Identification of the sex‐determining region in flathead grey mullet (Mugil cephalus)

Elucidation of the sex‐determination mechanism in flathead grey mullet (Mugil cephalus) is required to exploit its economic potential by production of genetically determined monosex populations and application of hormonal treatment to parents rather than to the marketed progeny. Our objective was to construct a first‐generation linkage map of the M. cephalus in order to identify the sex‐determining region and sex‐determination system. Deep‐sequencing data of a single male was assembled and aligned to the genome of Nile tilapia (Oreochromis niloticus). A total 245 M. cephalus microsatellite markers were designed, spanning the syntenic tilapia genome assembly at intervals of 10 Mb. In the mapping family of full‐sib progeny, 156 segregating markers were used to construct a first‐generation linkage map of 24 linkage groups (LGs), corresponding to the number of chromosomes. The linkage map spanned approximately 1200 cM with an average inter‐marker distance of 10.6 cM. Markers segregating on LG9 in two independent mapping families showed nearly complete concordance with gender (R² = 0.95). The sex determining locus was fine mapped within an interval of 8.6 cM on LG9. The sex of offspring was determined only by the alleles transmitted from the father, thus indicating an XY sex‐determination system.     

Genetic linkage map of the loach <Emphasis Type="Italic">Misgurnus anguillicaudatus</Emphasis> (Teleostei: Cobitidae)

In the present study, the first genetic linkage map of the loach Misgurnus anguillicaudatus was constructed with 164 microsatellite markers and a color locus, and it included 155 newly developed markers. A total of 159 microsatellite markers and a color locus were mapped in 27 linkage groups (LGs). The female map covered 784.5 cM with 153 microsatellite markers and a color locus, whereas the male map covered 662.2 cM with 119 microsatellite markers. The centromeric position in each LG was estimated by marker-centromere mapping based on half-tetrad analysis. In 4 LGs (LG2, LG3, LG4, and LG5), the centromere was estimated at the intermediate region. In LG1, LG11, and LG12, the centromere was estimated to shift from the sub-intermediate region to the end (telomeric). The number of these LGs (7) was identical to the collective number of bi-arm metacentric (5) and sub-metacentric chromosome (2) of the haploid chromosome set (n = 5) of the loach. In the other LGs, the position of the centromere was estimated at the end or outside. These results indicate satisfactory compliance between the linkage map and the chromosome set. Our map would cover approximately almost the entire loach genome because most markers were successfully mapped.     

A Consensus Microsatellite-Based Linkage Map for the Hermaphroditic Bay Scallop (Argopecten irradians) and Its Application in Size-Related QTL Analysis

Bay scallop (Argopecten irradians) is one of the most economically important aquaculture species in China. In this study, we constructed a consensus microsatellite-based genetic linkage map with a mapping panel containing two hybrid backcross-like families involving two subspecies of bay scallop, A. i. irradians and A. i. concentricus. One hundred sixty-one microsatellite and one phenotypic (shell color) markers were mapped to 16 linkage groups (LGs), which corresponds to the haploid chromosome number of bay scallop. The sex-specific map was 779.2 cM and 781.6 cM long in female and male, respectively, whereas the sex-averaged map spanned 849.3 cM. The average resolution of integrated map was 5.9 cM/locus and the estimated coverage was 81.3%. The proportion of distorted markers occurred more in the hybrid parents, suggesting that the segregation distortion was possibly resulted from heterospecific interaction between genomes of two subspecies of bay scallop. The overall female-to-male recombination rate was 1.13∶1 across all linked markers in common to both parents, and considerable differences in recombination also existed among different parents in both families. Four size-related traits, including shell length (SL), shell height (SH), shell width (SW) and total weight (TW) were measured for quantitative trait loci (QTL) analysis. Three significant and six suggestive QTL were detected on five LGs. Among the three significant QTL, two (qSW-10 and qTW-10, controlling SW and TW, respectively) were mapped on the same region near marker AiAD121 on LG10 and explained 20.5% and 27.7% of the phenotypic variance, while the third (qSH-7, controlling SH) was located on LG7 and accounted for 15.8% of the phenotypic variance. Six suggestive QTL were detected on four different LGs. The linkage map and size-related QTL obtained in this study may facilitate marker-assisted selection (MAS) in bay scallop.     

