Restriction Site-associated DNA Tags Research Articles

A Novel Strategy to Reveal the Landscape of Crossovers in an F1 Hybrid Population of Populus deltoides and Populus simonii.

Although the crossover (CO) patterns of different species have been extensively investigated, little is known about the landscape of CO patterns in Populus because of its high heterozygosity and long-time generation. A novel strategy was proposed to reveal the difference of CO rate and interference between Populus deltoides and Populus simonii using their F1 hybrid population. We chose restriction site-associated DNA (RAD) tags that contained two SNPs, one only receiving the CO information from the female P. deltoides and the other from the male P. simonii. These RAD tags allowed us to investigate the CO patterns between the two outbred species, instead of using the traditional backcross populations in inbred lines. We found that the CO rate in P. deltoides was generally greater than that in P. simonii, and that the CO interference was a common phenomenon across the two genomes. The COs landscape of the different Populus species facilitates not only to understand the evolutionary mechanism for adaptability but also to rebuild the statistical model for precisely constructing genetic linkage maps that are critical in genome assembly in Populus. Additionally, the novel strategy could be applied in other outbred species for investigating the CO patterns.

Morphometric and population genomic evidence for species divergence in the Chimarrichthys fish complex of the Tibetan Plateau

The uplift of the Tibetan Plateau altered the environmental conditions of the local area substantially. Here, we conducted a comprehensive investigation based on morphometrics, population genomics, and climatic factors to evaluate phenotypic and genome-level variations in a radiation of Chimarrichthys catfish endemic to the Plateau. Discriminant function analysis showed phenotypic differences of Chimarrichthys between rivers with respect to elevation. Genetic structure analysis based on 6606 single nucleotide polymorphisms (SNPs) deduced genetic differences between rivers, and species delimitation indicated that the Chimarrichthys fish complex could be divided into three species. Restriction site-associated DNA tags were mapped to the gene sets of Glyptosternon maculatum, and matches were searched against databases for Gene Ontology annotation. Genomic regions exhibiting marked differences among localities represented a range of biological functions, including growth (gdf11), bone development (bmp8a), cellular response to light stimulus (opn3), regulation of the rhodopsin-mediated signalling pathway (grk1), immune response (rag1 and ung), reproductive process (antxr2), and regulation of intracellular iron levels (ireb2). The tag44126, where gene gdf11 is located, was identified as an outlier exhibiting divergence between rivers with altitude differences, and the SNP is thymine (T) in Dadu and Yalong River (~2700 m), but guanine (G) in Jinsha and Qingyi rivers (~2200 and ~ 684 m), suggesting a possible effect of altitude on its differentiation.

Genome-wide single nucleotide polymorphism markers reveal population structure and dispersal direction of an expanding nuisance algal bloom species.

Species invasion and range expansion are currently under scrutiny due to increasing anthropogenic impact on the natural environment. This is also true for harmful algal blooms, which have been reported to have increased in frequency. However, this research is challenging due to the ephemeral nature, small size and mostly low concentrations of microalgae in the environment. One such species is the nuisance microalga Gonyostomum semen (Raphidophyceae), which has increased in occurrence in northern Europe in recent decades. The question of whether the species has expanded its habitat range or if it was already present in the lakes but was too rare to be detected remains unanswered. The aim of the present study was to determine the genetic structure and dispersal pathways of G. semen using RAD (restriction-site-associated DNA) tag sequencing. For G. semen, which has a huge genome (32Gbp), we faced particular challenges, but were nevertheless able to recover over 1000 single nucleotide polymorphisms at high coverage. Our data revealed a distinct population genetic structure, demonstrating a divide of western and eastern populations that probably represent different lineages. Despite significant genetic differentiation among lakes, we found only limited isolation-by-distance. While we had expected a pattern of recent expansion northwards, the data demonstrated gene flow from the northeast/east towards the southwest/west. This genetic signature suggests that the observed gene flow may be due to dispersal by autumn migratory birds, which act as dispersal vectors of resistant resting propagules that form at the end of the G. semen blooms.

