Rapid identification of thousands of copperhead snake (Agkistrodon contortrix) microsatellite loci from modest amounts of 454 shotgun genome sequence

Optimal integration of next-generation sequencing into mainstream research requires re-evaluation of how problems can be reasonably overcome and what questions can be asked. One potential application is the rapid acquisition of genomic information to identify microsatellite loci for evolutionary, population genetic and chromosome linkage mapping research on non-model and not previously sequenced organisms. Here, we report on results using high-throughput sequencing to obtain a large number of microsatellite loci from the venomous snake Agkistrodon contortrix, the copperhead. We used the 454 Genome Sequencer FLX next-generation sequencing platform to sample randomly ∼27 Mbp (128 773 reads) of the copperhead genome, thus sampling about 2% of the genome of this species. We identified microsatellite loci in 11.3% of all reads obtained, with 14 612 microsatellite loci identified in total, 4564 of which had flanking sequences suitable for polymerase chain reaction primer design. The random sequencing-based approach to identify microsatellites was rapid, cost-effective and identified thousands of useful microsatellite loci in a previously unstudied species.     

Characterization of 31 EST-derived microsatellite markers for the pearl oyster Pinctada martensii (Dunker)

We developed and characterized 31 microsatellite markers from expressed sequence tags of Pinctada martensii (Dunker). The number of alleles per locus ranged from 4 to 18 as determined in 44 individuals from a wild population. The expected heterozygosity ranged from 0.4121 to 0.9436, while the observed heterozygosity ranged from 0.4054 to 0.7273. Most of the loci are in Hardy-Weinberg equilibrium. These markers should be useful for population genetics studies, parentage and genome mapping in this species.     

马氏珠母贝雌雄同体和自体受精的研究

对马氏珠母贝(Pinctada martensiiDunker)成体的性腺进行外观和切片观察,可把马氏珠母贝雌雄同体的个体分为2种类型,其中A类型可以从性腺外观特征上辨别,而B类型需性腺穿刺或组织切片来确认。对试验群体的性腺的类型进行的初步统计表明,雌雄同体个体比例A类型为4.6%,B类型为3.4%,其中雌雄生殖细胞均接近成熟的个体分别约为1.8%和2.1%。通过解剖、0.07‰的氨海水处理,对雌雄同体个体的自体授精及其早期胚胎发育进行了研究,发现自体授精的受精率低于异体授精,同时在发育过程中有较多的卵裂球解体,但仍有部分胚胎可以发育成D型幼虫。     

Isolation and characterization of eight microsatellite loci from silver‐lipped pearl oyster Pinctada maxima

We have developed di‐ and tetra‐nucleotide microsatellite markers for the silver‐lipped pearl oyster Pinctada maxima. Screening of approximately 50 000 clones resulted in the identification of 74 microsatellites. Fluorescent PCR was optimized for eight polymorphic loci. In a sample of 1637 individuals, these eight loci possessed between 14 and 68 alleles (average 29.8), with observed and expected heterozygosity ranging from 0.479 to 0.891 and 0.872 to 0.972, respectively.     

基于粪便DNA 的藏狐微卫星位点筛选及个体识别

藏狐是我国青藏高原东部多房棘球绦虫和石渠棘球绦虫最主要的野生动物终末宿主。棘球绦虫会导致一类称为棘球绦虫病的致死性人兽共患疾病,青藏高原东部牧区是该病重要的流行区。因此作为终末宿主,评估藏狐种群的棘球绦虫感染率对于该病的流行病学研究意义明显。而要获取这方面信息,首先必须了解藏狐的种群数量。为此,我们基于非损伤取样的原则,使用藏狐新鲜粪便作为研究材料,从已发布的藏狐及近缘种的48个微卫星位点中筛选了11 个用于藏狐粪便DNA 多态性分析。对2011 -2012 年7 -8 月间收集的128 份有效藏狐粪便样品(2011 年68 份,2012 年60 份)进行特异性PCR 扩增,并用琼脂糖凝胶电泳和荧光引物标记法进行基因分型,根据各位点的等位基因频率计算出各位点的基因型数(N),期望杂合度(He )、观测杂合度(Ho)、多态信息含量(PIC)以及不同个体基因型相同概率值(PI)。结果发现,各位点N 介于4 - 7,H e为0.66 - 0. 80,H o为0.17 -0.68,PIC 为0.5496 - 0.7623。11 个位点的累积PI 值满足个体识别的需要(PIbiased = 1. 283 × 10 ^{- 11} ;PIsi bs =7.572 × 10 ^{- 5} )。但是,由于粪便DNA 质量差异较大,不同位点的扩增成功率差异较大(0.176 - 0. 926)。我们发现,按照扩增成功率由高到低排列,前6 个微卫星位点(P03,CXX172,CPH6,CPH8,P01i,P08)的扩增成功率均超过0.6,且累积PI 值小于0.004 (PIbiased =2.775 × 10 ^{- 7} ;PIsibs = 3. 606 ×10 ^{- 3} ),表明这6 个位点可以对藏狐进行个体识别。因此,针对本研究的数据,制定了如下的个体识别原则: (1)只有粪便DNA 至少成功扩增出前6 个微卫星位点的样品可以进入下一步分析; (2)所有位点的信息均相同的两个样品被认为是来自同一个体;(3)保险起见,如果仅有一对位点信息不相等,此两个样品依然被判定来自同一个体。在此基础上,我们从2011 年样品中识别出30 个藏狐个体,从2012 年样品中识别出21 个个体。     

