Computational protocol: Genetic Variability of Wild Cherry (Prunus avium L.) Seed Stands in Slovenia as Revealed by Nuclear Microsatellite Loci

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Protocol publication

[…] Total DNA was extracted with the DNeasy® Plant Mini Kit (Qiagen) from approximately 20 mg of dried leaves ground in an automatic grinding mill (Retsch MM200). All individuals were genotyped at ten nuclear microsatellite loci. Two multiplex polymerase chain reactions (PCR) were carried out as well as three simplex PCRs (EMPa15, UDP98-412 and PceGA34). The multiplex reactions were carried out using the following combinations of primers: Multiplex–A: EMPaS10, EMPaS12, and EMPaS14; Multiplex–B: EMPa4, EMPa5, EMPaS2, EMPaS6. The forward primers were labelled with fluorescent dyes FAM, HEX, or NED (Applied Biosystems). The Multiplex PCRs were performed according to the protocol of Vaughan and Russell . The simplex PCR for the locus EMPa15 followed the protocol of Clarke and Tobutt . The conditions for the remaining two simplex PCRs were: 94°C for 2 min. followed by 35 cycles of 94°C for 30 s, Ta°C for 45 s, and 72°C for 60 s with an elongation step of 72°C for 5 min. PCR products were genotyped with a capillary sequencer (SpectruMedix model SCE 960) and scored using GenoSpectrum software.For each of the microsatellite loci, we ascertained the total number of alleles, the range of allele sizes, and the gene diversity HE to assess overall polymorphisms. Additionally, for averages over loci we computed the average number of observed alleles A, the observed heterozygosity HO, the expected heterozygosity HE, and Wright's inbreeding coefficient FIS for each of the four seed stands. The software ARLEQUIN was applied for the analysis mentioned above while conformation of genotypic frequencies to the Hardy-Weinberg equilibrium was tested with the program GENEPOP version 4.0 . Differences between stands were analysed based on allele identity with the statistic FST and standardized values G′ST calculated according to Hedrick . The private allele method published in Barton and Slatkin was used to estimate the number of reproductively successful migrants per generation (Nm).Spatial genetic structure was assessed according to Rousset by testing the effect of distance on genetic divergence. The relationship between genetic distance, expressed as FST/(1−FST) and the natural logarithm of the geographic distance was analysed with Spearman's rank correlation and its significance was assessed through the Mantel test with the software program GENEPOP version 4.0 . […]

Pipeline specifications

Software tools Arlequin, Genepop
Application Population genetic analysis