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Intermediate outcrossing rates are theoretically predicted to maintain effective selection against harmful alleles, but few studies have empirically tested this prediction with the use of genomic data. We used whole-genome resequencing data from alpine rock-cress to study how genetic variation and purifying selection vary with mating system. We find that populations with intermediate outcrossing rates have similar levels of genetic diversity as outcrossing populations, and that purifying selection against harmful alleles is efficient in mixed-mating populations. In contrast, self-fertilizing populations from Scandinavia have strongly reduced genetic diversity and accumulate harmful mutations, likely as a result of demographic effects of postglacial colonization. Our results suggest that mixed-mating populations can avoid some of the negative evolutionary consequences of high self-fertilization rates.

The RCA procedure is robust. A. RCA does not introduce technical variability. Each individual plasmid DNA was run as four independent replicates for both conditions (with or without RCA). The technical reproducibility of the variant frequency call was evaluated by calculating the standard deviation within the four replicates. Value are plotted for each individual position of the reference sequence, for the two plasmids sequenced. Data are directly extracted from mpileup analysis applying a VarScan p-value threshold of 0.001. Left panels: plasmid 1, right panels: plasmid 2, top panels: crude plasmid DNA (no RCA amplification), bottom panels: RCA amplified plasmid DNA. B. Origin of the nonspecific RCA products. The sequences surrounding the unique position for which a large difference was observed between the crude plasmid DNAs and the RCA products were aligned. The sequence obtained from contaminating NTC reads is shown. The common (between the two plasmids) non-accurate position is boxed (“G” in the NTC, “A” in the two plasmids). This region is part of the plasmid DNA origin of replication. (PPTX 178 kb)

Coverage drop at the position 310 human C-stretch. The human sample #12878 from the CEPH family 1463 was sequenced either following the pipeline described in this paper or from a whole genome PCR free library (generated in our lab). The relative coverage reported by mpileup was plotted against each single position of the reference genome with a zoom in the C-stretch at position 310. (PPTX 233Â kb)

Primer sequences used for qPCR. (DOCX 37Â kb)

Sanger sequencing validation for the inheritance of high level heteroplasmy SNV. Slide 1. CEPH family 1463 pedigree chart. Slide 2. Level of heteroplasmy calculated from the MitoRS data, and the Sanger sequencing data in both forward and reverse orientation. MitoRS calculated frequencies are highlighted in red (homoplasmy, > 98%) or orange (high frequency heteroplasmy, between 10% and 98%). The corresponding Sanger frequencies are highlighted in green for easier visualization. Slides 3 to 10. Chromatograms highlighting a difference between the two family members. The positions considered are shown with a black arrowhead. Slide 10. Virtual gel visualization (on a LabChip GX - Perkin Elmer) of the PCR amplification products analyzed by Sanger sequencing. (PPTX 513Â kb)

Overview of the MitoRS wet lab method. Total DNA (5 ng) is amplified by RCA. Sequencing libraries are subsequently generated using the Nextera XT kit from Illumina, starting from 1 ng amplification product. Libraries are pooled at equimolar ratios and sequenced on a HiSeq 2500 (Illumina) using rapid mode for a paired-end run of 2 x 150 cycles. (PPTX 40 kb)

Overview of the MitoRS analysis methods. FastQ files are aligned with BWA to the original, and an origin-shifted version, of the DNA reference sequence (refer to Additional file 2 for details). Variant frequency is evaluated by samtools mpileup. Low frequency variants are finally identified using VarScan 2. Datasets generated from both the original reference and the shifted reference are merged, keeping only the per position values from the dataset with the highest coverage. Data are further processed to generate 1) a csv file summarizing the sequencing results observed at each individual mitochondrial DNA position and 2) a fastA file representing the corresponding consensus sequence. More details can be found in the Methods section. (PPTX 264Â kb)

Mouse SNV frequencies are homogenous within the 88 position analyzed. A. Individual SNV frequency. For the 12 mouse mtDNA mixture ratios tested, the frequencies measured for each SNV (88 in total) were plotted and summarized as a boxplot. The boxplot whiskers highlight the extreme values (min and max). Note that the scale is different for each plot. The eight positions showing the highest frequency underestimation are highlighted in blue. These data were used to build the Fig. 4. The raw data are available from the Additional file 7. B. The eight SNV with underestimated frequency are located into two dense clusters. For each 88 SNV, the deviation from the theoretical frequency was calculated and plotted versus their position in the mitochondrial genome. The eight positions showing a systematic frequency underestimation highlighted in A. are also shown in blue. They cluster into two very short genomic regions. (PPTX 484 kb)

Supporting Information. (DOCX 56Â kb)