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Background Crossostephium chinense is a traditional Chinese medicinal herb and it is often cultivated as an ornamental plant. Previous studies on this species mainly focused on its chemical composition and it was rarely represented in genetic studies, and thus genomic resources remain scarce. Methods and results Both chloroplast and nuclear polymorphic microsatellites of C. chinense were screened from genome skimming data of two individuals. 64 and 63 cpSSR markers were identified from two chloroplast genomes of C. chinense . A total of 133 polymorphic nSSRs were developed. Ten nSSRs were randomly selected to test their transferability across 35 individuals from three populations of C. chinense , and 20 individuals each of Artemisia stolonifera and A. argyi . Cross-amplifications were successfully done for C. chinense and were partially amplified for both Artemisia species. The number of alleles varied from two to nine. The observed heterozygosity and expected heterozygosity per locus ranged from 0.000 to 0.286 and from 0.029 to 0.755, respectively. Conclusions In this study, we developed polymorphic cpSSRs and nSSRs markers for C. chinense based on genome skimming sequencing. These genomic resources will be valuable for population genetics and conservation studies in C. chinense and Artemisia .

Rapid advancements in long-read sequencing technologies have transformed read lengths from bps to Mbps, which has enabled chromosome-scale genome assemblies. However, read lengths are now becoming limited by the extraction of pure high-molecular weight DNA suitable for long-read sequencing, which is particularly challenging in plants and fungi. To overcome this, we present a protocol collection; high-molecular weight DNA extraction, clean-up and size selection for long-read sequencing. We optimised a gentle magnetic bead based high-molecular weight DNA extraction, which is presented here in detail. The protocol circumvents spin columns and high-centrifugation, to limit DNA fragmentation. The protocol is scalable based on tissue input, which can be used on many species of plants, fungi, reptiles and bacteria. It is also cost effective compared to kit-based protocols and hence applicable at scale in low resource settings. An optional sorbitol wash is listed and is highly recommended for plant and fungal tissues. To further remove any remaining contaminants such as phenols and polysaccharides, optional DNA clean-up and size selection strategies are given. This protocol collection is suitable for all common long-read sequencing platforms, such as technologies offered by PacBio and Oxford Nanopore. Using these protocols, sequencing on the Oxford Nanopore MinION can achieve read length N50 values of 30–50 kb, with reads exceeding 200 kb and outputs ranging from 15–30 Gbp. This has been routinely achieved with various plant, fungi, animal and bacteria samples.

The laurel family within the Magnoliids has attracted attentions owing to its scents, variable inflorescences, and controversial phylogenetic position. Here, we present a chromosomelevel assembly of the Litsea cubeba genome, together with low-coverage genomic and transcriptomic data for many other Lauraceae. Phylogenomic analyses show phylogenetic discordance at the position of Magnoliids, suggesting incomplete lineage sorting during the divergence of monocots, eudicots, and Magnoliids. An ancient whole-genome duplication (WGD) event occurred just before the divergence of Laurales and Magnoliales; subsequently, independent WGDs occurred almost simultaneously in the three Lauralean lineages. The phylogenetic relationships within Lauraceae correspond to the divergence of inflorescences, as evidenced by the phylogeny of FUWA, a conserved gene involved in determining panicle architecture in Lauraceae. Monoterpene synthases responsible for production of specific volatile compounds in Lauraceae are functionally verified. Our work sheds light on the evolution of the Lauraceae, the genetic basis for floral evolution and specific scents.

DNA methylation is a ubiquitous chromatin feature, present in 25% of cytosines in the maize genome, but variation and evolution of the methylation landscape during maize domestication remain largely unknown. Here, we leverage whole-genome sequencing (WGS) and whole-genome bisulfite sequencing (WGBS) data on populations of modern maize, landrace, and teosinte ( Zea mays ssp. parviglumis) to estimate epimutation rates and selection coefficients. We find weak evidence for direct selection on DNA methylation in any context, but thousands of differentially methylated regions (DMRs) are identified population-wide that are correlated with recent selection. For two trait-associated DMRs, vgt1 -DMR and tb1 -DMR, HiChIP data indicate that the interactive loops between DMRs and respective downstream genes are present in B73, a modern maize line, but absent in teosinte. Our results enable a better understanding of the evolutionary forces acting on patterns of DNA methylation and suggest a role of methylation variation in adaptive evolution. Variation and evolution of DNA methylation during maize domestication remain largely unknown. Here, the authors generate genome and methylome sequencing data as well as HiChIP-based interactome data to investigate the adaptive and phenotypic consequences of methylation variations in maize.