Within‐population polymorphism of sex‐determination systems in the common frog (Rana temporaria)

In sharp contrast with birds and mammals, the sex chromosomes of ectothermic vertebrates are often undifferentiated, for reasons that remain debated. A linkage map was recently published for Rana temporaria (Linnaeus, 1758) from Fennoscandia (Eastern European lineage), with a proposed sex‐determining role for linkage group 2 (LG2). We analysed linkage patterns in lowland and highland populations from Switzerland (Western European lineage), with special focus on LG2. Sibship analyses showed large differences from the Fennoscandian map in terms of recombination rates and loci order, pointing to large‐scale inversions or translocations. All linkage groups displayed extreme heterochiasmy (total map length was 12.2 cM in males, versus 869.8 cM in females). Sex determination was polymorphic within populations: a majority of families (with equal sex ratios) showed a strong correlation between offspring phenotypic sex and LG2 paternal haplotypes, whereas other families (some of which with female‐biased sex ratios) did not show any correlation. The factors determining sex in the latter could not be identified. This coexistence of several sex‐determination systems should induce frequent recombination of X and Y haplotypes, even in the absence of male recombination. Accordingly, we found no sex differences in allelic frequencies on LG2 markers among wild‐caught male and female adults, except in one high‐altitude population, where nonrecombinant Y haplotypes suggest sex to be entirely determined by LG2. Multifactorial sex determination certainly contributes to the lack of sex‐chromosome differentiation in amphibians.     

A Preliminary Genetic Linkage Map of the Pacific Abalone Haliotis discus hannai Ino

Preliminary genetic linkage maps were constructed for the Pacific abalone (Haliotis discus hannai Ino) using amplified fragment length polymorphism (AFLP), randomly amplified polymorphic DNA (RAPD), and microsatellite markers segregating in a F₁ family. Nine microsatellite loci, 41 RAPD, and 2688 AFLP markers were genotyped in the parents and 86 progeny of the mapping family. Among the 2738 markers, 384 (including 365 AFLP markers, 10 RAPD markers, and 9 microsatellite loci) were polymorphic and segregated in one or both parents: 241 in the female and 146 in the male. The majority of these markers, 232 in the female and 134 in the male, segregated according to the expected 1:1 Mendelian ratio (α = 0.05). Two genetic linkage maps were constructed using markers segregating in the female or the male parent. The female framework map consisted of 119 markers in 22 linkage groups, covering 1773.6 cM with an average intermarker space of 18.3 cM. The male framework map contained 94 markers in 19 linkage groups, spanning 1365.9 cM with an average intermarker space of 18.2 cM. The sex determination locus was mapped to the male map but not to the female map, suggesting a XY-male determination mechanism. Distorted markers showing excess of homozygotes were mapped in clusters, probably because of their linkage to a gene that is incompatible between two parental populations.     

Construction of a High-Density Microsatellite Genetic Linkage Map and Mapping of Sexual and Growth-Related Traits in Half-Smooth Tongue Sole (Cynoglossus semilaevis)

