Sex Locus Research Articles

Thelytokous Parthenogenesis in Unmated Queen Honeybees (Apis mellifera capensis): Central Fusion and High Recombination Rates

The subspecies of honeybee indigenous to the Cape region of South Africa, Apis mellifera capensis, is unique because a high proportion of unmated workers can lay eggs that develop into females via thelytokous parthenogenesis involving central fusion of meiotic products. This ability allows pseudoclonal lineages of workers to establish, which are presently widespread as reproductive parasites within the honeybee populations of South Africa. Successful long-term propagation of a parthenogen requires the maintenance of heterozygosity at the sex locus, which in honeybees must be heterozygous for the expression of female traits. Thus, in successful lineages of parasitic workers, recombination events are reduced by an order of magnitude relative to meiosis in queens of other honeybee subspecies. Here we show that in unmated A. m. capensis queens treated to induce oviposition, no such reduction in recombination occurs, indicating that thelytoky and reduced recombination are not controlled by the same gene. Our virgin queens were able to lay both arrhenotokous male-producing haploid eggs and thelytokous female-producing diploid eggs at the same time, with evidence that they have some voluntary control over which kind of egg was laid. If so, they are able to influence the kind of second-division meiosis that occurs in their eggs post partum.

The cutthroat trout Y chromosome is conserved with that of rainbow trout

Five genetic markers previously shown to be located on the sex chromosomes of rainbow trout (Oncorhynchus mykiss) were tested for linkage with the sex locus of Yellowstone cutthroat trout (Oncorhynchus clarki bouvieri) in a genetic cross created from a rainbow × cutthroat male hybrid. We show that the sex locus of both rainbow and cutthroat trout is on the same homologous linkage group. Fluorescence in situ hybridization (FISH) using a probe for the microsatellite marker Omm1665, which maps close to the sex locus of Yellowstone cutthroat trout, was used to identify the Y chromosome of cutthroat trout in the hybrid. The Y chromosome of cutthroat trout is sub-telocentric and lacks a DAPI band found on the short arm of the Y chromosome of some rainbow trout males.

Identification of two markers linked to the sex locus in dioecious Asparagus officinalis plants

One hundred decamer primers of random-amplified polymorphic DNA were tested on dioecious Asparagus officinalis plants to identify sex-linked molecular markers. One primer (S368) produced two markers (S368-928 and S368-1178) in female plants. These two DNA markers were identified in 30 male and female plants, respectively, and a S368-928 marker was proved to be linked to the female sex locus. The female-linked S368-928 marker was sequenced and specific primers were synthesized to generate a 928 bp marker of sequence characterized amplified regions (SCAR) in female plants, SCAR928. SCAR928 could be used to correctly screen homozygous mm female plants of A. officinalis. However, results of Southern blot analysis suggest that the hybridization pattern of S368-928 was presented in both sex plants.

Genetic and physical maps around the sex-determining M-locus of the dioecious plant asparagus

Asparagus officinalis L. is a dioecious plant. A region called the M-locus located on a pair of homomorphic sex chromosomes controls the sexual dimorphism in asparagus. The aim of this work was to clone the region determining sex in asparagus from its position in the genome. The structure of the region encompassing M should be investigated and compared to the sex-determining regions in other dioecious model species. To establish an improved basis for physical mapping, a high-resolution genetic map was enriched with AFLP markers closely linked to the target locus by carrying out a bulked segregant analysis. By screening a BAC library with AFLP- and STS-markers followed by chromosome walking, a physical map with eight contigs could be established. However, the gaps between the contigs could not be closed due to a plethora of repetitive elements. Surprisingly, two of the contigs on one side of the M-locus did not overlap although they have been established with two markers, which mapped in a distance as low as 0.25 cM flanking the sex locus. Thus, the clustering of the markers indicates a reduced recombination frequency within the M-region. On the opposite side of the M-locus, a contig was mapped in a distance of 0.38 cM. Four closely linked BAC clones were partially sequenced and 64 putative ORFs were identified. Interestingly, only 25% of the ORFs showed sequence similarity to known proteins and ESTs. In addition, an accumulation of repetitive sequences and a low gene density was revealed in the sex-determining region of asparagus. Molecular cytogenetic and sequence analysis of BACs flanking the M-locus indicate that the BACs contain highly repetitive sequences that localize to centromeric and pericentromeric locations on all asparagus chromosomes, which hindered the localization of the M-locus to the single pair of sex chromosomes. We speculate that dioecious Silene, papaya and Asparagus species may represent three stages in the evolution of XX, XY sex determination systems. Given that asparagus still rarely produces hermaphroditic flowers and has homomorphic sex chromosomes, this species may be an ideal system to further investigates early sex chromosome evolution and the origins of dioecy.

