Genetic structure analysis of the cultivated blueberry (Vaccinium spp.) species and wild species in China based on EST-SSR markers

Abstract: 【Objective】Blueberries, belonging to the genus Vaccinium of family Ericaceae, including highbush blueberries (Vaccinium corymbosum) , rabbiteye blueberries (V. ashei) and low-bush blueberries (V.augustifolium) are the most recognized commercially cultivated species in the world. Some wild species of this genus are unique to cold resistance, drought resistance and fruit nutritions, and are valuable resources for the germplasm innovation and also broaden the genetic basis of the blueberry. An understanding of the genetic structural differences, relationship and genetic backgrounds among cultivated blueberries and wild species have crucial meanings, and guide the breeding programs accompanied by genetic exploration and utilization of the wild blueberry species.【Methods】Conducted a total of 84 tests including 58 cultivated varieties from abroad and 26 wild types from China. DNA was extracted from the young leaves of theplants using a CTAB method with modification. PCR reactions were carried out in a final volume of 15 μLcontaining 3 μL template DNA (20 mg·L^-1 ) , 0.2 μL forward primer (2.0 μmol·L^-1 ) , 1.0 μL M13 (labeledwith 6-FAM, NED, VIC or PET) , 1.2 μL reverse primer (2.0 μmol·L^-1 ) , 7.5 μL Mix-Taq (CWBIO, China) and 2.1 μL H2O (CWBIO, China) . PCR amplification conditions were as follows: 94 °C for 5 min, 35 cy-cles of 95 °C for 30 s, 52 °C for 45 s and 72 °C for 45 s, and a final extension at 72 °C for 10 min. All reac-tions were conducted using a thermal cycler (Veriti 96, Applied Biosystems, Foster City, USA) . The PCRproducts were separated by capillary electrophoresis using a ABI3730 xl DNA Analyzer (Applied Biosys-tems, Foster City, USA) . After PCR amplification confirmation with 2.0% agarosegel, nest-PCR multiplexsets were made based on fluorescence-labeled M13 primers. For nest-PCR multiplexing, 1 μL of 6-FAM, VIC-, NED-and 0.5 μL PET labeled PCR products representing different SSRs were combined in13 μL H2 O 1 μL mixed product which was then added to 7 μL Hi-Di formamide containing 0.07 μL Gen-e Scan™ 500 LIZ®as the internal size standard, denatured for 7 min at 95 °C, quick chilled on ice for 5min, and loaded on a ABI3730 xl DNA Analyzer. The fragment analysis was performed by using Gene Map-per v4.0 software (Applied Biosystems, Foster City, USA) . The genetic structure was investigated by usingsoftware STRUCTURE and Principal Coordinate Analysis (PCA) and a neighbor-joining tree clusterwhich was analyzed based on Nie's genetic distance and applied through software Power Marker.【Results】1) Totally, 101 polymorphic alleles were obtained with 8 EST-SSR primer pairs, which were screenedfrom a total of 54 EST-SSR primer pairs, with an average of 12.63 alleles per locus. The average of effec-tive alleles (Ne) , Shannon's index (I) , observed heterozygosity (Ho) and expected heterozygosity (He) perlocus were 8.711, 2.262, 0.523 and 0.860, respectively, and the polymorphic information content (PIC) ranged from 0.640 to 0.915 with an average of 0.845, indicating a high polymorphism. 2) Genetic structureanalysis based on the Bayesian method indicated that ΔK reached maximum when K=4, all the 84 testedblueberry accessions were clustered into 4 groups: S1, which is given priority to lingonberry (V. vitisidaea) and cranberry (V. macrocarpon) and characterized by red fruit; S2, which is given priority to bog bil-berry (V. uliginosum) and characterized by being highly cold-resistant; S3, which is given priority to south-ern highbush (V. corymbosum SHB) and rabbiterry (V. ashei) blueberry, and characterized by having a lowchilling requirement; S4, which is given priority to northern highbush (V. corymbosum NHB) and half-highbush (V. corymbosum HHB) blueberries and characterized by having a high chilling requirement, theabove results showed an obvious genetic structure within Vaccinium spp. 3) Both the results of neighbor-joining tree cluster analysis and principal component analysis (PCA) identified large genetic differencesthat existed between the cultivated species and wild species of China; 4) The Q value of the 84 tested blue-berry accessions higher than 0.6 accounted for 91.67%, which were considered an individual assigned un-equivocally to a cluster, with single genetic components.【Conclusion】Obvious genetic structure and dis-tantly relationships existed between the cultivated species and wild species of China, however, there was alimited amount of cultivated varieties with an ancestry of wild species (with S1 or S2 components) and thisindicates that there was introgression between the cultivated variety and wild species. This shows that youcan obtain variant plants through hybridization to innovated germplasm and broaden the genetic basis forblueberries, which also should enhance the exploitation and utilization of wild blueberry resources in China.