A galactose-specific lectin from the hemolymph of the pearl oyster, Pinctada fucata martensii

1. A lectin in the serum of Pinctada fucata martensii was purified by a combination of affinity chromatography on Sepharose 4B coupled with bovine submaxillary gland mucine, anion exchange chromatography on Mono Q and gel filtration on Superose 6. 2. The purified lectin was indicated to be homogeneous by polyacrylamide electrophoresis and rechromatography on Mono Q. 3. The purified lectin was approximately 440,000 in molecular weight and was composed of identical subunits with a molecular weight of approximately 20,000. 4. D-galactose and N-acetylgalactosamine gave a 50% inhibition of agglutination of horse erythrocytes by the lectin at 0.3 and 1.2 mM, respectively. 5. The antibody obtained from rabbit immunized with the purified lectin was monospecific to the lectin judged from the hemagglutination blocking test, immunoelectrophoresis and immunoblotting.     

马氏珠母贝3个野生种群及种群间杂交后代遗传多样性的ISSR分析

运用ISSR分子标记技术分析了马氏珠母贝(Pinctada martensii)广西北海(BW)、广东大亚湾(DW)和海南三亚(SW)野生种群及其自繁子一代(BB1、SS1、DD1)和杂交子一代(BS1、BD1、DS1)9个群体各50个个体的遗传多样性.结果表明:3个野生群体遗传多样性分别是0.2585、0.2607和0.2571;3个自繁群体子一代遗传多样性分别是0.2504、0.2545和0.2527,3个杂交子一代遗传多样性分别是0.2747、0.2659和0.2784.种群间杂交增加了子代的遗传多样性,同时增加了杂交子一代与杂交亲本间的遗传距离,而自繁群体的遗传多样性与其来源的野生种群比较,遗传多样性降低.本文指出利用野生群体进行杂交育种应该纯化亲本才能获得杂种优势,人工育苗或选择性育种需要保持足够数量的繁殖亲本以避免遗传多样性降低.     

Receptors for recombinant feline interferon-omega in hemocytes of the Japanese pearl oyster Pinctada fucata martensii

In a previous study we determined that administration of recombinant feline interferon-omega (rFeIFN-omega) could protect Japanese pearl oysters Pinctada fucata martensii from akoya-virus infection. Our results suggested that rFeIFN-omega enhanced the potential of agranulocytes to phagocytize necrotic cells and to produce collagen fibers that repair the lesions caused by the akoya-virus. In the present study, using an indirect immunofluorescence technique with an anti-rFeIFN-omega rabbit serum, we examined whether hemocytes bear receptors that bind rFeIFN-omega. rFeIFN-omega receptors were present on agranulocytes and bound rFeIFN-omega, and appeared as green fluorescent spots in the cytoplasm under a fluorescent microscope. Around 56% of agranulocytes in Japanese pearl oysters bore the rFeIFN-omega receptors.     

