A Molecular Approach to Identification of the Chinese Drug“pu Gong Ying”(Herba Taraxaci)and Six Adulterants byDNA Fingerprinting Using Random Primed PolymeraseChain Resaction(PCR)

DNA fingerprinting among members of the Chinese drug Pu Gong Ying(Taraxacum mongolicum Hand,-Mazz.)and six adulterants of Tu Gong Ying were demonstrated with random-primed polymerase chain reaction(PCR)including arbitrarily primed polymerase chain reaction(AP-PCR)and random amplified polymorphic DNA(RAPD).Distinctive,reproducible genomic fingerprints from DNA from 7 species belonged to Compositae were generated with two long(20 and 24 mer)and one short(10 mer)randomly chosen primers.The Pu Gong Ying can be differentiated from six species of Tu Gong Ying according to the banding pattems of their amplified DNA on agarose gels.The results showed that AP-PCR and RAPD methods can be used for identifying Chinese drugs.Moreover,the Similarity Indexes of the genomic DNA fingerprints showed that Pu Gong Ying and its adulterants are unrelated.Therefore,AP-PCR and RAPD methods can be used for identifying Chinese drugs.

Identification of Porphyra lines using computerized DNA fingerprinting

RAPD (Randomly Amplified Polymorphic DNA) analysis was performed with filaments of 15 Porphyra lines representing four important groups (P. yezoensis, P. haitanensis, P. katadai var. Hemiphylla and P. digospermatangia). Eight stable and repeatable RAPD bands amplified with two primers, OPN-02 and OPJ-18, were selected for the construction of DNA fingerprinting. The RAPD results were scored based on the presence or absence of each of the 8 bands and then converted to computer language expressed with two digitals, 1 and 0, which represented the presence (numbered as 1) or absence (numbered as 0) of each band, respectively. Based on these results, a model DNA fingerprint and a computerized DNA fingerprint were constructed. In the constructed DNA fingerprint, each Porphyra line has its unique fingerprinting pattern and can be easily distinguished from each other. Later, a software, named as PhGI, was designed based on this DNA fingerprinting. It can be used in practical Porphyra line identification.

Application of DNA Fingerprinting Technique in Adulterated Food Identification and Traceability

To further improvc the application of DNA fingerprinting technique in adulterated food identification and traceability, the paper briefly introduced the ap- plication of common DNA fingerprinting techniques, such as species-specific PCR, RAPD, AFLP, ISSR, SSR and SNP in adulterated food identification and traceability.

DNA Fingerprinting of Acer truncatum Clones Using SRAP Markers

[ Objective] This study aimed to establish DNA fingerprints of 23 Acer truncatum clones, thus providing the theoretical basis for selection, classification and identification of A. truncatum varieties. [Method] Sixteen pairs of SRAP primers with rich polymorphism and high specificity were used to establish DNA fin-gerprints. [Result] A total of 223 bands were amplified with 16 primer pairs, including 197 polymorphic bands. Averagely 13.9 loci and 12.3 polymorphic loci were amplified with each primer pair. The average percentage of polymorphic loci reached 88. 34% . [ Conclusion] The classification result drawn by cluster analy-sis is consistent with that obtained based on main characteristics and genetic relationships of A. truncatum, clones. By using DNA fingerprints established with prim-er pairs ME1-EM4 and ME2-EM1, 23 A. truncatum clones can be effectively distinguished, and the confidence probability is greater than 99.99%.

Plastid Inheritance in Sweet Potato as Revealed by DNA Restriction Fingerprinting

The inheritance of chloroplast DNA (cpDNA) in sweet potato (Ipomoea batatas Lain.) was analyzed using DNA restriction fingerprinting. The cpDNA fingerprints of hybrids from reciprocal crosses between Xushu18 and AB78-1 were found to be identical to those of their female parents, which reveals that cpDNA of sweet potato is maternally inherited in this intervarietal crossing. This maternal cpDNA transmission pattern does not accord with the putative one based on former cytological studies. The plastid inheritance in Convolvulaceae has been briefly reviewed in this study, and the utility of DNA restriction fingerprinting analysis in the study of plastid inheritance is also discussed.

