Genome-wide identification and expression pattern analysis of the GRF transcription factor family in Astragalus mongholicus.

BACKGROUND: Astragalus membranaceus is a plant of the Astragalus genus, which is used as a traditional Chinese herbal medicine with extremely high medicinal and edible value. Astragalus mongholicus, as one of the representative medicinal materials with the same origin of medicine and food, has a rising market demand for its raw materials, but the quality is different in different production areas. Growth-regulating factors (GRF) are transcription factors unique to plants that play important roles in plant growth and development. Up to now, there is no report about GRF in A. mongholicus. METHODS AND RESULTS: This study conducted a genome-wide analysis of the AmGRF gene family, identifying a total of nine AmGRF genes that were classified into subfamily V based on phylogenetic relationships. In the promoter region of the AmGRF gene, we successfully predicted cis-elements that respond to abiotic stress, growth, development, and hormone production in plants. Based on transcriptomic data and real-time quantitative polymerase chain reaction (qPCR) validation, the results showed that AmGRFs were expressed in the roots, stems, and leaves, with overall higher expression in leaves, higher expression of AmGRF1 and AmGRF8 in roots, and high expression levels of AmGRF1 and AmGRF9 in stems. CONCLUSIONS: The results of this study provide a theoretical basis for the further exploration of the functions of AmGRFs in plant growth and development.

Genome-wide identification of YABBY gene family and its expression pattern analysis in Astragalus mongholicus.

During plant growth and development, the YABBY gene plays a crucial role in the morphological structure, hormone signaling, stress resistance, crop breeding, and agricultural production of plant lateral organs, leaves, flowers, and fruits. Astragalus mongholicus is a perennial herbaceous plant in the legume family, widely used worldwide due to its high medicinal and edible value. However, there have been no reports of the YABBY gene family in A. mongholicus. This study used bioinformatics methods, combined with databases and analysis websites, to systematically analyze the AmYABBY gene family in the entire genome of A. mongholicus and verified its expression patterns in different tissues of A. mongholicus through transcriptome data and qRT-PCR experiments. A total of seven AmYABBY genes were identified, which can be divided into five subfamilies and distributed on three chromosomes. Two pairs of AmYABBY genes may be involved in fragment duplication on three chromosomes. All AmYABBY proteins have a zinc finger YABBY domain, and members of the same group have similar motif composition and intron - exon structure. In the promoter region of the genes, light-responsive and MeJa-response cis-elements are dominant. AmYABBY is highly expressed in stems and leaves, especially AmYABBY1, AmYABBY2, and AmYABBY3, which play important roles in the growth and development of stems and leaves. The AmYABBY gene family regulates the growth and development of A. mongholicus. In summary, this study provides a theoretical basis for in-depth research on the function of the AmYABBY gene and new insights into the molecular response mechanism of the growth and development of the traditional Chinese medicine A. mongholicus.

Cloning, Identification, and Functional Analysis of the Chalcone Isomerase Gene from Astragalus sinicus.

Astragalus sinicus is an important winter-growing cover crop. It is widely utilized, not only as a cover crop for its benefits in fertilizing the soil but also as a landscape ground cover plant. Anthocyanins are involved in the pigmentation of plants in leaves and flowers, which is a crucial characteristic trait for A. sinicus. The formation of anthocyanins depends significantly on the enzyme chalcone isomerase (CHI). However, research on the CHI gene of A. sinicus remains unexplored. The rapid amplification of cDNA ends (RACE) approach was used in this research to clone the CHI sequence from A. sinicus (AsiCHI). The expression profiles of the AsiCHI gene in multiple tissues of A. sinicus were subsequently examined by qRT-PCR (Quantitative Real-Time PCR). Furthermore, the function of the AsiCHI was identified by the performance of ectopic expression in Arabidopsis (Arabidopsis thaliana). The outcomes revealed that the full-length cDNA of the AsiCHI gene (GeneBank: OQ870547) measured 972 bp in length and included an open reading frame of 660 bp. The encoded protein contains 219 amino acids with a molecular weight of 24.14 kDa and a theoretical isoelectric point of 5.11. In addition, the remarkable similarity between the AsiCHI protein and the CHI proteins of other Astragalus species was demonstrated by the sequence alignment and phylogenetic analysis. Moreover, the highest expression level of AsiCHI was observed in leaves and showed a positive correlation with anthocyanin content. The functional analysis further revealed that the overexpression of AsiCHI enhanced the anthocyanidin accumulation in the transgenic lines. This study provided a better understanding of AsiCHI and elucidated its role in anthocyanin production.

