Madjid Delghandi

Identification and characterisation of novel SNP markers in Atlantic cod: Evidence for directional selection

BackgroundThe Atlantic cod (Gadus morhua) is a groundfish of great economic value in fisheries and an emerging species in aquaculture. Genetic markers are needed to identify wild stocks in order to ensure sustainable management, and for marker-assisted selection and pedigree determination in aquaculture. Here, we report on the development and evaluation of a large number of Single Nucleotide Polymorphism (SNP) markers from the alignment of Expressed Sequence Tag (EST) sequences in Atlantic cod. We also present basic population parameters of the SNPs in samples of North-East Arctic cod and Norwegian coastal cod obtained from three different localities, and test for SNPs that may have been targeted by natural selection.ResultsA total of 17,056 EST sequences were used to find 724 putative SNPs, from which 318 segregating SNPs were isolated. The SNPs were tested on Atlantic cod from four different sites, comprising both North-East Arctic cod (NEAC) and Norwegian coastal cod (NCC). The average heterozygosity of the SNPs was 0.25 and the average minor allele frequency was 0.18. FSTvalues were highly variable, with the majority of SNPs displaying very little differentiation while others had FSTvalues as high as 0.83. The FSTvalues of 29 SNPs were found to be larger than expected under a strictly neutral model, suggesting that these loci are, or have been, influenced by natural selection. For the majority of these outlier SNPs, allele frequencies in a northern sample of NCC were intermediate between allele frequencies in a southern sample of NCC and a sample of NEAC, indicating a cline in allele frequencies similar to that found at the Pantophysin I locus.ConclusionThe SNP markers presented here are powerful tools for future genetics work related to management and aquaculture. In particular, some SNPs exhibiting high levels of population divergence have potential to significantly enhance studies on the population structure of Atlantic cod.

Simultaneous analysis of six microsatellite markers in Atlantic cod ( Gadus morhua): a novel multiplex assay system for use in selective breeding studies.

A novel hexaplex assay system including Gmo8, Gmo19, Gmo35, Gmo37, Tch11, and Tch12 microsatellites from Atlantic cod, consisting of trinucleotide or tetranucleotide repeat units, is introduced. All 6 loci were coamplified in a single reaction employing dye-labeled primers. Alleles from these loci were sized using an internal standard by automated sample processing in an ABI 310 Genetic Analyser. Amplified alleles in profiles containing selected microsatellites were typed clearly, providing easily interpretable results. Sequencing data indicated that alleles at all loci consisted of simple repeat units. This may help minimize the likelihood of stuttering upon polymerase chain reaction amplification. The results suggest that the presented hexaplex assay system may be a useful tool in a selective breeding program in which genetic identification will allow different genotypes to be reared together from fertilization. This should have a great impact as it will make selective breeding more efficient.

Identification and characterisation of thirteen new microsatellites for Atlantic cod (Gadus morhua L.) from a repeat-enriched library

A total of 13 polymorphic microsatellite markers were developed for Atlantic cod (Gadus morhua L.) from a repeat-enriched library. Polymorphism of each locus was assessed in 96 unrelated individuals from a natural population. The number of alleles per locus varied from 8 to 45. The ranges of observed and expected heterozygosity were 0.122–0.907 and 0.673–0.965, respectively. Four of the loci (Gmo-G24, Gmo-G40, Gmo-G46 and Gmo-G49) followed Hardy–Weinberg expectation. No evidence for linkage disequilibrium between pairs of loci was found in any combination of loci pairs, except between Gmo-G40 and Gmo-G43. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and for constructing linkage maps of Atlantic cod.

Development of ten new EST-derived microsatellites in Atlantic cod (Gadus morhua L.)

Ten polymorphic microsatellite markers were developed from approximately 1,300 expressed sequence tags (ESTs) of Atlantic cod (Gadus morhua L.). Thirty two primer pairs were designed for EST sequences containing perfect di- tri- tetra- and pentanucleotide motifs and characterised in 96 unrelated fish. Ten markers were successfully amplified with number of alleles from 2 to 13 per locus and observed and expected heterozygosity ranging from 0.03 to 0.69 and 0.03 to 0.74, respectively. Loci Gmo-C131, C132 and C136 deviated from Hardy-Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C131 and Gmo-C132 and C128 and Gmo-C133. The gene identity was determined at five of the loci, confirming the associated microsatellites as Type I markers. The new microsatellites reported in this work can be used for conservation and enhancement of wild stocks for commercial harvesting.

Identification and characterisation of nine new gene-associated microsatellite markers for Atlantic cod (Gadus morhua L.)

Nine polymorphic microsatellite markers were identified by screening of 2464 ESTs derived from a cDNA library of Atlantic cod (Gadus morhua L.). About 35 novel microsatellite loci were selected and characterised in 96 individual cod. Nine markers were successfully amplified with number of alleles from 3 to 18 per locus and the average heterozygosity was 0.57 in the panel examined (range 0.29–0.86). All loci followed the Hardy–Weinberg expectation and no significant linkage disequilibrium was found in a test including all pairwise combinations. The gene identity was determined at four of the loci, confirming the associated microsatellites as Type I markers.

Development of twenty sequence-tagged microsatellites for the Atlantic cod (Gadus morhua L.)