Fine mapping of QTLs for resistance to Phytophthora nicotianae in flue-cured tobacco using a high-density genetic map

Tobacco (Nicotiana tabacum L.) is an important agronomic crop and model system for studies of plant-pathogen interactions. Black shank, caused by Phytophthora nicotianae, is an important disease affecting tobacco production worldwide. In this study, a mapping population of 177 F7:8-9 recombinant inbred lines was generated from a cross between the highly resistant cultivar ‘Yunyan 85’ and a susceptible line ‘Dabaijin 599’. A high-density genetic linkage map containing 7734 single-nucleotide polymorphic markers based on restriction site-associated DNA tag sequencing technology was used to finely map quantitative trait loci (QTL) for resistance to P. nicotianae. A total of 10 QTLs were detected as being associated with resistance to P. nicotianae across multiple environments, and two major QTL qBS7 and qBS14 were repeatedly identified under all five environments. They explained 16.48-62.20% and 3.94-11.29% of the phenotypic variance with high LOD score, respectively. One hundred thirty-eight candidate genes were identified for two major QTLs qBS7 and qBS14, and annotation analysis showing that several predicted genes encoded proteins associated with plant defense response to pathogens. This high-density single-nucleotide polymorphic genetic linkage map of flue-cured tobacco based on restriction site-associated DNA sequencing was useful in the QTL finely mapping of resistance to P. nicotianae. This study increases our understanding of the genetics of resistance to P. nicotianae and aids in marker-assisted selection.

Development of SNP markers and validation 24 SNPs in darkbarbel catfish (Pelteobagrus vachelli)

Darkbarbel catfish (Pelteobagrus vachelli) is an important commercial fish species in China. However, its wild populations have undergone a dramatic decline in recent years because of overfishing and habitat destruction for its spawning. Therefore, it is extremely urgent to develop sufficient molecular markers for the effective conservation and molecular-aided selection of this species. In the present study, 24 polymorphic single nucleotide polymorphism (SNP) markers were developed using restriction-site associated DNA tags sequencing (RAD-seq) in P. vachelli. Genetic diversity analysis revealed that the observed heterozygosity (Ho) and expected heterozygosity (He) ranged from 0.190 to 0.900 and 0.251 to 0.508, respectively, and the polymorphism informatin content (PIC) ranged from 0.215 to 0.375. Among these SNPs, 3 loci showed significant departures from Hardy–Weinberg equilibrium after Bonferroni adjustments. The panel of polymorphic SNPs developed here will be useful for genetic conservation and selective breeding in darkbarbel catfish.

Restriction-site associated DNA markers provide new insights into the evolutionary history of the bark beetle genus Dendroctonus

The bark beetle genus Dendroctonus contains some of the most economically important pests of conifers worldwide. Despite many attempts, there is no agreement today on the phylogenetic relationships within the genus, which limits our understanding of its evolutionary history. Here, using restriction-site associated DNA (RAD) markers from 70 specimens representing 17 species (85% of the known diversity) we inferred the phylogeny of the genus, its time of origin and biogeographic history, as well as the evolution of key ecological traits (host plants, larval behavior and adults’ attack strategies). For all combinations of tested parameters (from 6444 to 23,570 RAD tags analyzed), the same, fully resolved topology was inferred. Our analyses suggest that the most recent common ancestor (mrca) of all extant Dendroctonus species was widely distributed across eastern Palearctic and western Nearctic during the early Miocene, from where species dispersed to other Holarctic regions. A first main inter-continental vicariance event occurred during early Miocene isolating the ancestors of D. armandi in the Palearctic, which was followed by the radiation of the main Dendroctonus lineages in North America. During the Late Miocene, the ancestor of the ‘rufipennis’ species group colonized north-east Palearctic regions from western North America, which was followed by a second main inter-continental vicariance event isolating Pleistocene populations in Asia (D. micans) and western North America (D. murrayanae and D. punctatus). The present study supports previous hypotheses explaining intercontinental range disjunctions across the Northern Hemisphere by the fragmentation of a continuous distribution due to climatic cooling, host range fragmentation and geological changes during the late Cenozoic. The reconstruction of ancestral ecological traits indicates that the mrca bored individual galleries and mass attacked the boles of pines. The gregarious feeding behavior of the larvae as well as the individual attack of the base of trees have apparently independently evolved twice in North America (in the ‘rufipennis’ and the ‘valens’ species groups), which suggests a higher adaptive potential than previously thought and may be of interest for plant protection and biodiversity conservation in a rapidly changing world.