Identification of Four Gastrodia elata Bl. Variants with DNA Fingerprints

[Objective] This study aimed to investigate the four Gastrodia elata B1. variants at the DNA level. [Method] Primers with good reproducibility, abundant polymorphism and high resolution were selected from the 35 random primers with RAPD marker technology and the amplification results were analyzed. [ Result] By using RAPD technology, DNA fingerprints of four Gastrodia elata B1. variants with abundant polymorphism, good reproducibility and high resolution was successfully constructed; to be specific, each Gastrodia elata B1. variant has its unique fingerprint with primers S29 and S38 ; DNA bands of the Gastrodia elata BI. variants vary significantly, which is contributive to distinguish the four Gastrodia clara B1. variants. [Condusion] This study provided an effective means for the identification and protection of Gastrodia elata germplasms with intraspecific variations.

RAPD analysis of genetic diversity of nine strains of Auricularia auricular cultivated in Heilongjiang Province

Polymorphism of nine strains （CF05, CF09, 29, 916, AU9, Chang10, Chang7, 8808 and AU. Japanese） of A. auricular cultivated in Heilongjiang Province were analyzed by RAPD （Random Amplication polymorphic DNA）. Thirteen primers were selected from forty PCR primers with 10bp long random primer. The results showed that nine strains of A. auricular have a high level of genetic diversity and the percentage of DNA polymorphic was 96.05. The genotypes of 9 strains of Auricularia auricular were identified by the fingerprints from primer 27 and primer 46 by RAPD analysis. The results are helpful for quickly identifying strains of A. auricular in its early breeding time, and also provides a powerful theoretic basis to differentiate strains （Auricularia auricular） whose morphology is very similar in breeding programs of edible fungus.

Plant Variety Protection and DUS Testing Technique

DUS testing technique used for plant variety protection was reviewed in the paper, and some suggestions were made on how to establish the appropriate technology system in China. Meanwhile, the potential exploitation of the technique was discussed.

Applications of Molecular Markers in Fruit Crops for Breeding Programs—A Review

Selection and use of molecular markers for evaluation of DNA polymorphism in plants are couple of the most important approaches in the field of molecular genetics.The assessment of genetic diversity using morphological markers is not sufficient due to little differentiating traits among the species,genera or their individuals.Morphological markers are not only highly influenced by environmental factors but skilled assessment is also prerequisite to find the variations in plant genetic resources.Therefore,molecular markers are considered as efficient tools for detailed DNA based characterization of fruit crops.Molecular markers provide new directions to the efforts of plant breeders particularly in genetic variability,gene tags,gene localization,taxonomy,genetic diversity,phylogenetic analysis and also play an important role to decrease the time required for development of new and excellent cultivars.The success of molecular markers technology in genetic improvement programs depends on the close relationship among the plant breeders,biotechnologists,skilled manpower and good financial support.The present review describes application and success of molecular markers technology used for genetic improvement in different fruit crops.

Molecular Identification of Castanopsis hystrix,Castanopsis carlesii and Quercus griffithii Using ISSR-PCR Method

[Objective] This research aimed to develop molecular identification method for Castanopsis hystrix,Castanopsis carlesii and Quercus griffithii.[Method] DNA fingerprints of C.hystrix,C.carlesii and Q.griffithii were established by using ISSR-PCR method.Cluster Analysis was carried out by using UPGMA method based on Nei＇s genetic distances among each individual.[Result] Six polymorphic primers were selected from 50 ISSR primers for ISSR-PCR amplification,and totally 86 discernible DNA bands were amplified with 53 polymorphic bands,accounting for 61.2% of the total.The average number of DNA bands amplified by each primer was 10.75.Specifically,totally 5 primers had amplified differential bands and specific bands,which were able to accurately identify C.hystrix,C.carlesii and Q.griffithii.As calculated by DPS v3.01 software,the genetic distances among test materials were ranged from 0.166 67 to 0.809 52,with an average of 0.563 57.[Conclusion] ISSR-PCR method can be used to identify C.hystrix,C.carlesii and Q.griffithii effectively.

Advance of Study on SSR Molecular Marker in Citrus and Its Close Relatives

Simple sequences repeat （SSR） molecular maker, as a new type of DNA molecular marker, the second generation based on the polymerase chain reaction （PCR）, is valuable and of great potential as genetic markers for its characteristics of abundant quantity, high polymorphic, reproducibility, specific site amplification, high occurring frequency, and co-dominant inheritance etc. This paper outlined its principles and characteristics, and introduced its application to variety identification, phylogenetic relationship analysis, genetic diversity analysis, DNA fingerprinting and linkage map constructing etc. in recent years in Citrus and its close relatives.

