Sex-specific markers and meiosis
Extremely large male:female recombination rates were observed among all the linkage groups with the exception of SL12, making this one of the largest differences in sex-specific recombination rate reported for any animal species. The sex linkage group (SL15) was not included in the main analysis due to lack of markers genotyped in both sexes (N = 80). However, the data indicate that overall SL15 may have the highest sex-specific differences in recombination rate (29.2:1). Among all the other linkage groups the average male:female ratios were 10.49:1.
SL15 was also identified as the sex linkage group. A SNP marker localized to scaffold LSalAtl2s6658 associated with SNP AX-98427605 (#43100) has been identified as being close to the sex-specific marker in the salmon louse (Messmer et al. 2018), but differs from the exact SNP location of the RAD-seq sex marker Lsa101901 identified by Carmichael et al. (2013). This SNP which overlaps the prohibitin-2 gene is associated with segregation polymorphisms in the female parent indicating that sea lice have a ZW sex-determining system (Carmichael et al. 2013). Marker 43100 displayed unusual progeny genotype patterns that did not match Mendelian expectations across most of the families surveyed, with the exception of Dame 7, where it was observed that the marker localized to SL15. Dame 6 was also heterozygous at marker 43100 and expected genotypic patterns were observed in the progeny, but the marker was removed from the analysis due to segregation distortion. All males at marker 43100 were T/T homozygotes, whereas females were either G/G homozygotes or G/T heterozygotes. Marker 28506, along with marker 43100 were located within scaffold LSalAtl2s6658 and marker 28506 was localized to 53.35 cM on the composite male sire 5a SL15 map (overall composite map length = 106.61 cM Online Resources 2, 8). The weight adjusted map position would be 63.23 cM given that the overall map length among all male parents was 126.36 cM for SL15. One additional marker (49103) could have localized to this scaffold as well, but this marker although showing variation in all the mapping families was excluded from analysis as the progeny genotypes did not conform to Mendelian expectations. Given the large number of markers (1316) that showed unexpected genotypes in one or more of the mapping panels, and that were not observed to conform to Mendelian expectations in any of these families, we examined the possibility that some of these markers may be inherited in a hemizygous fashion. Hemizygous inheritance has been identified in invertebrate species (Kaiser and Bachtrog 2010; Lima 2014), and this mode of transmission is most often associated with markers on the sex chromosomes (Qvanström and Bailey 2009), especially highly evolved sex chromosomes that display heteromorphic differences. The 1316 markers that were refractory to demonstrating any type of normal inheritance pattern were queried across all 8 mapping families for conformation to possible hemizygous models of inheritance (Table 5) using the program Hemizygous_Recode (Danzmann 2018). It was observed that from 230 to 270 markers in the male parents, and from 113 to 155 markers in the female parents could result from resolved hemizygous inheritance in the alternate parent (Table 6). These values are actually conservative, as in many instances, one of the two parents, or both parents had overlapping genotype patterns that could potentially indicate hemizygosity, but due to confounding overlapping patterns of expressed alleles versus null alleles, the genotypes could not be resolved.
The new hemizygous marker set was first assembled into possible linkage groups using a LOD = 3.0 clustering in OneMap. In all parents examined this resulted in one large assemblage and several smaller clusters that ranged from 2 to 10 markers along with a few unlinked markers. The large cluster of markers constituted between 72.7–80.2% of all markers in females, and from 74.9 to 82.6% of the markers in males, if sire 9 is excluded. In the progeny from sire 9 it was observed that only 63.9% of the markers fell into 1 large cluster. A large number of markers were, however, excluded in sire 9 prior to analysis due to the detection of segregation distortion (P < 0.01)(Table 6). Subsequent assemblage of these markers with pre-existing markers on the map revealed that in all parents, the single large cluster of hemizygous markers was part of the sex linkage group SL15 (Online Resource 8). In total, 574 markers were placed onto SL15 in both sexes, with 494 markers having hemizygous inheritance patterns. By comparison, 419 hemizygous markers were localized to all the other linkage groups in the sea louse genome combined, with 27 hemizygous markers remaining unlinked to any linkage group at a LOD = 4.0 threshold (Online Resource 9). Two linkage groups (SL04, SL05) had more than 40 hemizygous markers localized, while only 1 hemizygous marker was detected within SL12. Other linkage groups had between 16 and 37 hemizygous markers assigned. Another feature of this analysis was the observation that the numerous markers (N = 147 within SL01-14 and N = 460 within SL15) were consistently identified as possessing hemizygous inheritance patterns across multiple parents. This attests to the widespread regulation of allelic heterogeneity at these genomic locations, and most notably within SL15. Another interesting feature of the hemizygous markers was that the SL15 markers exhibited weak linkage or co-segregation with some of the markers on SL01 and SL05 in sires 3a and 5a, respectively (data not shown).