Effects of cryopreservation methods on post-thaw motility of spermatozoa from the Japanese pearl oyster, Pinctada fucata martensii

In order to develop cryopreservation techniques for Japanese pearl oyster spermatozoa, the effects of various cryopreservation conditions on post-thaw motility were examined. Spermatozoa cryopreserved with 10% methanol (MET), dimethylformamide or dimethylacetamide plus 90% diluent comprising 80% seawater and 20% fetal bovine serum (FBS) showed higher percentages of post-thaw motility than those cryopreserved with 10% dimethylsulfoxide or glycerol. When spermatozoa were cryopreserved with various concentrations (0-20%) of MET and 100-80% diluent, 10% MET showed the highest percentages of post-thaw motility. When spermatozoa were cryopreserved with 10% MET and 90% diluent comprising various concentrations (0-100%) of FBS or Ringer solution mixed with seawater, the percentages of post-thaw motility peaked at 20% FBS or Ringer solution, and were significantly higher for 20% FBS than for 20% Ringer solution. The percentages of post-thaw motility increased with increasing dilution ratios from 2.5- to 50-fold. Spermatozoa cooled to -50 degrees C and then immersed in liquid nitrogen (LN) showed higher post-thaw motility than those cooled to -30 degrees C or -40 degrees C. When spermatozoa were cryopreserved to -50 degrees C at various cooling rates by changing the sample height above the LN surface, the post-thaw motilities of spermatozoa cooled at 10 cm (cooling rate: -21.3 degrees C/min) and 12.5 cm (-15.6 degrees C/min) from the LN surface were higher than those at 5, 7.5 or 15 cm. These results indicate that 10% MET plus 90% diluent comprising 80% seawater and 20% FBS is a suitable extender for cryopreservation of Japanese pearl oyster spermatozoa and that samples should be cooled to -50 degrees C at a cooling rate between -15 and -20 degrees C/min for efficient storage.     

Identification of EGFR in pearl oyster (Pinctada fucata martensii) and correlation analysis of its expression and growth traits

Marine pearl production is directly influenced by the growth speed of Pinctada fucata martensii. However, the slow growth rate of this organism remains the main challenge in aquaculture production. Epidermal growth factor receptor (EGFR), an important receptor of tyrosine kinases in animals, plays versatile functions in development, growth and tissue regeneration. In this study, we described the characteristic and function of an EGFR gene identified from P. f. martensii (PmEGFR). PmEGFR possesses a typical EGFR structure and is expressed in all studied tissues, with the highest expression level in adductor muscle. PmEGFR expression level is significantly higher in the fast-growing group than that in the slow-growing one. Correlation analysis represents that shell height and shell weight show positive correlation with PmEGFR expression (p < 0.05), and total weight and tissue weight exhibit positive correlation with it (p < 0.01). This study indicates that PmEGFR is a valuable functional gene associated with growth traits.     

The effects of cryoprotectants on sperm motility of the Chinese pearl oyster,Pinctada fucata martensii

Cryopreservation has been widely employed to preserve genetic material of aquatic animals. Although of common use in bivalves, resulting effects due to the toxicity of the cryoprotectants dimethyl sulfoxide (DMSO), propanediol (PG), methanol (MET) and ethylene glycol (EG), upon sperm motility in the Chinese pearl oyster, Pinctada fucata martensii, has remained undocumented. This study endeavors to identify the least toxic among the effective cryoprotectant agents by observing and comparing their toxic effects on sperm motility under varying concentrations and duration of exposure. Sperm samples were exposed during controlled experiments, for 1, 3, 6, 9, 12 and 15 min durations, to each of the listed cryoprotectants at 5, 10, 15, and 20% (volume:volume) concentrations. Sperm motility was observed to diminish when exposed to all cryoprotectant solutions, and observations demonstrated that toxicity increased relative to both concentration and equilibration time. After 6 min of exposure to the cryoprotectants, sperm motility was seen to have diminished significantly in DMSO at just 5% concentration, and in MET, PG and EG at 10% concentrations, respectively (the values of the lowest observed effect concentrations). The relationship between the quantity of immotile sperm and the cryoprotectant concentration was described using the logarithmic regression equation. MET exhibited the lowest effective concentration required to inhibit sperm motility by 50% (EC₅₀), followed by EG, PG and DMSO, in order. Therefore, MET proved most toxic under the test conditions for sperm of P. fucata martensii, whereas DMSO, PG and EG were observed as comparatively safer, suggesting that DMSO, PG and EG warrant further study in the application of cryopreservation of Chinese P. fucata martensii sperm.     

