An efficient pipeline for ancient DNA mapping and recovery of endogenous ancient DNA from whole‐genome sequencing data

Ancient DNA research has developed rapidly over the past few decades due to improvements in PCR and next‐generation sequencing (NGS) technologies, but challenges still exist. One major challenge in relation to ancient DNA research is to recover genuine endogenous ancient DNA sequences from raw sequencing data. This is often difficult due to degradation of ancient DNA and high levels of contamination, especially homologous contamination that has extremely similar genetic background with that of the real ancient DNA. In this study, we collected whole‐genome sequencing (WGS) data from 6 ancient samples to compare different mapping algorithms. To further explore more effective methods to separate endogenous DNA from homologous contaminations, we attempted to recover reads based on ancient DNA specific characteristics of deamination, depurination, and DNA fragmentation with different parameters. We propose a quick and improved pipeline for separating endogenous ancient DNA while simultaneously decreasing homologous contaminations to very low proportions. Our goal in this research was to develop useful recommendations for ancient DNA mapping and for separation of endogenous DNA to facilitate future studies of ancient DNA.     

The pollination of Habenaria rhodocheila (Orchidaceae) in South China: When butterflies take sides

Habenaria is one of the largest terrestrial genera in the family Orchidaceae. Most field studies on Habenaria species with greenish–white and nocturnal scented flowers are pollinated by nocturnal hawkmoths and settling moths. However, H. rhodocheila presents reddish flowers lacking a detectable scent and fails to fit the moth pollination syndrome. We investigated the pollinators, breeding system, and functional traits of H. rhodocheila in South China and found that two diurnal swallowtail butterflies Papilio helenus and Papilio nephelus (Papilionidae) were the effective pollinators. When butterflies foraged for nectar in the spur, the pollinia became attached between the palpi. A triangular projected median rostellar lobe was found at the entrance (sinus) of the spur of H. rhodocheila. This lobe divided the spur opening into two entrances forcing butterflies to enter their proboscides through the left or right side. When the projection of median rostellar lobe was removed, the site of pollinium attachment changed to the eyes of the butterflies, leading to a higher rate of pollinium removal but lower rate of pollinium deposition. Our quartz glass cylinder choice experiment suggested that visual rather than olfactory cues provided the major stimuli for butterflies to locate these flowers. Hand pollination experiments suggested this species was self‐compatible but pollinator‐dependent. However, the proportion of seeds with large embryos produced in self‐pollinated fruits was significantly lower than in cross‐pollinated fruits, indicating a significant inbreeding depression. Unlike many other orchid species, fruit set was higher than rates of pollinium removal, indicating a high level of pollination efficiency in a species with friable pollinia. Shifts from moth to butterfly pollination in the genus Habenaria parallel other orchid lineages providing insights into the potential for pollinator‐mediated floral trait selection.     

Home‐site fidelity and homing behavior of the big‐headed turtle Platysternon megacephalum

Site fidelity refers to the restriction of dispersal distance of an animal and its tendency to return to a stationary site. To our knowledge, the homing ability of freshwater turtles and their fidelity is reportedly very low in Asia. We examined mark–recapture data spanning a 4‐year period in Diaoluoshan National Nature Reserve, Hainan Province, China, to investigate the site fidelity and homing behavior of big‐headed turtles Platysternon megacephalum. A total of 11 big‐headed turtles were captured, and all individuals were used in this mark–recapture study. The site fidelity results showed that the adult big‐headed turtles (n = 4) had a 71.43% recapture rate in the original site after their release at the same site, whereas the juveniles (n = 1) showed lower recapture rates (0%). Moreover, the homing behavior results showed that the adults (n = 5) had an 83.33% homing rate after displacement. Adult big‐headed turtles were able to return to their initial capture sites (home) from 150 to 2,400 m away and precisely to their home sites from either upstream or downstream of their capture sites or even from other streams. However, none of the juveniles (n = 4) returned home, despite only being displaced 25–150 m away. These results indicated that the adult big‐headed turtles showed high fidelity to their home site and strong homing ability. In contrast, the juvenile turtles may show an opposite trend but further research is needed.     