The genetic map for SL15 was reconstructed with the inclusion of the hemizygous markers to assess whether the general 10-fold increase in marker numbers influenced map length estimates and recombination ratios. Similar to the methods described above, ZRCs were first established within SL15, and their relative ordering established using the record option in OneMap. However, given the smaller number of markers present in SL15 compared to the other linkage groups, singleton markers were also included in this build. With the inclusion of the hemizygous markers into the SL15 map, the sample size weight-averaged male linkage group size was 126.36 cM, while the estimate for the female map was 4.51 cM. As expected, average map length estimates increased by 29.79 cM in males, but only 1.17 cM in females, highlighting the extreme conservation of recombination in the heterogametic sex. Family specific male and female genetic maps and the overlap matrix of map positions for SL15 are given in Online Resource 8. The male map positions are also shown in a uniform ‘head–tail’ ordering among all the male maps. The revised estimate for male:female recombination differences (28.02:1) was the largest detected for any linkage group and was very similar to the original estimate based upon a much smaller number of markers.
The detection of such a large number of putative hemizygous markers that localize to both male and female maps is unusual. When sex chromosomes evolve, the homolog that does not contain the sex-determining region generally exhibits hemizygous segregation due to the loss of complementary alleles on the sex-bearing chromosome (Qvanström and Bailey 2009). For Z-W species, this type of segregation pattern may be expected for the heteromorphic or heterogametic sex, and, therefore, we would expect the prevalence of such markers to be much greater for female segregation patterns compared to males. Although the direction of this pattern was supported with the current dataset (i.e., a greater number of male markers were scored indicating hemizygous segregation in the female parent), there were also a very large number of female-scored markers indicating male hemizygosity. This suggests that sex chromosome diversification may be incipient in L. salmonis.
A survey of chromosome numbers in the orders Harpacticoida and Cyclopoida, which are more closely related to the Siphonostomatoida (Eyun 2017), to which L. salmonis belongs, reveals that the maximum haploid chromosome number is n = 12 (Yang et al. 2008). In contrast the higher chromosome number detected in L. salmonis, suggest that some of the smaller chromosomes within Siphonostomatoida may be derived from chromosomal rearrangements, similar to the evolution of sex chromosomes in some other invertebrates (Blackmon et al. 2017), involving fusions, fissions, small inversions, and translocations. Such rearrangements may lead to unequal and differential degrees of pairing during meiosis. During transitional states of sex chromosome evolution, varying states of hemizygosity may be detected due to varying and occasional rounds of recombination between the sex chromosomes (Qvarnström and Bailey 2009; Bachtrog et al. 2014). Recombination levels are higher between incompletely diverged sex-determining homologs (Lima 2014), and this retards the rate at which full sex determination becomes established following meiotic segregation. Incomplete sex-specific differentiation, may, however, be augmented via sex-specific differentiation in gene expression levels (Bachtrog et al. 2014; Jorden and Charlesworth 2012). Sex-specific differences in L. salmonis gene expression levels exist (Poley et al. 2016b), with higher levels detected in males when expression is detected in both sexes for certain genes, while for other transcripts male-only expression is evident. It will be of interest to couple the location of these differentially expressed transcripts to the genomic builds for sea lice and ascertain if a substantial portion align to the hemizygous markers on SL15.
