A microsatellite genetic map of the turbot (Scophthalmus maximus)

A consensus microsatellite-based linkage map of the turbot (Scophthalmus maximus) was constructed from two unrelated families. The mapping panel was derived from a gynogenetic family of 96 haploid embryos and a biparental diploid family of 85 full-sib progeny with known linkage phase. A total of 242 microsatellites were mapped in 26 linkage groups, six markers remaining unlinked. The consensus map length was 1343.2 cM, with an average distance between markers of 6.5 +/- 0.5 cM. Similar length of female and male maps was evidenced. However, the mean recombination at common intervals throughout the genome revealed significant differences between sexes, approximately 1.6 times higher in the female than in the male. The comparison of turbot microsatellite flanking sequences against the Tetraodon nigroviridis genome revealed 55 significant matches, with a mean length of 102 bp and high sequence similarity (81-100%). The comparative mapping revealed significant syntenic regions among fish species. This study represents the first linkage map in the turbot, one of the most important flatfish in European aquaculture. This map will be suitable for QTL identification of productive traits in this species and for further evolutionary studies in fish and vertebrate species.     

A genome-wide association study,supported by a new chromosome-level genome assembly,suggests sox2 as a main driver of the undifferentiatiated ZZ/ZW sex determination of turbot (Scophthalmus maximus)

BackgroundUnderstanding sex determination (SD) across taxa is a major challenge for evolutionary biology. The new genomic tools are paving the way to identify genomic features underlying SD in fish, a group frequently showing limited sex chromosome differentiation and high SD evolutionary turnover. Turbot (Scophthalmus maximus) is a commercially important flatfish with an undifferentiated ZW/ZZ SD system and remarkable sexual dimorphism. Here we describe a new long-read turbot genome assembly used to disentangle the genetic architecture of turbot SD by combining genomics and classical genetics approaches.ResultsThe new turbot genome assembly consists of 145 contigs (N50 = 22.9 Mb), 27 of them representing >95% of its estimated genome size. A genome wide association study (GWAS) identified a ~ 6.8 Mb region on chromosome 12 associated with sex in 69.4% of the 36 families analyzed. The highest associated markers flanked sox2, the only gene in the region showing differential expression between sexes before gonad differentiation. A single SNP showed consistent differences between Z and W chromosomes. The analysis of a broad sample of families suggested the presence of additional genetic and/or environmental factors on turbot SD.ConclusionsThe new chromosome-level turbot genome assembly, one of the most contiguous fish assemblies to date, facilitated the identification of sox2 as a consistent candidate gene putatively driving SD in this species. This chromosome SD system barely showed any signs of differentiation, and other factors beyond the main QTL seem to control SD in a certain proportion of families.     

Consolidation of the genetic and cytogenetic maps of turbot (Scophthalmus maximus) using FISH with BAC clones

Bacterial artificial chromosomes (BAC) have been widely used for fluorescence in situ hybridization (FISH) mapping of chromosome landmarks in different organisms, including a few in teleosts. In this study, we used BAC-FISH to consolidate the previous genetic and cytogenetic maps of the turbot (Scophthalmus maximus), a commercially important pleuronectiform. The maps consisted of 24 linkage groups (LGs) but only 22 chromosomes. All turbot LGs were assigned to specific chromosomes using BAC probes obtained from a turbot 5× genomic BAC library. It consisted of 46,080 clones with inserts of at least 100 kb and <5 % empty vectors. These BAC probes contained gene-derived or anonymous markers, most of them linked to quantitative trait loci (QTL) related to productive traits. BAC clones were mapped by FISH to unique marker-specific chromosomal positions, which showed a notable concordance with previous genetic mapping data. The two metacentric pairs were cytogenetically assigned to LG2 and LG16, and the nucleolar organizer region (NOR)-bearing pair was assigned to LG15. Double-color FISH assays enabled the consolidation of the turbot genetic map into 22 linkage groups by merging LG8 with LG18 and LG21 with LG24. In this work, a first-generation probe panel of BAC clones anchored to the turbot linkage and cytogenetical map was developed. It is a useful tool for chromosome traceability in turbot, but also relevant in the context of pleuronectiform karyotypes, which often show small hardly identifiable chromosomes. This panel will also be valuable for further integrative genomics of turbot within Pleuronectiformes and teleosts, especially for fine QTL mapping for aquaculture traits, comparative genomics, and whole-genome assembly.     