[Molecular mechanism underlying difference of astragaloside â £ content in imitating wild and cultivated Astragalus mongholicus].

The difference of astragaloside Ⅳ content and the expression of its biosynthesis related genes in imitating wild Astragalus mongolicus(IWA) and cultivated A.mongolicus(CA) under different growth years were systematically compared and analyzed.Then the key enzyme genes affected the difference of astragaloside Ⅳ content in the above two A.mongolicus were screened.High-perfo-rmance liquid chromatography(HPLC)was used to determine the content of astragaloside Ⅳ in A.mongolicusunderthe above two diffe-rent growth patterns.Based on the Illumina HiSeq and PacBio high-throughput sequencing platforms, thesecond-and third-generation transcriptome sequencing(RNA-Seq)databaseof the two A.mongolicuswas constructed.The related enzyme genes in the biosynthetic pathway of astragaloside Ⅳ were screened and verified byquantitative reverse transcriptase polymerase chain reaction(RT-qPCR).The RNA-sequencing(RNA-Seq) and RT-qPCR data of each gene were subjected to correlation analysis and trend analysis.The results showed that the variation trend of astragaloside Ⅳ contentby HPLC wasthe same as that of genes by RNA-Seq and RT-qPCR in 1-4 year IWA and 1-2 year CA.The trend level of astragaloside Ⅳ contentwas lower in 2-year IWA than 1-year IWA.Compared with 2-year IWA, 3-year IWA had an upward trend, while 4-year IWA hada downward trend versus 3-year IWA.Additionally, 1-year CA had increased trendthan 2-year CA.However, the content of astragaloside Ⅳ in 5-year IWA was higher than that of 6-year IWA, which wasinconsistent with the findings of RNA-Seq and RT-qPCR.This study preliminarily clarifiedthat the difference of astragaloside Ⅳ contentin 1-4 year IWA and 1-2 year CA wasclosely related to the expression of the upstream and midstream genes(MVK, CMK, PMK, MVD, SS) in the biosynthetic pathway.The results facilitate the production and planting of Radix Astragali seu Hedysari.

The chromosome-level genome assembly of Astragalus sinicus and comparative genomic analyses provide new resources and insights for understanding legume-rhizobial interactions.

The legume species Astragalus sinicus (Chinese milk vetch [CMV]) has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops for improving rice productivity and preventing soil degeneration. In this study, we generated the first chromosome-scale reference genome of CMV by combining PacBio and Illumina sequencing with high-throughput chromatin conformation capture (Hi-C) technology. The CMV genome was 595.52 Mb in length, with a contig N50 size of 1.50 Mb. Long terminal repeats (LTRs) had been amplified and contributed to genome size expansion in CMV. CMV has undergone two whole-genome duplication (WGD) events, and the genes retained after the WGD shared by Papilionoideae species shaped the rhizobial symbiosis and the hormonal regulation of nodulation. The chalcone synthase (CHS) gene family was expanded and was expressed primarily in the roots of CMV. Intriguingly, we found that resistance genes were more highly expressed in roots than in nodules of legume species, suggesting that their expression may be increased to bolster plant immunity in roots to cope with pathogen infection in legumes. Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.

Gene expression variation of Astragalus adsurgens Pall. through discharge plasma and its activated water.