Diploid males sire triploid daughters and sons in the parasitoid wasp Cotesia vestalis

In the Hymenoptera, males develop as haploids from unfertilized eggs and females develop as diploids from fertilized eggs. In species with complementary sex determination (CSD), however, diploid males develop from zygotes that are homozygous at a highly polymorphic sex locus or loci. We investigated mating behavior and reproduction of diploid males of the parasitoid wasp Cotesia vestalis (C. plutellae), for which we recently demonstrated CSD. We show that the behavior of diploid males of C. vestalis is similar to that of haploid males, when measured as the proportion of males that display wing fanning, and the proportion of males that mount a female. Approximately 29% of diploid males sired daughters, showing their ability to produce viable sperm that can fertilize eggs. Females mated to diploid males produced all-male offspring more frequently (71%) than females mated to haploid males (27%). Daughter-producing females that had mated to diploid males produced more male-biased sex ratios than females mated to haploid males. All daughters of diploid males were triploid and sterile. Three triploid sons were also found among the offspring of diploid males. It has been suggested that this scenario, that is, diploid males mating with females and constraining them to the production of haploid sons, has a large negative impact on population growth rate and secondary sex ratio. Selection for adaptations to reduce diploid male production in natural populations is therefore likely to be strong. We discuss different scenarios that may reduce the sex determination load in C. vestalis.

Sex determination in the haplodiploid wasp Nasonia vitripennis (Hymenoptera: Chalcidoidea): A critical consideration of models and evidence

Sex determining mechanisms are highly diverse. Like all Hymenoptera, the parasitic wasp Nasonia vitripennis reproduces by haplodiploidy: males are haploid and females are diploid. Sex in Nasonia is not determined by complementary alleles at sex loci. Evidence for several alternative models is considered. Recent studies on a polyploid and a gynandromorphic mutant strain point to a maternal product that is balanced against the number of chromosomal complements in the zygote and a parent-specific (imprinting) effect. Research is now focused on the molecular details of sex determination in Nasonia.

Complementary sex determination in the parasitoid wasp Cotesia vestalis (C. plutellae)

In the Hymenoptera, single locus complementary sex determination (sl-CSD) describes a system where males develop either from unfertilized haploid eggs or from fertilized diploid eggs that are homozygous at a single polymorphic sex locus. Diploid males are often inviable or sterile, and are produced more frequently under inbreeding. Within families where sl-CSD has been demonstrated, we predict that sl-CSD should be more likely in species with solitary development than in species where siblings develop gregariously (and likely inbreed). We examine this prediction in the parasitoid wasp genus Cotesia, which contains both solitary and gregarious species. Previous studies have shown that sl-CSD is absent in two gregarious species of Cotesia, but present in one gregarious species. Here, we demonstrate CSD in the solitary Cotesia vestalis, using microsatellite markers. Diploid sons are produced by inbred, but not outbred, females. However, frequencies of diploid males were lower than expected under sl-CSD, suggesting that CSD in C. vestalis involves more than one locus.

