Junjie Wang

Background: Astragalus membranaceus Mongholicus (Astragalus) is one of the most widely used medicinal plants in the world. It is a traditional Chinese medicine plant that has been used to treat various human diseases and conditions, such as inflammation, gastrointestinal bacterial infections and malignant tumors. With the expansion of artificial planting areas, the demand for high-quality Astragalus seedlings is also increasing. The traditional seedling-raising methods of direct seeding and induced callus have problems such as a long growth period, unstable genetic traits, strict sampling and rooting difficulties. Therefore, it is very important to multiply the excellent germplasm by optimizing the tissue culture regeneration system. Methods: In this study, Astragalus field cultivated plants, which are easily available, were used as materials and the method of tissue culture cuttings was used to select the direct inoculation method of stem segments as explant materials to establish a plant regeneration system of Astragalus and the best sampling period, sampling site and optimal medium for regeneration of field cultivated plants were screened. Result: The best sampling period for plant regeneration of Astragalus in the field was about 40 d after the emergence of the 3-year-old Astragalus and the rooting rate of the regenerated plants was 100.00%. The best sampling site is the middle section of the stem and the regeneration system using the middle section of the stem as the explant is better than using the top and bottom of the stem as the explant and the rooting rate of the regenerated plants is 100.00%; the best medium for the regenerated plants is N22: 1/2MS+1 mg/L NAA and T28: 1/2MS+1 mg/L ABT, the rooting rates were 76.67% and 96.67%, respectively. The regenerated plants established with the stems of the field cultivated plants of Astragalus as explants were all able to induce seedlings after 1 week and the regenerated seedlings start to take root after 3 weeks of growth and can be transplanted and propagated after 6 weeks of culture. The regeneration system established in this study by direct cuttings from cultivated Astragalus stems is more rapid and efficient, breaking through the difficulties of limited access to material and long growth period of regenerated seedlings in the traditional regeneration system and providing technical support for the conservation and utilization of wild resources of Astragalus and rapid expansion of new varieties and germplasm conservation.

Background: Astragalus membranaceus var. mongholicus (Astragalus), acknowledged as a pivotal “One Root of Medicine and Food”, boasts dual applications in both culinary and medicinal domains. The growth and metabolite accumulation of medicinal roots during the harvest period is intricately regulated by a transcriptional regulatory network. One key challenge is to accurately pinpoint the harvest date during the transition from conventional yield content of medicinal materials to high and to identify the core regulators governing such a critical transition. To solve this problem, we performed a correlation analysis of phenotypic, transcriptome, and metabolome dynamics during the harvesting of Astragalus roots. Results: First, our analysis identified stage-specific expression patterns for a significant proportion of the Astragalus root genes and unraveled the chronology of events that happen at the early and later stages of root harvest. Then, the results showed that different root developmental stages can be depicted by co-expressed genes of Astragalus. Moreover, we identified the key components and transcriptional regulation processes that determine root development during harvest. Furthermore, through correlating phenotypes, transcriptomes, and metabolomes at different harvesting periods, period D (Nov.6) was identified as the critical period of yield and flavonoid content increase, which is consistent with morphological and metabolic changes. In particular, we identified a flavonoid biosynthesis metabolite, isoliquiritigenin, as a core regulator of the synthesis of associated secondary metabolites in Astragalus. Further analyses and experiments showed that HMGCR, 4CL, CHS, and SQLE, along with its associated differentially expressed genes, induced conversion of metabolism processes, including the biosynthesis of isoflavones and triterpenoid saponins substances, thus leading to the transition to higher medicinal materials yield and active ingredient content. Conclusions: The findings of this work will clarify the differences in the biosynthetic mechanism of astragaloside IV and calycosin 7-O-β-D-glucopyranoside accumulation between the four harvesting periods, which will guide the harvesting and production of Astragalus.

Background: Astragalus is one of the largest angiosperms and has important economic value. The chloroplast genomes (Cp) of most plants in its family have been sequenced and annotated, but there are fewer studies on the characteristics of the chloroplast genomes and codon usage bias of Astragalus. In this study, we sequenced and annotated the complete chloroplast genomes of three Astragalus species, that systematically compared the chloroplast genome and codon usage characteristics of three Astragalus. Methods: In this study, we used three Astragalus species as materials. Firstly, we sequenced and assembled their chloroplast genomes. Subsequently, we analyzed the codon usage bias of the chloroplast genomes of the three medicinal Astragalus species using software such as CodonW, CUSP and SPSS. Result: The results illustrated that the length of Astragalus chloroplast genome ranges from 122,815 bp (A. dahuricus) to 123,729 bp (A. melilotoides Pall.). There were 107-110 genes, including 75-76 protein-coding genes (PCGs), four ribosomal RNA genes (rRNAs) and 28-30 transfer RNA genes (tRNAs). In addition, the results illustrated that codons in three Cp genomes from Astragalus had the same 11 Optimal codons and ending with A/U. The codon usage frequency of five model creatures were compared and the results revealed that the codon preferences of Astragalus and five model creatures were pretty significant, the natural selection was the main factor in codon preference. Our research provides some value for the researches of chloroplast genetic engineering and molecular breeding in Astragalus.

