Deoxyribonucleic Acid Barcoding for the Identification of Botanicals.

The Natural Herbal Products industry uses botanicals or herbs as raw materials for production of herbal products or dietary supplements. Recently, the demand for natural herbal products has increased tremendously and this has led to adulteration and to counterfeit herbal products. The present chapter deals with currently used molecular methods from "simple" single genomic regions to high-throughput whole genome or transcriptome sequencing methods used in the identification of botanicals.

Establishing DNA markers to differentiate Agastache rugosa and Pogostemon cablin, which are confusedly used as medicinal herbs, using real-time PCR.

Agastache rugosa and Pogostemon cablin are used as medicinal herbs and aromatic plants and belong to the family Lamiaceae. Despite differences in composition and physicochemical properties, both plants are frequently sold as the medical substance "Kwakhyang" in some Asian countries. Molecular markers were established to distinguish between the two plants using quantitative real-time PCR. Species-specific primers were designed in the nuclear internal transcribed spacer region of ribosomal DNA and in the chloroplast genes matK, rbcL, and rpoB. Six primer sets were tested, the correlation coefficient was higher than 0.99, and the slope was approximately - 3.36 to - 3.58. Efficiency ranged from 90.13 to 98.52%. The developed real-time PCR assay was validated with 14 off-target species, and its reliability was verified through blind testing of 14 commercial products. The assay developed here may help protect consumer rights, and the designed primers can be used to distinguish between the target species. Supplementary Information: The online version contains supplementary material available at 10.1007/s10068-022-01176-y.

Polyploid QTL-seq towards rapid development of tightly linked DNA markers for potato and sweetpotato breeding through whole-genome resequencing.

Potato (Solanum tuberosum L.) and sweetpotato (Ipomoea batatas L.), which are nutritionally and commercially important tuberous crops, possess a perplexing heredity because of their autopolyploid genomes. To reduce cross-breeding efforts for selecting superior cultivars from progenies with innumerable combinations of traits, DNA markers tightly linked to agronomical traits are required. To develop DNA markers, we developed a method for quantitative trait loci (QTL) mapping using whole-genome next-generation sequencing (NGS) in autopolyploid crops. To apply the NGS-based bulked segregant method, QTL-seq was modified. (1) Single parent-specific simplex (unique for one homologous chromosome) single-nucleotide polymorphisms (SNPs), which present a simple segregation ratio in the progenies, were exploited by filtering SNPs by SNP index (allele frequency). (2) Clusters of SNPs, which were inherited unevenly between bulked progenies with opposite phenotypes, especially those with an SNP index of 0 for the bulk that did not display the phenotypes of interest, were explored. These modifications allowed for separate tracking of alleles located on each of the multiple homologous chromosomes. By applying this method, clusters of SNPs linked to the potato cyst nematode resistance H1 gene and storage root anthocyanin (AN) content were identified in tetraploid potato and hexaploid sweetpotato, respectively, and completely linked DNA markers were developed at the site of the presented SNPs. Thus, polyploid QTL-seq is a versatile method that is free from specialized manipulation for sequencing and construction of elaborate linkage maps and facilitates rapid development of tightly linked DNA markers in autopolyploid crops, such as potato and sweetpotato.

A longitudinal study on morpho-genetic diversity of pathogenic Rhizoctonia solani from sugar beet and dry beans of western Nebraska.

BACKGROUND: Root and stem rot caused by Rhizoctonia solani is a serious fungal disease of sugar beet and dry bean production in Nebraska. Rhizoctonia root rot and crown rot in sugar beet and dry bean have reduced the yield significantly and has also created problems in storage. The objective of this study was to analyze morpho-genetic diversity of 38 Rhizoctonia solani isolates from sugar beet and dry bean fields in western Nebraska collected over 10 years. Morphological features and ISSR-based DNA markers were used to study the morphogenetic diversity. RESULTS: Fungal colonies were morphologically diverse in shapes, aerial hyphae formation, colony, and sclerotia color. Marker analysis using 19 polymorphic ISSR markers showed polymorphic bands ranged from 15 to 28 with molecular weight of 100 bp to 3 kb. Polymorphic loci ranged from 43.26-92.88%. Nei genetic distance within the population ranged from 0.03-0.09 and Shannon diversity index varied from 0.24-0.28. AMOVA analysis based on ΦPT values showed 87% variation within and 13% among the population with statistical significance (p < 0.05). Majority of the isolates from sugar beet showed nearby association within the population. A significant number of isolates showed similarity with isolates of both the crops suggesting their broad pathogenicity. Isolates were grouped into three different clusters in UPGMA based cluster analysis using marker information. Interestingly, there was no geographical correlation among the isolates. Principal component analysis showed randomized distribution of isolates from the same geographical origin. Identities of the isolates were confirmed by both ITS-rDNA sequences and pathogenicity tests. CONCLUSION: Identification and categorization of the pathogen will be helpful in designing integrated disease management guidelines for sugar beet and dry beans of mid western America.