To explore the effects of plasma-activated water (PAW) on gene expression, the combined treatment of PAW and discharge plasma on Astragalus adsurgens Pall seeds were performed, and then the gene expression of seedlings after treatmentwas analyzed at the molecular level. A needle array-plate dielectric-barrier discharge plasma was used to treat Astragalus adsurgens Pall seeds for 1, 2, and 3 h, and PAW was prepared at the same time to cultivate seeds. When the treatment time was 3 h, the survival rate of Plasma + PAW seedlings was only 9.2% of that of the CK. The Astragalus adsurgens Pall seedlings were analyzed using reactive oxygen species (ROS) and RNA-Seq. The ROS content of the seedlings in treatment group was significantly higher than that in the CK after 3 days of culture, that PAW cultivated can cause oxidative stress damage to Astragalus adsurgens Pall. The enzyme activity of the treated plant increased and the metabolic rate was accelerated. It helped to regulate the growth process of plants and improve the yield and quality of crops. This study discussed the gene expression of plasma and PAW induced Astragalus adsurgens Pall at the molecular level, and provided experimental data support for plasma and PAW treatment and selection of Astragalus adsurgens Pall.

Chloroplast phylogenomics and character evolution of eastern Asian Astragalus (Leguminosae): Tackling the phylogenetic structure of the largest genus of flowering plants in Asia.

Astragalus, as the largest genus of the flowering plants, is well-known for its high species richness and morphological diversity. Previous studies suggested that many of the subgenera of Astragalus are not monophyletic and the phylogenetic relationships within the genus are still poorly known. In this study, we sampled 117 accessions of Astragalus and its close relatives, covering 55 sections of the genus plus 30 outgroup taxa to recover the main clades of eastern Asian Astragalus based on sequences of the whole chloroplast genome and 65 chloroplast CDSs. Astragalus is supported to be monophyletic and it is sister to the Oxytropis + Coluteoid clade. Within Astragalus, we recovered ten clades, and the ten clades differ substantially from Bunge's subgenera. The former segregate genus Astracantha is also monophyletic, but embedded within Astragalus s. str., supporting the merge of the spiny former genus Astracantha with Astragalus. We detected the atpF intron losses in the chloroplast genome of the Oxytropis + Coluteoid clade, i.e., the sister clade to Astragalus. Furthermore, we estimated the ancestral states of the trichome morphology and habit via the Bayesian Binary Method. The medifixed hair type is inferred to have developed at least five times and the annual habit originated at least six times. In addition, Astragalus is estimated to have originated in the mid Miocene (stem age, 16.09 Ma, 95% HPD: 12.46-20.50 Ma). The divergence times of the medifixed hair groups ranged from 4.03 to 0.87 Ma, mostly 2-1 Ma, which are correlated with the estimated phased uplifts of the Qinghai-Tibetan Plateau (QTP). We hypothesize that the uplifts of the QTP, which contributed to aridification in eastern Asia and the adjacent regions, may have accelerated the rapid speciation of Astragalus, especially the xerophilous groups (i.e. the medifixed hair groups).

Using Relaxation Time to characterize biological effects of different mutagens.

All kinds of mutagenic factors may cause physiological, biochemical and genetic changes of all organisms. To characterize their characteristic biology effects, the concept of Relaxation Time (RT) was introduced for the first time, and the specific process was as follows. After mutation of organisms, the offsprings will be continuingly cultured (or cultivated) to the next generation (Rx). Once a biological effect began to show no significant difference compared to the untreated controls, the Rx was defined as the RT of the effect. In this paper, three kinds of mutagenic factors were selected to treat the seeds or seedlings of Astragalus sinicus L., subsequently, the corresponding RT was calibrated. The results showed that the RT was diverse not only among different biological effects but also among different mutagenic factors. For the RT of chemical mutagens and gamma rays, most of which are concentrated on R1, whereas the heavy ion beams have significant differences among different tracks. Among biological effects, the SOD activity and superoxide anion free radical content in the Peak region are more prominent, and their RT reaches R3 and R4, respectively. Thus, the RT may characterize the characteristic biological effects from differently mutagenic factors.