Development and application of perfect SSR markers in cotton

Background:This study aimed to develop a set of perfect simple sequence repeat(SSR)markers with a single copy in the cotton genome,to construct a DNA fingerprint database suitable for authentication of cotton cultivars.We optimized the polymerase chain reaction(PCR)system for multi-platform compatibility and improving detection efficiency.Based on the reference genome of upland cotton and 10×resequencing data of 48 basic cotton germplasm lines,single-copy polymorphic SSR sites were identified and developed as diploidization SSR markers.The SSR markers were detected by denaturing polyacrylamide gel electrophoresis(PAGE)for initial screening,then fluorescence capillary electrophoresis for secondary screening.The final perfect SSR markers were evaluated and verified using 210 lines from different sources among Chinese cotton regional trials.Results:Using bioinformatics techniques,1246 SSR markers were designed from 26626 single-copy SSR loci.Adopting a stepwise(primary and secondary)screening strategy,a set of 60 perfect SSR markers was selected with high amplification efficiency and stability,easy interpretation of peak type,multiple allelic variations,high polymorphism information content(PIC)value,uniform chromosome distribution,and single-copy characteristics.A multiplex PCR system was established with ten SSR markers using capillary electrophoresis detection.Conclusions:A set of perfect SSR markers of cotton was developed and a high-throughput SSR marker detection system was established.This study lays a foundation for large-scale and standardized construction of a cotton DNA fingerprint database for authentication of cotton varieties.

Genetic Diversity and Phylogenetic Relationships among Phytophthora capsici Isolates from Guizhou Province by RAPD

Ten random primers with clear amplification profile, significant and stable main band were screened from RAPD （Random Amplified Polymorphie DNAs） primers to analyze the genetic diversity among eight Phytophthora capsici isolates from Huaxi District, Wudang District and Kaiyang County of Guiyang City, and Zunyi County, Suiyang County and Luodian County of Zunyi City in Guizhou Province. A total of 70 DNA fingerprints were obtained, including 57 polymorphic bands, with a polymorphic percentage of 81.43%, suggesting abundant genetic diversity among experimental Phytophthora capsici isolates. According to the ampli- fied DNA fingerprint profiles, using genetic similarity coefficient 0.5 as the threshold, experimental Phytophthora capsici isolates were clustered into three genetic categories by UPGMA cluster analysis. The analysis result indicated that there was no direct correlation between the genetic similarity and cultivation areas of vari- ous Phytophthora caosici isolates.

Mechanistic insights toward identification and interaction of plant parasitic nematodes:A review

Nematode identification serves as an important param-eter to study their behaviour,importance and pathogenic-ity.Application of classical morphometric based identifica-tion methods prove to be lacking due to insufficient knowledge on morphological variations among closely related taxa.Molecular approaches such as DNA and protein-based information,microarray,probing,sequence-based methods and others have been used to supplement morphology-based methods for nematode identification.Ascarosides and certain protein-based nematode-associated molecular patterns(NAMPs),can be perceived by the host plants,and can initiate a signalling cascade.This review primarily emphasizes on an updated account of different classical and modern tools used for the identification of nematodes.Besides we also summa-rize the mechanism of some important signalling pathways which are involved in the different plant nematode interactions.Nematodes constitute most diverse and least studied group of soil inhabiting invertebrates.They are ecologically and physiologically important,however,wide range of nematodes show harmful impact on the individuals that live within their vicinity.Plant parasitic nematodes(PPNs)are transparent,pseudocoelomate,free living or parasitic microorganisms.PPNs lack morphometric identification methods due to insufficient knowl-edge on morphological variations among closely related taxa.As such,molecular approaches such as DNA and protein-based information,microarray,probing,sequence-based methods and others have been used to supplement morphology-based methods for their identification.To invade the defense response of different plant species,parasitic nematodes have evolved different molecular strategies.Ascarosides and certain protein-based nematode-associated molecular patterns(NAMPs),can be perceived by the host plants,and can initiate a signaling cascade.To overcome the host confrontation and develop certain nematode feeding sites,some members can inject effectors into the cells of susceptible hosts to reprogram the basal resistance signaling.This review primarily emphasizes on an updated account of different classical and modem tools used for the identification of PPNs.Besides we also summarize the mechanism of some important signaling pathways which are involved in the different plant nematode interactions.

