Cross-species amplification of 44 microsatellite loci developed for Chaetodon trifascialis, C. lunulatus and C. vagabundus in 22 related butterflyfish species

Forty‐four microsatellite primers developed for three species of butterflyfish were cross‐tested against 22 related confamilial species. Amplification success and cross‐species transferability of these markers were moderately high. Between 24 and 37 loci were amplified successfully in each species, with a mean success rate per species of 71.7% (± 1.8 SE). Rates of amplification success were comparable among primers designed for the three source species, ranging from a mean success rate per species of 16.9 loci (± 0.8 SE) for Chaetodon trifascialis source loci to 13.7 loci (± 1.5 SE) for C. vagabundus source loci. Polymorphism rates were high (76.1%± 3.1 SE of all successfully amplified loci), and 10 loci were polymorphic in all successfully amplified species (Tri14, B11, C5, D3, D113, D6, D117, D120, D111, D118). The number of alleles per polymorphic locus ranged from 2 to 8, and the average number of alleles across all polymorphic loci and all species was 3.6 (± 0.07 SE). Polymorphism rates were higher overall in primers designed for C. vagabundus (89.9%± 3.9 SE). Overall cross‐testing success was lowest for Heniochus chrysostomus, the most phylogenetically divergent species. The significant cross‐testing reported here provides a valuable resource that will enable population genetics studies to be undertaken on a range of butterflyfishes without the need for expensive and time‐consuming de novo microsatellite development.     

Characterization of eight microsatellite loci in Sea Otter, Enhydra lutris, and cross-species amplification in other Mustelidae

Herein we describe the development of eight microsatellite markers for the northern sea otter, Enhydra lutris kenyoni. A total of 45 primer pairs were developed and screened from enriched AAAT, CATC, TACA, and TAGA libraries derived from genomic DNA of E. lutris kenyoni. Of these, eight amplified successfully. The average observed heterozygosity, expected heterozygosity, and number of alleles per locus was 0.506, 0.537, and 3.4, respectively. These eight loci were tested across three additional genra; Vulpes lagopus, Martes americana, and Mustela nivalis. Based on the success of our results these loci will be useful for future studies across all sub-species of E. lutris.     

In Vivo RNA Interference of a Gonad-Specific Transforming Growth Factor-β in the Pacific Oyster Crassostrea gigas

We investigated the role of oyster gonadal TGFβ (og-TGFβ) in the reproduction of Crassostrea gigas, using an in vivo RNA interference approach. We designed double-stranded RNA targeting og-TGFβ, which is specifically expressed in the somatic cells surrounding germ cells in the gonad of both male and female oysters. In vivo injection of this og-TGFβ dsRNA into the gonad led to knock-down phenotypes for both sexes, with significant reduction (77.52% relative to controls) of the gonad area, lowered reproductive effort and germ cell under-proliferation. Interestingly, half of the injected females halted their vitellogenesis, since we were only able to observe pre-vitellogenic oocytes. In addition, apoptotic germ cells and haemocytes infiltrated into the gonad, likely as part of the active resorption of degenerating germ cells. Conversely, males showed a normal phenotype at the cellular level, with spermatids and spermatozoids observed in the gonads of control and injected males. As a result, og-TGFβ appears to play an essential role in C. gigas germ cell development by functioning as an activator of germ cell proliferation in both male and female oysters and vitellogenesis in females.     

Isolation and characterization of polymorphic microsatellite loci from a repeat-enriched genomic library of stone flounder (Kareius bicoloratus) and cross-species amplification

Stone flounder (Kareius bicoloratus) is a rare fish species in China. Here, we reported 11 polymorphic microsatellite loci isolated from a repeat-enriched genomic library of stone flounder. The number of alleles, observed and expected heterozygosity per locus in 32 individuals ranged from 3 to 6, from 0.1613 to 0.8667 and from 0.1549 to 0.7932, respectively. Two loci significantly deviated from Hardy–Weinberg equilibrium after Bonferroni correction and no significant linkage disequilibrium between pairs of loci was found. Cross-species amplification of these microsatellite loci in additional three fish species was performed. These polymorphic microsatellite loci would be useful for investigating genetic population structure and construction of genetic linkage map in Kareius bicoloratus. Yong-Sheng Tian and Gui-Dong Miao are contributed equally.     