A Methionine Sulfoxide Reductase B Is Required for the Establishment of Astragalus sinicus-Mesorhizobium Symbiosis.

Methionine sulfoxide reductase B (MsrB) is involved in oxidative stress or defense responses in plants. However, little is known about its role in legume-rhizobium symbiosis. In this study, an MsrB gene was identified from Astragalus sinicus and its function in symbiosis was characterized. AsMsrB was induced under phosphorus starvation and displayed different expression patterns under symbiotic and nonsymbiotic conditions. Hydrogen peroxide or methyl viologen treatment enhanced the transcript level of AsMsrB in roots and nodules. Subcellular localization showed that AsMsrB was localized in the cytoplasm of onion epidermal cells and co-localized with rhizobia in nodules. Plants with AsMsrB-RNAi hairy roots exhibited significant decreases in nodule number, nodule nitrogenase activity and fresh weight of the aerial part, as well as an abnormal nodule and symbiosome development. Statistical analysis of infection events showed that plants with AsMsrB-RNAi hairy roots had significant decreases in the number of root hair curling events, infection threads and nodule primordia compared with the control. The content of hydrogen peroxide increased in AsMsrB-RNAi roots but decreased in AsMsrB overexpression roots at the early stage of infection. The transcriptome analysis showed synergistic modulations of the expression of genes involved in reactive oxygen species generation and scavenging, defense and pathogenesis and early nodulation. In addition, a candidate protein interacting with AsMsrB was identified and confirmed by bimolecular fluorescence complementation. Taken together, our results indicate that AsMsrB plays an essential role in nodule development and symbiotic nitrogen fixation by affecting the redox homeostasis in roots and nodules.

De novo transcriptome characterisation of two auxin-related genes associated with plant growth habit in Astragalus adsurgens Pall.

Astragalus adsurgens Pall., a perennial legume native to China, is commonly used as a forage crop. And it has great value for sustainable development of grasslands in arid and semi arid regions. However, to date, little is known regarding the A. adsurgens genome, and no studies have determined whether it would be possible to improve the germplasm of A. adsurgens through genetic modification. In this study, we used an RNA-seq protocol to generate a de novo transcriptome including 151,516 unigenes of A. adsurgens. We compared the transcriptomes of A. adsurgens having different growth habits (prostrate/erect) and identified 14,133 single nucleotide polymorphism sites (SNP) in 8,139 unigenes. Differential expression gene (DEG) analysis suggested that 10,982 unigenes were up-regulated in the prostrate plant relative to the erect plant, while 10,607 unigenes were down-regulated. Of the 21,589 DEG, Unigene72782_All (LAX4) and CL12494.Contig3_All (TIR1), an auxin transporter gene and an auxin transport inhibitor gene, respectively, were predicted to influence the growth habit of A. adsurgens, which were verified by qRT-PCR in these phenotypes. These results suggest that auxin transport was more active in the prostrate plant than in the erect plant, resulting in asymmetric distribution of auxin that affects the growth habit of A. adsurgens. Overall, this study may provide a basis for future research on key genes in A. adsurgens and may deepen our understanding of the molecular mechanisms regulating plant growth habit.

Host Genotype and Precipitation Influence of Fungal Endophyte Symbiosis and Mycotoxin Abundance in a Locoweed.

Many plant endophytes produce mycotoxins, but how host genetic variation influences endophyte colonization and mycotoxin production under natural conditions is poorly understood. This interaction has not been fully considered in many previous studies which used controlled experiments with agronomic or model plant species. Here, we investigated this interaction in a naturally occurring forb (a locoweed species) Oxytropis ochrocephala, its symbiotic endophyte Alternaria oxytropis, and the mycotoxin swainsonine. Host genetic variation was characterized by microsatellite markers. Endophyte infection rate and swainsonine levels were determined by PCR and HPLC, respectively. Genetic markers defined two distinct host populations and revealed that host genetics were significantly correlated with geographical location, elevation, and precipitation. As the host diverged, symbiotic interactions were reduced or failed to produce detectable swainsonine in one host population. Host genotype and precipitation had a significant impact in shaping swainsonine production at the population level. This study highlights the effect of host genotype in influencing this interaction in locoweeds.

