Characteristics of chloroplast and mitochondrial genomes and intracellular gene transfer in the Korean endemic shrub, Sophora koreensis Nakai (Fabaceae).

Sophora koreensis Nakai, an endemic species distributed only in the Korean Peninsula, is of great geographical, economic, and taxonomic importance. Although its complete chloroplast (cp) genome sequence has been reported, its mitochondrial (mt) genome sequence has not yet been studied. Therefore, in this study, we aimed to investigate its mt genome sequence and compare it with those reported for other Fabaceae species. Total genomic DNA was extracted from fresh S. koreensis leaves collected from natural habitats in Gangwon-do Province, South Korea. This was followed by polymerase chain reaction (PCR) amplification of cpDNA insertions in the mt genome and the detection of microsatellites and dispersed repeats in the cp and mt genomes. Finally, the cp and mt genomes of S. koreensis were compared with those reported for other Fabaceae species. The cp sequence of S. koreensis showed identical gene orders and contents as those previously reported. Only six substitutions and one deletion were detected with 99 % homology. Conversely, the complete mt genome sequence, which was 517,845 bp in length and encoded 61 genes, including 43 protein-coding, 15 transfer RNAs, and 3 ribosomal RNA genes, was considerably different from that of S. japonica in terms of gene order and composition. Further, the mt genome of S. koreensis included ca. 7 and 3 kb insertions, representing an intracellular gene transfer (IGT) event, and the regions with these insertions were determined to be originally present in the cp genome. This IGT event was also confirmed via PCR amplification. IGT events can be induced via biological gene expression control or the use of repetitive sequences, and they provide important insights into the evolutionary lineage of S. koreensis. However, further studies are needed to clarify the gene transfer mechanisms between the two organelles.

Sophora genomes provide insight into the evolution of alkaloid metabolites along with small-scale gene duplication.

The genus Sophora (Fabaceae) includes medicinal plants that have been used in East Asian countries since antiquity. Sophora flavescens is a perennial herb indigenous to China, India, Japan, Korea, and Russia. Its dried roots have antioxidant, anti-inflammatory, antibacterial, apoptosis-modulating, and antitumor efficacy. The congeneric S. koreensis is endemic to Korea and its genome is less than half the size of that of S. flavescens. Nevertheless, this discrepancy can be used to assemble and validate the S. flavescens genome. A comparative genomic study of the two genomes can disclose the recent evolutionary divergence of the polymorphic phenotypic profiles of these species. Here, we used the PacBio sequencing platform to sequence and assemble the S. koreensis and S. flavescens genomes. We inferred that it was mainly small-scale duplication that occurred in S. flavescens. A KEGG analysis revealed pathways that might regulate the pharmacologically important secondary metabolites in S. flavescens and S. koreensis. The genome assemblies of Sophora spp. could be used in comparative genomics and data mining for various plant natural products.

Phylogeny, biogeography, and character evolution of the genus Sophora s.l. (Fabaceae, Papilionoideae).

The papilionoid legume genus Sophora (Fabaceae) exhibits a worldwide distribution, but a phylogenetic framework to understand the evolution of this group is lacking to date. Previous studies have demonstrated that Sophora is not monophyletic and might include Ammodendron, Ammothamnus, and Echinosophora, but the relationships among these four genera (defined as Sophora s.l.) are unclear. Here we used a nuclear DNA dataset (ETS, ITS, SQD1) and a plastid DNA dataset (matK, rbcL, rpl32-trnL, trnL-F) of 654 accession sequences to reconstruct the phylogenetic relationships, estimate the divergence times and ancestral range of Sophora s.l., and infer the evolution of chromosome number and morphological characteristics. Our major aim was to reconstruct phylogenetic relationships to test monophyly and elucidate relationships within the genus. Our results indicated that Ammodendron, Ammothamnus, and Echinosophora are embedded within Sophora s.s. and that nine well-supported clades can be recognized within comprise Sophora s.l. Ancestral character state estimation revealed that the most recent common ancestor of Sophora s.l. was a deciduous shrub that lacks rhizome spines and has unwinged legumes. Divergence times estimation and ancestral area reconstruction showed that Sophora s.l. originated in Central Asia and/or adjacent Southeast China in the early Oligocene (ca. 31 Mya) and dispersed from these regions into East and South Asia's adjacent areas and North America via the Bering land bridge. The analyses also supported a South American origin for S. sect. Edwardsia, which experienced rapid radiation with its major lineages diversifying over a relatively narrow timescale (8 Mya).