Fifty-four primer pairs were designed for expressed sequence tag (EST) sequences containing perfect di- and tri-nucleotide motifs and characterised in 96 unrelated fish. Twenty markers were successfully amplified with number of alleles from 2 to 10 per locus and observed and expected heterozygosity ranging from 0.01 to 0.56 and 0.03 to 0.70, respectively. Loci Gmo-C213, Gmo-C246 and Gmo-C247 deviated from Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C213 and Gmo-C222, Gmo-C233 and Gmo-C229, C223 and Gmo-C236 and C229 and Gmo-C236. The gene identity was determined at 10 of the loci, confirming the associated microsatellites as Type I markers. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and constructing linkage maps of Atlantic cod.

Isolation, characterization, and multiplexing of novel microsatellite markers for the tropical scalloped spiny lobster (Panulirus homarus)

Of the various spiny lobster species in the tropical and subtropical Indo-West Pacific region, the tropical scalloped spiny lobster (Panulirus homarus) supports one of the most commercially valuable fishery resources in many coastal African and Asian countries. The last decade has witnessed a serious decline in the wild populations of this species. Knowledge of the genetic basis of spiny lobster population structure is a prerequisite to achieve a sustainable fisheries management for this species. Here, we describe 13 novel polymorphic microsatellite markers developed for P. homarus, using a cross-species primer design strategy based on P. ornatus Roche 454 shot-gun generated sequencing. Microsatellite polymorphisms were assessed in 96 unrelated P. homarus individuals of a natural population, with the number of alleles per locus varying from 2 to 14, the observed and expected heterozygosity from 0.00 to 0.78 and from 0.03 to 0.79, respectively, and with only four loci (Pho-G27, Pho-G32, Pho-G36, and Pho-G58) deviating from Hardy- Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Pho-G22 - Pho-G30, and Pho-G30 - Pho-G35. The successful cross amplification of these microsatellites highlights the potential of the developed microsatellites for future population genetic research within the different Panulirus species.

Novel genomic microsatellite markers for genetic population and diversity studies of tropical scalloped spiny lobster (Panulirus homarus) and their potential application in related Panulirus species.

Fourteen polymorphic microsatellites with perfect di-, tri-, and tetra-nucleotide repeats were identified for Panulirus homarus using Roche 454 whole-genome sequencing method. Microsatellites were efficiently co-amplified in four multiplexes and a singleplex, providing consistent and easily interpretable genotypes. The number of alleles per locus ranged from 2 to 11 with the observed and expected heterozygosity ranging between 0.000-0.532 and 0.031-0.836, respectively. A significant deviation from Hardy-Weinberg equilibrium was observed for majority of the loci, probably due to homozygote excess. Genetic linkage disequilibrium analysis between all the possible pairs of the loci showed significant departure from the null hypothesis in the loci pairs Pho-G11-Pho-G33 and Pho-G33-Pho-G57. High success in primer cross-species amplification of these microsatellite markers indicates their utility for genetic studies of different Panulirus species.

New polymorphic di-nucleotide microsatellite markers for Atlantic cod (Gadus morhua L.)

Twenty three polymorphic microsatellite markers were developed from approximately 2,300 expressed sequence tags (ESTs) of Atlantic cod (Gadus morhua L.). Seventy two primer pairs were designed for EST sequences containing perfect di-nucleotide motifs and characterised in 96 unrelated fish. Twenty three markers were successfully amplified with number of alleles from 2 to 18 per locus and observed and expected heterozygosity ranging from 0.03 to 1.00 and 0.04 to 0.90, respectively. Loci Gmo-C280, Gmo-C283, Gmo-C290 and Gmo-C293 deviated from Hardy–Weinberg equilibrium. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between loci Gmo-C267 and Gmo-C269 and Gmo-C262 and Gmo-C291. The gene identity was determined at three of the loci, confirming the associated microsatellites as Type I markers. These microsatellite markers provide useful tools for studies of population genetics, reproductive ecology and constructing linkage maps of Atlantic cod.

Novel Polymorphic Microsatellite Markers for Panulirus ornatus and their Cross-species Primer Amplification in Panulirus homarus

ABSTRACT Polymorphic microsatellite loci were isolated for Panulirus ornatus using 454 GS-FLX Titanium pyrosequencing. Fifteen markers containing perfect di-, tri-, tetra-, and penta-nucleotide motifs were consistently co-amplified in five multiplexes in a panel of 91 randomly selected samples. Observed number of alleles varied from 2 to 14 per locus. Observed and expected heterozygosity ranged from 0.090 to 0.79 and 0.08 to 0.87, respectively. Ten loci deviated from Hardy-Weinberg equilibrium after sequential Bonferroni correction. Genetic linkage disequilibrium analysis between all pairs of the loci showed significant departure from the null hypothesis between 11 loci. The microsatellite markers were also amplified successfully in related Panulirus homarus species with adequate level of polymorphism. The successful cross-species primer amplification of the 15 microsatellites indicates the potential of the developed markers to be transferred to other Panulirus species. The 15 novel microsatellite markers reported in this work add to the previously characterized markers by our group, exhibit adequate levels of polymorphism for wide range of future studies investigating population structure, genetic diversity, and evolutionary relationships among Panulirus species.