Multiple auto- and allopolyploidisations marked the Pleistocene history of the widespread Eurasian steppe plant Astragalus onobrychis (Fabaceae).

The Eurasian steppes occupy a significant portion of the worldwide land surface and their biota have been affected by specific past range dynamics driven by ice ages-related climatic fluctuations. The dynamic alterations in conditions during the Pleistocene often triggered reticulate evolution and whole genome duplication events. Employing genomic, genetic and cytogenetic tools as well as morphometry we investigate the intricate evolution of Astragalus onobrychis, a widespread Eurasian steppe plant with diploid, tetraploid and octoploid cytotypes. To analyse the heteroploid RADseq dataset we employ both genotype-based and genotype-free methods that result in highly consistent results, and complement our inference with information from the plastid ycf1 region. We uncover a complex and reticulate evolutionary history, including at least one auto-tetraploidization event and two allo-octoploidization events; one of them involved also genetic contributions from other species, most likely A. goktschaicus. The present genetic structure points to the existence of four main clades within A. onobrychis, which only partly correspond to different ploidies. Time-calibrated diffusion models suggest that diversification within A. onobrychis was associated with ice age-related climatic fluctuations during the last million years. We finally argue for the usefulness of uniparentally inherited plastid markers, even in the genomic era, especially when investigating heteroploid systems.

Rarity does not limit genetic variation or preclude subpopulation structure in the geographically restricted desert forb Astragalus lentiginosus var. piscinensis.

PREMISE OF THE STUDY: Characteristics of rare taxa include small population sizes and limited geographical ranges. The genetic consequences of rarity are poorly understood for most taxa. A small geographical range could result in reduced opportunity for isolation by distance or environment, thereby limiting genetic structure and variation, but few studies explore genetic structure at small spatial scales with sufficient resolution to test this hypothesis. Moreover, few comparative genetic studies exist among infrataxa differing in rarity. Here, we compare genetic variation among varieties of Astragalus lentiginosus differing in range size. Additionally, we ask if genetic structure exists in A. lentiginosus var. piscinensis, a rare taxon consisting of several thousand individuals that persist on ~8 km2 of alkaline soil. METHODS: We compared genetic variation among 11 varieties of A. lentiginosus differing in range size using a genotyping by sequencing (GBS) approach, which generated 11,475 single nucleotide polymorphisms (SNPs). We characterized genetic structure among subpopulations of A. lentiginosus var. piscinensis using a second GBS data set of 7274 SNPs and explored associations between genetic structure and environmental variation. KEY RESULTS: We found no association between genetic variation and range size among varieties of A. lentiginosus. Additionally, despite the extremely small range of A. lentiginosus var. piscinensis, we report a well-defined genetic structure among subpopulations associated with microhabitat variation in soil composition. CONCLUSIONS: Our results suggest that fine scale genetic structure may exist within other rare Astragalus taxa and that rarity does not preclude the maintenance of genetic diversity in this genus.

Development and application of EST-SSRs markers for analysis of genetic diversity in erect milkvetch (Astragalus adsurgens Pall.).

Erect milkvetch (Astragalus adsurgens Pall.) is a major legume forage plant widely grown in Northern China. However, the lack of molecular markers has limited its research into its genetic diversity and work on germplasm improvement. In this study, a total of 39,163 EST-SSR loci were identified from 30,262 unigene sequences in the erect milkvetch transcriptome using Illumina sequencing. Moreover, 22,367 EST-SSR primer pairs (PPs) were successfully designed. In addition, 100 PPs were synthesized and preliminarily screened in two accessions; of these, 90 were determined to be clear and stable EST-SSR markers. Fifty-one PPs were randomly selected in order to assess the genetic diversity of 27 erect milkvetch accessions. The average polymorphism information content of the 51 PPs was 0.682. Greater genetic diversity was detected in accessions from Inner Mongolia and in the group of landrace and wild erect milkvetch accessions. This study provides an important resource for germplasm improvement and genetic diversity analysis in erect milkvetch.

