Efficient establishment of primary fibroblast cultures from the hawksbill sea turtle (Eretmochelys imbricata)

The hawksbill sea turtle (Eretmochelys imbricata) is a critically endangered species at a risk of extinction. Preservation of the genomic and cellular information of endangered animals is important for future genetic and biological studies. Here, we report the efficient establishment of primary fibroblast cultures from skin tissue of the hawksbill sea turtle. We succeeded in establishing 19 primary cultures from 20 hawksbill sea turtle individuals (a success rate of 95%). These cells exhibited a fibroblast-like morphology and grew optimally at a temperature of 26°C, but experienced a loss of viability when cultured at 37°C. Chromosomal analysis using the primary cells derived here revealed that hawksbill sea turtles have a 2n?=?56 karyotype. Furthermore, we showed that our primary cell cultures are free of several fish-related viruses, and this finding is important for preservation purposes. To our knowledge, this report is the first to describe primary cell cultures established from normal tissues of the hawksbill sea turtle. The results will contribute to the preservation of biodiversity, especially for the sea turtles that are critically endangered owing to human activities.     

Multiple paternity in egg clutches of hawksbill turtles (Eretmochelys imbricata)

We present the first data collected on the genetic mating system of the hawksbill turtle Eretmochelys imbricata, the only marine turtle not studied to date. We examined paternity within 12 egg clutches from ten female hawksbill turtles from Sabah Turtle Islands, Malaysia. A total of 375 hatchlings were analysed using five microsatellite markers. Results demonstrated that clutches from two out of ten females were sired by multiple males (maximum of two). Although at a low frequency, observation of multiple paternity indicates that hawksbill turtles exhibit the same genetic mating system (polyandry) as observed for other species of marine turtles. Consistent paternity across multiple clutches laid by individual females in one breeding season supports the hypothesis that sperm are stored from mating prior to nesting and are then used to fertilize all subsequent clutches of eggs that season.     

Isolation and characterization of polymorphic microsatellite loci in muskrat, Ondatra zibethicus

We describe the isolation and characterization of 12 highly polymorphic microsatellite loci for the muskrat, Ondatra zibethicus. Microsatellite markers from three other rodent species were cross-amplified in muskrat and one of them was polymorphic. We observed moderate to high levels of genetic variability in these 13 polymorphic loci (five to 22 alleles per locus) with observed heterozygosity ranging from 0.48 to 0.96. These markers will be useful for further studies on population genetic structure in muskrat and potentially in other rodent species.     

A mapped set of genetic markers for human chromosome 9

A genetic map of markers for human chromosome 9, spanning a genetic distance of 147 cM in males and 231 cM in females, has been constructed from linkage studies with 19 loci in a large panel of reference families. The markers included four classical systems previously assigned to chromosome 9, and restriction fragment length polymorphisms of two cloned genes, ABL oncogene and argininosuccinase synthetase pseudogene 3 (ASSP3). The remaining 13 marker loci, with an average heterozygosity of 42%, were defined by arbitrary DNA probes newly ascertained from genomic libraries; seven of them were variable number of tandem repeat (VNTR) loci. A subset of 7 of the 19 linked markers is proposed for a primary map that could detect linkage with a genetic defect within the covered region of chromosome 9, provided that at least 45 phase-known meioses were available for study in an affected family.     

A high-resolution consensus linkage map of the rat, integrating radiation hybrid and genetic maps

We have constructed a high-resolution consensus genetic map of the rat in a single large intercross, which integrates 747 framework markers and 687 positions of our whole-genome radiation hybrid (RH) map of the rat. We selected 136 new gene markers from the GenBank database and assigned them either genetically or physically to rat chromosomes to evaluate the accuracy of the integrated linkage-RH maps in the localization of new markers and to enrich existing comparative mapping data. These markers and 631 D-Got- markers, which are physically mapped but still uncharacterized for evidence of polymorphism, were tested for allele variations in a panel of 16 rat strains commonly used in genetic studies. The consensus linkage map constructed in the GK x BN cross now comprises 1620 markers of various origins, defining 840 resolved genetic positions with an average spacing of 2.2 cM between adjacent loci, and includes 407 gene markers. This whole-genome genetic map will contribute to the advancement of genetic studies in the rat by incorporating gene/EST maps, physical mapping information, and sequence data generated in rat and other mammalian species into genetic intervals harboring disease susceptibility loci identified in rat models of human genetic disorders.     

