Great deal of present-day research in biology is based on genomic data that are processed and analysed in the context of a linear reference genome. Typical examples of this are whole-genome sequencing studies where sequencing reads are mapped to the reference genome and the characteristics of interest are derived from local dissimilarities and statistics based on the alignments (Korneliussen et al., 2014;Schraiber & Akey, 2015). Reliability of those characteristics and the conclusions drawn from them depend not only on the quality of the sequencing data but also on the quality of the reference genome. Assembling and evaluating the quality of reference genomes is not easy (Baker, 2012;

The utility of genome-wide sequencing data in biological research depends heavily on the quality of the reference genome. Although the reference genomes have improved, it is evident that the assemblies could still be refined, especially in non-model study organisms. Here, we describe an integrative approach to improve contiguity and haploidy of a reference genome assembly. With two novel features of Lep-Anchor software and a combination of dense linkage maps, overlap detection and bridging long reads we generated an improved assembly of the nine-spined stickleback (Pungitius pungitius) reference genome. We were able to remove a significant number of haplotypic contigs, detect more genetic variation and improve the contiguity of the genome, especially that of X chromosome. However, improved scaffolding cannot correct for mosaicism of erroneously assembled contigs, demonstrated by a de novo assembly of a 1.7 Mbp inversion. Qualitatively similar gains were obtained with the genome of three-spined stickleback (Gasterosteus aculeatus).

Map distance is one of the key measures in genetics and indicates the expected number of crossovers between two loci. Map distance is estimated from the observed recombination frequency using mapping functions, the most widely used of those, Haldane and Kosambi, being developed at the time when the number of markers was low and unobserved crossovers had a substantial effect on the recombination fractions. In contemporary high-density marker data, the probability of multiple crossovers between adjacent loci is negligible and different mapping functions yield the same result, that is, the recombination frequency between adjacent loci is equal to the map distance in Morgans. However, high-density linkage maps contain an interpretation problem: the map distance over a long interval is additive and its association with recombination frequency is not defined. Here, we demonstrate with high-density linkage maps from humans and stickleback fishes that the inverses of Haldane’s and Kosambi’s mapping functions systematically underpredict recombination frequencies from map distance. To remedy this, we formulate a piecewise function that yields more accurate predictions of recombination frequency from map distance. Our results demonstrate that the association between map distance and recombination frequency is context-dependent and without a universal solution.

Crossovers in meiosis increase genetic variability in gamete pool by breaking existing and creating new haplotypes, resulting in novel combinations of alleles. Frequencies of crossovers are known to differ between sexes in many species, but possible sex differences in localization of crossovers have seldom been studied in detail. Here, we address several long-standing questions regarding frequency and distribution of crossovers, with aid of in-depth analyses of ultra-dense sex-specific linkage maps of nine-spined (Pungitius pungitius) and three-spined (Gasterosteus aculeatus) sticklebacks. We observe that males have typically one crossover per chromosome, independently of the chromosome length, whereas in females the number of crossovers increases with the chromosome length, being typically more than two. We show that these differences largely explain more uniform crossover distribution of females and the fact that females have crossovers closer to the centromere. Furthermore, highly constrained crossover localization reveals that the relationship between linkage and genetic distance varies inside the chromosome. Our analyses show that recombination rate is highly heritable in females (h^2=0.56-0.65), and thus, able to evolve in response to selection. Similar results for the two species that diverged 26 million years ago suggest that our findings are general, at least in the context of stickleback fishes.

Understanding what determines species and population differences in levels of genetic diversity has important implications for our understanding of evolution, as well as for the conservation and management of wild populations. Previous comparative studies have emphasized the roles of linked selection, life-history trait variation and genomic properties, rather than pure demography, as important determinants of genetic diversity. However, these findings are based on coarse estimates across a range of highly diverged taxa, and it is unclear how well they represent the processes within individual species. We assessed genome-wide genetic diversity (pi) in 45 nine-spined stickleback (Pungitius pungitius) populations and found that pi varied 15-fold among populations (pi min approx. 0.00015, pi max approx 0.0023) whereas estimates of recent effective population sizes varied 122-fold. Analysis of inbreeding coefficients (F ROH) estimated from runs of homozygosity revealed strong negative association between pi and F ROH. Genetic diversity was also negatively correlated with mean body size and longevity, but these associations were not statistically significant after controlling for demographic effects (F ROH). The results give strong support for the view that populations' demographic features, rather than life history differences, are the chief determinants of genetic diversity in the wild.