Development of germplasm identification markers and phylogenetics analysis of Ligusticum chuanxiong / 中草药

Objective: In this study, InDel markers were developed based on the high frequency Insertion/Deletion region of chloroplast genome of Ligusticum. Germplasm identification and phylogenetic development of Ligusticum chuanxiong and its common adulterants were studied with universal barcode. Methods: The 26 samples of L. chuanxiong and its common adulterants were amplified and sequenced by eight DNA universal barcodes: ycf1, matK, ITS2, rpoC1, rbcL, rpoB, trnK, and psbA-trnH. Genetic distance statistics, barcoding gap and phylogenetic tree analysis methods were used to study the phylogenetic relationship and phylogeny of L. chuanxiong. At the same time, the evolutionary tree was constructed to study molecular identification of L. chuanxiong and its common adulterants. Results: The results showed that rbcL conserved site was the highest (97.32%) with the highest GC content (44.9%). The rbcL+rpoB fragment had the smallest average intraspecific genetic distance (0.002 5). The psbA-trnH sequence fragment had the largest average interspecific genetic distance (0.429 2). The trnK and rbcL+rpoB sequence had the highest interspecific genetic distance. The overlap of the "barcoding gap" region of psbA-trnH was the least. The species of L. chuanxiong and other adulterated species were not accurately identified by the eight pairs of DNA barcodes. The cluster analysis of 24 InDel markers could accurately identify genuine L. chuanxiong and classify the species of L. chuanxiong and its adulterants into four categories, one of which was genuine L. chuanxiong collected from Sichuan. Conclusion: The ability of InDel markers to identify authentic L. chuanxiong and its common adulterants was higher than that of common barcode. According to the above studies, it is found that it is impossible to distinguish L. chuanxiong and its common adulterants by the traditional DNA barcodes because of the large difference in genetic components. The newly developed InDel molecular markers can effectively identify L. chuanxiong and its commonly used adulterants, and provide an effective method for the genuineness of L. chuanxiong at molecular level.

Germplasm identification of Lonicerae Japonicae Flos by DNA melt curve analysis / 中国中药杂志

To establish a new high resolution melting analytical method for identification of Lonicera japonica germplasm, the screening of 7 pairs of SSR (simple sequence repeats) primers, determining the suitable diagnostic primers by the differences of peak pattern and Tm was conducted. Then into the DNA template concentration, annealing temperature and the suitable range of cycle number were investigated. Combined with SIMCA-P software for data processing analysis, the results show that three main germplasm honeysuckle could be divided by four sets of primers. It provides methodology for improving L. japonica germplasm identification.

Tilling in Rice Breeding / 中国生物工程杂志

Targeting induced local lesions in genomes (TILLING) is a reverse genetics method for functional genomics research.It is possible to screen for point mutations in the populations of EMS mutagenesis with highthroughput and lowcost. EcoTILLING a method based on TILLING ,was developed for detecting multiple types of polymorphisms in germplasm collections,such as single nucleotide polymorphism,small deletion and insertion etc.Rice is a very important food crop and a model plant for genome research also. There are complete genome sequence and a lot of other bioinformatics resources about it.So the markerassisted breeding is becoming more and more important in rice breeding. Some issues based on TILLING about identifying germplasm based on gene sequence,EMS mutagenesis breeding,developing functional marker in rice breeding in future were discussed.

Application of AFLP markers for analyzing genetic difference among cultivated Pinellia ternata / 中草药

Objective The DNA fingerprints of cultivated Pinellia ternata collected from different geographic regions were generated by using AFLP markers to find the feasibility in analyzing their genetic diversity, relationship, and germplasm identification. Methods The DNA polymorphism of 51 cultivated germplasm of P. ternata collected from 17 different habitats and four cultivars of P. pedatisecta (outgroup) were detected by AFLP molecular markers. Results The DNA fingerprints of 51 individuals of P. ternata were obviously distinguished by eight pairs of high polymorphic and efficient primer combinations screened from 64 primer combinations. The phylogenetic clustering results revealed that all the tested cultivars were fully differentiated, and individuals from the same regions were mainly clustered together. Moreover, cultivars from East-China, including Zhejiang and Jiangsu Provinces, displayed clear genetic distinction from other regions. The clustering results were strongly supported by Bootstrap test. Conclusion AFLP Markers can be potentially used in analyzing of genetic diversity, relationship, and germplasm identification of this medicinal plant, and the germplasm from regions of East-China, including Zhejiang and Jiangsu Provinces, displays the relative separate genetic characters from other regions.