Rapid development of novel microsatellite markers from Mauremys reevesii (Testudines: Geoemydidae) using next-generation DNA sequencing technology

Mauremys reevesii (Gray, 1831), which belongs to Mauremys of Geoemydidae (Testudines), distributed in China, as well as Japan and Korea. Previous studies have developed several polymorphic microsatellite loci, but most of them were dinucleotide motifs. Here, we developed 15 polynucleotide-repeat microsatellite loci (including di-, tri, tetra-and pentanucleotide motifs) for M. reevesii through Restriction-site Associated DNA tags sequencing (RAD-seq). A total of 987 microsatellite loci with flanking sequences were suitable for setting primers for polymerase chain reactions (PCR). To verify the identified SSRs, 40 primer pairs were selected for PCR detection. In total, 32 primer sets produced strong PCR products matching their expected sizes, in which species amplification tests showed that 15 were polymorphic. And the number of alleles per locus ranged from 3 to 16. The observed and expected heterozygosity per locus varied from 0.3784 to 1.000 and from 0.3995 to 0.9700, respectively. The methodology of microsatellite isolation constructed in this study is not only cost-effective and time-saving in comparison to traditional approaches, but also can be served as useful tools which benefit population genetics studies and conservation management of M. reevesii.

Construction of a highly saturated Genetic Map for Vitis by Next-generation Restriction Site-associated DNA Sequencing

BackgroundHigh-saturate molecular linkage maps are an important tool in studies on plant molecular biology and assisted breeding. Development of a large set of single nucleotide polymorphisms (SNPs) via next-generation sequencing (NGS)-based methods, restriction-site associated DNA sequencing (RAD-seq), and the generation of a highly saturated genetic map help improve fine mapping of quantitative trait loci (QTL).ResultsWe generated a highly saturated genetic map to identify significant traits in two elite grape cultivars and 176 F1 plants. In total, 1,426,967 high-quality restriction site-associated DNA tags were detected; 51,365, 23,683, and 70,061 markers were assessed in 19 linkage groups (LGs) for the maternal, paternal, and integrated maps, respectively. Our map was highly saturated in terms of marker density and average “Gap ≤ 5 cM” percentage.ConclusionsIn this study, RAD-seq of 176 F1 plants and their parents yielded 8,481,484 SNPs and 1,646,131 InDel markers, of which 65,229 and 4832, respectively, were used to construct a highly saturated genetic map for grapevine. This map is expected to facilitate genetic studies on grapevine, including an evaluation of grapevine and deciphering the genetic basis of economically and agronomically important traits. Our findings provide basic essential genetic data the grapevine genetic research community, which will lead to improvements in grapevine breeding.

Fine-mapping of QTLs for individual and total isoflavone content in soybean (Glycine max L.) using a high-density genetic map.

Fifteen stable QTLs were identified using a high-density soybean genetic map across multiple environments. One major QTL, qIF5-1, contributing to total isoflavone content explained phenotypic variance 49.38, 43.27, 46.59, 45.15 and 52.50%, respectively. Soybeans (Glycine max L.) are a major source of dietary isoflavones. To identify novel quantitative trait loci (QTL) underlying isoflavone content, and to improve the accuracy of marker-assisted breeding in soybean, a valuable mapping population comprised of 196 F7:8-10 recombinant inbred lines (RILs, Huachun 2×Wayao) was utilized to evaluate individual and total isoflavone content in plants grown in four different environments in Guangdong. A high-density genetic linkage map containing 3469 recombination bin markers based on 0.2×restriction site-associated DNA tag sequencing (RAD-seq) technology was used to finely map QTLs for both individual and total isoflavone contents. Correlation analyses showed that total isoflavone content, and that of five individual isoflavone, was significantly correlated across the four environments. Based on the high-density genetic linkage map, a total of 15 stable quantitative trait loci (QTLs) associated with isoflavone content across multiple environments were mapped onto chromosomes 02, 05, 07, 09, 10, 11, 13, 16, 17, and 19. Further, one of them, qIF5-1, localized to chromosomes 05 (38,434,171-39,045,620bp) contributed to almost all isoflavone components across all environments, and explained 6.37-59.95% of the phenotypic variance, especially explained 49.38, 43.27, 46.59, 45.15 and 52.50% for total isoflavone. The results obtained in the present study will pave the way for a better understanding of the genetics of isoflavone accumulation and reveals the scope available for improvement of isoflavone content through marker-assisted selection.