Identification of Trichophyton rubrum by PCR-fingerprinting

OBJECTIVE: To observe the unique DNA profile and the relationship between DNA profile and phenotype of Trichophyton rubrum,and establish an effective molecular method to identify T. rubrum. METHODS: Three primers, including (GACA)(4), (GTG)(5) and M13 core sequence (5'-GAGGGTG-GCGGTTCT-3'), were used to distinguish variations among 20 clinical isolates of T. rubrum and Trichophyton mentagrophytes. RESULTS: Different PCR-fingerprinting was seen between T. rubrum and T. mentagrophytes using three different primers. 2 stains of T. rubrum were identified among 6 supposed T. mentagrophytes. CONCLUSIONS: T. rubrum can be distinguished using PCR, and (GACA)(4) is the most suitable primer.

Development of Insertion and Deletion Markers for Bottle Gourd Based on Restriction Site-associated DNA Sequencing Data

Bottle gourd is an important cucurbit crop worldwide. To provide more available molecular markers for this crop, a bioinformatic approach was employed to develop insertion–deletions(In Dels) markers in bottle gourd based on restriction site-associated DNA sequencing(RAD-Seq)data. A total of 892 Indels were predicted, with the length varying from 1 bp to 167 bp. Single-nucleotide In Dels were the predominant types of In Dels. To validate these In Dels, PCR primers were designed from 162 loci where In Dels longer than 2 bp were predicated. A total of 112 In Dels were found to be polymorphic among 9 bottle gourd accessions under investigation. The rate of prediction accuracy was thus at a high level of72.7%. DNA fingerprinting for 4 cultivars were performed using 8 selected Indels markers, demonstrating the usefulness of these markers.

Authentication of Medicinal Plants by DNA-based Markers and Genomics

For the protection of consumers and developments of relevant industry, authentication of medicinal plants is a critical issue. This review covers various aspects of authentication methods and techniques based on molecular biology and genomics with special emphasis on molecular biology techniques including genome-based authentication, microchip-based authentication, DNA barcoding, and their applications.

SSR Analysis of Genetic Diversity Among 192 Diploid Potato Cultivars

In potato breeding,it is difficult to improve the traits of interest at the tetraploid level due to the tetrasomic inheritance.A promising alternative is diploid breeding.Thus it is necessary to assess the genetic diversity of diploid potato germplasm for efficient exploration and deployment of desirable traits.In this study,we used SSR markers to evaluate the genetic diversity of diploid potato cultivars.To screen polymorphic SSR markers,55 pairs of SSR primers were employed to amplify 39 cultivars with relatively distant genetic relationships.Among them,12 SSR markers with high polymorphism located at 12 chromosomes were chosen to evaluate the genetic diversity of 192 diploid potato cultivars.The primers produced 6 to 18 bands with an average of 8.2 bands per primer.In total,98 bands were amplified from 192 cultivars,and 97 of them were polymorphic.Cluster analysis using UPGMA showed the genetic relationships of all accessions tested:186 of the 192 accessions could be distinguished by only 12 pairs of SSR primers,and the 192 diploid cultivars were divided into 11 groups,and 83.3% constituted the first group.Clustering results showed relatively low genetic diversity among 192 diploid cultivars,with closer relationship at the molecular level.The results can provide molecular basis for diploid potato breeding.

Combination of ARDRA and RAPD genotyping techniques in identification of Acinetobacter spp. genomic species

A total of 10 non-repetitive multi-drug-resistant Acinetobacter strains were collected.With reference to A.calcoaceticus(ATCC23055),A.baumannii(ATCC19606),A.lwoffii(ATCC17986),and A.junii(NCTC5866),DNA fingerprint technique,amplified ribosomal DNA restriction analysis(ARDRA),and random amplified polymorphism DNA(RAPD)were carried out to identify the genomic species of Acinetobacter spp.The distances between them were calculated by the unweighted pair group method with arithmetic(UPGMA).Genotypes of Acinetobacter spp.were effectively classified and an A.junii together with nine A.baumannii isolates was genomically identified.The combination of ARDRA and RAPD DNA-fingerprint technique shows high complementarity,and could be a useful tool in Acinetobacter genomic species identification.