Sex determination and inbreeding depression in an ant with regular sib-mating

Haplodiploidy is one of the most widespread mechanisms of sex determination in animals. In many Hymenoptera, including all hitherto investigated social species, diploid individuals, which are heterozygous at the sex locus, develop as females, whereas haploid, hemizygous individuals develop as males (single-locus complementary sex determination, sl-CSD). Inbreeding leads to homozygosity at the sex locus, resulting in the production of diploid males, which are usually sterile and constitute a considerable fitness cost. Nevertheless, regular inbreeding without diploid male production is known from several solitary wasps, suggesting that in these species sex is not determined by sl-CSD but alternative mechanisms. Here, we examine sex determination in an ant with regular inbreeding, Cardiocondyla obscurior. The almost complete absence of diploid males after 10 generations of brother-sister mating in the laboratory documents for the first time the absence of sl-CSD and CSD with two or a few unlinked sex loci in a species of social Hymenoptera. Queens, which mated with a brother, appeared to decrease the number of males in their brood, as expected from the relatedness relationships under inbreeding. In contrast, some colonies began to show signs of an inbreeding depression after several generations of sib-mating, such as shortened queen life span, higher brood mortality, and a shift to more male-biased sex ratios in some colonies, presumably due to lower insemination capability of sperm.

Single-locus complementary sex determination absent in Heterospilus prosopidis (Hymenoptera: Braconidae)

In the haplodiploid Hymenoptera, haploid males arise from unfertilized eggs, receiving a single set of maternal chromosomes while diploid females arise from fertilized eggs and receive both maternal and paternal chromosomes. Under single-locus complementary sex determination (sl-CSD), sex is determined by multiple alleles at a single locus. Sex locus heterozygotes develop as females, while hemizygous and homozygous eggs develop as haploid and diploid males, respectively. Diploid males, which are inviable or sterile in almost all cases studied, are therefore produced in high frequency under inbreeding or in populations with low sex allele diversity. CSD is considered to be the ancestral form of sex determination within the Hymenoptera because members of the most basal taxa have CSD while some of the more derived groups have other mechanisms of sex determination that produce the haplo-diploid pattern without penalizing inbreeding. In this study, we investigated sex determination in Heterospilus prosopidis Viereck, a parasitoid from a relatively primitive subfamily of the Braconidae, a hymenopteran family having species with and without CSD. By comparing sex ratio and mortality patterns produced by inbred and outbred females, we were able to rule out sl-CSD as a sex determination mechanism in this species. The absence of sl-CSD in H. prosopidis was unexpected given its basal phylogenetic position in the Braconidae. This and other recent studies suggest that sex determination systems in the Hymenoptera may be evolutionary labile.

Identification of the sex chromosome pair in coho salmon (Oncorhynchus kisutch): lack of conservation of the sex linkage group with chinook salmon (Oncorhynchus tshawytscha)

Fluorescence in situ hybridization (FISH) using a probe to the male-specific GH-Y (growth hormone pseudogene) was used to identify the Y chromosome in coho salmon (Oncorhynchus kisutch). The sex chromosome pair is morphologically similar to chinook salmon (Oncorhynchus tshawytscha) with the GH-Y localized to the small short arm of the largest subtelocentric chromosome pair. FISH experiments with probes containing sex-linked genes in rainbow trout (Oncorhynchus mykiss) (SCAR163) and chinook salmon (Omy7INRA) showed that the coho sex linkage group is different from chinook and rainbow trout and this was confirmed by segregation analysis for the Omy7INRA locus. The telomeric location of the SEX locus, the presence of shared male-specific markers in coho and chinook salmon, and the lack of conservation of sex-linkage groups suggest that transposition of a small male-specific region may have occurred repeatedly in salmonid fishes of the genus Oncorhynchus.