Ten polymorphic microsatellite DNA loci for paternity and population genetics analysis in the fen raft spider (Dolomedes plantarius)

Ten polymorphic di‐ and trinucleotide microsatellite loci were developed in the fen raft spider Dolomedes plantarius from a partial phagemid genomic library enriched for microsatellite inserts. The expected heterozygosity at these loci ranges from 0.62 to 0.9, with the observed allele numbers varying from four to 15 in the 22 individuals tested. Average paternity exclusion probabilities ranged between 0.290 and 0.686. In combination, the 10 polymorphic loci elicit an exclusion probability of 0.999. The high level of polymorphism of these microsatellite loci makes them ideal genetic markers for paternity and population genetics analysis in this endangered species.     

Targeted development of informative microsatellite (SSR) markers

We describe a novel approach, selectively amplified microsatellite (SAM) analysis, for the targeted development of informative simple sequence repeat (SSR) markers. A modified selectively amplified microsatellite polymorphic loci assay is used to generate multi-locus SSR fingerprints that provide a source of polymorphic DNA markers (SAMs) for use in genetic studies. These polymorphisms capture the repeat length variation associated with SSRs and allow their chromosomal location to be determined prior to the expense of isolating and characterising individual loci. SAMs can then be converted to locus-specific SSR markers with the design and synthesis of a single primer specific to the conserved region flanking the repeat. This approach offers a cost-efficient and rapid method for developing SSR markers for predetermined chromosomal locations and of potential informativeness. The high recovery rate of useful SSR markers makes this strategy a valuable tool for population and genetic mapping studies. The utility of SAM analysis was demonstrated by the development of SSR markers in bread wheat.     

Sixteen novel microsatellite loci developed for the giant panda (Ailuropoda melanoleuca)

Sixteen novel microsatellite DNA loci were developed from the giant panda (Ailuropoda melanoleuca) using a magnetic-bead capture method. A total of 115 alleles were obtained for these markers, ranging from 4 to 12 alleles per locus (average 7.188). These loci exhibited high levels of polymorphic information content and expected heterozygosity, 0.558–0.855 (average 0.729) and 0.628–0.885 (average 0.778), respectively. Therefore, the allelic polymorphism and heterozygosity show that the giant pandas raised in China Research and Conservation Center possess abundant genetic variation. In addition, if the three markers showing null alleles were excluded, the remaining 13 microsatellite loci still presented extremely low non-exclusion probabilities of parentage (0.002), paternity (0.000) and identity (0.000). As a result, this new suit of microsatellite markers would be a very informative tool for the genetic and conservation studies of giant pandas.     

Concordance of genetic divergence among sockeye salmon populations at allozyme, nuclear DNA, and mitochondrial DNA markers

Abstract.— We examined genetic variation at 21 polymorphic allozyme loci, 15 nuclear DNA loci, and mitochondrial DNA in four spawning populations of sockeye salmon ( Oncorhynchus nerka ) from Cook Inlet, Alaska, to test for differences in the patterns of divergence among different types of markers. We were specifically interested in testing the suggestion that natural selection at allozyme loci compromises the effectiveness of these markers for describing the amount and patterns of gene flow among populations. We found concordance among markers in the amount of genetic variation within and among populations, with the striking exception of one allozyme locus ( sAH ), which exhibited more than three times the amount of among-population differentiation as other loci. A consideration of reports of discordance between allozymes and other loci indicates that these differences usually result from one or two exceptional loci. We conclude that it is important to examine many loci when estimating genetic differentiation to infer historical amounts of gene flow and patterns of genetic exchange among populations. It is less important whether those loci are allozymes or nuclear DNA markers.     

Nuclear microsatellite loci for Arabidopsis halleri (Brassicaceae), a model species to study plant adaptation to heavy metals

? Premise of the study: Arabidopsis halleri is a model species to study the adaptation of plants to soils contaminated by zinc, cadmium, and lead. To provide a neutral genetic background with which adaptive genetic markers could be compared, we developed highly polymorphic neutral microsatellite markers. ? Methods and Results: Using a microsatellite-enriched library method, we identified 120 microsatellite loci for quantitative trait locus (QTL) mapping analysis, of which eight primer pairs were developed in a single multiplex for population genetic studies. Analyses were performed on 508 individuals from 26 populations. All loci were polymorphic with six to 23 alleles per locus. Genetic diversity varied between 0.56 and 0.76. ? Conclusions: Our results demonstrated the value of these eight microsatellite markers to investigate neutral population genetic structure in A. halleri. To increase the resolution of population genetic analyses, we suggest adding them to the 11 markers previously developed independently.     

