurs. These germplasms are difficult to distinguish with traditional methods due to the morphological similarity of leaf or fruit shape. Molecular biological methods, especially DNA fingerprinting techniques, have promising applications in the identification of plant species and cultivars. Molecular marker has been used as an effective approach to identify plant variety. The study aimed to to carry out the preliminary evaluation and identification of white loquat germplasms based on genic SSR molecular markers in order to set up an efficient method for cultivar registration in Zhejiang.【Methods】Ten polymorphic genic SSR primers were used to perform PCR amplification of 26 different germplasms of white loquat including 14 new accessions of Zhejiang and 12 existing cultivars. PCR amplification was carried out in a 20 μL reaction system, containing 2 μL of genomic DNA (30 mg · L-1) , 0.8 μL of 10 μmol of each primer, 10 μL of 2×Taq Mix, 6.4 μL of dd H2 O. Amplification reactions were performed in a Huayue Biometra Thermalcycler, initial denaturation for 5 minat 94 ℃, 30 cycles of denaturation at 94 ℃ for 30 sec, annealing for 30 sec, and extension at 72 ℃ for 1 min was performed, a final extension step at 72 ℃ for 10 min was done. PCR products were put on the automatic nucleic acid protein analyzer for testing the peak figures after agarose gel checking. The manual cultivar identification diagram (MCID) was taken to identify all the loquat germplasms. Only clear unambiguous peak figures were manually scored from photographic prints of gels for each cultivar. We classified these cultivars into different groups according to the fingerprint amplified by each primer. The cultivars shared the same band patterns were put into the same group. More primers were then employed to further distinguish the cultivars in each group. As more primers were used, more specific amplified bands were generated and used for differentiation of all the cultivars separately by, the cultivar identification diagram (CID) .【Results】All the 26 loquat germplasms were divided into 10 groups by polymorphic bands with sizes of 334, 318, 291, 276, 262, and 241 bp amplified by primer 3842.‘Ninghaibai'in group one was separate by the presence of 318 bp and 291 bp. Nine varieties including‘Ningbai 12'‘Huaifanbaisha'‘Tangjiaaobaisha'‘Gaowanbaisha 1'‘Ruantiaobaisha'‘Yingtiaobaisha'‘Gaowanbaisha 2'‘Tangshuangpipa'‘Mutongaobaisha'were classified into group two with polymorphic bands of 318 bp and 262 bp.‘Biqizhong'was grouped into the third group, by the presence of 276 bp, 262 bp.‘Qingzhong'was grouped into the fourth group with 291 bp and 262 bp. Eight germplasms of‘Shabubaisha'‘Guanyu'‘Changlü 5'‘Wuerbaisha'‘Tangkebairou'‘Gangbeibaisha'‘Lanxibaisha'‘Muwubaisha'were classified into the fifth group with 262 bp and 241 bp.‘Changlü 4'was grouped into the sixth group by the presence of 334 bp and 262 bp.‘Zhaozhong'in group seven was separated by the bands of 318 bp and 241 bp.‘Xishangbaisha'and‘Dabai'in group eight was separated by 241 bp.‘Bingtangzhong'was put into the ninth group with 262 bp band.‘Taipingbai'in group ten was separated by three bands of 334 bp, 276 bp and 241 bp. Four feature bands of 207 bp, 195 bp, 183 bp and 178 bp were further utilized to identify the different varieties of the second, fifth and eight groups. In the same way, the primers 4019, 4297, 8296 and corresponding polymorphic bands were used for identification of the remaining varieties. White loquat CID was constructed and of the 26 germplasms could be rapidly separated from each other, according to the polymorphic bands and corresponding primers marked in the correct position. The verified results of six cultivars from inter-or intra subgroup was consistent with the obtained CID.【Conclusion】MCID method proved reliable and practicable for rapid identification of white flesh loquat germplasmss. The obtained clusters of the 14 new white loquat germplasms and 12 known varieties basically accorded with the origins and genetic background of these white flesh loquat resources.