High-Density Genetic Linkage Mapping in Turbot (Scophthalmus maximus L.) Based on SNP Markers and Major Sex- and Growth-Related Regions Detection

This paper describes the development of a high density consensus genetic linkage map of a turbot (Scophthalmus maximus L.) family composed of 149 mapping individuals using Single Nucleotide Polymorphisms (SNP) developed using the restriction-site associated DNA (RAD) sequencing technique with the restriction enzyme, PstI. A total of 6,647 SNPs were assigned to 22 linkage groups, which is equal to the number of chromosome pairs in turbot. For the first time, the average marker interval reached 0.3958 cM, which is equal to approximately 0.1203 Mb of the turbot genome. The observed 99.34% genome coverage indicates that the linkage map was genome-wide. A total of 220 Quantitative Traits Locus (QTLs) associated with two body length traits, two body weight traits in different growth periods and sex determination were detected with an LOD > 5.0 in 12 linkage groups (LGs), which explained the corresponding phenotypic variance (R²), ranging from 14.4–100%. Among them, 175 overlapped with linked SNPs, and the remaining 45 were located in regions between contiguous SNPs. According to the QTLs related to growth trait distribution and the changing of LGs during different growth periods, the growth traits are likely controlled by multi-SNPs distributed on several LGs; the effect of these SNPs changed during different growth periods. Most sex-related QTLs were detected at LG 21 with a linkage span of 70.882 cM. Additionally, a small number of QTLs with high feasibility and a narrow R² distribution were also observed on LG7 and LG14, suggesting that multi LGs or chromosomes might be involved in sex determination. High homology was recorded between LG21 in Cynoglossus semilaevis and turbot. This high-saturated turbot RAD-Seq linkage map is undoubtedly a promising platform for marker assisted selection (MAS) and flatfish genomics research.     

Exploitation of a turbot (Scophthalmus maximus L.) immune-related expressed sequence tag (EST) database for microsatellite screening and validation

In this study, we identified and characterized 160 microsatellite loci from an expressed sequence tag (EST) database generated from immune-related organs of turbot (Scophthalmus maximus). A final set of 83 new polymorphic microsatellites were validated after the analysis of 40 individuals of Atlantic origin including both wild and farmed individuals. The allele number and the expected heterozygosity ranged from 2 to 18 and from 0.021 to 0.951, respectively. Evidences of null alleles at moderate-high frequencies were detected at six loci using population data. None of the analysed loci showed deviations from Mendelian segregation after the analysis of five full-sib families including approximately 92 individuals/family. The markers are used to consolidate the turbot genetic map, and because they are mostly EST-derived, they will be very useful for comparative genomic studies within flatfishes and with model fish species. Using an in silico approach, we detected significant homologies of microsatellite sequences with the EST databases of the flatfish species with highest genomic resources (Senegalese sole, Atlantic halibut, bastard halibut) in 31% of these turbot markers. The conservation of these microsatellites within Pleuronectiformes will pave the way for anchoring genetic maps of different species and identifying genomic regions related to productive traits.     

0-group flatfish growth conditions on a nursery ground (Bay of Canche, Eastern English Channel)

The settlement and growth conditions of three 0-group flatfish species, sole (Solea solea L.), plaice (Pleuronectes platessa L.) and dab (Limanda limanda L.) in the Bay of Canche, French coast of the Eastern English Channel, were investigated during summer 1997. 0-group dab were the more abundant of the three species (75.4%) followed by plaice (21%) and sole (3.6%). Analyses of fish size distribution indicated that settlement of plaice occurred before sole and dab. Growth of sole was faster than that of plaice, which in turn was faster than that of dab (0.68, 0.43 and 0.33 mm d^?1 respectively). Sole differed from plaice and dab by their larger mean length attained at the end of their first summer stay in the nursery ground. Growth performances of 0-group sole and plaice were analysed by comparing estimated growth in the field with predicted maximum growth according to temperature-growth rate models from experimental studies of growth with unlimited food supply. The data of this study suggest that plaice growth may have been limited during late summer, whereas sole growth does appear to have been determined only by water temperature.     