Characterization of eight microsatellite loci in the Galápagos endemic land snail Bulimulus reibischi, and their cross-species amplification

Variation in and amplification conditions for eight polymorphic microsatellite loci initially identified from Bulimulus akamatus, a pulmonate land snail from Galápagos, are described. Intraspecific polymorphism and heterozygosity of the eight markers were studied in 19 populations of Bulimulus reibischi, a closely related species of B. akamatus. Furthermore, the eight loci were also cross-amplified in six other closely related bulimulid species. The number of alleles across populations of B. reibischi at six loci is moderate (three to 10), but considerable for two other loci (19 and 20). There is no strong evidence for linkage among any of the loci examined.     

Viral gametocytic hypertrophy of Crassostrea gigas in France: from occasional records to disease emergence?

Viral gametocytic hypertrophy was reported for the first time in 2001 in Pacific oyster Crassostrea gigas in France. Since this date, the number of reported cases and the distribution area have increased every year; however, the cases are not associated with macroscopic signs or increased mortality rates. Both male and female gametes were hypertrophied and basophilic inclusions were observed in gamete nuclei. Transmission electron microscopy revealed the presence of viral particles in these intranuclear basophilic inclusions. These particles had characteristics similar to those of the Papillomaviridae and Polyoma viridae families: they were small, non-enveloped, icosahedral, and 44 to 56 nm in diameter. The viral particles were found in male, female and hermaphrodite oysters and no significant difference in viral infection was observed between those groups. The frequency of detection and the intensity of infection were low and no host defence reaction was recognised, suggesting that the viral particles had a weak impact on C. gigas. The viral particles described in the present study seem to be similar to these described in C. virginica in the USA and Canada and in C. gigas in Korea, but further studies are required to confirm their identity. The issue of a possible emergence of this infection is discussed.     

Novel microsatellites from the European plaice (Pleuronectes platessa)––identification by data mining and cross-species amplification in other flatfishes

The European plaice (Pleuronectes platessa; Pleuronectidae) is considered the most important flatfish for fisheries in Europe. Overall catches have been reduced drastically throughout the past decades, indicating a severe decline in the natural populations. However, a limited set of genetic tools are available to improve stock assessment and management for the species. Sequences of European plaice and its congener, the winter flounder (Pleuronectes americanus) deposited in the public database were searched for short tandem repeats. Out of 22 loci derived from database sequences, eight were amplified successfully and genotyping of 23 wild-caught individuals showed expected heterozygosities ranging from 0.18 to 0.84. The average allele number was 4.75 per locus (range 2–11). Successful cross-species amplification of the markers was demonstrated from two related flatfishes. All eight markers were successfully amplified from the European flounder (Platichthys flesus), a phylogenetically closely related species, whereas three of them worked in Achiroides melanorhynchus, an endemic species of Southeast Asia for which no microsatellites have been described to date.     

Hybridization and introgression in Carex aquatilis and C. paleacea

The diversification and distribution of Abies species throughout the Mediterranean region has led to a complex of species which are difficult to classify. An open question is whether these mainly allopatric taxa have exchanged genetic information via secondary contact. We studied the variation and geographic distribution of paternally inherited chloroplast DNA markers in nine Mediterranean Abies taxa. Markers with high and low mutation rates were applied in order to differentiate among a scenario of secondary genetic contact and a scenario of complete isolation after speciation. The observed molecular variation was analysed using statistical parsimony, geostatistics, and measures of population genetic differentiation. Ancient paternal lineages, represented by markers with low mutation rates, were shared among species. The central and widespread A. alba retained all ancient lineages whereas other species exhibited fewer, down to a single lineage. In contrast, modern lineages, depicted by markers with high mutation rates, were largely separated among species. The western Mediterranean A. pinsapo and A. numidica were clearly separated from each other and from the remaining Abies species. This indicates the absence of secondary contact. The same scenario applies to the eastern Mediterranean Abies species. An exception is the parapatric complex of A. alba, A. cephalonica, and their supposed hybrid A. borisii regis, which exhibited evidence of secondary contact.     

Asymmetry and growth rate of the shell valves in juveniles of Crassostrea gigas Thünberg

Thanks to a sampling of juvenile oysters with different growth rates (slow, medium and fast), a subtle biometrical study of their shell evolution is described distinguishing the two valves. We conclude that all the phenomena are dominated by the early establishment of a directional asymmetry, which rapidly becomes so large that we should consider dissymmetry. The clearest relationship is revealed to be that between the weakness of the growth rate and the degree of the relative dissymetry between the weights of the valves. This result is to be compared with the highly debated relationship between the asymmetry and the growth rate. The existence and interpretation of both of these phenomena have led to many publications.     