An AFLP-based approach for the identification of sex-linked markers in rainbow trout ( Oncorhynchus mykiss)

A DNA AFLP-fingerprinting approach has been used in this study for the identification of sex-linked markers in rainbow trout. Using a sex-typed pool strategy we analyzed a total of 486 different primer combinations of which 19 gave sex-associated amplification across the pooled DNA samples. Segregation analysis showed that 18 out of 19 AFLPs (95%) were informative for individual sexing and genetic mapping. Fifteen AFLPs that showed sex-associated amplification in the pooled segregant analysis were converted into single locus markers (SCARs). One out of 15, the SCAR Omy-163, was Y chromosome-linked and mapped near the sex locus in all of four genetically diverse male clonal lines of rainbow trout examined. Three microsatellites, Ots517NWFSC, OmyFGT19TUF and Omm1026, which were previously mapped on the sex linkage group in rainbow trout, were also analyzed. The SCAR Omy-163, Ots517NWFC and OmyFGT19TUF showed low levels of intraspecific allelic polymorphism. Y chromosome-linked AFLP markers were relatively strain-specific suggesting an intraspecific genetic polymorphism of the rainbow trout Y-chromosome. Estimation of the level of polymorphism for AFLPs using one of our strains revealed that approximately 34,506 putative loci were tested in our study of which about 4374 were polymorphic. Considering 11 Y-linked polymorphic loci in this strain, we estimated that 0.25% of polymorphic loci (11/4374) were confined to a single sex. Assuming that the length of the rainbow trout genome is estimated to be 2627.5 cM (2.4×10 6 kb), the male-determining region detected using our screen may span 6.6 cM (6100 kb). The AFLP technique provides an efficient marker system for revealing polymorphic loci and for linkage map construction. An AFLP approach in combination with a sex-type pool strategy resulted in a quick analysis for identifying sex-linked loci in rainbow trout. Similar approaches may be useful for molecular mapping of markers linked to genes of interest in other species.

Evolutionary genetics: Stickleback's view of sex chromosome evolution

Evolutionary genetics: Stickleback's view of sex chromosome evolution

Polymorphism and differentiation of rainbow trout Y chromosomes

Most fish species show little morphological differentiation in the sex chromosomes. We have coupled molecular and cytogenetic analyses to characterize the male-determining region of the rainbow trout (Oncorhynchus mykiss) Y chromosome. Four genetically diverse male clonal lines of this species were used for genetic and physical mapping of regions in the vicinity of the sex locus. Five markers were genetically mapped to the Y chromosome in these male lines, indicating that the sex locus was located on the same linkage group in each of the lines. We also confirmed the presence of a Y chromosome morphological polymorphism among these lines, with the Y chromosomes from two of the lines having the more common heteromorphic Y chromosome and two of the lines having Y chromosomes morphologically similar to the X chromosome. The fluorescence in situ hybridization (FISH) pattern of two probes linked to sex suggested that the sex locus is physically located on the long arm of the Y chromosome. Fishes appear to be an excellent group of organisms for studying sex chromosome evolution and differentiation in vertebrates because they show considerable variability in the mechanisms and (or) patterns involved in sex determination.

Effective population size in Hymenoptera with complementary sex determination.

Complementary sex determination in the haplodiploid Hymenoptera leads to the production of inviable or effectively sterile diploid males when diploid progeny are homozygous at the sex-determining locus. The production of diploid males reduces the number of females in a population and biases the effective breeding sex ratio in favor of haploid males. This in turn will reduce the effective population size (Ne) of hymenopteran populations with complementary sex determination relative to the expected reductions due to haplodiploidy alone. The effects of diploid male production on Ne in hymenopterans with complementary sex determination when diploid males are either inviable or effectively sterile are assessed theoretically. In both models, low allelic diversity at the sex locus reduces the Ne of hymenopteran populations, and this effect is largest when diploid males are effectively sterile.

Isolation and Physical Mapping of Sex-Linked AFLP Markers in Nile Tilapia (Oreochromis niloticus L.)

