基于转录组测序的枫香EST-SSR引物开发及有效性评价

枫香是广西重要的乡土树种之一，具有较高的用材、观赏及药用价值。为验证枫香SSR引物的实际应用效果，该研究基于转录组测序技术，检测枫香SSR位点并设计引物，通过PCR扩增和聚丙烯酰胺凝胶电泳筛选出具有较高多态性的枫香EST-SSR引物，并对1个枫香天然群体进行遗传多样性分析。结果表明：(1)共发掘到23 777个SSR位点，单核苷酸重复类型SSR位点占总位点比例最高(46.54%),在重复次数上5～12次之间的SSR位点占比最高(72.36%)。(2)共开发出262对SSR引物，有效扩增率为53.1%,最终筛选出扩增稳定、条带清晰的引物18对。(3)多态性检测结果显示所有位点均具有多态性，天然群体遗传多样性结果显示该天然群体中等位基因数量(Na)、有效等位基因数量(Ne)、Shannon多样性指数(I)、观测杂合度(Ho)变化范围分别为2～4、1.112 8～2.609 6、0.208 9～1.112 7和0.275 9～1.000 0,平均值分别为2.333 3、1.957 4、0.708 5和0.722 6。综上认为，枫香中占优势的SSR位点重复类型和重复基序与其他物种基本相同，所开...     

Identification of random amplified polymorphic DNA (RAPD) markers highly linked to sex determination in the red alga Gracilaria gracilis

The random amplified polymorphic DNA (RAPD) technique was employed in the haplo-diploid dioecious species Gracilaria gracilis to identify sex-linked PCR markers. Sixty-nine decamer oligonucleotide primers were tested on two bulks of DNA, one from five haploid males and the other from five haploid females. One of these primers (OPD13) generated a 430-bp fragment specific to males and a 620-bp fragment specific to females. The diploid individuals (tetrasporophytes) showed the co-occurrence of these two fragments. In order to verify the linkage between the sexual phenotypes and these markers, a progeny array of 59 haploid individuals (male and female) born on a diploid individual was analysed, in all of which the two markers produced by the OPD13 primer segregated perfectly with sex.     

An examination of hybridization between the cattail species typha latifolia and typha angustifolia using random amplified polymorphic DNA and chloroplast DNA markers

Typha glauca represents a significant portion of the biomass of the wetlands surrounding the Great Lakes, USA. It is generally accepted to be a form of hybrid between T. latifolia and T. angustifolia, which itself appears to be an exotic introduction from Europe. Based on morphological and isozyme data, conflicting theories have been proposed for the hybrid nature of T. glauca: it has been described as a hybrid swarm, a distinct hybrid species and an F1 hybrid. Therefore, we developed random amplified polymorphic DNA (RAPD) and chloroplast DNA markers, specific to the parental species, to assess hybrids. Ten RAPD primers gave 17 fragments specific to T. angustifolia and 13 fragments specific to T. latifolia. All of the interspecific hybrids contained each of the species-specific markers, indicating an F1 hybrid status. Furthermore, all hybrids tested contained the T. angustifolia chloroplast haplotype, which is consistent with differential interspecific crossing success found previously. Additional confirmation of an F1 hybrid status was gained by examining seedlings from T. glauca. These progeny were expected to be advanced-generation hybrids, as opposed to the F1 hybrid parent. Analysis of the seedlings revealed segregating marker patterns consistent with patterns observed in experimental advanced-generation hybrids, although these advanced hybrids do not appear to be a significant part of mature stands. Our data do not provide support for extensive gene flow between T. latifolia and T. angustifolia. However, our results suggest that hybridization between the native and introduced Typha species has impacted the native population through the spread of the F1 hybrid, T. glauca.     

Development of sequence amplified characterized region (SCAR) markers of helicoverpa armigera: a new polymerase chain reaction-based technique for predator gut analysis

A method is described for the development of DNA markers for detection of Helicoverpa armigera (Hubner) (Lepidoptera: Noctuidae) in predator gut analysis, based on sequence characterized amplified regions (SCARs) derived from a randomly amplified polymorphic DNA (RAPD) band. A 1200-bp DNA fragment of H. armigera, absent in the predator band pattern and in other closely related prey species, was identified by RAPD analysis. This fragment was cloned and its extremes sequenced to design extended strand-specific 20-mer oligonucleotide primers. Three pairs of SCAR primers, which amplified three different DNA fragments, were used to study the effect of fragment length on detection of prey in the predator gut. Using the pair of primers that amplified the longest fragment of H. armigera DNA, a single band of 1100 bp was obtained, but its detection was not possible in the predator gut. Detection of the ingested prey was possible with the other two pairs of SCAR primers, obtaining bands of 600 and 254 bp, respectively. Detection of H. armigera DNA in the gut of the predator Dicyphus tamaninii was evaluated immediately after ingestion (t = 0) and after 4 h. Detection of H. armigera DNA after 4 h was only possible using the pair of primers that amplified the shortest fragment (254 bp). The test for specificity, using these last pair of primers, showed that H. armigera was the only species detected. The detection threshold was defined at a 1:8192 dilution of a H. armigera whole egg in all samples.     