[N-terminal truncation of prenyltransferase enhances the biosynthesis of prenylnaringenin].

8-prenylnaringenin (8-PN) is a potent estrogen with high medicinal values. It also serves as an important precursor for many prenylated flavonoids. Microbial synthesis of 8-PN is mainly hindered by the low catalytic activity of prenyltransferases (PTS) and insufficient supply of precursors. In this work, a SfN8DT-1 from Sophora flavescens was used to improve the efficiency of (2S)-naringenin prenylation. The predicted structure of SfN8DT-1 showed that its main body is comprised of 9 α-helices and 8 loops, along with a long side chain formed by nearly 120 amino acids. SfN8DT-1 mutants with different side-chain truncated were tested in Saccharomyces cerevisiae. A mutant expressing the truncated enzyme at K62 site, designated as SfND8T-1-t62, produced the highest 8-PN titer. Molecular docking of SfN8DT-1-t62 with (2S)-naringenin and dimethylallyl diphosphate (DMAPP) showed that K185 was a potentially crucial residue. Alanine scanning within a range of 0.5 nm around these two substrates showed that the mutant K185A may decrease its affinity to substrates, which also indicated K185 was a potentially critical residue. Besides, the mutant K185W enhanced the affinity to ligands implied by the simulated saturation mutation, while the saturated mutation of K185 showed a great decrease in 8-PN production, indicating K185 is vital for the activity of SfN8DT-1. Subsequently, overexpressing the key genes of Mevalonate (MVA) pathway further improved the titer of 8-PN to 31.31 mg/L, which indicated that DMAPP supply is also a limiting factor for 8-PN synthesis. Finally, 44.92 mg/L of 8-PN was produced in a 5 L bioreactor after 120 h, which is the highest 8-PN titer reported to date.

Chromosome-level genome assembly and characterization of Sophora Japonica.

Sophora japonica is a medium-size deciduous tree belonging to Leguminosae family and famous for its high ecological, economic and medicinal value. Here, we reveal a draft genome of S. japonica, which was ∼511.49 Mb long (contig N50 size of 17.34 Mb) based on Illumina, Nanopore and Hi-C data. We reliably assembled 110 contigs into 14 chromosomes, representing 91.62% of the total genome, with an improved N50 size of 31.32 Mb based on Hi-C data. Further investigation identified 271.76 Mb (53.13%) of repetitive sequences and 31,000 protein-coding genes, of which 30,721 (99.1%) were functionally annotated. Phylogenetic analysis indicates that S. japonica separated from Arabidopsis thaliana and Glycine max ∼107.53 and 61.24 million years ago, respectively. We detected evidence of species-specific and common-legume whole-genome duplication events in S. japonica. We further found that multiple TF families (e.g. BBX and PAL) have expanded in S. japonica, which might have led to its enhanced tolerance to abiotic stress. In addition, S. japonica harbours more genes involved in the lignin and cellulose biosynthesis pathways than the other two species. Finally, population genomic analyses revealed no obvious differentiation among geographical groups and the effective population size continuously declined since 2 Ma. Our genomic data provide a powerful comparative framework to study the adaptation, evolution and active ingredients biosynthesis in S. japonica. More importantly, our high-quality S. japonica genome is important for elucidating the biosynthesis of its main bioactive components, and improving its production and/or processing.

Morphological, transcriptomic and metabolomic analyses of Sophora davidii mutants for plant height.

Sophora davidii is an important plant resource in the karst region of Southwest China, but S. davidii plant-height mutants are rarely reported. Therefore, we performed phenotypic, anatomic structural, transcriptomic and metabolomic analyses to study the mechanisms responsible for S. davidii plant-height mutants. Phenotypic and anatomical observations showed that compared to the wild type, the dwarf mutant displayed a significant decrease in plant height, while the tall mutant displayed a significant increase in plant height. The dwarf mutant cells were smaller and more densely arranged, while those of the wild type and the tall mutant were larger and loosely arranged. Transcriptomic analysis revealed that differentially expressed genes (DEGs) involved in cell wall biosynthesis, expansion, phytohormone biosynthesis, signal transduction pathways, flavonoid biosynthesis and phenylpropanoid biosynthesis were significantly enriched in the S. davidii plant-height mutants. Metabolomic analysis revealed 57 significantly differential metabolites screened from both the dwarf and tall mutants. A total of 8 significantly different flavonoid compounds were annotated to LIPID MAPS, and three metabolites (chlorogenic acid, kaempferol and scopoletin) were involved in phenylpropanoid biosynthesis and flavonoid biosynthesis. These results shed light on the molecular mechanisms of plant height in S. davidii mutants and provide insight for further molecular breeding programs.

