Cylindrotheca closterium Is a Species Complex as Was Evidenced by the Variations of rbcL Gene and SSU rDNA

The genus Cylindrotheca consists of a small group of marine diatoms with a few species described. Eleven isolates of diatoms identified as Cylindrotheca closterium morphologically were obtained from Jiaozhou Bay with their nuclear-encoded small-subunit ribosomal RNA （SSU rDNA） and chloroplast-encoded rbcL gene sequences determined in this study. Interestingly, very high sequence divergences of SSU rDNA and rbcL gene were found among these isolates, and numerous nucleotide variation of rbcL gene caused relatively few variation of deduced amino acid sequence. Phylogenetic analyses based on SSU rDNA and rbcL gene, respectively, grouped the isolates into 6 clades. Phylogenetic tree of SSU rDNA placed all the Cylindrotheca isolates together, separating them into two lineages clearly. LineageⅠ was composed of the eleven C. closterium isolates obtained in this study together with another C. closterium isolate, but some clades were not well supported. LineageⅠwas contained two C. closterium isolates and one C. fusiformis isolate. Phylogenetic analysis of rbcL gene also separated the Cylindrotheca isolates into two well-defined lineages. The eleven C. closterium isolates formed a lineage and all clades were supported strongly. Statistical comparisons of SSU rDNA indicated that the average distance within lineageⅠwas significantly higher than that of other microalgae species （P 〈0.01）. These results suggested the existence of cryptic species within C. closterium.

基于SSU rDNA序列的网状车轮虫群体遗传结构及多样性研究

基于SSU rDNA序列对当前分布于我国的网状车轮虫(Trichodina reticulata Hirschman&Partsch,1955)的群体遗传结构与多样性进行了研究。遗传结构研究结果表明:20个样本共检测到9个单倍型,含4个共享单倍型与5个特有单倍型,其中鲫来源的Hap3是最大的共享单倍型;草鱼来源的Hap8和小黄黝鱼来源的Hap9暂被视为湖北武汉和西藏地区各自特有的单倍型;同时推测鲫来源的单倍型Hap1为祖先单倍型。结合ML系统发育树分析推测,在多宿主的进化历程中,鲫寄生的网状车轮虫可能是分化最早的群体,且草鱼寄生的网状车轮虫在起源上来自于鲫。遗传多样性结果显示,所有群体均呈现较高的单倍型多样性(Hd≥0.5)与较低的核苷酸多样性(P_(i)<0.005),且鲫来源的单倍型多样性显著高于草鱼来源,但核苷酸多样性(P_(i))明显低于后者。遗传分化(F_(st))与基因流(N_(m))研究结果表明,Group A(鲫来源)与Group B(草鱼来源)群体间相对独立且已经达到了极度分化程度,群体内基因层面的交流较少。综合中性检验与核苷酸单倍型错配分析认为,Group A(鲫来源)未发生过种群扩张,Group B(草鱼来源)则存在过早期的种群扩张历史。

基于TaqMan-MGB探针的虾肝肠胞虫荧光定量PCR检测方法

【目的】基于TaqMan-MGB探针建立虾肝肠胞虫(EHP)荧光定量PCR检测方法,为EHP的快速定量检测及实时监测提供技术手段。【方法】以EHP的SSU rDNA基因为检测靶基因,在其保守区设计特异性的扩增引物和TaqMan-MGB探针,将PCR扩增目标片段克隆至pMD18-T载体构建重组质粒pMD18-T-SSU^EHP(标准品),以标准品DNA为模板优化反应体系,建立检测EHP的TaqMan-MGB探针荧光定量PCR;以10倍梯度稀释的标准品DNA为模板构建扩增标准曲线,并进行特异性、敏感性、重复性及临床应用试验。【结果】制备的重组质粒pMD18-T-SSU^EHP DNA稳定性好,可满足作为标准品和阳性对照的要求;标准品(pMD18-T-SSU^EHP)起始模板范围为2.0×10^1~2.0×10^9拷贝/反应时,建立的标准曲线线性关系良好,对应的线性回归方程为y=-3.232×lg(x)+39.51。基于TaqMan-MGB探针建立的EHP荧光定量PCR对标准品的检测灵敏度可达20拷贝/反应,对临床虾样品EHP的检测灵敏度约10拷贝/mg,较农业农村部推荐的套式PCR约提高10倍;对虾类其他常见病原均无扩增反应,其组内和组间变异系数分别为0.26%~0.72%和0.56%~0.92%,整个检测过程可在1 h内完成。采用建立的TaqMan-MGB探针荧光定量PCR与套式PCR同时对276份临床虾样品进行检测比较,发现两种方法的符合率为96.7%,检测低拷贝数虾样品时前者具有更高的灵敏度;2018和2019年广西虾样品的EHP阳性检出率分别为16.2%和30.6%。【结论】基于TaqMan-MGB探针建立的EHP荧光定量PCR检测方法具有特异性强、灵敏度高、重复性好、定量范围宽及简单快速等优点,适用于虾类样品的EHP定量检测,可为EHP实时监测及EHPD快速诊断提供一种新的技术手段。