Chromatin diminution has also been observed within copepod species (Wyngaard and Gregory 2001; McKinnon and Drouin 2013) which involves the complete deletion of portions of the genome during early mitotic divisions following pronuclei fusions post-fertilization. The deletion of genomic DNA is reportedly only expected to occur in somatic cell lines with gonadal cell lines retaining a complete complement of nuclear DNA (Standiford 1989). Deletion of entire segments of DNA could result in a mosaic pattern of DNA retention whereby an entire locus could be lost in one parent and wholly or partially retained in another parent at the DNA breakpoint. To investigate this possibility, we re-examined the segregation patterns of loci initially excluded due to non-conformance to Mendelian segregation patterns for the presence of null locus inheritance in one of the parents using the program Homozygous_Null_Recode (Danzmann 2018). This program identifies normal Mendelian or hemizygous inheritance modes in one parent, coupled with the complete loss of allelic segregation at the same locus in the alternate parent (see Table 7). Such patterns could result from the process of chromatin diminution. A total of 176 markers were observed to comply with these models of inheritance, and 129 of these markers were localized to the existing salmon louse linkage groups (Online Resource 10). Interestingly, only a single marker matching these models was located within SL15. While the higher proclivity to possess hemizygous markers on SL15 compared to homozygous null markers on the sex linkage group is not entirely clear at present, it may suggest that there is a need to retain at least one copy of the sex chromosome complement in all cells within the species. It will be of interest to couple the location of these differentially expressed transcripts to the genomic builds for sea lice and ascertain if a substantial portion align to the hemizygous markers on SL15. Future research should also determine the genomic location of these markers, and whether genes closely linked to genomic regions with a potential for complete or partial loss display differential sex gene expression.
Mate Selection, differential family survivorship, and recombination
An unexpected finding from the current study was the lack of detection of multiple half-sib families despite having mating arenas established with 10 or more virgin females in each arena. In five lots, all copepodids genotyped were derived from a single family, while in 3 lots, only two paternal half-sib families were detected. In one of these lots, the number of surviving progeny was very low in one of the half-sib families (5b) and was not included in the initial map builds. When final average map lengths were calculated within each parent (which reflect recombination levels), it was evident that bi-parental averages in linkage group lengths were very similar to one another across the families (Fig. 6). This indicates that males possessing either very low, intermediate, or high recombination rates also mated with females possessing these intrinsic recombination rates. This suggests that some type of assortative mating may be occurring in the salmon louse, or that survivorship of the progeny is favoured if the parents have similar intrinsic recombination rates. Although the current dataset is small it does indicate reduced progeny viability if the parental genomic backgrounds differ too greatly in their intrinsic recombination levels. In fact, from a preliminary assessment of the viability of surviving copepodids sampled among families, it appears that an inverse relationship may exist between the number of surviving progeny produced and the level of recombination in their parents (Fig. 7). Families with higher levels of recombination in both the male and female parent produce lower numbers of surviving offspring while the opposite is true for families where both parents have lower recombination rate. This latter observation must be treated with caution, however, as too few families and populations have been tested to assess the general validity of the association.
Assortative mating has been reported to occur in the purple sea urchin (Strongylocentrotus purpuratus) to account for the differences observed in sperm and egg binding protein patterns that exist in this species (Stapper et al. 2015), and mosquitos (Anopheles gambiae) due to X-chromosomal genomic islands of selection (Aboagye-Antwi et al. 2015). Further testing in the sea louse would be needed to confirm assortative mating using pedigreed lines of low (L) and high (H) recombination rate families. If four types of mating chambers were established (i.e., L × L, L × H, H × L, and H × H) and the highest production of egg strings with viable progeny among multiple females occurred within L × L chambers it would confirm that assortative mating does occur based upon background genomic structure within L. salmonis, and that viability may be inversely related to recombination differences between the parents. Conversely, If mating is at random, but survivorship is poorer among crosses with parents possessing higher or more divergent recombination rates, then a census of the proportion of females in mating arenas with developing egg strings would need to be performed. In other words, if initial fertilization levels were random among test arenas, with subsequent lower rates of offspring viability among H × L, L × H and H × H groups it would suggest some form of selection against genomes where recombination levels differed greatly. Attempted mating could first be assessed by examining the virgin females placed into a mating arena for the presence of spermatophore plugs, after a set time period. If all females have been potentially inseminated (i.e., all possess a spermatophore plug) but only copepodids from a small number of females are censused then it would imply that some type of differential selection has influenced family survival. Conversely, the presence of spermatophores in only a subset of the females present would indicate a degree of assortative mating by males.