A putative AGAMOUS ortholog is a candidate for the gene determining ease of dehulling in Tartary buckwheat (Fagopyrum tataricum).

MAIN CONCLUSION: Tartary buckwheat rice-type cultivars, which allow easy dehulling, lacked periclinal cell divisions that proceed underneath the epidermis in the proximity of ovary midribs in non-rice-type cultivars. The easy dehulling in these cultivars was associated with a G→A substitution in an AGAMOUS ortholog. Ease of dehulling in Tartary buckwheat (Fagopyrum tataricum) can affect the quality of its products. Tartary buckwheat cultivars that allow easy dehulling are called rice-type cultivars. The rice and non-rice hull types are determined by a single gene, but this gene is unclear. Here, we show that cells underneath the epidermis in the proximity of ovary midribs undergo periclinal cell divisions in non-rice-type cultivars but do not in a rice-type cultivar. The cells that arose from the periclinal cell divisions later underwent lignification, which should increase mechanical strength of hulls. In RNA sequencing, a partial mRNA of an AGAMOUS ortholog in Tartary buckwheat (FtAG) was found to be absent in the rice-type cultivar. Cloning of this gene revealed that this is a 42-bp deletion due to a G→A substitution at a splice acceptor site in the FtAG genomic region. In F2 progeny derived from a cross between non-rice-type and rice-type cultivars, all the rice-type plants exhibited the homozygous A/A allele at this site, whereas all the Tartary-type plants exhibited either the homozygous G/G allele or the heterozygous A/G allele. These results suggest that FtAG is a candidate for the gene that determines ease of dehulling in Tartary buckwheat. The DNA marker that we developed to distinguish the FtAG alleles can be useful in breeding Tartary buckwheat cultivars.

Identification and Monitoring of Amomi Fructus and its Adulterants Based on DNA Barcoding Analysis and Designed DNA Markers.

Amomi Fructus is one of the traditional medicines derived from the ripe fruits of the Zingiberaceae family of plants, which include Amomum villosum, A. villosum var. xanthioides, and A. longiligulare. Owing to their highly similar morphological traits, several kinds of adulterants of Amomi Fructus have been reported. Therefore, accurate and reliable methods of identification are necessary in order to ensure drug safety and quality. We performed DNA barcoding using five regions (ITS, matK, rbcL, rpoB, and trnL-F intergenic spacer) of 23 Amomi Fructus samples and 22 adulterants. We designed specific DNA markers for Amomi Fructus based on the single nucleotide polymorphisms (SNPs) in the ITS. Amomi Fructus was well separated from the adulterants and was classified with the species of origin based on the detected SNPs from the DNA barcoding results. The AVF1/ISR DNA marker for A. villosum produced a 270 bases amplified product, while the ALF1/ISF DNA marker produced a 350 bases product specific for A. longiligulare. Using these DNA markers, the monitoring of commercially distributed Amomi Fructus was performed, and the monitoring results were confirmed by ITS analysis. This method identified samples that were from incorrect origins, and a new species of adulterant was also identified. These results confirmed the accuracy and efficiency of the designed DNA markers; this method may be used as an efficient tool for the identification and verification of Amomi Fructus.

Development of a DNA marker for variety discrimination specific to 'Manten-Kirari' based on an NGS-RNA sequence in Tartary buckwheat (Fagopyrum tataricum).