Identification of massive molecular markers in Echinochloa phyllopogon using a restriction-site associated DNA approach

Echinochloa phyllopogon proliferation seriously threatens rice production worldwide. We combined a restriction-site associated DNA (RAD) approach with Illumina DNA sequencing for rapid and mass discovery of simple sequence repeat (SSR) and single nucleotide polymorphism (SNP) markers for E. phyllopogon. RAD tags were generated from the genomic DNA of two E. phyllopogon plants, and sequenced to produce 5197.7 Mb and 5242.9 Mb high quality sequences, respectively. The GC content of E. phyllopogon was 45.8%, which is high for monocots. In total, 4710 putative SSRs were identified in 4132 contigs, which permitted the design of PCR primers for E. phyllopogon. Most repeat motifs among the SSRs identified were dinucleotide (>82%), and most of these SSRs were four motif-repeats (>75%). The most frequent motif was AT, accounting for 36.3%–37.2%, followed by AG and AC. In total, 78 putative polymorphic SSR loci were found. A total of 49,179 SNPs were discovered between the two samples of E. phyllopogon, 67.1% of which were transversions and 32.9% were transitions. We used eight SSRs to study the genetic diversity of four E. phyllopogon populations collected from rice fields in China and all eight loci tested were polymorphic.

Restriction site‐associated DNA sequencing allows for the rapid identification of simple sequence repeat markers in Echinochloa crus‐galli

Echinochloa crus‐galli is a serious weed worldwide. Microsatellite markers (simple sequence repeats, SSRs) are important molecular markers that are used widely for studying genetic diversity in plants. However, a limited number of SSRs is available for E. crus‐galli. The restriction site‐associated DNA (RAD) sequencing approach was combined with Illumina DNA sequencing for the rapid and mass detection of SSRs in E. crus‐galli. The RAD tags were generated from the genomic DNA of E. crus‐galli and were sequenced in order to produce 6921.6 Mb of high‐quality sequences with 45.1% guanine–cytosine content. In total, 3081 putative SSRs were detected, of which 82.2% were dinucleotide motif‐repeats. AT was the most frequent motif, accounting for 35.0% of the SSRs. In order to test the validity of the SSRs that were developed here, eight SSRs that were selected from putative SSRs were used to study the genetic diversity and structures of 20 E. crus‐galli populations that had been collected from rice fields in eastern China. Ninety‐seven alleles were amplified from the eight microsatellite loci among the 20 E. crus‐galli populations. These populations showed low genetic diversity and were classified on the basis of their genetic structures into three distinct groups that corresponded to the three regions of population sampling. The SSRs that were identified in this study represent a valuable resource for studying the genetic diversity, population biology and evolution of E. crus‐galli.

New view of population genetics of zooplankton: RAD-seq analysis reveals population structure of the North Atlantic planktonic copepod Centropages typicus.

Detection of population genetic structure of zooplankton at medium-to-small spatial scales in the absence of physical barriers has remained challenging and controversial. The large population sizes and high rates of gene flow characteristic of zooplankton have made resolution of geographical differentiation very difficult, especially when using few genetic markers and assuming equilibrium conditions. Next-generation sequencing now allows simultaneous sampling of hundreds to thousands of genetic markers; new analytical approaches allow studies under nonequilibrium conditions and directional migration. Samples of the North Atlantic Ocean planktonic copepod, Centropages typicus, were analysed using restriction site-associated DNA (RAD) sequencing on a PROTON platform. Although prior studies revealed no genetic differentiation of populations across the geographical range of the species, analysis of RAD tags showed significant structure across the North Atlantic Ocean. We also compared the likelihood for models of connectivity among NW Atlantic populations under various directional flow scenarios that replicate oceanographic conditions of the sampled domain. High-density marker sampling with RAD sequencing markedly outperformed other technical and analytical approaches in detection of population genetic structure and characterization of connectivity of this high geneflow zooplankton species.