Genetic linkage map of olive flounder, Paralichthys olivaceus

Olive flounder, Paralichthys olivaceus, is an important fish species in Asia, both for fisheries and aquaculture. As the first step for better understanding the genomic structure and functional analysis, we constructed a genetic linkage map for olive flounder based on 180 microsatellites and 31 expressed sequence tag (EST)-derived markers. Twenty-four linkage groups were identified, consistent with the 24 chromosomes of this species. The total map distance was 1,001.3 cM based on Kosambi sex-average mapping, and the average inter-locus distance was 4.7 cM. Linkage between the loci was identified by an LOD score of ≥3. This linkage map may be used to map quantitative trait loci associated with important traits of the species and may assist in breeding programs.     

A sex-associated sequence identified by RAPD screening in gynogenetic individuals of turbot (Scophthalmus maximus)

Understanding the genetic basis of sex determination mechanisms is essential for improving the productivity of farmed aquaculture fish species like turbot (Scophthalmus maximus). In culture conditions turbot males grow slower than females starting from eight months post-hatch, and this differential growth rate is maintained until sexual maturation is reached, being mature females almost twice as big as males of the same age. The goal of this study was to identify sex-specific DNA markers in turbot using comparative random amplified polymorphism DNA (RAPD) profiles in males and females to get new insights of the genetic architecture related to sex determination. In order to do this, we analyzed 540 commercial 10-mer RAPD primers in male and female pools of a gynogenetic family because of its higher inbreeding, which facilitates the detection of associations across the genome. Two sex-linked RAPD markers were identified in the female pool and one in the male pool. After the analysis of the three markers on individual samples of each pool and also in unrelated individuals, only one RAPD showed significant association with females. This marker was isolated, cloned and sequenced, containing two sequences, a microsatellite (SEX01) and a minisatellite (SEX02), which were mapped in the turbot reference map. From this map position, through a comparative mapping approach, we identified Foxl2, a relevant gene related to initial steps of sex differentiation, and Wnt4, a gene related with ovarian development, close to the microsatellite and minisatellite markers, respectively. The position of Foxl2 and Wnt4 was confirmed by linkage mapping in the reference turbot map.     

New microsatellite markers in turbot (Scophthalmus maximus) derived from an enriched genomic library and sequence databases

Scophthalmus maximus is an important commercially aquaculture fish species. We tackle the search for new microsatellites using two different approaches: an enriched partial genomic library and a screening of all turbot DNA sequences deposited in GenBank. Out of 15 genomic library derived loci, five gave working primer pairs, with expected heterozygosities (H_E) ranging from 0.13 to 0.91. Out of seven loci derived from database sequences, two amplified successfully with an H_E ranging from 0.56 to 0.63. The average allele number of these microsatellites was 5.7 per locus with a range between two and 15.     

Polymorphic dinucleotide microsatellites in tongue sole (Cynoglossus semilaevis)

The tongue sole, Cynoglossus semilaevis, is a rare marine flatfish distributed in Chinese coastal waters. From a (GT)_n‐enriched genomic library, 57 microsatellites were isolated and characterized. Seventeen of these loci were polymorphic in a test population with alleles ranging from three to 13, and observed and expected heterozygosities from 0.1613 to 1.0000 and from 0.2126 to 0.8983, respectively. Five loci deviated from the Hardy–Weinberg equilibrium in the sampled population, and linkage disequilibrium between two loci was significant after applying Bonferroni correction. Three additional fish species assessed for cross‐species amplification revealed that only one locus was also polymorphic in one species. These polymorphic microsatellite loci should provide sufficient level of genetic diversity to evaluate the breeding strategy and investigate the fine‐scale population structure in C. semilaevis.     