Isolation and characterization of microsatellite loci in Pratt’s Leaf-nosed Bat (<Emphasis Type="Italic">Hipposideros pratti</Emphasis>) and cross-species amplification in closely related taxa

We developed nine microsatellite loci using an enriched library method from the genomic DNA of Pratt’s leaf-nosed bat (Hipposideros pratti). These loci were tested on 96 individuals sampled from Sichuan Province, China. The number of alleles per locus varied from 4 to 14. The expected and observed heterozygosity values ranged from 0.513 to 0.886 and from 0.375 to 0.966, respectively. Three microsatellite loci departed significantly from Hardy–Weinberg equilibrium (HWE). No linkage disequilibrium was found. These microsatellite loci will be used in studies of conservation genetics in this species.     

Microsatellite loci for tucumã of Amazonas (Astrocaryum aculeatum) and amplification in other Arecaceae

• Premise of the study: Microsatellite loci were developed for tucumã of Amazonas (Astrocaryum aculeatum), and cross-species amplification was performed in six other Arecaceae, to investigate genetic diversity and population structure and to provide support for natural populations management. • Methods and Results: Fourteen microsatellite loci were isolated from a microsatellite-enriched genomic library and used to characterize two wild populations of tucumã of Amazonas (Manaus and Manicoré cities). The investigated loci displayed high polymorphism for both A. aculeatum populations, with a mean observed heterozygosity of 0.498. Amplification rates ranging from 50% to 93% were found for four Astrocaryum species and two additional species of Arecaceae. • Conclusions: The information derived from the microsatellite markers developed here provides significant gains in conserved allelic richness and supports the implementation of several molecular breeding strategies for the Amazonian tucumã.     

Two new Cladophialophora species,C. tumbae sp. nov. and C. tumulicola sp. nov., and chaetothyrialean fungi from biodeteriorated samples in the Takamatsuzuka and Kitora Tumuli

During dismantling and relocation of the Takamatsuzuka Tumulus stone chamber, many Cladophialophora and chaetothyrialean black fungi, such as Exophiala and Phialophora, were isolated from samples taken from the joints between the stone walls. However, inside the stone chamber of the Kitora Tumulus, after intermittent UV irradiation in 2009, these black fungi were also isolated from samples taken from the stone walls. Molecular phylogenetic analyses based on only nrLSU and the concatenated (nrLSU D1/D2 + ITS) sequences revealed that the 35 Takamatsuzuka and Kitora Tumuli isolates of Cladophialophora and the chaetothyrialean black fungi were divergent. Two new species of Cladophialophora are described herein: C. tumulicola from the viscous gels and various substrates on the stone walls of the Takamatsuzuka and Kitora Tumuli and C. tumbae from black substances on the plastic cover over the “thief hole,” soil and plaster pieces between the stone walls, and the exterior of the Takamatsuzuka Tumulus chamber. Also, molecular phylogenetic placements for the remaining eight Takamatsuzuka and Kitora Tumuli isolates of chaetothyrialean black fungi have been determined or suggested.     

Loss of intestinal nuclei and intestinal integrity in aging C. elegans

The roundworm C. elegans is widely used as an aging model, with hundreds of genes identified that modulate aging (Kaeberlein et al., 2002. Mech. Ageing Dev. 123 , 1115–1119). The development and bodyplan of the 959 cells comprising the adult have been well described and established for more than 25 years ( Sulston & Horvitz, 1977 . Dev. Biol. 56 , 110–156; Sulston et al., 1983. Dev. Biol. 100 , 64–119.). However, morphological changes with age in this optically transparent animal are less well understood, with only a handful of studies investigating the pathobiology of aging. Age‐related changes in muscle ( Herndon et al., 2002 . Nature 419 , 808–814), neurons ( Herndon et al., 2002 ), intestine and yolk granules ( Garigan et al., 2002 . Genetics 161 , 1101–1112; Herndon et al., 2002 ), nuclear architecture ( Haithcock et al., 2005 . Proc. Natl Acad. Sci. USA 102 , 16690–16695), tail nuclei ( Golden et al., 2007 . Aging Cell 6 , 179–188), and the germline ( Golden et al., 2007 ) have been observed via a variety of traditional relatively low‐throughput methods. We report here a number of novel approaches to study the pathobiology of aging C. elegans. We combined histological staining of serial‐sectioned tissues, transmission electron microscopy, and confocal microscopy with 3D volumetric reconstructions and characterized age‐related morphological changes in multiple wild‐type individuals at different ages. This enabled us to identify several novel pathologies with age in the C. elegans intestine, including the loss of critical nuclei, the degradation of intestinal microvilli, changes in the size, shape, and cytoplasmic contents of the intestine, and altered morphologies caused by ingested bacteria. The three‐dimensional models we have created of tissues and cellular components from multiple individuals of different ages represent a unique resource to demonstrate global heterogeneity of a multicellular organism.     