TMT based proteomic profiling of Sophora alopecuroides leaves reveal flavonoid biosynthesis processes in response to salt stress.

Salt stress is the major abiotic stress worldwide, adversely affecting crop yield and quality. Utilizing salt tolerance genes for the genetic breeding of crops is one of the most effective measures to withstand salinization. Sophora alopecuroides is a well-known saline-alkaline and drought-tolerant medicinal plant. Understanding the underlying molecular mechanism for Sophora alopecuroides salt tolerance is crucial to identifying the salt-tolerant genes. In this study, we performed tandem mass tag (TMT) based proteomic profiling of S. alopecuroides leaves under 150 mM NaCl induced salt stress condition for 3 d and 7 d. Data are available on ProteomeXchange (PXD027627). Furthermore, the proteomic findings were validated through parallel reaction monitoring (PRM). We observed that the expression levels of several transporter proteins related to the secondary messenger signaling pathway were altered under salt stress conditions induced for 3 d. However, the expression of the certain transferase, oxidoreductase, dehydrogenase, which are involved in the biosynthesis of flavonoids, alkaloids, phenylpropanoids, and amino acid metabolism, were mainly alerted after 7 d post-salt-stress induction. Several potential genes that might be involved in salt stress conditions were identified; however, it demands further investigation. Although salt stress affects the level of secondary metabolites, their correlation needs to be investigated further. SIGNIFICANCE: Salinization is the most severe abiotic adversity, which has had a significant negative effect on world food security over the time. Excavating salt-tolerant genes from halophytes or medicinal plants is one of the important measures to cope with salt stress. S. alopecuroides is a well-known medicinal plant with anti-tumor, anti-inflammatory, and antibacterial effects, anti-saline properties, and resistance to drought stress. Currently, only a few studies have explored the S. alopecuroides' gene function, and regulation and these studies are mostly related to the unpublished genome sequence information of S. alopecuroides. Recently, transcriptomics and metabolomics studies have been carried on the abiotic stress in S. alopecuroides roots. Multiple studies have shown that altered gene expression at the transcript level and altered metabolite levels do not correspond to the altered protein levels. In this study, TMT and PRM based proteomic analyses of S. alopecuroides leaves under salt stress condition induced using 150 mM NaCl for 3 d and 7 d was performed. These analyses elucidated the activation of different mechanisms in response to salt stress. A total of 434 differentially abundant proteins (DAPs) in salt stress conditions were identified and analyzed. For the first time, this study utilized proteomics technology to dig out plentiful underlying salt-tolerant genes from the medicinal plant, S. alopecuroides. We believe that this study will be of great significance to crop genetics and breeding.

Integrated transcriptome and small RNA sequencing analyses reveal a drought stress response network in Sophora tonkinensis.

BACKGROUND: Sophora tonkinensis Gagnep is a traditional Chinese medical plant that is mainly cultivated in southern China. Drought stress is one of the major abiotic stresses that negatively impacts S. tonkinensis growth. However, the molecular mechanisms governing the responses to drought stress in S. tonkinensis at the transcriptional and posttranscriptional levels are not well understood. RESULTS: To identify genes and miRNAs involved in drought stress responses in S. tonkinensis, both mRNA and small RNA sequencing was performed in root samples under control, mild drought, and severe drought conditions. mRNA sequencing revealed 66,476 unigenes, and the differentially expressed unigenes (DEGs) were associated with several key pathways, including phenylpropanoid biosynthesis, sugar metabolism, and quinolizidine alkaloid biosynthesis pathways. A total of 10 and 30 transcription factors (TFs) were identified among the DEGs under mild and severe drought stress, respectively. Moreover, small RNA sequencing revealed a total of 368 miRNAs, including 255 known miRNAs and 113 novel miRNAs. The differentially expressed miRNAs and their target genes were involved in the regulation of plant hormone signal transduction, the spliceosome, and ribosomes. Analysis of the regulatory network involved in the response to drought stress revealed 37 differentially expressed miRNA-mRNA pairs. CONCLUSION: This is the first study to simultaneously profile the expression patterns of mRNAs and miRNAs on a genome-wide scale to elucidate the molecular mechanisms of the drought stress responses of S. tonkinensis. Our results suggest that S. tonkinensis implements diverse mechanisms to modulate its responses to drought stress.