RAD Capture (Rapture): Flexible and Efficient Sequence-Based Genotyping.

Massively parallel sequencing has revolutionized many areas of biology, but sequencing large amounts of DNA in many individuals is cost-prohibitive and unnecessary for many studies. Genomic complexity reduction techniques such as sequence capture and restriction enzyme-based methods enable the analysis of many more individuals per unit cost. Despite their utility, current complexity reduction methods have limitations, especially when large numbers of individuals are analyzed. Here we develop a much improved restriction site-associated DNA (RAD) sequencing protocol and a new method called Rapture ( R: AD c APTURE: ). The new RAD protocol improves versatility by separating RAD tag isolation and sequencing library preparation into two distinct steps. This protocol also recovers more unique (nonclonal) RAD fragments, which improves both standard RAD and Rapture analysis. Rapture then uses an in-solution capture of chosen RAD tags to target sequencing reads to desired loci. Rapture combines the benefits of both RAD and sequence capture, i.e., very inexpensive and rapid library preparation for many individuals as well as high specificity in the number and location of genomic loci analyzed. Our results demonstrate that Rapture is a rapid and flexible technology capable of analyzing a very large number of individuals with minimal sequencing and library preparation cost. The methods presented here should improve the efficiency of genetic analysis for many aspects of agricultural, environmental, and biomedical science.

Identification of Laying-Related SNP Markers in Geese Using RAD Sequencing.

Laying performance is an important economical trait of goose production. As laying performance is of low heritability, it is of significance to develop a marker-assisted selection (MAS) strategy for this trait. Definition of sequence variation related to the target trait is a prerequisite of quantitating MAS, but little is presently known about the goose genome, which greatly hinders the identification of genetic markers for the laying traits of geese. Recently developed restriction site-associated DNA (RAD) sequencing is a possible approach for discerning large-scale single nucleotide polymorphism (SNP) and reducing the complexity of a genome without having reference genomic information available. In the present study, we developed a pooled RAD sequencing strategy for detecting geese laying-related SNP. Two DNA pools were constructed, each consisting of equal amounts of genomic DNA from 10 individuals with either high estimated breeding value (HEBV) or low estimated breeding value (LEBV). A total of 139,013 SNP were obtained from 42,291,356 sequences, of which 18,771,943 were for LEBV and 23,519,413 were for HEBV cohorts. Fifty-five SNP which had different allelic frequencies in the two DNA pools were further validated by individual-based AS-PCR genotyping in the LEBV and HEBV cohorts. Ten out of 55 SNP exhibited distinct allele distributions in these two cohorts. These 10 SNP were further genotyped in a goose population of 492 geese to verify the association with egg numbers. The result showed that 8 of 10 SNP were associated with egg numbers. Additionally, liner regression analysis revealed that SNP Record-111407, 106975 and 112359 were involved in a multiplegene network affecting laying performance. We used IPCR to extend the unknown regions flanking the candidate RAD tags. The obtained sequences were subjected to BLAST to retrieve the orthologous genes in either ducks or chickens. Five novel genes were cloned for geese which harbored the candidate laying-related SNP, including membrane associated guanylate kinase (MAGI-1), KIAA1462, Rho GTPase activating protein 21 (ARHGAP21), acyl-CoA synthetase family member 2 (ACSF2), astrotactin 2 (ASTN2). Collectively, our data suggests that 8 SNP and 5 genes might be promising candidate markers or targets for marker-assisted selection of egg numbers in geese.

Construction of a high density SNP linkage map of kelp (Saccharina japonica) by sequencing Taq I site associated DNA and mapping of a sex determining locus.

