High-throughput single-cell RNA sequencing (scRNA-seq) technology has developed rapidly in recent years (Xie et al., 2022). However, due to differentiated cell types resistant to protoplasting, and cell spatial information restoration dependent on the wellstudied marker genes and other reasons, which are limited the application of scRNA-seq in non-model species (Shaw et al., 2021). Peanut possesses a unique feature to embed its fertilized ovary into the soil through a specialized organ known as the peg, which has a stem-like morphology and anatomy but behaves like a root with positive gravitropism (Moctezuma, 2003). Here, we successfully established the SpaTial Enhanced REsolution Omics-sequencing (Stereo-seq) in this species and revealed the complex cell typespecific and spatial gene expression features of the peg by comparison with other three tissues (root, stem and hypocotyl, which have some common anatomical structures). Schematic representationof theStereo-seqprocedure forpeanut tissues was shown in Figure 1a. Sample preparation, RNA-seq and data analysis were improved according to Chen et al. (2022). A two-step cryo-embedding method was developed to minimize the tissue contraction for sample preparation of roots, hypocotyls, stems and pegs. Cryo-sections at 10 lm thickness were mounted on the 100 mm Stereo-seq chips designed for plant tissues. In the two-step permeabilization method, the solutions were sprayed on the section surface with an ultrasonic atomizer to avoid lateral diffusion of mRNA solution (see details in supplemental methods). The fluorescence intensity of in situ synthesized cDNA was significantly enhanced by the improved methods (Figures 1a and S1). Totally, 1 347 263 645 valid raw reads and 938 764 561 clean readswereobtained fromthechipsof the four tissues. Byaligning to Tifrunner genome, 35 970, 47 304, 47 096 and 44 825 genes were identified in roots, hypocotyls, stems and pegs, respectively. Basedon the statistics of cell size andgene capture at bin size in four tissues (Tables S1, S2 and Figure S2), bin50-100 was used for furtheranalysis, and theaverageuniquemolecular identifiers (UMIs) and gene number captured in the four tissues at bin80 is 1858 and 871, respectively, which is about half of that of single-cell sequencing technique (Zhang et al., 2019). The transcriptome profiles were projected in an unsupervised analysis by Seurat and the clusterswere visualized by the uniformmanifold approximation and projection (UMAP) (Stuart et al., 2019). 18 clusters (0–17) were visualized and their spatial distribution corresponding to the section structure was optimal at bin80 (Figure 1b). The epidermis and exodermis fromhypocotyl and stem were clustered together as cluster11, while the epidermis and exodermis from roots and pegswere classified into cluster12, 2 and 13, respectively. The cortex regions from roots, hypocotyls, stems, and pegs were classified into cluster5, 1, 3 and 9, respectively. Notably, although the epidermis and exodermis of stem and hypocotyl were classified as the same cluster (cluster11), the UMAP showed the cluster11 can further be divided into two subclusters (Figure 1b). Dots of subcluster11-1 and 11-2 are from the stem and hypocotyl, respectively (Figure S3). We obtained 282 subclusterenriched genes (¦log2¦ ≧ 0.25) among the two subclusters. Gene ontology (GO) enrichment analysis showed that the enriched genes in subcluster11-1 were related to photosynthesis and carbon fixation, while those in subcluster11-2 were related to cell wall modification and plant pathogen resistance (Tables S3 and S4). These results are consistent with their development and adaptation to environments where stems requiremore carbohydrates for rapid growth, while hypocotyls require predominant expression of disease-resistant and cell wall genes to cope with plant diseases and stresses. Our data provided new insights into the cell heterogeneity in peanut, where the same type cells according to classical anatomy from different tissues may retain a high divergence of gene expression. For instance, the epidermis and exodermis from the tip of the pegs (cluster13) were separated from that of the basal part (cluster2). GO enrichment analysis showed that the enriched genes in cluster2 are mainly related to the stimulation of sensing environmental signals, which is consistent with the function divergence to respond to mechanical stimulus to initiate the ovule development. However, the cluster13 is related to the synthesis of glycoside and saponin, which may protect the ovules from underground pests (Table S5). For the stem individually, nine cell clusters (S0-S8)were definedat bin75,which is optimally consistentwith the distribution of cell type visualized in stemanatomywhen restored (Figure 1c). Compared to bin80 (Figure 1b), cluster6 was further divided into 3 subclusters (S2, S5 and S7), cluster10was divided into 2 subclusters (S4 and S6), which is more consistent with the stem anatomy of peanut. Ordering cells of the clusterS4-S7 by pseudo-time analysis revealed two-directional differentiation trajectory of cambium cells. As expected, most cells from clusterS7 (cambium cells) assembled at the beginning of pseudo-time, while xylem vessel and xylem
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