Identification of interspecific hybrids derived from Fragaria × ananassa × F. viridis by morphological features and SSR markers

Abstract: 【Objective】To identify the interspecific hybrids derived from Fragaria × ananassa Duch. × F.viridis Duch. and to analyze the genetic relationships between these hybrids and their parents by using themorphological characteristics and SSR markers.【Methods】Thirty F1 progenies were obtained from an interspecies cross of‘Ningyu’(Fragaria × ananassa) × Fragaria viridis. According to descriptors and datastandard for strawberry (Fragaria spp.), the main botanical characteristics including the plant traits, leaftraits, floral traits and stolon traits were investigated of the F1 progenies and their parents. The genomicDNAs of leaves were extracted by rapid genomic DNA extraction Reagent kit of BioTeke Corporation, andthe quality and concentration of genomic DNA were detected by 1% agarose gel electrophoresis. In thisstudy, 40 pairs of SSR primers were randomly selected. The PCR reaction cycling profile was 94 ℃ for 4min followed by 35 cycles at 94 ℃ for 30 s, 55 ℃ for 45 s, 72 ℃ for 60 s, and a final extension at 72 ℃ for5 min. PCR amplifications were conducted in a final volume of 25 μL containing 50 ng template DNA,PCR buffer mix, 0.4 μL DNA Taq polymeraseand and 0.4 μmol·L^-1 of each primer. The genetic diversityanalysis was performed on a 6% denaturing polyacrylamide gel electrophoresis for strip analysis. Gelswere stained with silver staining. The polymorphisms of the materials of the parents were analyzed by the40 pairs of SSR primers. The discrepant primers were selected to identify the hybrids of the offspring. Theclear specific bands possessing genes of male parent were used to identify the hybrids. The matrix was obtained by the statistical data on the amplification chart, which was analyzed by using the ntsys-pc soft⁃ware. The similarity data were analyzed by the Dice coefficient and UPGMA cluster.【Results】The vigorof F1 generation was stronger than that of the male parent, but similar to the female parent according to theinvestigation and analysis of the main morphological characteristics of parents and F1 generation. The off⁃springs had some heterosis based on the cacullation of the mid-parent heterosis of the quantitative traits.The 20 pairs of primers selected could distinguish the parents, of which 12 pairs of primers were used toidentify the hybrids in the 30 progenies. The results showed that the offsprings possessed the same frag⁃ment as their male parent. All the F1 individuals were the interspecific hybrids between the Fragaria ×ananassa × Fragaria viridis. At the same time, some offsprings showed variation for the appearance of newbands or the disappearance of some bands. The genetic relationship between the parents and the off⁃springs was further analyzed. According to the results of morphology and UPGMA cluster analysis, the offsprings could be divided into three groups. The first group (group I) contained 5 materials including the female parent‘Ningyu’. The second group (Group II) consisted of 7 materials including the male parentFragaria viridis. The remaining 20 offsprings belonged to the third group, accounting for 2/3 of the proportion of offspring.【Conclusion】This study confirmed that the individuals of hybrid progeny of the cultivatedstrawberry and the wild strawberry were true hybrids by morphological features and SSR analysis. 12 pairsof SSR primers could be effectively used for identifying the hybrids derived from ( Fragaria ×ananassa Duch.)×Fragaria viridis. The primers EMFvi109, EMFvi108, EMFvi025, EMFvi072, EMFvi146, EMF⁃vi133, EMFvi092 and other ones could be used as important primers in the study of the classification ofwild strawberry and cultivated strawberry. The analysis of the morphological characteristics showed thatthe descendants had some heterosis. Six of the offspring were gathered together with Fragaria viridis bythe cluster analysis.