If the lowest viability occurred in the female (H) × male (L) crosses, and possibly H × H backgrounds, it would support the hypothesis of accelerated evolutionary change in ZW species (Ritchie 2007; Saether et al. 2007). Recombination among mate choice loci is generally accepted as being an impediment to population diversification (Nei 1969; Felsenstein 1981) due to the fact that if many such loci are involved and they are randomly scattered among chromosomes, then no clear assemblage of distinct ‘trait choice’ loci are expected to arise within populations. Clustering of such loci into a region of linkage disequilibrium or their concentration within sex chromosomes has been postulated to be a way of accelerating evolutionary rates based upon mate choice; as such regions would be largely non-recombining. Evidence is accumulating that in ZW determining species evolutionary rates may be elevated due to tracking of male-specific traits through the female-derived Z-chromosome, upon which females subsequently make mate choice decisions (Ritchie 2007). Females of a specific Z-type prefer to mate with males of a similar Z-type. Recombination rates will be crucial in determining the extent of differentiation among family lines. These models predict that in species with higher rates of recombination, the Z-chromosome will change at a faster rate compared to ancestral populations and female mate choice patterns will seek to match males with the highest concordance to their own intrinsic Z-chromosome configuration. Otherwise, chromosomal rearrangements between fast and slow-evolving Z chromosomes will cause incompatibilities leading to lower ZZ male viability. Therefore, another caveat that arises from the proposed experiment described above is that skewed sex ratios should be observed in the progeny from hybrid recombination-type tanks, with a lower percentage of males being produced. Therefore, in H × H background crosses a greater range of variation may be observed. In certain families the Z/W allelic combination may be largely incompatible, while in others they may be highly compatible leading to a greater array of surviving progeny levels derived from H x H parental backgrounds. Variable and significant sex ratio differences have been observed among family lines of T. californicus (Voordouw et al. 2005), suggesting that skewed family sex ratios can occur in copepods which may relate to the differences in genomic compatibility conditioned upon recombination ancestry among parental pairs.
In contrast to birds, male mate choice appears to be prevalent across copepod species (Burton 1985; Heuch and Schram 1996) including L. salmonis (Ritchie et al. 1996a; Hull et al. 1998; Birkett et al. 2002; Todd et al. 2005). Male preferences are highest for unfertilized virgin females followed by later advanced copepodid stages (Hull et al. 1998), which is similar to findings in other copepods (Burton 1985). How assortative mating based upon recombination differences within individuals operates remains unclear, but may involve chemosensory cues. Indeed, L. salmonis males react, and are directionally motivated towards female extracts conditional upon the female’s sexual stage (Birkett et al. 2002) and these responses are removed by ablation of male antennae (Todd et al. 2005). Therefore, smaller demic groups of sea lice possessing intrinsically different basal levels of recombination may differ sufficiently in their chemical composition due to shared past ancestry, that mate selection may be ‘fine tuned’ among these demes. Since mate choice appears predominantly male-driven in copepods (Hull et al. 1998; Ritchie et al. 1996a), chemosensory matching across the sexes is suggested to play a role. Here, lack of recombination within chromosomes may drive “species” or even “demi-specific” traits for selection. The current study has shown that the largest male:female recombination rate differences occur with the sex chromosome SL15 making it tempting to speculate that the Z-chromosome or even the undifferentiated W-chromosome may preferentially house such “mate choice specific” genes. It will be of interest in the future, once the current linkage map is aligned to genomic scaffolds, to ascertain if the sex chromosomes possess higher complements of chemosensory genes such as olfactory G-protein coupled receptors.
Although monogamy was once thought to be prevalent across copepods, because it was thought that sperm plugs would prevent successful fertilizations by successive males (Ritchie et al. 1996b), the report of polyandry occurring in L. salmonis (Todd et al. 2005) has cast doubt upon this supposition. If polyandry does occur to a wide degree among L. salmonis populations, it could suggest some degree of female mate choice also occurs. It will be of interest to assess if females prone to polyandry are a result of more divergent recombination background in initial pairings and if subsequent pairings more closely match their own ‘background recombination level’. Future studies could include mating arena trials where both males and females of known recombination background are placed together after the females have been inseminated by another male. Higher levels of polyandrous spermatophores detected within females introduced to males of similar recombination background would suggest some degree of female mate choice. In addition, higher copepodid survival derived from ‘similar’ sperm, versus ‘dissimilar sperm’ in such crosses could also support the concept of adaptive selection occurring in such crosses.