To discriminate the trace-rutinosidase variety of Tartary buckwheat 'Manten-Kirari', we developed DNA markers based on RNA polymorphism. Specifically, we mapped 17.76 GB RNA sequences, obtained using HiSeq2000, to create 11,358 large contigs constructed de novo from 'Manten-Kirari' RNA derived from GS-FLX+ titanium. From these, we developed eight DNA markers corresponding to single- to four-nucleotide polymorphisms between 'Manten-Kirari' and 'Hokkai T8', which is representative of normal rutinosidase content varieties in Japan. Using these markers, 'Manten-Kirari' was discriminated from 'Hokkai T8' by eight markers, from major Tartary buckwheat varieties by three markers, and from common buckwheats by two markers. We also performed direct PCR from flour and dried noodle made with 'Manten-Kirari' and 'Hokkai T8'. Based on the results, the DNA markers developed are promising for discriminating 'Manten-Kirari'. This is the first study to develop a DNA marker to discriminate varieties in the Polygonaceae family including buckwheat species.

Nuclear ribosomal DNA - ITS region based molecular marker to distinguish Gmelina arborea Roxb. Ex Sm. from its substitutes and adulterants.

Roots of Gmelina arborea (Gambhari) is a medicinally important raw drug traded in India. However, Gmelinaasiatica and Mallotus nudiflorus are also found in the raw drug markets as Gambhari. The current study aims to identify molecular markers based on the nuclear ribosomal DNA - ITS1 region to distinguish the authentic species from substitute/adulterants. The nuclear ribosomal internal transcribed spacer 1 (ITS1) was amplified to identify species-specific markers using universal primers. Based on the sequence of the ITS region, specific primers were designed for G. arborea, G. asiatica and M. nudiflorus which efficiently amplified 142 bp, 93 bp and 150 bp of the ITS1 region of the respective species. The notable feature of this molecular method is that it is technically accurate, practically convenient and suitable for analyzing large numbers of samples. This study demonstrates that the ITS1 region can be used for reliable authentication of medicinal plants and detection of adulterants and substitutes of Gambhari.

[DNA marker-assisted selection of medicinal plants (â ) .Breeding research of disease-resistant cultivars of Panax notoginseng].

DNA marker-assisted selection of medicinal plants is based on the DNA polymorphism, selects the DNA sequences related to the phenotypes such as high yields, superior quality, stress-resistance and so on according to the technologies of molecular hybridization, polymerase chain reaction and high-throughput sequencing, and assists the breeding of new cultivars. This study bred the first disease-resistant cultivar of notoginseng "Miaoxiang Kangqi 1" using the technology of DNA marker-assisted selection of medicinal plants and systematic breeding. The disease-resistant cultivar of notoginseng contained 12 special SNPs based on the analysis of Restriction-site Associated DNA Sequencing (RAD-Seq). Among the SNP (record_519688) was related to the root rot-resistant characteristics, which indicated this SNP could serve as genetic markers of disease-resistant cultivars and assist the systematic breeding. Compared to the conventional cultivated cultivars, the incidence rate of root-rot and rust-rot in notoginseng seedlings decreased by 83.6% and 71.8%, respectively. The incidence rate of root-rot respectively declined by 43.6% and 62.9% in notoginseng cultivation for 2 and 3 years compared with those of the conventional cultivated cultivars. Additionally, the potential disease-resistant groups were screened based on the relative SNP, and this model enlarged the target groups and advanced the breeding efficiency. DNA marker-assisted selection of medicinal plants accelerated the breeding and promotion of new cultivars, and guaranteed the healthy development of Chinese medicinal materials industry.

DNA marker-assisted selection of medicinal plants (Ⅰ) .Breeding research of disease-resistant cultivars of Panax notoginseng / 中国中药杂志