BackgroundKelp (Saccharina japonica) has been intensively cultured in China for almost a century. Its genetic improvement is comparable with that of rice. However, the development of its molecular tools is extremely limited, thus its genes, genetics and genomics. Kelp performs an alternative life cycle during which sporophyte generation alternates with gametophyte generation. The gametophytes of kelp can be cloned and crossed. Due to these characteristics, kelp may serve as a reference for the biological and genetic studies of Volvox, mosses and ferns.ResultsWe constructed a high density single nucleotide polymorphism (SNP) linkage map for kelp by restriction site associated DNA (RAD) sequencing. In total, 4,994 SNP-containing physical (tag-defined) RAD loci were mapped on 31 linkage groups. The map expanded a total genetic distance of 1,782.75 cM, covering 98.66% of the expected (1,806.94 cM). The length of RAD tags (85 bp) was extended to 400–500 bp with Miseq method, offering us an easiness of developing SNP chips and shifting SNP genotyping to a high throughput track. The number of linkage groups was in accordance with the documented with cytological methods. In addition, we identified a set of microsatellites (99 in total) from the extended RAD tags. A gametophyte sex determining locus was mapped on linkage group 2 in a window about 9.0 cM in width, which was 2.66 cM up to marker_40567 and 6.42 cM down to marker_23595.ConclusionsA high density SNP linkage map was constructed for kelp, an intensively cultured brown alga in China. The RAD tags were also extended so that a SNP chip could be developed. In addition, a set of microsatellites were identified among mapped loci, and a gametophyte sex determining locus was mapped. This map will facilitate the genetic studies of kelp including for example the evaluation of germplasm and the decipherment of the genetic bases of economic traits.Electronic supplementary materialThe online version of this article (doi:10.1186/s12864-015-1371-1) contains supplementary material, which is available to authorized users.

Characterization of 19 polymorphic microsatellite markers for Asian yellow pond turtle (Mauremys mutica)

Asian yellow pond turtle (Mauremys mutica) is an endangered species. In this study, we developed and characterized 19 novel polymorphic microsatellite markers for M. mutica through the Restriction-site Associated DNA tags sequencing (RAD-seq). 60 individuals were used to assess the level of polymorphism of each locus. The number of alleles ranged from 4 to 25, the observed and expected heterozygosity ranged from 0.40000 to 0.90000 and 0.39572 to 0.93201, respectively. These markers would be used in genetic studies helping conservation and management of M. mutica.

Genetic architecture of a hormonal response to gene knockdown in honey bees.

Variation in endocrine signaling is proposed to underlie the evolution and regulation of social life histories, but the genetic architecture of endocrine signaling is still poorly understood. An excellent example of a hormonally influenced set of social traits is found in the honey bee (Apis mellifera): a dynamic and mutually suppressive relationship between juvenile hormone (JH) and the yolk precursor protein vitellogenin (Vg) regulates behavioral maturation and foraging of workers. Several other traits cosegregate with these behavioral phenotypes, comprising the pollen hoarding syndrome (PHS) one of the best-described animal behavioral syndromes. Genotype differences in responsiveness of JH to Vg are a potential mechanistic basis for the PHS. Here, we reduced Vg expression via RNA interference in progeny from a backcross between 2 selected lines of honey bees that differ in JH responsiveness to Vg reduction and measured JH response and ovary size, which represents another key aspect of the PHS. Genetic mapping based on restriction site-associated DNA tag sequencing identified suggestive quantitative trait loci (QTL) for ovary size and JH responsiveness. We confirmed genetic effects on both traits near many QTL that had been identified previously for their effect on various PHS traits. Thus, our results support a role for endocrine control of complex traits at a genetic level. Furthermore, this first example of a genetic map of a hormonal response to gene knockdown in a social insect helps to refine the genetic understanding of complex behaviors and the physiology that may underlie behavioral control in general.

Rapid SNP discovery and a RAD-based high-density linkage map in jujube (Ziziphus Mill.).