A genome‐wide linkage map for the house sparrow (Passer domesticus) provides insights into the evolutionary history of the avian genome

The house sparrow is an important model species for studying physiological, ecological and evolutionary processes in wild populations. Here, we present a medium density, genome wide linkage map for house sparrow (Passer domesticus) that has aided the assembly of the house sparrow reference genome, and that will provide an important resource for ongoing mapping of genes controlling important traits in the ecology and evolution of this species. Using a custom house sparrow 10 K iSelect Illumina SNP chip we have assigned 6,498 SNPs to 29 autosomal linkage groups, based on a mean of 430 informative meioses per SNP. The map was constructed by combining the information from linkage with that of the physical position of SNPs within scaffold sequences in an iterative process. Averaged between the sexes; the linkage map had a total length of 2,004 cM, with a longer map for females (2,240 cM) than males (1,801 cM). Additionally, recombination rates also varied along the chromosomes. Comparison of the linkage map to the reference genomes of zebra finch, collared flycatcher and chicken, showed a chromosome fusion of the two avian chromosomes 8 and 4A in house sparrow. Lastly, information from the linkage map was utilized to conduct analysis of linkage disequilibrium (LD) in eight populations with different effective population sizes (N_e) in order to quantify the background level LD. Together, these results aid the design of future association studies, facilitate the development of new genomic tools and support the body of research that describes the evolution of the avian genome.     

The genomics of incompatibility factors and sex determination in hybridizing species of Cottus (Pisces)

Cottus rhenanus and Cottus perifretum have formed hybrid lineages and narrow hybrid zones that can be best explained through the action of natural selection. However, the underlying selective forces as well as their genomic targets are not well understood. This study identifies genomic regions in the parental species that cause hybrid incompatibilities and tests whether these manifest in a sex-specific manner to learn about processes that affect natural hybridization in Cottus. Interspecific F₂ crosses were analyzed for 255 markers for genetic mapping and to detect transmission distortion as a sign for genetic incompatibilities. The Cottus map consists of 24 linkage groups with a total length of 1575.4 cM. A male heterogametic (XY) sex determination region was found on different linkage groups in the two parental species. Genetic incompatibilities were incomplete, varied among individuals and populations and were not associated with the heterogametic sex. The variance between populations and individuals makes it unlikely that there are species-specific incompatibility loci that could affect the gene pool of natural hybrids in a simple and predictable way. Conserved synteny with sequenced fish genomes permits to genetically study the Cottus genome through the transfer of genomic information from the model fish species. Homology relationships of candidate genomic regions in Cottus indicate that sex determination is not based on the same genomic regions found in other fish species. This suggests a fast evolutionary turnover of the genetic basis of sex determination that, together with the small size of the heterogametic regions, may contribute to the absence of fitness effects related to the Haldane''s rule.     

A Gene-Based Linkage Map for Bicyclus anynana Butterflies Allows for a Comprehensive Analysis of Synteny with the Lepidopteran Reference Genome

Lepidopterans (butterflies and moths) are a rich and diverse order of insects, which, despite their economic impact and unusual biological properties, are relatively underrepresented in terms of genomic resources. The genome of the silkworm Bombyx mori has been fully sequenced, but comparative lepidopteran genomics has been hampered by the scarcity of information for other species. This is especially striking for butterflies, even though they have diverse and derived phenotypes (such as color vision and wing color patterns) and are considered prime models for the evolutionary and developmental analysis of ecologically relevant, complex traits. We focus on Bicyclus anynana butterflies, a laboratory system for studying the diversification of novelties and serially repeated traits. With a panel of 12 small families and a biphasic mapping approach, we first assigned 508 expressed genes to segregation groups and then ordered 297 of them within individual linkage groups. We also coarsely mapped seven color pattern loci. This is the richest gene-based map available for any butterfly species and allowed for a broad-coverage analysis of synteny with the lepidopteran reference genome. Based on 462 pairs of mapped orthologous markers in Bi. anynana and Bo. mori, we observed strong conservation of gene assignment to chromosomes, but also evidence for numerous large- and small-scale chromosomal rearrangements. With gene collections growing for a variety of target organisms, the ability to place those genes in their proper genomic context is paramount. Methods to map expressed genes and to compare maps with relevant model systems are crucial to extend genomic-level analysis outside classical model species. Maps with gene-based markers are useful for comparative genomics and to resolve mapped genomic regions to a tractable number of candidate genes, especially if there is synteny with related model species. This is discussed in relation to the identification of the loci contributing to color pattern evolution in butterflies.     