Understanding muscle markers: lower limbs

Musculoskeletal markers are frequently used to reconstruct past lifestyles and activity patterns. Yet the reliability of muscle marker measurements has been called into question because they may be confounded by body size. In this study, an aggregate muscle marker variable was calculated using 20 insertion sites (14 femoral, 6 tibial), and I examined their effects on lower limb size (as a proxy for body size), age, and sex. Analyses were made of a sample of 77 (57 males, 20 females) Native British Columbians (3,500-1,500 years BP) and 18th century Quebec prisoners. Muscle markers were measured using two-point observer rating scales; size was measured by standard methods; and age and sex were determined through pelvic, cranial, and dental morphology. Lower limb muscle markers correlated with: age, r=0.61; lower limb size, r=0.52; and sex, r=0.49; P <0.001. Older individuals had higher muscle marker scores, as did larger individuals and males. Based on partial correlations and regression analyses, age was the best overall predictor of lower limb muscle markers.     

Expression of functionally relevant cell surface markers in dibutyltin-exposed human natural killer cells

Butyltin (BT) compounds are known for their worldwide contamination. Dibutyltin (DBT) is used as a stabilizer in plastic products, and as a deworming agent in poultry. Poultry products have been shown to contain measurable levels of DBT. Drinking water has also been reported to contain BTs due to leaching from PVC pipes. We, and others, have found measurable levels of DBT in human blood. BTs appear to increase the risk of cancer and other viral infections in exposed individuals. In previous studies we have shown that the tumor killing function of natural killer (NK) lymphocytes was greatly diminished after as little as a 1 h exposure to DBT and the inhibition continued even after removal of the compound. We also showed that there was a significant decrease in NK cell lysis of K562 target cells after an exposure to 1.5 microM DBT for 24 h. This 24 h exposure also decreased the ability of NK cells to bind to tumor cells. Loss of binding function was not seen when NK cells were exposed to 5-10 microM DBT for 1 h. However, NK cells exposed to 5 microM DBT for 1 h and then incubated in DBT-free media for 24, 48, or 96 h, showed a significant loss of tumor-binding function within 24 h. The effects of DBT exposure on seven cell surface molecules that are involved in NK-cell interactions with target cells were investigated. The results indicated that the exposure of NK cells to 1.5 microM DBT for 24 h decreased the expression of CD2, CD11a, CD16, CD11c. There was no decrease in expression of any of the markers studied when NK cells were exposed to 5 microM DBT for 1 h, consistent with the fact that a 1-h exposure had no effect on the ability of NK cells to bind tumor cells. However, when NK cells were exposed to 5 microM DBT for 1 h followed by 24, 48 or 96 h incubations in DBT-free media there was decreased expression of several of the cells surface molecules with the most dramatic decreases being in CD16 and CD56.     

L-ascorbic acid (vitamin C) induces the apoptosis of B16 murine melanoma cells via a caspase-8-independent pathway

l-Ascorbic acid (vitamin C) has been reported to play a role in the treatment and prevention of cancer. However, its specific mechanistic pathways remain obscure. This study was carried out to identify the sodium ascorbate–induced apoptotic pathway in B16F10 murine melanoma cells. Sodium ascorbate was found to induce the apoptosis of B16F10 murine melanoma in a time- and dose-dependent manner, and this was prevented by pretreatment with N-acetyl-l-cysteine (NAC), a well-known antioxidant. In fact, sodium ascorbate–treated B16F10 melanoma cells showed increased intracellular reactive oxygen species generation (ROS) levels. These results indicate that sodium ascorbate induced apoptosis in B16F10 murine melanoma cells by acting as a prooxidant. We examined the involvement of caspase-8 using a specific caspase-8 inhibitor (z-IETD-fmk) on the sodium ascorbate–induced apoptotic pathway. Cell death was found not to be inhibited by z-IETD-fmk treatment, indicating that sodium ascorbate–induced apoptosis is not mediated by caspase-8. In addition, we detected a reduction in the mitochondrial membrane potential during apoptosis and confirmed cytochrome-c release from mitochondria by immunoblotting. Taken together, it appears that the induction of a prooxidant state by sodium ascorbate and a subsequent reduction in mitochondrial membrane potential are involved in the apoptotic pathway of B16F10 murine melanoma cells, and that this occurs in a caspase-8–independent manner.Abbreviations NAC N-acetyl-l-cysteine - ROS reactive oxygen species - _m mitochondrial membrane potentialJae Seung Kang and Daeho Cho contributed equally to this work.