Background: Transplantation of Astragalus Mongholicus seedlings, compare the growth and development of astragalus and its yield and quality after graded transplantation, to provide the basis for improving the yield and quality of astragalus medicinal materials. Methods: With the planting of Astragalus Mongholicus seedlings for one year as the material were divided into four grades according to different lengths and thicknesses and the indexes of plant height, thick stem, stem and leaf weight, root length, thick root, yield, Astragaloside IV and Calycosin7-O-β-D-Glucopyranoside content after different seedlings grades were determined. Result: There is a close connection between the grade of Astragalus Mongholicus seedlings and the quality of the yield of medicinal materials. After graded transplantation, the yield of medicinal materials improves 38.52kg/667m2 compared to the traditional transplantation method. The content of Astragaloside IV and Calycosin7-O-b-D-Glucopyranoside is also closely related to the seedling grade, the content of Astragaloside IV in the medicinal materials after transplanting the second and third grade seedlings was more than 0.02% higher than that of the first and fourth grades, after transplantationof the grade I seedlings, the content of Calycosin7-O-b-D-Glucopyranoside in the medicinal materials was more than 0.01% higher than that of the other three grades. The yield of astragalus medicinal materials is the highest after the seedlings are transplanted by grade and the content of the main active components of the medicinal materials is quite different after the different grades of seedlings are transplanted. Therefore, in actual production, the seedlings should be transplanted according to different specifications and grades to improve the yield and quality of astragalus.

Background: Using genomic DNA from 79 F1 plants resulted from a crossing between parents with strong and weak grazing tolerance inMedicago falcataL., we generated anEcoRIrestriction site-associated DNA (RAD) sequencing library. After sequencing and assembly, a high-density genetic map with high-quality SNP markers was constructed, with a total length of 1312.238 cM and an average density of 0.844 SNP/cM.Methods: The phenotypic traits of 79 F1 families were observed and the QTLS of 6 traits were analyzed by interval mapping.Results: Sixty three QTLs were identified for seven traits with LOD values from 3 to 6 and the contribution rates from 15% to 30%. Among the 63 QTLs, 17 were for natural shoot height, 12 for rhizome Length, 10 for Shoot canopy diameter, 9 for Basal plant diameter, 6 for stem number, 5 for absolute shoot height, and 4 for rhizome width. These QTLs were concentrated on LG2, LG4, LG5, LG7, and LG8. LG6 had only 6 QTLs. According to the results of QTL mapping, comparison of reference genomes, and functional annotation, 10 candidate genes that may be related to grazing tolerance were screened. qRT-PCR analysis showed that two candidate genes (LOC11412291 and LOC11440209) may be the key genes related to grazing tolerance ofM. falcata.Conclusion: The identified trait-associated QTLs and candidate genes in this study will provide a solid foundation for future molecular breeding for enhanced grazing-tolerance inM. falcata.

Tolerance of alfalfa (Medicago sativa L.) to animal grazing varies widely within the species. However, the molecular mechanisms influencing the grazing tolerant phenotype remain uncharacterized. The objective of this study was to identify genes and pathways that control grazing response in alfalfa. We analyzed whole-plant de novo transcriptomes from grazing tolerant and intolerant populations of M. sativa ssp. falcata subjected to grazing by sheep. Among the Gene Ontology terms which were identified as grazing responsive in the tolerant plants and differentially enriched between the tolerant and intolerant populations (both grazed), most were associated with the ribosome and translation-related activities, cell wall processes and response to oxygen levels. Twenty-one grazing responsive pathways were identified that also exhibited differential expression between the tolerant and intolerant populations. These pathways were associated with secondary metabolite production, primary carbohydrate metabolic pathways, shikimate derivative dependent pathways, ribosomal subunit composition, hormone signaling, wound response, cell wall formation and anti-oxidant defense. Sequence polymorphisms were detected among several differentially expressed homologous transcripts between the tolerant and intolerant populations. These differentially responsive genes and pathways constitute potential response mechanisms for grazing tolerance in alfalfa. They also provide potential targets for molecular breeding efforts to develop grazing-tolerant cultivars of alfalfa.