Effects of calcium on the activities of cytosolic antioxidative enzymes and IAA oxidase during in vitro adventitious rooting of mung bean seedlings

The effects of Ca²⁺ on antioxidative enzymes and indole-3-acetic acid (IAA) oxidase during adventitious rooting were investigated in mung bean (Vigna radiata). CaCl₂ significantly promoted the formation and growth of adventitious roots. EGTA (a Ca²⁺ chelator) or ruthenium red (a Ca²⁺-channel blocker) significantly inhibited root formation and growth, but these inhibitory effects could be partially reversed by CaCl₂. Furthermore, inclusion of 5 mM CaCl₂ significantly increased superoxide dismutase (SOD) activity by 10% at 3 h and catalase (CAT) activity by an average of 29.6% at each time point. CaCl₂ decreased peroxidase (POD) activity by 9.4% and 21% at 12 and 24 h, respectively, and ascorbate peroxidase (APX) activity by an average of 13.9% at each time point. These CaCl₂-induced changes in enzymatic activities were similar to changes caused by indole-3-butyric acid (IBA). Treatment with EGTA or ruthenium red decreased SOD activity by an average of 18.4% and 15.2%, respectively; POD activity by 27.4% and 57.6%, respectively; APX activity by 10.3% and 15.6%, respectively; and CAT activity by 19.3% and 5.2%, respectively, when compared with CaCl₂. In addition, CaCl₂ increased IAA oxidase activity by an average of 5.5% beginning at 6 h, whereas EGTA significantly decreased IAA oxidase activity by 29.2%, 22.9%, and 13.5% at 6, 9, and 12 h, respectively. The inhibitory effects of EGTA could be partially suppressed by addition of CaCl₂. These results imply that the stimulative effect of Ca²⁺ on adventitious rooting is partially related to Ca²⁺-induced changes in the activities of antioxidative enzymes and IAA oxidase.     

快速STR分型在法医DNA分析中的应用研究进展

STR分型是目前世界通用的DNA指纹分析方法.传统的分型方法包括DNA提取、DNA定量、PCR扩增和对扩增后的STR片段进行检测和分析,耗时较长(8¹0 h).一些情况下很难满足案件的实际需求.法医学家们在加快STR分型的方法研究一直不懈努力.现从快速提取DNA、无提取直接PCR、快速PCR、微流控芯片技术在STR快速分型方面的应用四方面,对近年来出现的可进行快速STR分型的新技术、新方法进行综述.     

靶向白介素-1α基因沉默的Hela229稳定细胞系的构建

目的：构建针对IL-1α基因的shRNA表达载体,筛选能够抑制Hela229细胞内源性IL-1α表达的shRNA,建立无内源性IL-1d表达的Hela229稳定细胞系.方法：根据shRNA的设计原则,以IL-1 αcDNA oligo为模板设计一段21 bp核苷酸目标序列,构建成siRNA的DNA模板并克隆到shRNA表达载体pRNAT-U6.1/Neo中,获得靶向抑制IL-1α基因的重组shRNA质粒,转染Hela229细胞,经G418筛选后获得单克隆稳定细胞株,用ELISA方法在蛋白水平上检测IL-1α基因的沉默效果.结果：经酶切鉴定和测序分析确定IL-1 α-shRNA重组质粒构建正确,ELISA筛选出能够显著抑制内源性IL-1α表达的shRNA,获得沉默内源性IL-1 α表达的单克隆稳定的Hela229细胞株.结论：靶向IL-1α基因的重组shRNA表达质粒可显著抑制Hela229细胞内源性IL-1α的表达,成功构建靶向IL-1α基因沉默的Hela229稳定细胞系.     

福建尤溪九阜山省级自然保护区自然植被类型调查研究

采用路线踏查与典型样地相结合的调查方法,按照《中国植被》的划分,结果表明:尤溪九阜山省级自然保护区自然植被类型十分丰富,植被保存完整,共有8个植被型36个群系85个群丛;植被分布水平规律变化界限和植被类型的垂直带谱分布不明显,常绿阔叶林遍布从山麓到山顶,针阔混交林多分布于中上坡,常绿针叶林主要分布于山脊山顶附近,毛竹林多分布在局部常绿阔叶林下缘,有的与常绿阔叶林呈带状或团块状混生,是中亚热带典型的常绿阔叶林森林生态系统.     

沙眼衣原体持续感染对Hela细胞TLR4/IL-6/STAT3信号通路的影响

目的：分析沙眼衣原体（Chlamydia trachomatis,Ct）持续感染对靶细胞TLR4/IL-6/STAT3信号通路的影响.方法：利用Hela细胞分别建立Ct急性感染及持续性感染模型,通过qRT-PCR、ELISA等方法比较Ct感染过程中靶细胞TLR4、STAT3、IL-6转录水平及细胞因子IL-6分泌量的变化.结果：Ct感染后靶细胞TLR4、IL-6、STAT3转录水平及细胞因子IL-6分泌量均呈现时间相关性上调,且持续性感染状态下比急性感染状态下的上调更为显著;IL-6/STAT3的表达量与TLR4转录水平正相关.结论：Ct持续感染过程中TLR4 的持续活化可大幅上调IL-6/STAT3信号通路表达,可能参与了Ct持续感染后慢性炎性损伤过程.