High-density genetic linkage maps of half-smooth tongue sole were developed with 1007 microsatellite markers, two SCAR markers and an F1 family containing 94. The female map was composed of 828 markers in 21 linkage groups, covering a total of 1447.3 cM, with an average interval 1.83 cM between markers. The male map consisted of 794 markers in 21 linkage groups, spanning 1497.5 cM, with an average interval of 1.96 cM. The female and male maps had 812 and 785 unique positions, respectively. The genome length of half-smooth tongue sole was estimated to be 1527.7 cM for the females and 1582.1 cM for the males. Based on estimations of the map lengths, the female and male maps covered 94.74 and 94.65% of the genome, respectively. The consensus map was composed of 1007 microsatellite markers and two SCAR markers in 21 linkage groups, covering a total of 1624 cM with an average interval of 1.67 cM. Furthermore, 159 sex-linked SSR markers were identified. Five sex-linked microsatellite markers were confirmed in their association with sex in a large number of individuals selected from different families. These sex-linked markers were mapped on the female map LG1f with zero recombination. Two QTLs that were identified for body weight, designated as We-1 and We-2, accounted for 26.39% and 10.60% of the phenotypic variation. Two QTLs for body width, designated Wi-1 and Wi-2, were mapped in LG4f and accounted for 14.33% and 12.83% of the phenotypic variation, respectively. Seven sex-related loci were mapped in LG1f, LG14f and LG1m by CIM, accounting for 12.5–25.2% of the trait variation. The results should prove to be very useful for improving growth traits using molecular MAS.     

An EST-SSR-based linkage map for Persea americana Mill. (avocado)

Recent enhancement of the pool of known molecular markers for avocado has allowed the construction of the first moderately dense genetic map for this species. Over 300 SSR markers have been characterized and 163 of these were used to construct a map from the reciprocal cross of two Florida cultivars 'Simmonds' and 'Tonnage'. One hundred thirty-five primer pairs amplified 163 usable loci with 20 primer pairs amplifying more than one locus. 'Tonnage' was heterozygous for 152 (93%) loci, whereas 'Simmonds' was heterozygous for 64 (39%). Null alleles were identified at several loci. Linkage maps were produced for both reciprocal crosses and combined to generate a composite linkage map for the F₁ population of 715 individuals. The composite map contains 12 linkage groups. Linkage groups ranged in size from 157.3 cM (LG2) to 2.4 cM (LG12) and the number of loci mapped per group ranged from 29 (LG1) to two (LG12). The total map length was 1,087.4 cM. Only seven markers were observed to have segregation distortion (α ≤ 0.05) across both sub-composite (reciprocal) maps. Phenotypic data from traits of horticultural interest are currently being collected on this population with the ultimate goal of identifying useful quantitative trait loci and the development of a marker-assisted selection program.     

High‐density interspecific genetic linkage mapping provides insights into genomic incompatibility between channel catfish and blue catfish

Catfish is the leading aquaculture species in the United States. The interspecific hybrid catfish produced by mating female channel catfish with male blue catfish outperform both of their parent species in a number of traits. However, mass production of the hybrids has been difficult because of reproductive isolation. Investigations of genome structure and organization of the hybrids provide insights into the genetic basis for maintenance of species divergence in the face of gene flow, thereby helping develop strategies for introgression and efficient production of the hybrids for aquaculture. In this study, we constructed a high‐density genetic linkage map using the hybrid catfish system with the catfish 250K SNP array. A total of 26 238 SNPs were mapped to 29 linkage groups, with 12 776 unique marker positions. The linkage map spans approximately 3240 cM with an average intermarker distance of 0.25 cM. A fraction of markers (986 of 12 776) exhibited significant deviation from the expected Mendelian ratio of segregation, and they were clustered in major genomic blocks across 15 LGs, most notably LG9 and LG15. The distorted markers exhibited significant bias for maternal alleles among the backcross progenies, suggesting strong selection against the blue catfish alleles. The clustering of distorted markers within genomic blocks should lend insights into speciation as marked by incompatibilities between the two species. Such findings should also have profound implications for understanding the genomic evolution of closely related species as well as the introgression of hybrid production programs in aquaculture.     