Microsatellite loci for studies of wild and hatchery Australian Murray cod Maccullochella peelii peelii (Percichthyidae)

Murray cod is a species of conservation concern that is subject both to wild harvest and to aquaculture production. Six polymorphic microsatellite loci have been developed for this species, which will facilitate studies of wild‐stock structure, will help the management of hatchery diversity, and will aid genetic improvement programmes. The markers exhibited nonindependence of genotypes between loci and deviations from Hardy–Weinberg expectations, although these most probably reflect practices at the hatchery from which the genotyped individuals were sourced.     

Genetic variation in remnant populations of Dalbergia nigra (Papilionoideae), an endangered tree from the Brazilian Atlantic Forest

BACKGROUND AND AIMS: Dalbergia nigra, known as Brazilian rosewood, is an endangered tree species restricted to the Brazilian Atlantic Forest and has been intensively logged for five centuries due to its high-quality wood. The objective of the present study was to assess the genetic variation and structure in adults and saplings of the species from a large reserve of the Atlantic Forest, the Rio Doce State Park, and from two small surrounding fragments, one better preserved and another with a high degree of anthropogenic disturbance. METHODS: Analyses of genetic variation and structure were conducted by studying allozyme markers. Seven putative enzymatic loci were resolved, five of them being polymorphic. KEY RESULTS: The mean numbers of alleles per locus (A) were 1.93 and 1.73, while the percentages of polymorphic loci (P) were 93 and 73 % for adults and saplings, respectively. Saplings from the fragment with high anthropogenic disturbance exhibited the lowest values of A and P. The fragment that constitutes a conservation area exhibited genetic variation similar to the population from the large reserve. The observed (H(o)) and expected (H(e)) heterozygosities were not significantly different among the three populations. Only sapling populations showed F(ST) values (divergence among populations) significantly different from zero over all studied loci. The fragment with high anthropogenic disturbance exhibited considerable genetic divergence in relation to the above-cited populations. CONCLUSIONS: The evaluated populations displayed mean levels of genetic variation intermediate to those expected for narrow and widespread species. The results suggest that fragments with similar area and geographical distance from a large protected reserve can exhibit different levels of genetic variation, depending on the degree of anthropogenic disturbance. The considerable genetic variation in the protected fragment points to the importance of adequate conservation of small fragments for the preservation of genetic variation in D. nigra.     

Microsatellite markers for the June sucker (Chasmistes liorus mictus), Utah sucker (Catostomus ardens), and five other catostomid fishes of western North America

We developed and optimized five new microsatellite markers for the genetic management of the endangered June sucker. We report the cross‐amplification of these markers, and seven microsatellites previously developed for Klamath Basin suckers, in seven catostomid species of western North America. No linkage disequilibrium was detected between pairs of loci. Since most of these loci exhibited conserved priming sites, they may be useful for landscape‐scale studies of speciation and patterns of gene flow among multiple sucker lineages.     

Development of high transferability cpSSR markers for individual identification and genetic investigation in Cupressaceae species

Given the low substitution rate in plastomes, the polymorphic and codominant nature of chloroplast SSRs (cpSSRs) makes them ideal markers, complementing their nuclear counterpart. In Cupressaceae, cpSSRs are mostly paternally inherited, thus, they are useful in mating systems and pollen flow studies. Using e‐PCR, 92 SSR loci were identified across six Cupressaceae plastomes, and primers were designed for 26 loci with potential interspecific transferability. The 26 developed cpSSRs were polymorphic in four genera, Platycladus, Sabina, Juniperus, and Cupressus and are suitable for Cupressaceae molecular genetic studies and utilization. We genotyped 192 Platycladus orientalis samples from a core breeding population using 10 of the developed cpSSRs and 10 nuclear SSRs, and these individuals were identified with high confidence. The developed cpSSRs can be used in (1) a marker‐assisted breeding scheme, specifically when paternity identification is required, (2) population genetics investigations, and (3) biogeography of Cupressaceae and unraveling the genetic relationships between related species.     

Development and characterization of 70 novel microsatellite markers for the sea cucumber (Apostichopus japonicus)

The sea cucumber (Apostichopus japonicus) is an important item in Asian cuisine. It is currently produced through aquaculture, especially in China, after being overexploited in the wild in the 1990s. We isolated 70 novel polymorphic microsatellite loci using an enrichment-colony hybridization protocol. All loci were characterized in 48 individuals from a natural population in Rongcheng (Shandong, China) using genomic DNA isolated from muscle tissue. The number of alleles ranged from 2 to 17 (mean 7.0), and the observed and expected heterozygosities varied from 0.0010 to 1.0000 and from 0.2125 to 0.9477, respectively. Thirty-one of the 70 loci exhibited departure from Hardy-Weinberg equilibrium. These microsatellite markers should be useful resources for population genetic studies and for molecular marker-assisted breeding of A. japonicus.     