[Purification and biochemical function of Astragalus membtanaceus pathogenesis-related protein 10].

Astragalus membranaceus pathogenesis-related protein 10 (AmPR-10) is largely expressed in case of environmental pressure and pathogen invasion. This study aims to explore the biochemical functions of AmPR-10. The dried root of Astragalus membranaceus was mechanically homogenized and extracted by Tris-HCl buffer to obtain its crude extract, which was then purified by anion exchange chromatography and gel filtration chromatography to obtain electrophoretically pure AmPR-10. The nuclease activity of AmPR-10 was tested with different RNAs by detecting the absorption value at 260 nm. The results demonstrated potent nuclease activity toward yeast tRNA, yeast RNA, Poly (A) and Poly (C). The optimum reaction temperature was 50 °C and pH was 7-8. EDTA showed no effect on its activity, while Mg²âº exhibited potent activation effect on the activity, and Co²âº, Ca²âº and Zn²âº manifested moderately inhibition of the activity. Since AmPR-10 had no sequence homology with other known nucleases, AmPR-10 was probably a novel nuclease. The inhibition kinetic data against papain was analyzed by Lineweaver-Burk plots, and the results showed that the inhibition of papain followed noncompetitive-type kinetics. AmPR-10 played an important role in Astragalus membranaceus defense mechanism against environmental pressure and pathogen invasion, which may be achieved by inhibiting cycteine enzymes activity.

Genetic analyses of Astragalus sect. Humillimi (Fabaceae) resolve taxonomy and enable effective conservation.

PREMISE OF THE STUDY: Astragalus sect. Humillimi is distributed across the southwestern United States and contains two endangered taxa, A. cremnophylax var. cremnophylax and A. humillimus. The former was originally described from the South Rim of the Grand Canyon. Analysis of individuals discovered on the North Rim of the Grand Canyon yielded some evidence that the population represented a distinct species. To enable effective conservation, we clarify the group's taxonomy and characterize the genetic diversity of A. cremnophylax and A. humillimus. METHODS: We used AFLPs to genotype most species in sect. Humillimi, focusing on the two endangered forms. We examined patterns of genetic diversity using complementary analytical approaches. KEY RESULTS: Our results demonstrate that North Rim populations group with A. c. var. cremnophylax. We found low levels of genetic diversity at certain localities and strong differentiation among populations. Astragalus humillimus, which has suffered recent and severe population declines, exhibits weak differentiation among and low diversity within populations. CONCLUSIONS: Our results clarify the taxonomy of sect. Humillimi and define the boundaries of A. c. var. cremnophylax, which is shown to inhabit both rims of the Grand Canyon. This clarification, and detailed analysis of genetic variation within both endangered taxa, may advance ongoing efforts to conserve these taxa. Our results suggest that range-wide genetic analysis of A. humillimus may inform recovery strategies for this taxon.

The Different Resistance of Two Astragalus Plants to UV-B Stress is Tightly Associated with the Organ-specific Isoflavone Metabolism.

In this work, the changes in isoflavone levels and the expression of genes involved in their biosynthesis were studied in two Astragalus by UPLC-MS and real-time PCR after 10 days of UV-B treatment (λmax  = 313 nm, 804 J m-2 ). Isoflavones were significantly induced by UV-B irradiation. The influence might be activated by the regulation of these target genes. Our results indicate that (1) the resistance of Astragalus membranaceus might not be as good as Astragalus mongholicus in the enhanced UV-B radiation environment; (2) the enhanced accumulation of calycosin and calycosin-7-glucoside with UV-B treatment in roots of A. mongholicus might be derived from formononetin which is synthesized in the leaves; (3) the glycosylation process could be stimulated and activated by the enhanced UV-B radiation in both A. mongholicus and A. membranaceus. In other words, glycosylation of isoflavones might play a crucial role for two Astragalus plants in response to UV-B stress. Overall, this study offered a feasible elicitation strategy to understand the accumulation pattern of isoflavone in A. mongholicus and A. membranaceus, and also provided a reference for the changes in isoflavone levels of Astragalus in UV-B enhanced environment in the future.