Sex-specific sex chromosome recombination rates, in general, tend to be higher within the sex chromosome itself, than found within other autosomal pairs (Burt et al. 1991), and this was also observed in this study where the female-specific SL15 W-chromosome had extremely low levels resulting in the highest linkage group specific male:female recombination ratio. This difference was 29:1 when only the Mendelian compliant markers were assessed and was only slightly adjusted to a ratio of 28:1 (male:female) when the addition of hemizygous markers was considered. Biological reasons for the suppression of recombination in the heterogametic sex are still unclear, but the suggestion that a suppression of recombination maintains linked loci critical to sex-determination is the most powerful (Nei 1969; Burt et al. 1991). The extension of suppressed recombination levels to the autosomal pairs is, however, more of an enigma. Researchers have suggested that gametic selection in plant species differs between the sexes and that this drives recombination levels in general (Lenormand and Dutheil 2005), but empirical evidence for this hypothesis is still generally lacking in animal species.
If gametic selection occurs in animals, evolutionary rates may be accelerated in ZW species due to the fact that gametic selection is likely to be more accelerated in females compared to males. Only 1 of 4 meiosis products survives in females, whereas all 4 are potentially viable in males. If some degree of autosomal meiotic drive accompanies the segregation of the Z and W chromosomes at meiosis I, then quite significant differences (i.e., non-random) should exist in the phase configuration of gametes across linkage groups.
The large sex-specific recombination levels detected in this study appear to be unusual among crustacean species studied to date, with recombination ratio differences approximating 1:1 ratios between the sexes (Perez et al. 2004; You et al. 2010; Cui et al. 2015). Admittedly, the number of species with available sex-specific maps is limited, and, therefore, general assessments of these differences are unwarranted at the present time. However, the Chinese mitten crab (Eriocheir sinensis) has also been reported to have a ZW sex-determining mechanism and in this species, the female sex linkage ratio was actually larger than observed in males (Cui et al. 2015). The most recent genetic map for this species estimates female:male recombination rates at 1.13:1 (Qiu et al. 2017). A genetic map is currently available for the copepod species Tigriopus californicus but this map is entirely male-based (Foley et al. 2011; Pritchard et al. 2011), as female meiosis have been reported to be achiasmate in this species (Ar-Rushdi 1963; Burton et al. 1981). Environmental sex determination is also presumed to occur in this species (Burton et al. 1981; Foley et al. 2011). Our findings confirm that although female recombination levels are extremely low, they do not appear to be achiasmatic in L. salmonis. Some evidence for low levels of female recombination were also incidentally reported in Tigriopus (Foley et al. 2011) but were excluded as genotyping errors. We cannot entirely exclude the possibility of some unusual mode of female-specific gamete transmission influencing recombination events. Although most meioses generated in female copepods appear to be achiasmate, recombination may be possible through extended multiple chromosomal ring formations similar to those that have been observed during female meioses in copepod species such as Mesocyclops edax (Chinnappa and Victor 1979). Such formations could lead to ectopic recombination within homologs which may account for the poor congruence in male and female map orders observed in the current study. Current cytological studies are lacking in L. salmonis such that the extent to which bivalents or different types of meiosis I configurations exist in this species is unknown.
Crossover rates; segregation distortion; and heterozygosity
Although the current estimates of recombination differences in L. salmonis are not aligned with estimates of genomic build distances, the inferences made on the distribution of recombination events is still insightful. Plots showing the total recombination counts per recombination domain (which roughly span 10 cM intervals on the references maps) (e.g., Fig. 8 and Online Resource 7) indicate that crossover hotspots are fairly randomly distributed along the lengths of all male chromosomes. However, for all linkage groups with the exceptions of SL01, SL02 and SL06, a recombination hotspot was localized to one end of the chromosome (occurring in either the ultimate or penultimate domain). For SL01/06 a region of very high recombination was only detected in the centre of the linkage group, while for SL02, both ends of the linkage group possessed terminal domains with higher recombination levels. The predominant pattern is more consistent with the pattern of recombination observed in another crustacean Daphnia magna, where recombination rates were all reported to be elevated towards the terminal ends of the linkage groups (Dukic et al. 2016). This suggests that the ends of linkage groups surveyed in L. salmonis with elevated recombination rates may be the telomeric ends of the chromosomes.
