基于板蓝根及其混伪品叶绿体基因组的mini-barcode开发及其定性能力的研究

[目的]基于板蓝根及其混伪品的叶绿体基因组序列开发mini-barcode并验证其鉴定能力,为板蓝根的质量控制和科学评价提供方法。[方法]从GenBank数据库中下载板蓝根及其混伪品所有已发表的叶绿体基因组序列,导入PhyloSuite v1.2.1软件提取其共有蛋白编码基因并分别对齐,后续使用DnaSP 6软件进行核苷酸多态性分析以筛选出候选DNA mini-barcode区域。通过Geneious软件多序列比对,筛选高变区用于mini-barcode开发。针对候选条形码区域设计引物。采用试剂盒法提取板蓝根药材总DNA分别利用通用ITS2引物和该实验设计引物进行聚合酶链式反应(PCR)和测序。[结果]基于叶绿体基因组的核苷酸多态性分析结果,选择accD基因作为候选mini-barcode开发区域并设计了扩增引物。通过遗传距离和测序数据分析,该分子标记能被成功扩增并实现鉴定。[结论]开发了用于鉴定板蓝根及其混伪品的mini-barcode,为实现DNA降解的板蓝根药材及含板蓝根的中药制剂等混合样本的真伪鉴别奠定基础。

DNA barcoding in herbal medicine: Retrospective and prospective

DNA barcoding has been widely used for herb identification in recent decades,enabling safety and innovation in the field of herbal medicine.In this article,we summarize recent progress in DNA bar-coding for herbal medicine to provide ideas for the further development and application of this tech-nology.Most importantly,the standard DNA barcode has been extended in two ways.First,while conventional DNA barcodes have been widely promoted for their versatility in the identification of fresh or well-preserved samples,super-barcodes based on plastid genomes have rapidly developed and have shown advantages in species identification at low taxonomic levels.Second,mini-barcodes are attractive because they perform better in cases of degraded DNA from herbal materials.In addition,some mo-lecular techniques,such as high-throughput sequencing and isothermal amplification,are combined with DNA barcodes for species identification,which has expanded the applications of herb identification based on DNA barcoding and brought about the post-DNA-barcoding era.Furthermore,standard and high-species coverage DNA barcode reference libraries have been constructed to provide reference se-quences for species identification,which increases the accuracy and credibility of species discrimination based on DNA barcodes.In summary,DNA barcoding should play a key role in the quality control of traditional herbal medicine and in the international herb trade.

Low Genetic Polymorphism at the Cytochrome C Oxidase I in Silkworm Strains of the Brazilian Germplasm Bank

Nucleotide sequences have been used to distinguish species and specimens for many years. More recently, the use of a partial sequence of 650 bp of the cytochrome c oxidase I, COI mitochondrial gene, has been proposed for species identification, known as DNA barcodes. In this work, a short sequence of the DNA barcode is described—approximately 250 bp, named as “DNA mini-barcode”—to molecularly identify different silkworm strains maintained at the unique public Germplasm Bank of Bombyx mori, at the Universidade Estadual de Maringá, UEM, Brazil. Analysis revealed no significant differences among the silkworm strains. The phylogenetic tree obtained by the neighbor-joining method and K2P distance, in which specimens of B. mandarina were used as outgroup, clustered all the specimens of B. mori in a unique clade. Genetic variability detect within B. mori was low or nonexistent. In conclusion, the partial region of 250 bp of the mitochondrial gene COI herein analyzed may not be efficient to discriminate silkworm strains from the UEM Germplasm Bank of Bombyx mori.

Identification of processed Chinese medicinal materials using DNA mini-barcoding

Most of Chinese medicinal herbs are subjected to traditional processing procedures, including stir-frying, charring, steaming, boiling, and calcining before they are released into dispensaries. The marketing and identification of processed medicinal materials is a growing issue in the marketplace. However, conventional methods of identification have limitations, while DNA mini-barcoding, based on the sequencing of a short-standardized region, has received considerable attention as a new potential means to identify processed medicinal materials. In the present study, six DNA barcode loci including ITS2, psb A-trn H, rbc L, mat K, trnL(UAA) intron and its P6 loop, were employed for the authentication of 45 processed samples belonging to 15 species. We evaluated the amplification efficiency of each locus. We also examined the identification accuracy of the potential mini-barcode locus, of trnL(UAA) intron P6 loop. Our results showed that the five primary barcode loci were successfully amplified in only 8.89%——20% of the processed samples, while the amplification rates of the trnL(UAA) intron P6 loop were higher, at 75.56% successful amplification. We compared the mini-barcode sequences with Genbank using the Blast program. The analysis showed that 45.23% samples could be identified to genus level, while only one sample could be identified to the species level. We conclude that trnL(UAA) p6 loop is a candidate mini-barcode that has shown its potential and may become a universal mini-barcode as complementary barcode for authenticity testing and will play an important role in medicinal materials control.

A novel biological sources consistency evaluation method reveals high level of biodiversity within wild natural medicine:A case study of Amynthas earthworms as"Guang Dilong"

For wild natural medicine,unanticipated biodiversity as species or varieties with similar morphological characteristics and sympatric distribution may co-exist in a single batch of medical materials,which affects the efficacy and safety of clinical medication.DNA barcoding as an effective species identification tool is limited by its low sample throughput nature.In this study,combining DNA minibarcode,DNA metabarcoding and species delimitation method,a novel biological sources consistency evaluation strategy was proposed,and high level of interspecific and intraspecific variations were observed and validated among 5376 Amynthas samples from 19 sampling points regarded as"Guang Dilong"and 25 batches of proprietary Chinese medicines.Besides Amynthas aspergillum as the authentic source,8 other Molecular Operational Taxonomic Units(MOTUs)were elucidated.Significantly,even the subgroups within A.aspergillum revealed here differ significantly on chemical compositions and biological activity.Fortunately,this biodiversity could be controlled when the collection was limited to designated areas,as proved by 2796"decoction pieces"samples.This batch biological identification method should be introduced as a novel concept regarding natural medicine quality control,and to offer guidelines for in-situ conservation and breeding bases construction of wild natural medicine.

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 abun-dance,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 par-asites,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 as-sess 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 cover-age 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.