Transcriptomic Analysis and Identification of Candidate Genes Involved in Rhizome Development in Agropyron michnoi

Agropyron michnoi is a perennial grass with rhizomes in the genus Agropyron. It has a strong tolerance to drought and low temperature, and it is an established species in sandy flat and hilly slope lands, which constitute sandy grassland. So, it is an important forage species in dry grassland and desert steppes. Rhizomes not only enable asexual reproducibility but also confer strong resilience to stresses in A. michnoi. However, during production and utilization, it has been found that there are significant differences in the development of rhizomes among individuals of A. michnoi, yet the regulatory mechanism remains unclear. Therefore, in this study, the A. michnoi &lsquo:Baiyinxile&rsquo: was used as the material, and the anatomical structures of the rhizomes, roots, and stems were analyzed using the paraffin sectioning technique. The results showed that the anatomical structure composition of the cross-section of the rhizome was similar to that of the root, while the arrangement of the vascular bundles in the stele was different from that of the root but similar to that of the stem. Subsequently, the Agropyron michnoi plants were classified into two types: plants with rhizomes and plants without rhizomes. Root, stem, and rhizome samples were collected from each type, and RNA sequencing was conducted. De novo transcriptomic analysis was performed to identify the candidate genes involved in rhizome development. From the RNA sequencing, a total of 103.73 Gb clean bases were obtained, from which 215,282 unigenes with an average length of 905.67 bp were assembled. Among these unigenes, 161,175 (74.87%) were functionally annotated based on seven common public databases. From pairwise comparisons of differentially expressed genes between the five samples, 129 candidate genes that are potentially specifically expressed in rhizomes were selected. Pathway enrichment analysis revealed that the rhizome-expressed genes are highly enriched in pathways of phenylpropanoid biosynthesis and starch and sucrose metabolism. The rhizome-specific expression pattern of 10 of the 129 candidate genes was further validated using qRT-PCR. Through the analysis of metabolites, 11 metabolites closely related to rhizome development, such as choline and betaine, were successfully identified. CYP family genes were selected for functional verification, and phylogenetic analysis revealed that CYP86B1 was grouped with CYP 86B1 of species such as Triticum aestivum and Lolium rigidum and was named AmrCYP86B1. The cloning results showed that its size was 1599 bp, and its subcellular localization was in the endoplasmic reticulum. Through stable genetic transformation, the study found that AmrCYP86B1 can promote the development of plant roots and stems and increase the dry matter content of the roots. Hormone detection showed that overexpression of AmrCYP 86B1 decreased the content of ABA hormone and increased the content of GA3 hormone in the plants. Combined with previous studies, it was determined that AmrCYP 86B1 promoted rhizome elongation by regulating ABA and GA3 hormones. The selected candidate genes involved in rhizome development, along with the preliminary functional verification, provide a preliminary mechanistic interpretation of rhizome development. This will contribute to in-depth research on the molecular mechanism of rhizome development in A. Michnoi.