Computational protocol: Range shift and introgression of the rear and leading populations in two ecologically distinct Rubus species

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Protocol publication

[…] Primers for nuclear genes were designed based on the R. palmatus EST libraries. The EST libraries were constructed from cDNA synthesized from R. palmatus mRNA samples from Yakushima Island. Sequences were generated using the GS FLX (454 Life Sciences, Branford, CT). The GS De Novo assembler version 2.3 (provided by 454 Life Sciences) was used for sequence assembly, and the quality trimming was performed with default settings. The function of homologous genes was predicted using Arabidopsis RNA protein reference sequences (, threshold E-value 10-20 ). We found a homolog sequence as a hypothetical protein for some loci in the Arabidopsis reference sequences and in the reference sequences of other species (Populus and Vitis). Primers were designed based on the aligned sequences between R. palmatus and R. grayanus using Primer3 version 0.4.0 []. Twelve nuclear loci were sequenced for the populations at the species boundaries to infer past population dynamics of the two species. Since the contact zones on Yakushima Island are geographically limited, we sequenced 5 additional nuclear loci (giving a total of 17 nuclear loci) to increase the resolution of their genetic structure analysis, in addition to one chloroplast region (trnH-psbA) used to trace their maternal species. Loci information was described in Additional file : Table S1 in the Additional file of Supporting Information.Genomic DNA was isolated from dried tissues using the modified CTAB method []. PCR reaction mixes contained 0.2 mM dNTPs, 1 × reaction buffer, 0.5 mM of each primer, 1 U of Taq DNA polymerase (TAKARA BIO) and approximately 20 ng of DNA. PCR amplifications were performed under the following conditions: 5 min at 94°C, followed by 30 cycles of 30 sec. at 94°C, 30 sec. at 55°C, and 1 min at 72°C. The amplified products were sequenced using ABI Prism BigDye Terminator v. 3.1 on an ABI3730 DNA Analyzer. Individual haplotypes were reconstructed using the PHASE algorithm []. [...] We estimated the number of segregating sites, S, the total nucleotide diversity, and the nucleotide diversity within populations for the total (π (total), θ (total)), synonymous (π (s)), and non-synonymous (π (a)) sites []. The HKA test [] was conducted to test for neutral evolution across loci: we used a maximum likelihood ratio test [] to evaluate a model, assuming that one of the genes is not neutral, against a null model assuming that all genes are neutral. Statistical significance of Tajima’s D [] and Fu and Li’s D (Fu and Li, []) was also tested with 1000 coalescent simulations using a standard neutral model. Simulations of neutrality tests were performed using the programs SITES and HKA (J. Hey Bayesian clustering program STRUCTURE var. 2.3.3 [] was used to assess the genetic clustering of individuals from the two species and the contact zones on Yakushima Island. We randomly collected one SNP from each locus. We performed five independent runs with a burn-in of 5.0 × 105 and additional 5.0 × 105 steps with the admixture model, and estimated log-likelihoods for each number of clusters (1 < K < 6) for the populations from the species’ ranges. For the contact zones on the island, we assumed K = 2, two gene pools from the two putative parental species. […]

Pipeline specifications

Software tools Newbler, Primer3, HKA
Applications Population genetic analysis, qPCR