Comparative analyses within Gyrodactylus (Platyhelminthes: Monogenea) mitochondrial genomes and conserved polymerase chain reaction primers for gyrodactylid mitochondrial DNA

In this study, we describe the complete mitochondrial genomes of Gyrodactylus brachymystacis and Gyrodactylus parvae infecting rainbow trout (Oncorhynchus mykiss) and the invasive topmouth gudgeon (Pseudorasbora parva), respectively. The two circular genomes have a common genome organization found in other Gyrodactylus species. Comparative analyses of mitochondrial genomes from six Gyrodactylus species were carried out to determine base composition, codon usage, transfer RNA and ribosomal RNA genes, major non‐coding regions, and nucleotide diversity within the genus. We also provide the first universal models of the secondary structures of rrnS and rrnL for this group thereby promoting utilization of these genetic markers. Universal primers provided herein can be used to obtain more mitochondrial information for pathogen identification and may reveal different levels of molecular phylogenetic inferences for this lineage.     

基于环境DNA的岩原鲤检测及生物量评估

为建立岩原鲤基于环境DNA （eDNA）的实时荧光定量PCR （qPCR）检测方法,准确鉴别岩原鲤并探讨e DNA浓度与其生物量的定量关系,实验根据岩原鲤mtDNA中12S rRNA基因序列设计eDNA引物和TaqMan探针,利用PCR扩增出岩原鲤12S rRNA基因的序列,克隆入pMD19-T载体,构建重组质粒作为qPCR标准;使用梯度稀释质粒标准品作为模板,进行qPCR扩增,制作标准曲线,建立岩原鲤qPCR检测方法,并评价其特异性、灵敏性和应用效果。结果显示,引物和TaqMan探针对供试的岩原鲤样品出现荧光增长曲线,显示阳性扩增,而其他鱼类和空白对照均未得到扩增信号,表现为阴性;qPCR的阈值循环数（C_t）与标准品拷贝数的线性关系好,且线性范围广,获得的标准曲线相关系数（R²）达到0.999,检测限位DNA浓度为5×10^-6 ng/μL,扩增效率为94.7%;检测养殖不同数量岩原鲤的水体中eDNA浓度,目标DNA浓度和岩原鲤数量存在线性正相关性（R²=0.957）,得到岩原鲤DNA浓度与其个体数...     

不同引物RT-PCR方法检测猪繁殖与呼吸综合征病毒的比较研究

用3对分别针对猪繁殖与呼吸综合征病毒(PRRSV)的ORF7、ORF5和ORF5的PCR引物N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2进行RT-PCR,检测PRRSV,从其敏感性、特异性和临床样品检出率等方面进行比较,在此基础上进一步建立一步法RT-PCR检测方法。结果显示:3对引物对PRRSV均有很高的特异性;应用N1/N2引物病毒最低检测量为7.9 TC ID50,而AdGP5.1/AdGP5.2引物和RFLP5.1/RFLP5.2引物PCR最低检测量为79 TC ID50;运用N1/N2、AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物分别进行RT-PCR扩增检测临床样品,PRRSV检出率分别为28/48、27/48和25/48,且用AdGP5.1/AdGP5.2和RFLP5.1/RFLP5.2引物检测的阳性样品,用N1/N2引物检测也都呈阳性。运用N1/N2引物,通过一步法RT-PCR成功地从PRRSV S1株中扩增出374 bp的目的基因片段。结果表明,用N1/N2引物扩增PRRSV目的基因,其敏感性和临床样品检出率更高,更适合临床样品PRRSV的检测。     

Simultaneous analysis of the intestinal parasites and diet through eDNA metabarcoding