Screening and identification of salt-tolerance genes in Sophora alopecuroides and functional verification of SaAQP.

MAIN CONCLUSION: Overexpression of SaAQP can improve the salt tolerance of transgenic soybean hairy roots and A. thaliana. Salt stress severely affects crop yield and food security. There is a need to improve the salt tolerance of crops, but the discovery and utilization of salt-tolerance genes remains limited. Owing to its strong stress tolerance, Sophora alopecuroides is ideal for the identification of salt-tolerance genes. Therefore, we aimed to screen and identify the salt-tolerance genes in S. alopecuroides. With a yeast expression library of seedlings, salt-tolerant genes were screened using a salt-containing medium to simulate salt stress. By combining salt-treatment screening and transcriptome sequencing, 11 candidate genes related to salt tolerance were identified, including genes for peroxidase, inositol methyltransferase, aquaporin, cysteine synthase, pectinesterase, and WRKY. The expression dynamics of candidate genes were analyzed after salt treatment of S. alopecuroides, and salt tolerance was verified in yeast BY4743. The candidate genes participated in the salt-stress response in S. alopecuroides, and their overexpression significantly improved the salt tolerance of yeast. Salt tolerance mediated by SaAQP was further verified in soybean hairy roots and Arabidopsis thaliana, and it was found that SaAQP might enhance the salt tolerance of A. thaliana by participating in a reactive oxygen species scavenging mechanism. This result provides new genetic resources in plant breeding for salt resistance.

Analysis of Phytohormone Signal Transduction in Sophora alopecuroides under Salt Stress.

Salt stress seriously restricts crop yield and quality, leading to an urgent need to understand its effects on plants and the mechanism of plant responses. Although phytohormones are crucial for plant responses to salt stress, the role of phytohormone signal transduction in the salt stress responses of stress-resistant species such as Sophora alopecuroides has not been reported. Herein, we combined transcriptome and metabolome analyses to evaluate expression changes of key genes and metabolites associated with plant hormone signal transduction in S. alopecuroides roots under salt stress for 0 h to 72 h. Auxin, cytokinin, brassinosteroid, and gibberellin signals were predominantly involved in regulating S. alopecuroides growth and recovery under salt stress. Ethylene and jasmonic acid signals may negatively regulate the response of S. alopecuroides to salt stress. Abscisic acid and salicylic acid are significantly upregulated under salt stress, and their signals may positively regulate the plant response to salt stress. Additionally, salicylic acid (SA) might regulate the balance between plant growth and resistance by preventing reduction in growth-promoting hormones and maintaining high levels of abscisic acid (ABA). This study provides insight into the mechanism of salt stress response in S. alopecuroides and the corresponding role of plant hormones, which is beneficial for crop resistance breeding.

Combined Transcriptomic and Metabolomic Analysis Reveals the Role of Phenylpropanoid Biosynthesis Pathway in the Salt Tolerance Process of Sophora alopecuroides.

Salt stress is the main abiotic stress that limits crop yield and agricultural development. Therefore, it is imperative to study the effects of salt stress on plants and the mechanisms through which plants respond to salt stress. In this study, we used transcriptomics and metabolomics to explore the effects of salt stress on Sophora alopecuroides. We found that salt stress incurred significant gene expression and metabolite changes at 0, 4, 24, 48, and 72 h. The integrated transcriptomic and metabolomic analysis revealed that the differentially expressed genes (DEGs) and differential metabolites (DMs) obtained in the phenylpropanoid biosynthesis pathway were significantly correlated under salt stress. Of these, 28 DEGs and seven DMs were involved in lignin synthesis and 23 DEGs and seven DMs were involved in flavonoid synthesis. Under salt stress, the expression of genes and metabolites related to lignin and flavonoid synthesis changed significantly. Lignin and flavonoids may participate in the removal of reactive oxygen species (ROS) in the root tissue of S. alopecuroides and reduced the damage caused under salt stress. Our research provides new ideas and genetic resources to study the mechanism of plant responses to salt stress and further improve the salt tolerance of plants.