Construction of a Genetic Linkage Map and Mapping of a Female-Specific DNA Marker in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

The half-smooth tongue sole (Cynoglossus semilaevis, hereafter, “tongue sole”) is a marine flatfish with great commercial importance for fisheries and aquaculture in China. It has also been a promising model for the study of sex determination mechanisms in fish. Here, we report the construction of a genetic linkage map for the tongue sole, based on 137 markers including 103 AFLP markers, 33 microsatellite markers, and one female-specific DNA marker. Twenty-six linkage groups (LGs) were found. The total map length was 934.6 cM (Kosambi), with an average spacing of 8.4 cM, covering 64.4% of the estimated genome size. Furthermore, a female-specific SCAR marker, CseF-382, was mapped on LG5. This study represents the first genetic linkage map in the tongue sole. This map has great potential in the identification of quantitative traits loci and sex-related genes and marker-assisted selection in the tongue sole. Meanwhile, the new set of polymorphic microsatellite markers developed in this study is not only useful for genetic mapping but also of critical importance for studies on genetic diversity and broodstock management in tongue sole.     

Identification of Microsatellite Repeats in Turbot (Scophthalmus maximus) and Dover Sole (Solea solea) using a RAPD-Based Technique: Characterization of Microsatellite Markers in Dover Sole

We have used a RAPD-based technique to identify several microsatellite repeats in turbot (Scophthalmus maximus) and Dover sole (Solea solea) and report the characterization of six novel polymorphic microsatellite markers for Dover sole. These are the first such markers to be developed for this flatfish species. They exhibit much higher levels of heterozygosity than those previously observed with allozyme loci and should prove useful in addressing population genetic questions as well as more fundamental aquaculture-related questions. Received ; accepted July 13, 1999.     

The genetics of domestication of rice bean, Vigna umbellata

Background and Aims

The Asian genus Vigna, to which four cultivated species (rice bean, azuki bean, mung bean and black gram) belong, is suitable for comparative genomics. The aims were to construct a genetic linkage map of rice bean, to identify the genomic regions associated with domestication in rice bean, and to compare these regions with those in azuki bean.

Methods

A genetic linkage map was constructed by using simple sequence repeat and amplified fragment length polymorphism markers in the BC₁F₁ population derived from a cross between cultivated and wild rice bean. Using this map, 31 domestication-related traits were dissected into quantitative trait loci (QTLs). The genetic linkage map and QTLs of rice bean were compared with those of azuki bean.

Key Results

A total of 326 markers converged into 11 linkage groups (LGs), corresponding to the haploid number of rice bean chromosomes. The domestication-related traits in rice bean associated with a few major QTLs distributed as clusters on LGs 2, 4 and 7. A high level of co-linearity in marker order between the rice bean and azuki bean linkage maps was observed. Major QTLs in rice bean were found on LG4, whereas major QTLs in azuki bean were found on LG9.

Conclusions

This is the first report of a genetic linkage map and QTLs for domestication-related traits in rice bean. The inheritance of domestication-related traits was so simple that a few major QTLs explained the phenotypic variation between cultivated and wild rice bean. The high level of genomic synteny between rice bean and azuki bean facilitates QTL comparison between species. These results provide a genetic foundation for improvement of rice bean; interchange of major QTLs between rice bean and azuki bean might be useful for broadening the genetic variation of both species.     

Mapping quantitative trait loci (QTL) in sheep. I. A new male framework linkage map and QTL for growth rate and body weight

A male sheep linkage map comprising 191 microsatellites was generated from a single family of 510 Awassi-Merino backcross progeny. Except for ovine chromosomes 1, 2, 10 and 17, all other chromosomes yielded a LOD score difference greater than 3.0 between the best and second-best map order. The map is on average 11% longer than the Sheep Linkage Map v4.7 male-specific map. This map was employed in quantitative trait loci (QTL) analyses on body-weight and growth-rate traits between birth and 98 weeks of age. A custom maximum likelihood program was developed to map QTL in half-sib families for non-inbred strains (QTL-MLE) and is freely available on request. The new analysis package offers the advantage of enabling QTL × fixed effect interactions to be included in the model. Fifty-four putative QTL were identified on nine chromosomes. Significant QTL with sex-specific effects (i.e. QTL × sex interaction) in the range of 0.4 to 0.7 SD were found on ovine chromosomes 1, 3, 6, 11, 21, 23, 24 and 26.