Genetic dissection of sex determinism, inflorescence morphology and downy mildew resistance in grapevine

A genetic linkage map of grapevine was constructed using a pseudo-testcross strategy based upon 138 individuals derived from a cross of Vitis vinifera Cabernet Sauvignon × Vitis riparia Gloire de Montpellier. A total of 212 DNA markers including 199 single sequence repeats (SSRs), 11 single strand conformation polymorphisms (SSCPs) and two morphological markers were mapped onto 19 linkage groups (LG) which covered 1,249 cM with an average of 6.7 cM between markers. The position of SSR loci in the maps presented here is consistent with the genome sequence. Quantitative traits loci (QTLs) for several traits of inflorescence and flower morphology, and downy mildew resistance were investigated. Two novel QTLs for downy mildew resistance were mapped on linkage groups 9 and 12, they explain 26.0–34.4 and 28.9–31.5% of total variance, respectively. QTLs for inflorescence morphology with a large effect (14–70% of total variance explained) were detected close to the Sex locus on LG 2. The gene of the enzyme 1-aminocyclopropane-1-carboxylic acid synthase, involved in melon male organ development and located in the confidence interval of all QTLs detected on the LG 2, could be considered as a putative candidate gene for the control of sexual traits in grapevine. Co-localisations were found between four QTLs, detected on linkage groups 1, 14, 17 and 18, and the position of the floral organ development genes GIBBERELLIN INSENSITIVE1, FRUITFULL, LEAFY and AGAMOUS. Our results demonstrate that the sex determinism locus also determines both flower and inflorescence morphological traits.     

Construction of an Integrated Map of Haliotis diversicolor Using Microsatellite Markers

Small abalone, Haliotis diversicolor, is naturally distributed along the coastal waters of East Asia from Japan to the Philippines. It is an economically important maricultured species in southern China and Taiwan. Genetic linkage maps for small abalone were constructed using a total of 308 simple sequence repeat markers including 297 novel markers. Segregation data on 96 progeny were genotyped using a pseudo-testcross strategy. Sixteen linkage groups were identified in both female and male maps, consistent with the haploid chromosome number. The female linkage map covered 758.3 cM, with an average interval of 5.2 cM. The male linkage map spanned a total genetic distance of 676.2 cM, with an average interval of 4.5 cM. An integrated map was constructed by incorporating the homologous parental linkage groups, resulting in 16 linkage groups with a total of 762.1 cM. Genome coverage of the integrated linkage map was approximately 80.7%. The genetic linkage maps of small abalone may facilitate marker-assisted selection and quantitative trait loci mapping.     

Construction of a sequence-tagged high-density genetic map of papaya for comparative structural and evolutionary genomics in brassicales

A high-density genetic map of papaya (Carica papaya L.) was constructed using microsatellite markers derived from BAC end sequences and whole-genome shot gun sequences. Fifty-four F(2) plants derived from varieties AU9 and SunUp were used for linkage mapping. A total of 707 markers, including 706 microsatellite loci and the morphological marker fruit flesh color, were mapped into nine major and three minor linkage groups. The resulting map spanned 1069.9 cM with an average distance of 1.5 cM between adjacent markers. This sequence-based microsatellite map resolved the very large linkage group 2 (LG 2) of the previous high-density map using amplified fragment length polymorphism markers. The nine major LGs of our map represent papaya's haploid nine chromosomes with LG 1 of the sex chromosome being the largest. This map validates the suppression of recombination at the male-specific region of the Y chromosome (MSY) mapped on LG 1 and at potential centromeric regions of other LGs. Segregation distortion was detected in a large region on LG 1 surrounding the MSY region due to the abortion of the YY genotype and in a region of LG6 due to an unknown cause. This high-density sequence-tagged genetic map is being used to integrate genetic and physical maps and to assign genome sequence scaffolds to papaya chromosomes. It provides a framework for comparative structural and evolutional genomic research in the order Brassicales.     