[Association analysis of phenotypic traits of alkaloid content in Sophora alopecuroides with SSR molecular markers].

To further study and fully exploit the medicinal plant Sophora alopecuroides, the molecular markers related with the phenotypic traits of alkaloid content in S. alopecuroides should be detected. In this study, SSR molecular markers were used to analyze the genetic diversity and genetic structure of 23 S. alopecuroides populations, in combination with the association analysis between molecular markers and the alkaloid contents. The results showed that P, H, I, G_(st) and N_m values were 40.10%, 0.335 3, 0.504 5, 0.433 7 and 0.625 9 respectively, in 23 S. alopecuroides populations. This indicated that there was less gene exchange and higher genetic differentiation among different S. alopecuroides populations. The results of SSR unweighted pair-group method with arithmetic means(UPGMA) cluster showed that the S. alopecuroides populations relationship from Xinjiang was far from the populations of other regions, but the populations of S. alopecuroides from Gansu, Inner Mongolia and Qinghai were closely relevant to those from Ningxia. The 23 populations were further divided into 2 genetic subpopulations by the population structure analysis. Through association analysis, a total of 26 loci in 13 SSR markers were found to be significantly associated(P<0.005)with the content of MA, OMA, SC and OSC, and the rate of explanation on the phenotype variance of related markers ranged from 36.45% to 77.93%. Among the locus, 1 each were related with MA and OSC content at interpretation rate reached as high as 50% with high threshold(P<0.000 1). These results could provide support for the discovery of important genes in the alkaloid biosynthetic and metabolic pathway of S. alopecuroides.

The complete chloroplast genome sequence of the medicinal plant Sophora tonkinensis.

Sophora tonkinensis belongs to genus Sophora of the Fabaceae family. It is mainly distributed in the ridge and peak regions of limestone areas in western China and has high medicinal value and important ecological functions. Wild populations of S. tonkinensis are in danger and need urgent conservation. Furthermore, wild S. tonkinensis resources are very limited relative to the needs of the market, and many adulterants are present on the market. Therefore, a method for authenticating S. tonkinensis and its adulterants at the molecular level is needed. Chloroplast genomes are valuable sources of genetic markers for phylogenetic analyses, genetic diversity evaluation, and plant molecular identification. In this study, we report the complete chloroplast genome of S. tonkinensis. The circular complete chloroplast genome was 154,644 bp in length, containing an 85,810 bp long single-copy (LSC) region, an 18,321 bp short single-copy (SSC) region and two inverted repeat (IR) regions of 50,513 bp. The S. tonkinensis chloroplast genome comprised 129 genes, including 83 protein-coding genes, 38 transfer RNA (tRNA) genes, and 8 ribosomal RNA (rRNA) genes. The structure, gene order and guanine and cytosine (GC) content of the S. tonkinensis chloroplast genome were similar to those of the Sophora alopecuroides and Sophora flavescens chloroplast genomes. A total of 1,760 simple sequence repeats (SSRs) were identified in the chloroplast genome of S. tonkinensis, and most of them (93.1%) were mononucleotides. Moreover, the identified SSRs were mainly distributed in the LSC region, accounting for 60% of the total number of SSRs, while 316 (18%) and 383 (22%) were located in the SSC and IR regions, respectively. Only one complete copy of the rpl2 gene was present at the LSC/IRB boundary, while another copy was absent from the IRA region because of the incomplete structure caused by IR region expansion and contraction. The phylogenetic analysis placed S. tonkinensis in Papilionoideae, sister to S. flavescens, and the genera Sophora and Ammopiptanthus were closely related. The complete genome sequencing and chloroplast genome comparative analysis of S. tonkinensis and its closely related species presented in this paper will help formulate effective conservation and management strategies as well as molecular identification approaches for this important medicinal plant.

De novo transcriptome sequencing and analysis of salt-, alkali-, and drought-responsive genes in Sophora alopecuroides.

