A Rapid PCR-RFLP Method for Assessing Heterozygosity of Murraya paniculata Germplasm / 中国实验方剂学杂志

ObjectiveTo establish a rapid method for evaluating the heterozygosity of Murraya paniculata germplasm materials and provide as a foundation for developing germplasm breeding and innovation measures for M. paniculata. MethodSingle nucleotide polymorphisms （SNPs） were screened from the genome resequencing data of 65 plants of M. paniculata. A self-written script was used to transform 20 SNPs into restriction fragment length polymorphism （RFLP） markers. Polymerase chain reaction-restriction fragment length polymorphism （PCR-RFLP） was employed to detect the 20 RFLP markers in 12 M. paniculata germplasm accessions， and the heterozygosity of M. paniculata germplasm accessions was calculated based on the number of enzyme-cutting bands at the 20 RFLP marker sites. Plink was used to calculate the whole genome heterozygosity of 12 M. paniculata germplasm accessions， and the results obtained with different methods were compared. ResultThere was no significant difference in the heterozygosity calculated by the PCR-RFLP method and the genome resequencing method. The PCR-RFLP and genome resequencing methods identified 8 and 9 germplasm accessions， respectively， with a heterozygosity level less than 30%. Seven germplasm accessions with heterozygosity less than 30.00% were calculated by both methods. ConclusionThe PCR-RFLP method established in this study for evaluating the heterozygosity of M. paniculata germplasm demonstrates the precision of 87.5% and the accuracy of 77.8%. This method serves as a reference for developing heterozygosity evaluation methods in other medicinal plant germplasm resources.

Specific PCR for Identification of Astragalus membranaceus var. mongholicus Seeds， A. membranaceus Seeds， and Adulterants / 中国实验方剂学杂志

ObjectiveTo establish a method based on specific polymerase chain reaction （PCR） that can accurately and rapidly identify Astragalus membranaceus var. mongholicus （AMM） seeds and A. membranaceus （AM） seeds. MethodThe Chloroplast Genome Information Resource （CGIR） and IdenDSS were used to obtain the characteristic DNA fragments of AMM and AM， and the specific single nucleotide polymorphism （SNP） sites of AMM and AM were screened out， on the basis of which the specific primers MG-F/MG-R of AMM and MJ-F/MJ-R of AM were designed. The specific PCR method for identifying AMM and AM was established and optimized， and the specificity and applicability of the method were investigated. ResultThe specific PCR conditions for the identification of AMM were primers MG-F/MG-R， annealing at 62 ℃， and 28 cycles. After PCR amplification and gel electrophoresis， the specific band appeared at about 220 bp， with no band for the seeds of AM or adulterants. The specific PCR conditions for identifying the AM were primers MJ-F/MJ-R， annealing at 58 ℃， and 28 cycles. After PCR amplification and gel electrophoresis， the band appeared at about 150 bp， with no band of AMM or adulterants. ConclusionThe specific PCR method established in this study can accurately and quickly identify the seeds of AMM and AM， which provides a basis for the classification and accurate identification of Astragalus seeds and adulterants.

Specific PCR Identification of Artemisia absinthium， A New Foreign Medicinal Resource of Artemisia / 中国实验方剂学杂志

ObjectiveTo establish a specific polymerase chain reaction （PCR） method for the identification of Artemisia absinthium to allow accurate and convenient identification of A. absinthium and its related species. MethodThe chloroplast genome sequences of A. absinthium and its related species were searched from Chloroplast Genome Information Resource （CGIR）， and the specific single nucleotide polymorphism （SNP） sites of A. absinthium were screened out. A pair of specific identification primers （zykh1-F and zykh1-R） of A. absinthium was designed. The original plant samples of A. absinthium and its related species were collected. The specific PCR method was established and optimized， and the tolerance and feasibility of this method were investigated and verified. The method was used to identify A. absinthium samples purchased from Xinjiang medicinal materials market. ResultA 210 bp bright band was obtained from A. absinthium after PCR amplification and gel electrophoresis under the following conditions： specific primers zykh1-F and zykh1-R， annealing temperature of 54 ℃， and the number of cycles of 33. No such band was observed from its relative species， such as A. argyi， A. annua， A. leucophylla， and A. lavandulaefolia. ConclusionThe specific PCR identification method of established in this study can accurately identify A. absinthium and its common related species with high specificity. The method can save time and cost and allows a convenient and fast species identification for the introduction and utilization of A. absinthium resources.

Identification of Murrayae Folium et Cacumen by Multiplex Allele-Specific PCR / 中国实验方剂学杂志

ObjectiveTo establish a polymerase chain reaction（PCR） method to accurately discriminate the crude materials of Murrayae Folium et Cacumen， Murraya exotica and M. paniculata. MethodBased on the difference in chloroplast genome sequences of M. exotica and M. paniculata， species-specific identification primers P03 and P04 of M. exotica and M. paniculata were designed according to single nucleotide polymorphism （SNP） on the chloroplast genome. A multiplex allele-specific PCR identification method was established for the identification of M. exotica and M. paniculata following the optimization of annealing temperature， number of cycles， and primer concentration ratio. The established PCR method for identification was explored and verified in terms of tolerance and feasibility by investigating the type of Taq polymerases and PCR system model. ResultIn this multiplex allele-specific PCR identification method， about 330 and 230 bp of specific fragments were amplified from DNA templates of M. exotica and M. paniculata， respectively， under the following conditions：cycle number of 31， annealing temperature of 60 ℃， and primer concentration ratio of P03 and P04 of 1∶2. Consistent results were obtained for samples from different sources. ConclusionThe multiplex allele-specific PCR identification method established in this study can accurately identify the origin of Murrayae Folium et Cacumen， which can be used for the simultaneous identification of M. exotica and M. paniculata by the length of fragments in a single identification assay.

Improved fluorescence spectrometric determination of lipid content in Botryococcus braunii / 生物工程学报

Botryococcus braunii is a unique colonial green microalga and a great potential renewable resource of liquid fuel because of its ability to produce lipids. Due to the dense cell colonies and rigidly thick cell wall of B. braunii, the traditional Nile red method is usually of low sensitivity and bad repeatability and hard for the determination of lipid content in the cells. By dispersing the colony with ultrasonic, assisting permeation of Nile red across the cell wall with dimethyl sulfoxide and optimizing the staining conditions, we established an improved detection method. The details were as follows: after the colonial algal sample was treated by ultrasonic at 20 kHz for 20 s, 100 W transmitting power and with 1 s on/1 s off intermittent cycle, the equivoluminal 15% (V/V) dimethyl sulfoxide and 3 microg/mL Nile red were successively added and mixed evenly, then the staining system was incubated in dark at 40 degrees C for 10 min, and subsequently was measured by fluorescence spectroscopy detection with an excitation wavelength of 490 nm. Compared with the traditional method, the improved one not only had higher detection sensitivity which was increased by 196.6%, but also had obviously better detection repeatability whose characteristic parameter - relative standard deviation (RSD) was decreased from 10.91% to 1.84%. Therefore, the improved method could provide a rapid and sensitive detection of lipid content for B. braunii breeding and cultivation.