DNA-based testing has been gaining acceptance as a tool for authentication of a wide range of food products; however, its applicability for testing of herbal supplements remains contentious. We utilized Sanger and Next-Generation Sequencing (NGS) for taxonomic authentication of fifteen herbal supplements representing three different producers from five medicinal plants: Echinacea purpurea, Valeriana officinalis, Ginkgo biloba, Hypericum perforatum and Trigonella foenum-graecum. Experimental design included three modifications of DNA extraction, two lysate dilutions, Internal Amplification Control, and multiple negative controls to exclude background contamination. Ginkgo supplements were also analyzed using HPLC-MS for the presence of active medicinal components. All supplements yielded DNA from multiple species, rendering Sanger sequencing results for rbcL and ITS2 regions either uninterpretable or non-reproducible between the experimental replicates. Overall, DNA from the manufacturer-listed medicinal plants was successfully detected in seven out of eight dry herb form supplements; however, low or poor DNA recovery due to degradation was observed in most plant extracts (none detected by Sanger; three out of seven-by NGS). NGS also revealed a diverse community of fungi, known to be associated with live plant material and/or the fermentation process used in the production of plant extracts. HPLC-MS testing demonstrated that Ginkgo supplements with degraded DNA contained ten key medicinal components. Quality control of herbal supplements should utilize a synergetic approach targeting both DNA and bioactive components, especially for standardized extracts with degraded DNA. The NGS workflow developed in this study enables reliable detection of plant and fungal DNA and can be utilized by manufacturers for quality assurance of raw plant materials, contamination control during the production process, and the final product. Interpretation of results should involve an interdisciplinary approach taking into account the processes involved in production of herbal supplements, as well as biocomplexity of plant-plant and plant-fungal biological interactions.

Herbarium collections are potentially an enormous resource for DNA studies, but the use of herbarium specimens in molecular studies has thus far been slowed down by difficulty in obtaining amplifiable DNA. Here we compare a set of commercially available DNA extraction protocols and their performance in terms of DNA purity and yield, and PCR amplification success as measured by using three differentially sized markers, the rbcL barcoding marker (cpDNA), the LEAFY exon 3 (nrDNA), and the trnL((UAA)) P6 loop (cpDNA). Results reveal large differences between extraction methods, where DNA purity rather than yield is shown to be strongly correlated with PCR success. Amplicon size shows similarly strong correlation with PCR success, with the shortest fragment showing the highest success rate (78%, P6 loop, 10-143 base pairs (bp)) and the largest fragment the lowest success (10%, rbcL, 670 bp). The effect of specimen preparation method on PCR success was also tested. Results show that drying method strongly affects PCR success, especially the availability of fragments longer than 250 bp, where longer fragments are more available for PCR amplification in air dried material compared to alcohol dried specimens. Results from our study indicate that projects relying on poor-quality starting material such as herbarium or scat samples should focus on extracting pure DNA and aim to amplify short target regions (<200-300 bp) in order to maximise outcomes. Development of shorter barcoding regions, or mini-barcodes within existing ones should be of high importance as only a few options are currently available; this is particularly important if we hope to incorporate the millions of herbarium samples available into barcoding initiatives and other molecular studies.

Transferable DNA markers are essential for breeding and genetics. Grapevine ( Vitis ) breeders utilize disease resistance alleles from congeneric species ~20 million years divergent, but existing Vitis marker platforms have cross-species transfer rates as low as 2%. Here, we apply a marker strategy targeting the inferred Vitis core genome. Incorporating seven linked-read de novo assemblies and three existing assemblies, the Vitis collinear core genome is estimated to converge at 39.8 Mb (8.67% of the genome). Adding shotgun genome sequences from 40 accessions enables identification of conserved core PCR primer binding sites flanking polymorphic haplotypes with high information content. From these target regions, we develop 2,000 rhAmpSeq markers as a PCR multiplex and validate the panel in four biparental populations spanning the diversity of the Vitis genus, showing transferability increases to 91.9%. This marker development strategy should be widely applicable for genetic studies in many taxa, particularly those ~20 million years divergent. Trait introgression requires universal markers, but cross-species transferability of current SNP markers can be as low as 2%. Here, the authors use an AmpSeq haplotype strategy targeting the collinear core genome for marker development and show transferability increases to 91.4% in the Vitis genus.