DNA marker-assisted selection of medicinal plants is based on the DNA polymorphism, selects the DNA sequences related to the phenotypes such as high yields, superior quality, stress-resistance and so on according to the technologies of molecular hybridization, polymerase chain reaction and high-throughput sequencing, and assists the breeding of new cultivars. This study bred the first disease-resistant cultivar of notoginseng "Miaoxiang Kangqi 1" using the technology of DNA marker-assisted selection of medicinal plants and systematic breeding. The disease-resistant cultivar of notoginseng contained 12 special SNPs based on the analysis of Restriction-site Associated DNA Sequencing (RAD-Seq). Among the SNP (record_519688) was related to the root rot-resistant characteristics, which indicated this SNP could serve as genetic markers of disease-resistant cultivars and assist the systematic breeding. Compared to the conventional cultivated cultivars, the incidence rate of root-rot and rust-rot in notoginseng seedlings decreased by 83.6% and 71.8%, respectively. The incidence rate of root-rot respectively declined by 43.6% and 62.9% in notoginseng cultivation for 2 and 3 years compared with those of the conventional cultivated cultivars. Additionally, the potential disease-resistant groups were screened based on the relative SNP, and this model enlarged the target groups and advanced the breeding efficiency. DNA marker-assisted selection of medicinal plants accelerated the breeding and promotion of new cultivars, and guaranteed the healthy development of Chinese medicinal materials industry.

Cytoplasmic male sterility (CMS) in hybrid breeding in field crops.

KEY MESSAGE: A comprehensive understanding of CMS/Rf system enabled by modern omics tools and technologies considerably improves our ability to harness hybrid technology for enhancing the productivity of field crops. Harnessing hybrid vigor or heterosis is a promising approach to tackle the current challenge of sustaining enhanced yield gains of field crops. In the context, cytoplasmic male sterility (CMS) owing to its heritable nature to manifest non-functional male gametophyte remains a cost-effective system to promote efficient hybrid seed production. The phenomenon of CMS stems from a complex interplay between maternally-inherited (mitochondrion) and bi-parental (nucleus) genomic elements. In recent years, attempts aimed to comprehend the sterility-inducing factors (orfs) and corresponding fertility determinants (Rf) in plants have greatly increased our access to candidate genomic segments and the cloned genes. To this end, novel insights obtained by applying state-of-the-art omics platforms have substantially enriched our understanding of cytoplasmic-nuclear communication. Concomitantly, molecular tools including DNA markers have been implicated in crop hybrid breeding in order to greatly expedite the progress. Here, we review the status of diverse sterility-inducing cytoplasms and associated Rf factors reported across different field crops along with exploring opportunities for integrating modern omics tools with CMS-based hybrid breeding.

Efficient callus formation and plant regeneration are heritable characters in sugar beet (Beta vulgaris L.).

BACKGROUND: Obtaining dedifferentiated cells (callus) that can regenerate into whole plants is not always feasible for many plant species. Sugar beet is known to be recalcitrant for dedifferentiation and plant regeneration. These difficulties were major obstacles for obtaining transgenic sugar beets through an Agrobacterium-mediated transformation procedure. The sugar beet line 'NK-219mm-O' is an exceptional line that forms callus efficiently and is easy to regenerate, but the inheritance of these characters was unknown. Another concern was whether these characters could coexist with an annual habitat that makes it possible to breed short life-cycle sugar beet suitable for molecular genetic analysis. FINDINGS: Five sugar beet lines including NK-219mm-O were crossed with each other and subjected to in vitro culture to form callus. F1s with a NK-219mm-O background generally formed callus efficiently compared to the others, indicating that efficient callus formation is heritable. The regeneration potential was examined based on the phenotypes of calli after placement on regeneration medium. Five phenotypes were observed, of which two phenotypes regenerated shoots or somatic embryo-like structures. Vascular differentiation was evident in regenerable calli, whereas non-regenerable calli lacked normally developed vascular tissues. In a half-diallel cross, the callus-formation efficiency and the regeneration potential of reciprocal F1s progeny having a NK-219mm-O background were high. Finally, we crossed NK-219mm-O with an annual line that had a poor in vitro performance. The callus-formation efficiency and the regeneration potential of reciprocal F1 were high. The regenerated plants showed an annual habitat. CONCLUSIONS: Efficient callus formation and the high plant regeneration potential of NK-219mm-O were inherited and expressed in the F1. The annual habitat does not impair these high in vitro performances.

Practical application of DNA markers for high-throughput authentication of Panax ginseng and Panax quinquefolius from commercial ginseng products.