Linkage Map of the Honey Bee, Apis Mellifera, Based on Rapd Markers

A linkage map was constructed for the honey bee based on the segregation of 365 random amplified polymorphic DNA (RAPD) markers in haploid male progeny of a single female bee. The X locus for sex determination and genes for black body color and malate dehydrogenase were mapped to separate linkage groups. RAPD markers were very efficient for mapping, with an average of about 2.8 loci mapped for each 10-nucleotide primer that was used in polymerase chain reactions. The mean interval size between markers on the map was 9.1 cM. The map covered 3110 cM of linked markers on 26 linkage groups. We estimate the total genome size to be ~3450 cM. The size of the map indicated a very high recombination rate for the honey bee. The relationship of physical to genetic distance was estimated at 52 kb/cM, suggesting that map-based cloning of genes will be feasible for this species.     

Linkage analysis and identification of quantitative trait loci associated with freeze tolerance and turf quality traits in St. Augustinegrass

St. Augustinegrass [Stenotaphrum secundatum (Walt.) Kuntze] is a warm-season turfgrass commonly grown in the southern USA. In this study, the first linkage map for all nine haploid chromosomes of the species was constructed for cultivar ‘Raleigh’ and cultivar ‘Seville’ using a pseudo-F₂ mapping strategy. A total of 160 simple sequence repeat markers were mapped to nine linkage groups (LGs) covering a total distance of 1176.24 cM. To demonstrate the usefulness of the map, quantitative trait loci (QTL) were mapped controlling field winter survival, laboratory-based freeze tolerance, and turf quality traits. Multiple genomic regions associated with these traits were identified. Moreover, overlapping QTL were found for winterkill and spring green up on LG 3 (99.21 cM); turf quality, turf density, and leaf texture on LG 3 (68.57–69.50 cM); and surviving green tissue and regrowth on LGs 1 (38.31 cM), 3 (77.70 cM), 6 (49.51 cM), and 9 (34.20 cM). Additional regions, where QTL identified in both field and laboratory-based/controlled environment freeze testing co-located, provided strong support that these regions are good candidates for true gene locations. These results present the first complete linkage map produced for St. Augustinegrass, providing a template for further genetic mapping. Additionally, markers linked to the QTL identified may be useful to breeders for transferring these traits into new breeding lines and cultivars.     

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.     

Linkage maps for the Pacific abalone (genus Haliotis) based on microsatellite DNA markers

This study presents linkage maps for the Pacific abalone (Haliotis discus hannai) based on 180 microsatellite DNA markers. Linkage mapping was performed using three F1 outbred families, and a composite linkage map for each sex was generated by incorporating map information from the multiple families. A total of 160 markers are placed on the consolidated female map and 167 markers on the male map. The numbers of linkage groups in the composite female and male maps are 19 and 18, respectively; however, by aligning the two maps, 18 linkage groups are formed, which are consistent with the haploid chromosome number of H. discus hannai. The female map spans 888.1 cM (Kosambi) with an average spacing of 6.3 cM; the male map spans 702.4 cM with an average spacing of 4.7 cM. However, we encountered several linkage groups that show a high level of heterogeneity in recombination rate between families even within the same sex, which reduces the precision of the consolidated maps. Nevertheless, we suggest that the composite maps are of significant potential use as a scaffold to further extend the coverage of the H. discus hannai genome with additional markers.     

Identification of Sex-determining Loci in Pacific White Shrimp <Emphasis Type="Italic">Litopeneaus vannamei</Emphasis> Using Linkage and Association Analysis

The Pacific white shrimp Litopenaeus vannamei is a predominant aquaculture shrimp species in the world. Like other animals, the L. vannamei exhibited sexual dimorphism in growth trait. Mapping of the sex-determining locus will be very helpful to clarify the sex determination system and further benefit the shrimp aquaculture industry towards the production of mono-sex stocks. Based on the data used for high-density linkage map construction, linkage-mapping analysis was conducted. The sex determination region was mapped in linkage group (LG) 18. A large region from 0 to 21.205 cM in LG18 showed significant association with sex. However, none of the markers in this region showed complete association with sex in the other populations. So an association analysis was designed using the female parent, pool of female progenies, male parent, and pool of male progenies. Markers were de novo developed and those showing significant differences between female and male pools were identified. Among them, three sex-associated markers including one fully associated marker were identified. Integration of linkage and association analysis showed that the sex determination region was fine-mapped in a small region along LG18. The identified sex-associated marker can be used for the sex detection of this species at genetic level. The fine-mapped sex-determining region will contribute to the mapping of sex-determining gene and help to clarify sex determination system for L. vannamei.     