Gynogenetically produced XX and YY Nile tilapia (Oreochromis niloticus) and diploid control groups were screened for amplified fragment length polymorphisms (AFLPs) to search for sex-linked or sex-specific markers. Family-level bulked segregant analysis (XX and YY gynogenetic family pools) and individual screening (XX and YY gynogenetics and XX and XY control individuals) identified 3 Y-linked (OniY425, OniY382, OniY227) and one X-linked (OniX420) AFLP markers. OniX420 and OniY425 were shown to be allelic. Single locus polymerase chain reaction assays were developed for these markers. Tight linkage was demonstrated between the AFLP markers and the sex locus within the source families. However, these markers failed to consistently identify sex in unrelated individuals, indicating recombination between the markers and the sex-determining loci. O. niloticus bacterial artificial chromosome clones, containing the AFLP markers, hybridized to the long arm of chromosome 1. This confirmed previous evidence, based on meiotic chromosome pairing and fluorescence in situ hybridization probes obtained through chromosome microdissection, that chromosome pair 1 is the sex chromosomes.

Does differential selection on the 5S rDNA explain why the rainbow trout sex chromosome heteromorphism is not linked to the SEX locus?

Many but not all rainbow trout strains have morphologically distinguishable sex chromosomes. In these strains, the short arm of the X has multiple copies of 5S rDNA and a bright DAPI band near the centromere, both of which are missing from the Y chromosome, which has a very small short arm. We examined the presence of these markers using fluorescence in situ hybridization (FISH) in four different YY clonal lines derived from different strains and compared the results with sexed fish of the Donaldson strain with the normal X/Y heteromorphism. The Y chromosome in two of the YY clonal lines (Arlee and Swanson) is indistinguishable from the X chromosome and it is positive for 5S rDNA and the DAPI bright band. On the other hand, both 5S rDNA sequences and the DAPI band were not found on the Y chromosome in Hot Creek and Clearwater which have the normal Y. Thus the presence of these two cytogenetic markers may account for the size difference between the short arm of the X and Y chromosome found in most rainbow trout strains. In fishes the expression of one type of 5S rRNA is restricted to oocytes and previous work suggests that although XX males are fairly common, XY females are rare, implying a selective disadvantage for XY females. A hypothesis is presented to explain why this sex chromosome heteromorphism is not closely linked to the SEX locus, which is found on the long arm of the Y chromosome in rainbow trout.

Identification of quantitative trait loci affecting sex determination in the Eastern treehole mosquito (Ochlerotatus triseriatus).

Laboratory colonies of the eastern treehole mosquito (Ochlerotatus triseriatus (Say)) exhibit a consistent female-biased sex ratio. This is unusual among mosquito species, in which heritable sex ratio distortion is usually male biased and mediated by meiotic drive. Quantitative trait loci (QTL) affecting sex were mapped in an F(1) intercross to better understand the genetics underlying this female bias. In P(1) and F(1) parents and in 146 F(2) individuals with a female-biased sex ratio (106 females:40 males), regions of seven cDNA loci were analyzed with single-strand conformation polymorphism (SSCP) analysis to identify and orient linkage groups. Genotypes were also scored at 73 random amplified polymorphic DNA (RAPD)-SSCP loci. In addition to the sex locus, at least four QTL affecting sex determination were detected with interval mapping on linkage groups I and II. Alleles at the sex locus cumulatively accounted for approximately 61-77% of the genetic variance in sex. Alleles at QTL adjacent to the sex locus and at a QTL on the opposite end of linkage group I increased the proportion of females, but alleles at a QTL on linkage group I and a second QTL on linkage group II increased the proportion of males. The female-biased sex ratio observed in laboratory colonies of O. triseriatus is most easily explained by the existence of multiple female biased distorter loci, as have been observed in other Diptera.