BACKGROUND: Salinity, alkalinity, and drought stress are the main abiotic stress factors affecting plant growth and development. Sophora alopecuroides L., a perennial leguminous herb in the genus Sophora, is a highly salt-tolerant sand-fixing pioneer species distributed mostly in Western Asia and northwestern China. Few studies have assessed responses to abiotic stress in S. alopecuroides. The transcriptome of the genes that confer stress-tolerance in this species has not previously been sequenced. Our objective was to sequence and analyze this transcriptome. RESULTS: Twelve cDNA libraries were constructed in triplicate from mRNA obtained from Sophora alopecuroides for the control and salt, alkali, and drought treatments. Using de novo assembly, 902,812 assembled unigenes were generated, with an average length of 294 bp. Based on similarity searches, 545,615 (60.43%) had at least one significant match in the Nr, Nt, Pfam, KOG/COG, Swiss-Prot, and GO databases. In addition, 1673 differentially expressed genes (DEGs) were obtained from the salt treatment, 8142 from the alkali treatment, and 17,479 from the drought treatment. A total of 11,936 transcription factor genes from 82 transcription factor families were functionally annotated under salt, alkali, and drought stress, these include MYB, bZIP, NAC and WRKY family members. DEGs were involved in the hormone signal transduction pathway, biosynthesis of secondary metabolites and antioxidant enzymes; this suggests that these pathways or processes may be involved in tolerance towards salt, alkali, and drought stress in S. alopecuroides. CONCLUSION: Our study first reported transcriptome reference sequence data in Sophora alopecuroides, a non-model plant without a reference genome. We determined digital expression profile and discovered a broad survey of unigenes associated with salt, alkali, and drought stress which provide genomic resources available for Sophora alopecuroides.

Complete chloroplast genome of Sophora alopecuroides (Papilionoideae): molecular structures, comparative genome analysis and phylogenetic analysis.

Sophora alopecuroides belongs to the genus Sophora of the family Papilionoideae. It is mainly distributed in the desert and semidesert areas of northern China, and has high medicinal value and ecological function. Previous studies have reported the chemical composition and ecological functions of S. alopecuroides. However, only a few reports are available on the genomic information of S. alopecuroides, especially the chloroplast genome, which greatly limits the study of the evolutionary relationship between other species of Papilionoideae. Here, we report the complete chloroplast genome of S. alopecuroides. The size of the chloroplast genome is 155,207 bp, and the GC content is 36.44%. The S. alopecuroides chloroplast genome consists of 132 genes, including 83 protein-coding genes, 41 transfer RNA (tRNA) genes,and eight ribosomal RNA (rRNA) genes. Phylogenetic analysis revealed the taxonomic position of S. alopecuroides in Papilionoideae, and the genus Sophora and the genus Ammopiptanthus were highly related. Comparative genomics analysis revealed the gene rearrangement in the evolution of S. alopecuroides. The comparison between S. alopecuroides and the species of the Papilionoideae identified a novel 23 kb inversion between the trnC-GCA and trnF-GAA which occurred before the divergence of Sophora and Ammopiptanthus of Thermopsideae. This study provided an essential data for the understanding of phylogenetic status of S. alopecuroides.

The genetic diversity and population structure of Sophora alopecuroides (Faboideae) as determined by microsatellite markers developed from transcriptome.

Sophora alopecuroides (Faboideae) is an endemic species, mainly distributed in northwest China. However, the limited molecular markers range for this species hinders breeding and genetic studies. A total of 20,324 simple sequence repeat (SSR) markers were identified from 118,197 assembled transcripts and 18 highly polymorphic SSR markers were used to explore the genetic diversity and population structure of S. alopecuroides from 23 different geographical populations. A relatively low genetic diversity was found in S. alopecuroides based on mean values of the number of effective alleles (Ne = 1.81), expected heterozygosity (He = 0.39) and observed heterozygosity (Ho = 0.55). The results of AMOVA indicated higher levels of variation within populations than between populations. Bayesian-based cluster analysis, principal coordinates analysis and Neighbor-Joining phylogeny analysis roughly divided all genotypes into four major groups with some admixtures. Meanwhile, geographic barriers would have restricted gene flow between the northern and southern regions (separated by Tianshan Mountains), wherein the two relatively ancestral and independent clusters of S. alopecuroides occur. History trade and migration along the Silk Road would together have promoted the spread of S. alopecuroides from the western to the eastern regions of the northwest plateau in China, resulting in the current genetic diversity and population structure. The transcriptomic SSR markers provide a valuable resource for understanding the genetic diversity and population structure of S. alopecuroides, and will assist effective conservation management.