Korean ginseng (Panax ginseng) and American ginseng (Panax quinquefolius) are widely used medicinal plants with similar morphology but different medicinal efficacy. Roots, flowers, and processed products of Korean and American ginseng can be difficult to differentiate from each other, leading to illegal trade in which one species is sold as the other. This study was carried out to develop convenient and reliable chloroplast genome-derived DNA markers for authentication of Korean and American ginseng in commercial processed products. One codominant marker could reproducibly identify both species and intentional mixtures of the two species. We further developed a set of species-unique dominant DNA markers. Each species-specific dominant marker could detect 1% cross contamination with other species by low resolution agarose gel electrophoresis or quantitative polymerase chain reaction. Both markers were successfully applied to evaluate the original species from various processed ginseng products purchased from markets in Korea and China. We believe that high-throughput application of this marker system will eradicate illegal trade and promote confident marketing for both species to increase the value of Korean as well as American ginseng in Korea and worldwide.

From crossbreeding to biotechnology-facilitated improvement of banana and plantain.

The annual harvest of banana and plantain (Musa spp.) is approximately 145 million tons worldwide. About 85% of this global production comes from small plots and kitchen or backyard gardens from the developing world, and only 15% goes to the export trade. Musa acuminata and Musa balbisiana are the ancestors of several hundreds of parthenocarpic Musa diploid and polyploid cultivars, which show multiple origins through inter- and intra-specific hybridizations from these two wild diploid species. Generating hybrids combining host plant resistance to pathogens and pests, short growth cycles and height, high fruit yield, parthenocarpy, and desired quality from the cultivars remains a challenge for Musa crossbreeding, which started about one century ago in Trinidad. The success of Musa crossbreeding depends on the production of true hybrid seeds in a crop known for its high levels of female sterility, particularly among polyploid cultivars. All banana export cultivars grown today are, however, selections from somatic mutants of the group Cavendish and have a very narrow genetic base, while smallholders in sub-Saharan Africa, tropical Asia and Latin America use some bred-hybrids (mostly cooking types). Musa improvement goals need to shift to address emerging threats because of the changing climate. Innovative cell and molecular biology tools have the potential to enhance the pace and efficiency of genetic improvement in Musa. Micro-propagation has been successful for high throughput of clean planting materials while in vitro seed germination assists in obtaining seedlings after inter-specific and across ploidy hybridization. Flow cytometry protocols are used for checking ploidy among genebank accessions and breeding materials. DNA markers, the genetic maps based on them, and the recent sequencing of the banana genome offer means for gaining more insights in the genetics of the crops and to identifying genes that could lead to accelerating Musa betterment. Likewise, DNA fingerprinting has been useful to characterize Musa diversity. Genetic engineering provides a complementary tool to Musa breeders who can introduce today transgenes that may confer resistance to bacteria, fungi and nematodes, or enhance pro-vitamin A fruit content. In spite of recent advances, the genetic improvement of Musa depends on a few crossbreeding programs (based in Brazil, Cameroon, Côte d'Ivoire, Guadeloupe, Honduras, India, Nigeria, Tanzania and Uganda) or a handful of genetic engineering endeavors (Australia, Belgium, India, Kenya, Malaysia and Uganda). Development investors (namely international aid and philanthropy) should therefore increase their funding to genetically enhance this crop that ranks among the 10-top staple foods of the developing world.

Key Techniques and Application Progress of Molecular Pharmacognosy / 中草药·英文版

At the boundary between pharmacognosy and molecular biology, molecular pharmacognosy has developed as a new borderline discipline. This paper reviews the methods, application, and prospect of molecular pharmacognosy. DNA marker is one of genetic markers and some molecular marker methods which have been successfully used for genetic diversity identification and new medicinal resources development. Recombinant DNA technology provides a powerful tool that enables scientists to engineer DNA sequences. Gene chip technique could be used in determination of gene expression profiles, analyses of polymorphisms, construction of genomic library, analysis of mapping, and sequencing by hybridization. Using the methods and theory of molecular biology and pharmacognosy, molecular pharmacognosy represents an extremely prospective branch of pharmacognosy and focuses on the study of systemic growth of medicinal plants, identification and evaluation of germplasm resources, plant metabolomics and production of active compounds. Furthermore, the great breakthrough of molecular pharmacognosy could be anticipated on DNA fingerprint analysis, cultivar improvement, DNA identification, and a global DNA barcoding system in the future.