Bacteria and fungi form complex communities (microbiomes) in above- and below-ground organs of plants, contributing to hosts’ growth and survival in various ways. Recent studies have suggested that host plant genotypes control, at least partly, plant-associated microbiome compositions. However, we still have limited knowledge of how microbiome structures are determined in/on grafted crop plants, whose above-ground (scion) and below-ground (rootstock) genotypes are different with each other. By using eight varieties of grafted tomato plants, we examined how rootstock genotypes could determine the assembly of leaf endophytic microbes in field conditions. An Illumina sequencing analysis showed that both bacterial and fungal community structures did not significantly differ among tomato plants with different rootstock genotypes: rather, sampling positions in the farmland contributed to microbiome variation in a major way. Nonetheless, a further analysis targeting respective microbial taxa suggested that some bacteria and fungi could be preferentially associated with particular rootstock treatments. Specifically, a bacterium in the genus Deinococcus was found disproportionately from ungrafted tomato individuals. In addition, yeasts in the genus Hannaella occurred frequently on the tomato individuals whose rootstock genotype was “Ganbarune”. Overall, this study suggests to what extent leaf microbiome structures can be affected/unaffected by rootstock genotypes in grafted crop plants.

DNA extraction is a fundamental technique in molecular biology. For Chimonanthus praecox— a winter-flowering tree species — extensive and rapid DNA extraction is necessary to support genetic analyses. Currently, DNA extraction in C. praecox primarily relies on the traditional cetyltrimethylammonium bromide (CTAB) method, which is time-consuming and labor-intensive, hindering large-scale DNA extraction work. In this study, the different steps in the CTAB method are compared and evaluated to optimize the C. praecox leaf DNA extraction process. The water bath duration significantly impacts DNA extraction efficiency; the longer the water bath, the higher the DNA concentration. However, a 10-min water bath is sufficient to yield DNA of ideal concentration and purity. Additionally, a single extraction step is appropriate, with a 10-min precipitation at − 20 °C yielding high-quality DNA. Additionally, the pre-treatment step was modified by using a frozen pipette tip to crush samples directly in a centrifuge tube, reducing operational complexity and minimizing liquid nitrogen and sample consumption. Only 25 mg of sample is required, and high-quality DNA from C. praecox leaves can be extracted within 1 h. The amounts of required sample and liquid nitrogen were reduced by 75% and 90%, respectively. Moreover, the time required for the simplified extraction step was reduced by 77.14%. The applicability of the simplified scheme was evaluated using different C. praecox tissues, Calycanthaceae family members, and species of other families. The simplified scheme extracted DNA from the tepals and leaves of C. praecox with higher concentration purity. However, this protocol was biased toward the Chimonanthus family, Nicotiana tabacum , Populus tomentosa , and Lilium brownii . The proposed method enables the rapid and efficient extraction of high-quality DNA from 25 mg of plant leaves and is suitable for multiple species. This method reduces sample and liquid nitrogen consumption, lowering costs while significantly shortening the procedure time and enhancing extraction efficiency. This method is highly suitable for applications involving the extraction of large amounts of low-concentration DNA across various plant species.

Plants and their associated insect herbivores, represent more than 50% of all known species on earth. The first step in understanding the mechanisms generating and maintaining this important component of biodiversity is to identify plant-herbivore associations. In this study we determined insect-host plant associations for an entire guild of insect herbivores using plant DNA extracted from insect gut contents. Over two years, in a tropical rain forest in Costa Rica (La Selva Biological Station), we recorded the full diet breadth of rolled-leaf beetles, a group of herbivores that feed on plants in the order Zingiberales. Field observations were used to determine the accuracy of diet identifications using a three-locus DNA barcode (rbcL, trnH-psbA and ITS2). Using extraction techniques for ancient DNA, we obtained high-quality sequences for two of these loci from gut contents (rbcL and ITS2). Sequences were then compared to a comprehensive DNA barcode library of the Zingiberales. The rbcL locus identified host plants to family (success/sequence = 58.8%) and genus (success/sequence = 47%). For all Zingiberales except Heliconiaceae, ITS2 successfully identified host plants to genus (success/sequence = 67.1%) and species (success/sequence = 61.6%). Kindt's sampling estimates suggest that by collecting ca. four individuals representing each plant-herbivore interaction, 99% of all host associations included in this study can be identified to genus. For plants that amplified ITS2, 99% of the hosts can be identified to species after collecting at least four individuals representing each interaction. Our study demonstrates that host plant identifications at the species-level using DNA barcodes are feasible, cost-effective, and reliable, and that reconstructing plant-herbivore networks with these methods will become the standard for a detailed understanding of these interactions.