GENETIC MAPPING OF THE LAMINARIA JAPONICA (LAMINARALES,PHAEOPHYTA) USING AMPLIFIED FRAGMENT LENGTH POLYMORPHISM MARKERS1

To establish a molecular‐marker‐assisted system of breeding and genetic study for Laminaria japonica Aresch., amplified fragment length polymorphism (AFLP) was used to construct a genetic linkage map of L. japonica featuring 230 progeny of F₂ cross population. Eighteen primer combinations produced 370 polymorphic loci and 215 polymorphic loci segregated in a 3:1 Mendelian segregation ratio (P ≤ 0.05). Of the 215 segregated loci, 142 were ordered into 27 linkage groups. The length of the linkage groups ranged from 6.7 to 90.3 centimorgans (cM) with an average length of 49.6 cM, and the total length was 1,085.8 cM, which covered 68.4% of the estimated 1,586.9 cM genome. The number of mapped markers on each linkage group ranged from 2 to 12, averaging 5.3 markers per group. The average density of the markers was 1 per 9.4 cM. Based on the marker density and the resolution of the map, the constructed linkage map can satisfy the need for quantitative trait locus (QTL) location and molecular‐marker‐assisted breeding for Laminaria.     

Mapping the d1 and d2 dwarfing genes and the purple foliage color locus P in pearl millet

The d(1) and d(2) dwarfing genes and the P purple foliage color gene were placed on the restriction fragment length polymorphism (RFLP)-based molecular marker linkage map of pearl millet [Pennisetum glaucum (L.) R. Br.] using a mapping population based on a cross of inbred lines IP 18293 (D(1)/D(1), d(2)/d(2), P/P) and Tift 238D1 (d(1)/d(1) D(2)/D(2) p/p). A skeleton genetic linkage map of 562 cM (Haldane function) was constructed using 33 RFLP markers and these three morphological markers. The D(1)/d(1) plant height locus mapped to pearl millet linkage group 1, while the D(2)/d(2) plant height locus and the P/p foliage color locus mapped to pearl millet linkage group 4. Loose genetic linkage was observed between the D(2)/d(2) and P/p loci, with 42% repulsion-phase recombination corresponding to 92 cM (Haldane). This loose linkage of morphological marker loci detected on pearl millet LG4 can likely find use in applied pearl millet breeding programs, as host plant resistances to both downy mildew and rust have previously been identified in this genomic region. Such exploitation of these morphological markers in an applied disease resistance breeding program would require development of appropriate genetic stocks, but the relatively loose genetic linkage between d(2) and P suggests that this should not be difficult.     

High-density linkage mapping revealed suppression of recombination at the sex determination locus in papaya

A high-density genetic map of papaya (Carica papaya L.) was constructed using 54 F(2) plants derived from cultivars Kapoho and SunUp with 1501 markers, including 1498 amplified fragment length polymorphism (AFLP) markers, the papaya ringspot virus coat protein marker, morphological sex type, and fruit flesh color. These markers were mapped into 12 linkage groups at a LOD score of 5.0 and recombination frequency of 0.25. The 12 major linkage groups covered a total length of 3294.2 cM, with an average distance of 2.2 cM between adjacent markers. This map revealed severe suppression of recombination around the sex determination locus with a total of 225 markers cosegregating with sex types. The cytosine bases were highly methylated in this region on the basis of the distribution of methylation-sensitive and -insensitive markers. This high-density genetic map is essential for cloning of specific genes of interest such as the sex determination gene and for the integration of genetic and physical maps of papaya.