Exploration and mapping of microsatellite markers from subtracted drought stress ESTs in Sorghum bicolor (L.) Moench

Molecular variation within defined genes underlying specific biochemical or physiological functions provide candidate gene-based markers which show very close association with the trait of interest and thus should enable to design superior genotypes. We explored microsatellite loci in a total of 9,892 subtracted drought stress ESTs of sorghum (6,295 after flowering ESTs and 3,597 before flowering ESTs) available in the NCBI dbEST database. Analysis of 9,892 ESTs identified 221 non-redundant ESTs with SSRs, from which 109 functional SSRs were developed. Among them 62 EST-microsatellites (56.8%) exhibited polymorphism for at least one sorghum genotype among the five tested and yielded a total of 161 alleles, with an average of 2.59 alleles per marker. We present a microsatellite linkage map using a RIL population derived from the cross 296B and IS18551. The map contains 128 microsatellite loci distributed over 15 linkage groups, and spanning a genetic distance of 1,074.5 cM. The map includes map positions of 28 drought EST-microsatellites developed and seven new genomic-SSRs, and are distributed throughout the map. The developed EST markers include genes coding for important regulatory proteins and functional proteins that are involved in stress related metabolism. The drought EST-microsatellites will have applications in functional diversity studies, association studies, QTL studies for drought, and other agronomically important traits in sorghum, and comparative genomics studies between sorghum and other members of the Poaceae family.     

凡纳滨对虾微卫星位点在两个选育家系中遗传的初步研究

利用两个选育凡纳滨对虾全同胞家系研究了10个微卫星位点的遗传特征。通过ABI310或3100测序仪检测, 在所观察到的20个基因型比例（genotypic ratios）(10个微卫星位点 X 2个家系)中,有17个基因型比例符合孟德尔遗传。微卫星位点TUMXLv8.220在两个家系中均存在无效等位基因,从而3个不符合孟德尔遗传基因型中2个可由无效等位基因来解释。TUMXLv 3.1在06家系偏离了1：1：1：1的孟德尔预期比。3个微卫星位点（TUMXLv5.66,TUMXLv7.74,TUMXLv8.224）在两个家系中均表现单态。3个微卫星位点（TUMXLv5.45,TUMXLv7.56,TUMXLv8.256）在两个家系均既表现多态又遵循孟德尔共显性遗传, 是亲子鉴定和种群遗传分析的较好选择。结果显示在应用微卫星标记进行遗传分析之前利用全同胞家系进行遗传模式研究是非常必要的。     

Development of microsatellite markers for the red tide‐forming harmful species Chattonella antiqua,C. marina,and C. ovata (Raphidophyceae)

We have developed 11 microsatellite markers that are specific to Chattonella antiqua, C. marina, and C. ovata, the red tide‐forming harmful phytoplanktons. The 11 loci were amplified in the three species. The number of alleles per locus ranged from 5 to 16. The three species shared most microsatellite regions, although the genetic differences in specific loci were detected among them. These markers of the Chattonella species will be beneficial for biogeographical, detailed taxonomic, studies.     

Chicken microsatellite primers are not efficient markers for Japanese quail

Domestic fowl or chicken (Gallus gallus) and Japanese quail (Coturnix japonica) belong to the family Phasianidae. The exchange of marker information between chicken and quail is an important step towards the construction of a high-resolution comparative genetic map in Phasianidae, which includes several poultry species of agricultural importance. We tested chicken microsatellite markers to see if they would be suitable as genetic linkage markers in Japanese quail. Twenty-six per cent (31/120) of chicken primers amplified individual loci in Japanese quail and 65% (20/31) of the amplified loci were found to be polymorphic. Eleven of the polymorphic loci were excluded as uninformative because of the lack of amplification in some individuals or high frequency of nonspecific amplification. The sequence information of the remaining nine loci revealed six of them to contain microsatellites that were nearly identical with those of the orthologous regions in chicken. For these six loci, allele frequencies were estimated in 50 unrelated quails. Although the very few chicken markers that do work well in quail could be used as anchor points for a comparative mapping, most chicken markers are not useful for studies in quail. Therefore, more effort should be committed to developing quail-specific markers rather than attempting to adapt chicken markers for work in quail.