Transcriptomic analysis and identification of candidate genes involved in rhizome development in Agropyron michnoi Roshev.

Agropyron michnoi Roshev. is a perennial grass with rhizomes in the genus Agropyron. Agropyron michnoi has strong tolerance to drought and low temperature. It is an establishing species in sandy flat and hilly slope lands, where it constitutes sandy grassland. Therefore, Agropyron michnoi is an important forage species in dry grassland and desert steppe. Rhizomes not only enables asexual reproducibility but also confers strong resilience to stresses in Agropyron michnoi. However, in production practice, the rhizome trait is not stable, with some Agropyron michnoi plants having no rhizomes. In addition, the genomic resources for this species are scarce, which limits the studies on rhizome development. Therefore, the anatomical structure of rhizomes, roots and stems was analyzed by paraffin sectioning technique. The results showed that rhizomes were both similar to roots and stems, and had partial functions of roots and stems. Than Agropyron michnoi plants were divided into two groups: Group Y (with rhizomes) and Group W (without rhizomes). From each plant group, root (YG and WG), stem (YJ and WJ) and rhizome (YGJ) samples were harvested and subjected for RNA sequencing. De novo transcriptomic analysis was performed to identify 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 ten of the 129 candidate genes were further validated using qRT-PCR. Results from this study enriched the genomic resources and the morphology information in Agropyron michnoi. The selected candidate genes involved in rhizome development provide preliminary mechanistic interpretation of rhizome development and will be informative for in-depth investigation of the molecular mechanism of rhizome development in Agropyron michnoi.

米氏冰草（Agropyron michnoi Roshev.）是冰草属（Agropyron）的多年生根茎型禾本科植物，具备极强的耐旱与耐低温特性。其为沙质平原与丘陵坡地的建群物种，可形成沙质草原群落，因此是干旱草原与荒漠草原中的重要饲用物种。米氏冰草的根茎不仅赋予植株无性繁殖能力，还能显著提升其逆境耐受能力。但在生产实践中，其根茎性状并不稳定，部分米氏冰草植株并不具备根茎。此外，该物种的基因组资源较为匮乏，限制了对其根茎发育机制的研究。为此，本研究通过石蜡切片技术，对米氏冰草的根茎、根与茎的解剖结构进行了分析。结果显示，根茎兼具根与茎的部分结构特征，并具备二者的部分生理功能。随后，将米氏冰草植株分为两组：Y组（具根茎植株）与W组（无根茎植株）。分别从两组植株中采集根（YG、WG）、茎（YJ、WJ）以及根茎（YGJ）样本，进行RNA测序（RNA-seq）。随后开展从头转录组分析，以筛选参与根茎发育的候选基因。本次RNA测序共获得103.73 Gb的清洁碱基数据，经组装得到215282条单基因簇（unigenes），平均长度为905.67 bp。其中，161175条单基因簇（占比74.87%）通过7个通用公共数据库完成了功能注释。通过对5个样本间的差异表达基因进行两两比较，最终筛选得到129个可能在根茎中特异性表达的候选基因。通路富集分析结果显示，根茎特异性表达基因显著富集于苯丙烷生物合成及淀粉与蔗糖代谢通路。采用实时荧光定量PCR（qRT-PCR）对129个候选基因中的10个的根茎特异性表达模式进行了验证。本研究的结果丰富了米氏冰草的基因组资源与形态学相关数据。筛选得到的参与根茎发育的候选基因，为解析米氏冰草根茎发育的分子机制提供了初步的理论依据，同时也为后续深入研究其根茎发育的分子调控机理提供了重要参考。