Cotesia flavipes Cameron (Hymenoptera: Braconidae) does not exhibit complementary sex determination (ii) Evidence from laboratory experiments

Sex determination in the order Hymenoptera is based on haplodiploid arrhenotoky; in which males develop from unfertilized eggs and are haploid, whereas females develop from fertilized eggs and are diploid. However, some hymenopteran species produce diploid males through a mechanism known as single-locus complementary sex determination (sl-CSD). In these species, heterozygous individuals at a single sex locus develop into females, whereas hemizygotes (haploids) and homozygotes (diploids) develop into males. Inbreeding leads to homozygosity and consequent production of diploid males. We investigated the presence of sl-CSD in the braconid Cotesia flavipes using a series of inbreeding crosses among five isofemale lines. Sex ratio (proportion of females) did not differ among the within-line crosses, between-line crosses and crosses carried out between isofemale lines and a mixed (outbred) colony. Brood size of within-line and between-line crosses did not differ. Culturing populations for 25 generations did not result in changes to more male-biased sex ratios. We conclude that CSD does not exist in C. flavipes. The implications of absence of CSD in C. flavipes are discussed in context of mass rearing for classical biological control programmes.

Single-locus complementary sex determination in the inbreeding wasp Euodynerus foraminatus Saussure (Hymenoptera: Vespidae)

The Hymenoptera have arrhenotokous haplodiploidy in which males normally develop from unfertilized eggs and are haploid, while females develop from fertilized eggs and are diploid. Multiple sex determination systems are known to underlie haplodiploidy, and the best understood is single-locus complementary sex determination (sl-CSD) in which sex is determined at a single polymorphic locus. Individuals heterozygous at the sex locus develop as females; individuals that are hemizygous (haploid) or homozygous (diploid) at the sex locus develop as males. sl-CSD can be detected with inbreeding experiments that produce diploid males in predictable proportions as well as sex ratio shifts due to diploid male production. This sex determination system is considered incompatible with inbreeding because the ensuing increase in homozygosity increases the production of diploid males that are inviable or infertile, imposing a high cost on matings between close relatives. However, in the solitary hunting wasp Euodynerus foraminatus, a species suspected of having sl-CSD, inbreeding may be common due to a high incidence of sibling matings at natal nests. In laboratory crosses with E. foraminatus, we find that sex ratios and diploid male production (detected as microsatellite heterozygosity) are consistent with sl-CSD, but not with other sex determination systems. This is the first documented example of sl-CSD in a hymenopteran with an apparent natural history of inbreeding, and thus presents a paradox for our understanding of hymenopteran genetics.

Population structure, mating system, and sex-determining allele diversity of the parasitoid wasp Habrobracon hebetor.

Besides haplo-diploid sex determination, where females develop from fertilized diploid eggs and males from unfertilized haploid eggs, some Hymenoptera have a secondary system called complementary sex determination (CSD). This depends on genotypes of a 'sex locus' with numerous sex-determining alleles. Diploid heterozygotes develop as females, but diploid homozygotes become sterile or nonviable diploid males. Thus, when females share sex-determining alleles with their mates and produce low fitness diploid males, CSD creates a genetic load. The parasitoid wasp Habrobracon hebetor has CSD and displays mating behaviours that lessen CSD load, including mating at aggregations of males and inbreeding avoidance by females. To examine the influence of population structure and the mating system on CSD load, we conducted genetic analyses of an H. hebetor population in Wisconsin. Given the frequency of diploid males, we estimated that the population harboured 10-16 sex-determining alleles. Overall, marker allele frequencies did not differ between subpopulations, but frequencies changed dramatically between years. This reduced estimates of effective size of subpopulations to only N3 approximately 20-50, which probably reflected annual fluctuations of abundance of H. hebetor. We also determined that the mating system is effectively monogamous. Models relating sex-determining allele diversity and the mating system to female productivity showed that inbreeding avoidance always decreased CSD loads, but multiple mating only reduced loads in populations with fewer than five sex-determining alleles. Populations with N3 less than 100 should have fewer sex-determining alleles than we found, but high diversity could be maintained by a combination of frequency-dependent selection and gene flow between populations.