Chemical and genetic diversity of Astragalus mongholicus grown in different eco-climatic regions.

Astragalus mongholicus Bunge (Fabaceae) is an important plant source of the herbal drug known as Radix Astragali, which is used worldwide as a medicinal ingredient and a component of food supplement. Russian Federation, Mongolia, Kazakhstan, and China are the main natural distribution areas of A. mongholicus in the world. However, the quality of medicinal plant varies among different locations. As for A. mongholicus, limited literature focused on its biodiversity mechanism. Here, we combined the chemometric analysis of chemical components with genetic variation, as well as climatic and edaphic traits, to reveal the biodiversity mechanism of A. mongholicus. Results showed that the detected chemical, genetic and climatic traits comprehensively contributed to the quality diversity of A. mongholicus. The eight main chemical components, as well as the inorganic elements of P, B and Na were all significant chemical factors. The precipitation and sunshine duration were the main distinguishing climatic factors. The inorganic elements As, Mn, P, Se and Pb were the distinguishing edaphic factors. The systematic method was firstly established for this medicinal plant in order to illustrate the formation of diversity in terms of quality, and provide scientific evidence for geographic indications and climatic adaptation in production and in the clinical application of herbal medicinal plants.

Small RNA and degradome sequencing reveals important microRNA function in Astragalus chrysochlorus response to selenium stimuli.

Selenium (Se), an essential element, plays important roles in human health as well as environmental sustainability. Se hyperaccumulating plants are thought as an alternative selenium resource, recently. Astragalus species are known as hyperaccumulator of Se by converting it to nonaminoacid compounds. However, Se-metabolism-related hyperaccumulation is not elucidated in plants yet. MicroRNAs (miRNAs) are key molecules in many biological and metabolic processes via targeting mRNAs, which may also play an important role in Se accumulation in plants. In this study, we identified 418 known miRNAs, belonging to 380 families, and 151 novel miRNAs induced by Se exposure in Astragalus chyrsochlorus callus. Among known miRNAs, the expression of 287 families was common in both libraries, besides 71 families were expressed only in Se-treated sample, whereas 60 conserved families were expressed in control tissue. miR1507a, miR1869 and miR2867-3p were mostly up-regulated, whereas miR1507-5p and miR8781b were significantly down-regulated by Se exposure. Computational analysis shows that the targets of miRNAs are involved in different types of biological mechanisms including 47 types of cellular component, 103 types of molecular function and 144 types of biological process. Degradome analysis shows that 1256 mRNAs were targeted by 499 miRNAs. We conclude that some known and novel miRNAs such as miR167a, miR319, miR1507a, miR4346, miR7767-3p, miR7800, miR9748 and miR-n93 target transcription factors, disease resistance proteins and some specific genes like cysteine synthase and might be related to plant hormone signal transduction, plant-pathogen interaction and sulphur metabolism pathways.

[Astragali Radix and Hedysari Radix molecular identification of SSR primers screening and fingerprints code].

Leguminous related SSR primers were collected, core primers used for Astragali Radix and Hedysari Radix identification were screened and validated by using molecular marker techniques. 6 core primers were selected from 101 pairs of primers, the molecular weight of PCR products was 100-500 bp, which formed 7-12 electrophoresis bands with 55 amplified loci. The percentage of polymorphic loci was 100%, and the average polymorphism information content was 0.371. According to the results of cluster analysis, obtained core primer could completely distinguish 62 mixture samples of Astragali Radix and Hedysari Radix in similarity coefficient of 0.46. Core primers and the corresponding characteristics from gel electrophoresis were tagged. The results provide identification basis for Astragali Radix and Hedysari Radix.