Agricultural expansion and intensification are having a huge impact on plant and arthropod diversity and abundance, affecting food availability for farmland birds. Difficult food access, in turn, can lead to immunosuppression and a higher incidence of parasites. In the studies designed to examine changes in the diet of birds and their parasites, metabarcoding is proving particularly useful. This technique requires mini-barcodes capable of amplifying the DNA of target organisms from fecal environmental DNA. To help to understand the impact of agricultural expansion on biodiversity, this study sought to design and identify mini-barcodes that might simultaneously assess diet and intestinal parasites from the feces of farmland birds. The capacity to identify diet and parasites of 2 existing and 3 newly developed mini-barcodes was tested “in silico” in relation to the behavior of a reference eukaryotic barcode. Among the newly designed mini-barcodes, MiniB18S_81 showed the higher taxonomic coverage of eukaryotic taxa and a greater amplification and identification capacity for diet and parasite taxa. Moreover, when it was tested on fecal samples from 5 different steppe bird species, MiniB18S_81 showed high taxonomic resolution of the most relevant diet and parasite phyla, Arthropoda, Nematoda, Platyhelminthes, and Apicomplexa at the order level. Thus, the mini-barcode developed emerges as an excellent tool to simultaneously provide detailed information regarding the diet and parasites of birds, essential for conservation and management.     

中国南方稻田及其周边环境根结线虫种类鉴定

为了解根结线虫在中国南方稻区的分布和种类, 从15个省(直辖市/自治区)采集1 181份土样, 经生物测定发现312份土样中种植的水稻或番茄幼苗根系会产生根结, 阳性总检出率为26.42%。利用线虫特异性引物从171份阳性样品中鉴定出6种根结线虫:拟禾本科根结线虫Meloidogyne graminicola、北方根结线虫M.hapla、花生根结线虫M.arenaria、爪哇根结线虫M.javanica、象耳豆根结线虫M.enterolobii和南方根结线虫M.incognita, 分别占鉴定总样品数的88.89%、41.52%、29.82%、10.53%、4.09%和3.51%。被1、2、3、4、5、6种根结线虫混合侵染的土样比例分别为42.11%、41.52%、12.87%、2.92%、0.58%和0, 其中最常见的为拟禾本科根结线虫单独侵染(34.50%)、拟禾本科根结线虫与北方根结线虫(22.81%)或花生根结线虫(13.45%)混合侵染。本研究结果将为制定相应的传播阻断策略和防控措施提供参考。     

桂花RAPD分析的引物筛选

从桂花4个品种群(银桂品种群、金桂品种群、丹桂品种群和四季桂品种群)中选取8个桂花样品,用于桂花RAPD分析的引物筛选试验。从Operon公司的OP系统的AB、AC、AD、Y、Z5组共80个10碱基引物中筛选出20个扩增条带多且多态性好的引物,20个引物共扩增116条DNA条带,用100～3000bp的DNAmarker标记片断长度在200～2000bp,多态性条带有72条,多态性位点比率为62.0%。不同引物的扩增带数不同,最多的可扩增出8条DNA条带。     

Genetic instability of bermudagrass (Cynodon) cultivars 'Tifgreen' and 'Tifdwarf' detected by DAF and ASAP analysis of accessions and off-types

DNA amplification fingerprinting (DAF) and arbitrary signatures from amplification profiles (ASAP) were used to evaluate the genetic stability of two important bermudagrass (Cynodon) cultivars, the interspecific cross Tifgreen and its somatic mutant Tifdwarf, and study genetic diversity and origin of derived bermudagrass off-types that exhibit patches of contrasting morphology and performance. Mini-hairpin primers produced complex and reproducible DAF and ASAP profiles with high levels of polymorphic DNA, and established genetic relationships between 11 Tifgreen and 8 Tifdwarf turf plot accessions and 16 off-types. DAF analysis revealed an average 14.1 ± 5.6 (SE) polymorphic bands/primer within cultivar accessions. In contrast, the higher resolving power of ASAP detected 24.5 ± 2.1 polymorphic bands/primer. Similarly, DAF and ASAP produced 13.0 ± 5.5 (SE) and 20 ± 2.8 polymorphic bands/primer within off-types, respectively. Phenetic analysis using cluster (UPGMA) and ordination (PCO) techniques showed that both Tifdwarf and Tifgreen were genetically unstable. The analysis showed that almost all cultivar accessions and one-half of the off-types studied were genetically distinct, but very close to each other. In this case, genetic variation was probably the result of somatic mutations. The other off-types and some Tifgreen accessions represented a genetically distant and diverse bermudagrass group of interspecific hybrid (n=27) origin. Off-types were probably the result of sod contamination. Results complement a previous study that established that the interspecific Tifway bermudagrass was genetically stable whereas derived off-types were contaminants rather than somatic mutants. Tifgreen and Tifdwarf showed genetic instabilities that were readily detected by DNA amplification with mini-hairpin primers. The present study offers a direct molecular alternative capable of evaluating the genetic stability of selected cultivars. This revised version was published online in July 2006 with corrections to the Cover Date.     