A novel carbonic anhydrase from the mantle of the pearl oyster (Pinctada fucata)

A novel carbonic anhydrase (CA) has been purified from the mantle of the pearl oyster, Pinctada fucata, by ammonium sulfate precipitation and affinity chromatography. Its molecular mass was determined by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) to be approximately 38 kDa. Native-PAGE shows that the novel CA can bind a fluorescent probe, 5-dimethylamino-1-naphthalenesulfonamide (DNSA), known to specifically bind carbonic anhydrase. Compared to carbonic anhydrase I (CAI) from human erythrocytes, the novel CA migrates faster indicating that it is more acidic. The effect of an inhibitor on the enzyme activity was also examined. The CA from the mantle showed a weak resistance to acetazolamide (AZ), a specific inhibitor of CA. When DNSA was bound to CA, it caused the wavelength of emission maximum intensity to blue shift to 454 nm upon excitation at 326 nm. Histochemical data indicates that the enzyme is distributed widely throughout the mantle tissue, being concentrated at the edge of the mantle. The evidence presented indicates a function for CA in the process of pearl formation and biomineralization.     

Realized heritability and genetic gains of three generation for superior growth in the pearl oyster Pinctada martensii

The pearl oyster Pinctada martensii is an important economic shellfish species and mainly cultured in the southern provinces of China. The species is cultured for marine pearl production. However, pearl quality has recently decreased because the cultured stocks had slow growth and mass mortalities caused by inbreeding depression. We have initiated a selective breeding program to improve the cultivated stocks since 2002. In 2004, a base population was developed by collecting the breeders from Beibuwan and Liushagang stocks. During the period of 2005–2007, a two successive generation selection for shell length in the base population was carried out to produce the second generation selected and unselected lines. In May of 2008, three types of lines were produced by selecting the breeders from the second generation selected and unselected lines. The three types of lines were designated as SS (Selected for three generations), SC (selected for the two generations) and CC (unselected for three generations). Realized heritability for the third generation, cumulative (over three generation) and current (for the third generation) genetic gains were evaluated by comparing the growth performance of three types of lines at days 8, 16, 45, 75, 180 and 360. It was found that the SS lines had significant larger mean shell length and shell height than the SC and CC lines at all sampling ages (P < 0.05). The realized heritability estimate for adult shell length for the third generation was 0.41, similar with those detected for the first and second generation. The cumulative and current genetic gains for adult shell length were 20.94% and 13.27%, respectively. The present results indicate that there exists a high genetic variation in the population and mass selection is potential to improve pearl oyster stocks.     

Isolation and characterization of microsatellite loci in endangered Cycas changjiangensis (Cycadaceae)

Eight microsatellite loci were isolated from repetitive DNA enriched libraries for Cycas changjiangensis, an endangered endemic species in Hainan Island, China. The number of allele ranged from three to seven. The observed (H _O) and expected (H _E) heterozygosities ranged from 0.0000 to 0.8750 and from 0.2359 to 0.7582, respectively. These microsatellite loci will enrich our scientific understanding for C. changjiangensis conservation.     

Development and characterization of six new microsatellite markers for the silver‐ or gold‐lipped pearl oyster,Pinctada maxima (Pteriidae)

Six di‐, tri‐ and tetranucleotide microsatellite loci were developed for the silver‐ or gold‐lipped pearl oyster Pinctada maxima using a linker‐ligated, magnetic bead enrichment protocol. Based on a minimum of 134 Indonesian pearl oyster samples, number of alleles and observed heterozygosity at each locus ranged from six to 17 alleles and from 0.172 to 0.813 (mean = 0.448), respectively. Mean polymorphic information content for the six loci was 0.562. These loci should be very useful in DNA parentage analyses and population differentiation of P. maxima in Australia and Indonesia.     

A novel putative tyrosinase involved in periostracum formation from the pearl oyster (Pinctada fucata)

Tyrosinase (monophenol, L-DOPA: oxygen oxidoreductase, EC 1.14.18.1), a kind of copper-containing phenoloxidase, arouses great interests of scientists for its important role in periostracum formation. A cDNA clone encoding a putative tyrosinase, termed OT47 because of its estimated molecular mass of 47kDa, was isolated from the pearl oyster, Pinctada fucata. This novel tyrosinase shares similarity with the cephalopod tyrosinases and other type 3 copper proteins within two conserved copper-binding sites. RT-PCR analysis showed that OT47 mRNA was expressed only in the mantle edge. Further in situ hybridization analysis and tyrosinase activity staining revealed that OT47 was expressed at the outer epithelial cells of the middle fold, different from early histological results in Mercenaria mercenaria, suggesting a different model of periostracum secretion in P. fucata. Taken together, these results suggest that OT47 is most likely involved in periostracum formation. The identification and characterization of oyster tyrosinase also help to further understand the structural and functional properties of molluscan tyrosinase.