Background Ziziphus Mill. (jujube), the most valued genus of Rhamnaceae, comprises of a number of economically and ecologically important species such as Z. jujuba Mill., Z. acidojujuba Cheng et Liu and Z. mauritiana Lam. Single nucleotide polymorphism (SNP) markers and a high-density genetic map are of great benefit to the improvement of the crop, mapping quantitative trait loci (QTL) and analyzing genome structure. However, such a high-density map is still absent in the genus Ziziphus and even the family Rhamnaceae. The recently developed restriction-site associated DNA (RAD) marker has been proven to be most powerful in genetic map construction. The objective of this study was to construct a high-density linkage map using the RAD tags generated by next generation sequencing.ResultsAn interspecific F1 population and their parents (Z. jujuba Mill. ‘JMS2’ × Z. acidojujuba Cheng et Liu ‘Xing 16’) were genotyped using a mapping-by-sequencing approach, to generate RAD-based SNP markers. A total of 42,784 putative high quality SNPs were identified between the parents and 2,872 high-quality RAD markers were grouped in genetic maps. Of the 2,872 RAD markers, 1,307 were linked to the female genetic map, 1,336 to the male map, and 2,748 to the integrated map spanning 913.87 centi-morgans (cM) with an average marker interval of 0.34 cM. The integrated map contained 12 linkage groups (LGs), consistent with the haploid chromosome number of the two parents.ConclusionWe first generated a high-density genetic linkage map with 2,748 RAD markers for jujube and a large number of SNPs were also developed. It provides a useful tool for both marker-assisted breeding and a variety of genome investigations in jujube, such as sequence assembly, gene localization, QTL detection and genome structure comparison.

Lep-MAP: fast and accurate linkage map construction for large SNP datasets

Current high-throughput sequencing technologies allow cost-efficient genotyping of millions of single nucleotide polymorphisms (SNPs) for hundreds of samples. However, the tools that are currently available for constructing linkage maps are not well suited for large datasets. Linkage maps of large datasets would be helpful in de novo genome assembly by facilitating comprehensive genome validation and refinement by enabling chimeric scaffold detection, as well as in family-based linkage and association studies, quantitative trait locus mapping, analysis of genome synteny and other complex genomic data analyses. We describe a novel tool, called Lepidoptera-MAP (Lep-MAP), for constructing accurate linkage maps with ultradense genome-wide SNP data. Lep-MAP is fast and memory efficient and largely automated, requiring minimal user interaction. It uses simultaneously data on multiple outbred families and can increase linkage map accuracy by taking into account achiasmatic meiosis, a special feature of Lepidoptera and some other taxa with no recombination in one sex (no recombination in females in Lepidoptera). We demonstrate that Lep-MAP outperforms other methods on real and simulated data. We construct a genome-wide linkage map of the Glanville fritillary butterfly (Melitaea cinxia) with over 40 000 SNPs. The data were generated with a novel in-house SOLiD restriction site-associated DNA tag sequencing protocol, which is described in the online supplementary material. Java source code under GNU general public license with the compiled classes and the datasets are available from http://sourceforge.net/users/lep-map.

New SNPs for population genetic analysis reveal possible cryptic speciation of eastern Australian sea mullet (Mugil cephalus)

Sustainable management of sea mullet (Mugil cephalus) fisheries needs to account for recent observations of regional-scale differentiation. Population genetic analysis is sought to assess the situation of this ecologically and economically important fish species in eastern Australian waters. Here, we report (i) new population genetic markers [single nucleotide polymorphisms (SNPs) and potential microsatellites], (ii) first estimates of spatial genetic differentiation and (iii) prospective power tests for designing more comprehensive studies. Six DNA samples from three sampling regions (North Queensland, South Queensland and central New South Wales) on the eastern coast of Australia were used to prepare restriction site associated DNA (RAD) tag libraries from genomic DNA digested with EcoRI and MseI. A pooled sample of regional RAD tag libraries was sequenced using the Roche GS-FLX Titanium platform. A total of 172,837 raw reads (17.4 Mbp) were retrieved, 95,500 of which were used to discover 1267 SNPs and 1417 microsatellites. A subset of 161 SNPs was validated based on 63 additional DNA samples genotyped using the Sequenom MassArray (iPLEX Gold chemistry). Altogether 92 SNPs (57%) were confirmed, with 40% of these marking fixed variants between northern and southern sampling regions. Our preliminary findings indicate a multispecies fishery stock of M. cephalus in eastern Australian waters, but suggest that strong genetic differentiation occurs north of major fishing grounds. Low potential differentiation within major fishing grounds (e.g. FST = 0.0025) can be resolved with a likely power ≥67% by using standard sample sizes of 50 and validated subsets of available markers.