A molecular detection and identification method for papaya mealybug （Paracoccus marginatus）, the first recorded invasive pest in Fujian province of China北大核心CSCD

【Objective】Paracoccus marginatus Williams and Granara de Willink is an invasive pest with strong diffusion and fecundity. It has caused serious damage to the papaya industry in Central America, Florida (USA), Guam (USA) and India. Pa. marginatus was first discovered in Fujian Province (Fuzhou and Zhangzhou) in 2017, showing great potential risks to papaya and other fruit crops, as well as flower industry in Fujian province. Because of the small body size and similar morphological characteristics, the morphological identification of mealybugs was inefficient. Rapid molecular identification of different species could be achieved through the use of DNA barcoding technology. Therefore, a technology for rapid molecular identification of Pa. marginatus was established based on the species-specific PCR method. 【Methods】A species-specific PCR method based on ribosomal DNA-28S gene fragment (28S rDNA) was exploited to establish one technology for rapid detection and identification of Pa. marginatus. The additional 10 species of mealybugs (Phenacoccus solenopsis, Dysmicoccus boninsis, Nipaecoccus viridis, Phenacoccus solani, Pseudococcus comstocki, Pseudococcus cryptus, Planococcus lilacinus, Pseudococcus odermatti, Planococcus minor and Phenacoccus madeirensi) were collected in the fields as the contrast. In order to ensure the uniqueness of the source of DNA, the DNA templates were all extracted from one single female adult of these 11 species of mealybugs, respectively. 28S rDNA of the 11 species was amplified by a pair of universal primers (S3660/A335). The obtained partial fragments of 28S rDNA were sequenced. And the phylogenetic tree was established by using a Neighbor-joining (NJ) method. According to the obtained 28S rDNA gene partial sequence of the 11 species and 28S rDNA gene sequences of Paracoccus galzerae in GeneBank database, the sequence alignment and analysis were performed on DNAMAN. 28S rDNA species-specific primers (28S-ParF/ 28S-MarR) for Pa. marginatus were designed by selecting the sites with large differences in the sequence. And then, the specific effects, versatility and sensitivity of the specific primers were examined. 【Results】The comparative results showed that the similarity between Pa. marginatus and Paracoccus marginatus isolate S3-668, KP692333 in the GenBank database was 100%. It was also indicated that the mealybugs were identified as Pa. marginatus by molecular identification. Phylogenetic analysis indicated that Pa. marginatus from Fuzhou and Zhangzhou was clustered in a clade. And that combined with Paracoccus galzerae (inter-species genetic distance is 0.058) to form a clade of the genus Paracoccus. The results of specificity tests showed that all Pa. marginatus specimens could be detected positively and a 446 bp fragment of the 28S rDNA of Pa. marginatus was obtained by the species-specific primers, while there was no cross reactions with other 10 species of mealybugs. The species-specific primers not only had a stable amplification effect on female adults, but also were proved to be applicable for the 2nd instar nymphs and the 3rd instar nymphs. Pa. marginatus from three different regions (Fuzhou and Zhangzhou in Fujian province, Jinghong in Yunnan province) and six different host plants (Carica papaya, Solanum melongena, Plumeria rubra, Solanum tuberosum, Tithonia diversifolia and Duranta erecta) was also successfully detected by the species-specific primers.【Conclusion】Molecular identification of Pa. marginatus first reported in Fujian province was carried out based on 28S rDNA molecular markers. It was proved by experiments that the 28S rDNA species-specific primers had ideal and stable specificity for Pa. marginatus and could be used to identify Pa. marginatus accurately. A rapid molecular detection technique for Pa. marginatus was established based on the species-specific PCR method. The technology has the characteristics of accuracy, rapidity, sensitivity and simplicity. Our present results indicated that the rapid detection technique should be useful in quarantine at ports, in pest detection and in monitoring during transportation of papaya and other fruit tree seedlings, as well as flowers. However, in view of the fact that no other mealybugs of the genus Paracoccus has been reported in China, this study can provide a reference for the molecular identification for the closely related species of Pa. marginatus. © 2019 Journal of Fruit Science