Development and characterization of microsatellite markers for rice leaffolder, Cnaphalocrocis medinalis (Guenée) and cross-species amplification in other Pyralididae

Rice leaffolder, Cnaphalocrocis medinalis (Guenée), is a destructive and widespread pest on rice. In this study, 20 microsatellite markers were isolated and characterized from C. medinalis partial genomic libraries using the method of fast isolation by AFLP of sequence containing repeats. Of these markers, 18 markers displayed polymorphisms. Polymorphisms were evaluated in 48 individuals from two natural populations. The number of alleles per locus ranged from 2 to 15, and the expected and observed heterozygosities ranged from 0.324 to 0.934 and from 0.304 to 0.917, respectively. Cross-species amplification was also performed to test the transferability of the 20 microsatellite markers and a moderate level of cross amplication was observed across the three species of Pyralididae (26.67 %). These microsatellite loci would facilitate the future study on population genetics and molecular genetics of rice leaffolder and would also be useful for study in Chilo suppressalis, Scirpophaga incertulas and Pyrausta nubilalis.     

Development and integration of EST–SSR markers into an established linkage map in switchgrass

Switchgrass (Panicum virgatum L.) is a model cellulosic biofuel crop in the United States. Simple sequence repeat (SSR) markers are valuable resources for genetic mapping and molecular breeding. A large number of expressed sequence tags (ESTs) of switchgrass are recently available in our sequencing project. The objectives of this study were to develop new SSR markers from the switchgrass EST sequences and to integrate them into an existing linkage map. More than 750 unique primer pairs (PPs) were designed from 243,600 EST contigs and tested for PCR amplifications, resulting in 538 PPs effectively producing amplicons of expected sizes. Of the effective PPs, 481 amplifying informative bands in NL94 were screened for polymorphisms in a panel consisting of NL94 and its seven first-generation selfed (S1) progeny. This led to the selection of 117 polymorphic EST–SSRs to genotype a mapping population encompassing 139 S1 individuals of NL94. Of 83 markers demonstrating clearly scorable alleles in the mapping population, 79 were integrated into a published linkage map, with three linked to accessory loci and one unlinked. The newly identified EST–SSR loci were distributed in 17 of 18 linkage groups with 27 (32.5 %) exhibiting distorted segregations. The integration of EST–SSRs aided in reducing the average marker interval (cM) to 3.7 from 4.2, and reduced the number of gaps (each >15 cM) to 10 from 23. Developing new EST–SSRs and constructing a higher density linkage map will facilitate quantitative trait locus mapping and provide a firm footing for marker-assisted breeding in switchgrass.     

Mo–Cu metal cluster formation and binding in an orange protein isolated from Desulfovibrio gigas

The orange protein (ORP) isolated from the sulfate-reducing bacterium Desulfovibrio gigas (11.8 kDa) contains a mixed-metal sulfide cluster of the type [S₂MoS₂CuS₂MoS₂]^3- noncovalently bound to the polypeptide chain. The D. gigas ORP was heterologously produced in Escherichia coli in the apo form. Different strategies were used to reconstitute the metal cluster into apo-ORP and obtain insights into the metal cluster synthesis: (1) incorporation of a synthesized inorganic analogue of the native metal cluster and (2) the in situ synthesis of the metal cluster on the addition to apo-ORP of copper chloride and tetrathiomolybdate or tetrathiotungstate. This latter procedure was successful, and the visible spectrum of the Mo–Cu reconstituted ORP is identical to the one reported for the native protein with absorption maxima at 340 and 480 nm. The ¹H–¹⁵N heteronuclear single quantum coherence spectra of the reconstituted ORP obtained by strategy 2, in contrast to strategy 1, exhibited large changes, which required sequential assignment in order to identify, by chemical shift differences, the residues affected by the incorporation of the cluster, which is stabilized inside the protein by both electrostatic and hydrophobic interactions.