More than half of the 13 linkage groups surveyed (SL01, 03, 05, 06, 08, 09, 10, 11, 14) had a recombination hotspot immediately adjacent to a recombination coldspot, and within SL06, the region of very high recombination (domain 7) was flanked both up- and downstream by recombination coldspots. Regions of low recombination often flank hotspots in mammalian genomes (Lichten and Goldman 1995; Hey 2004) and often coincide with regions of lower gene density. In humans, approximately 25,000 hotspots have been identified (Myers et al. 2005) through linkage disequilibrium mapping, and display a great deal of heterogeneity on a fine scale, that is not readily evident from broad scale surveys such as the ones depicted in this study. Future alignments of the current genetic map to genomic scaffolds in the salmon louse will be able ascertain if crossover frequency is related to higher gene density.
Levels of overall nucleotide variability have been reported to be directly related to recombination rate differences in the genomes of animals (Begun and Aquadro 1992; Nachman 2002). Increased genetic variability is found in regions of higher recombination. This suggests that overall levels of genetic variability detected among individuals (i.e., total number of heterozygous individuals) should also be coupled to higher regions of recombination. We investigated this relationship in L. salmonis by documenting the total number of genetically variable SNPs found within each recombination domain within a linkage group, using the program Polymorph_Count_X_Recom_Domains (Danzmann 2018). This census included not only the polymorphic male markers that were used to construct the map, but also all of the polymorphic female markers occurring within a domain as well as doubly heterozygous markers of the type ab x ab. Such markers were initially excluded from the analysis since the phase of doubly heterozygous progeny for these markers cannot be ordered within phase maps. For this analysis, heterozygous markers that occurred wholly within a recombination domain were scored a value of 1.0 while markers that occurred in comparison parent recombination bins that overlapped either an upstream or downstream domain were scored a value of 0.5 within each overlapping domain (Online Resource 11). Results from this analysis weakly supported the association between increased recombination levels and genetic variability within a linkage group domain (Fig. 9; Online Resource 7; Online Resource 12). An increased level of genetic variability was significantly coupled to higher crossover levels in six (SL01, 06, 09, 10, 13, and 14) of 13 linkage groups examined, while this was marginally true (P < 0.1) for another two linkage groups (SL02 and SL08). Overall, however, in all linkage groups with the exception of SL04, the overall relationship between these two variables was positive, albeit extremely weak for SL05 and SL07.
The high levels of segregation distortion detected in linkage groups SL04, SL07, and SL11 (Fig. 5) may have contributed to the lack of a strong coupling between observed genetic variability and recombination rate. Markers exhibiting distortion would not have been included in map ordering and this could slightly bias the observed associations given that they were included in the polymorphism counts. Similarly, for SL06, the strong association between genetic variability and recombination level is due to the extremely high levels of variation observed within domain 7, which also possesses very high crossing-over levels. If this domain is removed from the analysis no association between the two variables is detected. SL01 also possessed an extreme polymorphism peak (> 15,000 polymorphic SNP markers in domain 9) that was coupled to extremely high crossover rates. When this point was removed, however, the association between variability and crossover rate was still significant (data not shown). Interestingly, SL06 also possessed regions of extreme segregation distortion which could have influenced map order structure (Fig. 5).
Recombination coldspots have been empirically and theoretically linked to regions that may contain higher numbers of coadapted genes related to species survival. These so-called “genomic islands of selection” have been postulated to arise through various evolutionary forces, but are recognized as regions more refractory to change that may result from recombination events that disrupt favourable allelic combinations (Yeaman 2013). Genomic rearrangements have been predicted to be important in localizing coadapted genes into smaller genomic regions that more readily ameliorate the disruptive effects of recombination. Researchers have reported that gene densities are reduced in regions of lower recombination, and that these regions may also accumulate higher densities of transposable elements (TEs) (Fontinallas et al. 2007; Dolgin and Charlesworth 2008). In contrast, other studies have reported elevated levels of recombination in regions of high TE density (Everitt et al. 2014), or have found no association between recombination and TE density (Adrion et al. 2017). The current study provides a framework map that has identified recombination hot- and coldspots within linkage groups. Future coupling of these map regions to genomic scaffolds will facilitate an in-depth analysis of the genomic regions for differences in transposable element and gene abundance in relation to differences in recombination levels within the chromosomes. Alignment of markers to scaffold builds will also help to resolve discrepancies in the ordering of markers among multiple mapping parents (see Lien et al. 2016).