Assembly and comparative analysis of the complete mitochondrial genome sequence of Sophora japonica 'JinhuaiJ2'.

Sophora japonica L. (Faboideae, Leguminosae) is an important traditional Chinese herb with a long history of cultivation. Its flower buds and fruits contain abundant flavonoids, and therefore, the plants are cultivated for the industrial extraction of rutin. Here, we determined the complete nucleotide sequence of the mitochondrial genome of S. japonica 'JinhuaiJ2', the most widely planted variety in Guangxi region of China. The total length of the mtDNA sequence is 484,916 bp, with a GC content of 45.4%. Sophora japonica mtDNA harbors 32 known protein-coding genes, 17 tRNA genes, and three rRNA genes with 17 cis-spliced and five trans-spliced introns disrupting eight protein-coding genes. The gene coding and intron regions, and intergenic spacers account for 7.5%, 5.8% and 86.7% of the genome, respectively. The gene profile of S. japonica mitogenome differs from that of the other Faboideae species by only one or two gene gains or losses. Four of the 17 cis-spliced introns showed distinct length variations in the Faboideae, which could be attributed to the homologous recombination of the short repeats measuring a few bases located precisely at the edges of the putative deletions. This reflects the importance of small repeats in the sequence evolution in Faboideae mitogenomes. Repeated sequences of S. japonica mitogenome are mainly composed of small repeats, with only 20 medium-sized repeats, and one large repeat, adding up to 4% of its mitogenome length. Among the 25 pseudogene fragments detected in the intergenic spacer regions, the two largest ones and their corresponding functional gene copies located in two different sets of medium-sized repeats, point to their origins from homologous recombinations. As we further observed the recombined reads associated with the longest repeats of 2,160 bp with the PacBio long read data set of just 15 × in depth, repeat mediated homologous recombinations may play important role in the mitogenomic evolution of S. japonica. Our study provides insightful knowledge to the genetic background of this important herb species and the mitogenomic evolution in the Faboideae species.

Rapid Authentication of Ginkgo biloba Herbal Products Using the Recombinase Polymerase Amplification Assay.

Species adulteration in herbal products (HPs) exposes consumers to health risks. Chemical and morphological methods have their own deficiencies when dealing with the detection of species containing the same active compounds in HPs. In this study, we developed a rapid identification method using the recombinase polymerase amplification (RPA) assay to detect two species, Ginkgo biloba and Sophora japonica (as adulteration), in Ginkgo biloba HPs. Among 36 Ginkgo biloba HP samples, 34 were found to have Ginkgo biloba sequences, and 9 were found to have Sophora japonica sequences. During the authentication process, the RPA-LFS assay showed a higher specificity, sensitivity and efficiency than PCR-based methods. We initially applied the RPA-LSF technique to detect plant species in HPs, demonstrating that this assay can be developed into an efficient tool for the rapid on-site authentication of plant species in Ginkgo biloba HPs.

Profiling of the Major Phenolic Compounds and Their Biosynthesis Genes in Sophora flavescens Aiton.

Sophorae Radix (Sophora flavescens Aiton) has long been used in traditional medicine in East Asia due to the various biological activities of its secondary metabolites. Endogenous contents of phenolic compounds (phenolic acid, flavonol, and isoflavone) and the main bioactive compounds of Sophorae Radix were analyzed based on the qualitative HPLC analysis and evaluated in different organs and at different developmental stages. In total, 11 compounds were detected, and the composition of the roots and aerial parts (leaves, stems, and flowers) was significantly different. trans-Cinnamic acid and p-coumaric acid were observed only in the aerial parts. Large amounts of rutin and maackiain were detected in the roots. Four phenolic acid compounds (benzoic acid, caffeic acid, ferulic acid, and chlorogenic acid) and four flavonol compounds (kaempferol, catechin hydrate, epicatechin, and rutin) were higher in aerial parts than in roots. To identify putative genes involved in phenolic compounds biosynthesis, a total of 41 transcripts were investigated. Expression patterns of these selected genes, as well as the multiple isoforms for the genes, varied by organ and developmental stage, implying that they are involved in the biosynthesis of various phenolic compounds both spatially and temporally.