*2.6. Prediction of Endogenous Target Mimics for miRNAs*

MicroRNA data for *S. lycopersicum* were obtained from miRbase (http://www.mirbase. org/) (accessed on 26 July 2019). WGCNA was used to predict the relationships between mRNAs, lncRNAs, and miRNAs. A threshold weight value of >0.15 was used for the selection of lncRNA-miRNA-mRNA triplets. Cytoscape (https://cytoscape.org/) (accessed on 26 July 2019) was used to plot the networks of lncRNAs, miRNAs, and mRNAs.

#### *2.7. GenBank Accession Numbers*

CK\_1: GSM4238348; CK\_2: GSM4238349; CK\_3: GSM4238350; T1\_1: GSM4238351; T1\_2: GSM4238352; T1\_3: GSM4238353; T2\_1: GSM4238354; T2\_2: GSM4238355; T2\_3: GSM4238356. The RNA sequencing read data have been deposited in the Short Read Archive (SRA) data library (accession number: SRP239079).

#### **3. Results**

#### *3.1. Identification of lncRNAs in S. lycopersicum*

Substantial numbers of novel transcripts could be discovered via the combination of high-throughput RNA-seq and de novo assembly. In this study, the 'Diana' cultivar of *S. lycopersicum* was used for RNA-seq. As a new cultivar of *S. lycopersicum*, 'Diana' is widely cultivated in China. *S. lycopersicum*; an important crop species, it is also affected by continuous cropping obstacles, which are mainly caused by allelopathy. PHBA, a

common allelochemical, was studied in depth, and the results showed that photosynthesis, respiratory effects, and gene expression in the leaves were affected by PHBA. Thus, the 'Diana' cultivar of *S. lycopersicum* was chosen to study gene expression in PHBA-induced allelopathy. We built the unigene library with three samples, including leaves undergoing three different treatments (CK: leaves of *S. lycopersicum* under 0 mmol/L PHBA treatment for 2 days; T1: leaves of *S. lycopersicum* under 15 mmol/L PHBA treatment for 2 days; and T2: leaves of *S. lycopersicum* under 30 mmol/L PHBA treatment for 2 days) (Figure 1A).

A total of 82,126 unigenes with an overall length greater than 200 bp and poly(A) tails were identified after de novo assembly. Following the protocol represented in Figure S1, 65,178 unigenes were acquired as credible lncRNAs, among which 61,729 lncRNAs were expressed during the plant allelopathy progression in *S. lycopersicum* in response to PHBA (Table S2). Ten lncRNAs were randomly chosen, and their expression was verified by qRT-PCR; all results were consensual (Figure S2).

The average length of the 61,729 assumed lncRNAs was 565 bp, the minimum length was 201 bp, and the maximum length was 19,161 bp (Figure 1B and Table S2). Of these lncRNAs, 80.68% were shorter than 600 bp, while 9.59% were longer than 1000 bp. About two-thirds of them expressed at low levels, with FPKM values (log2) ranging from 0 to 3 (Figure 1C and Table S2). Interestingly, the trends in the data for the lncRNAs corresponded to those for the PGs across all of the samples (Figure 1D and Table S3).

#### *3.2. Expression of lncRNAs in* S. lycopersicum *Leaves under Different PHBA Treatments*

As we knew, 61,729 lncRNAs were expressed in *S. lycopersicum* leaves during the different PHBA treatments, among which 34,000 lncRNAs were expressed throughout all three treatments. Almost half of the lncRNAs were expressed exclusively in two stages, while a large number of lncRNAs were expressed in only one stage (Figure 2A).

**Figure 2.** Expression of lncRNAs in *S. lycopersicum* leaves under different PHBA treatments. (**A**) Venn diagram detailing shared and distinct lncRNA expression patterns in *S. lycopersicum* leaves under different treatments of PHBA. (**B**) Differential expressions of lncRNAs and PGs between the PHBAtreated and untreated lines of *S. lycopersicum*.

We compared the differences in expression between the lncRNAs and PGs in *S. lycopersicum* leaves under different PHBA treatments (Table S4). The numbers of differentially expressed PGs and lncRNAs between the treated and untreated *S. lycopersicum* leaves increased gradually with increasing PHBA concentration and reached an apex under the T2 treatment (Figure 2B).

#### *3.3. Prediction of Cis-Regulated Target PGs (CTPGs) of Lncrnas*

The expression of proximal and distal PGs has been found to be regulated by lncRNAs through cis- and trans-acting mechanisms [55]. The regulation of genes located on the same chromosome as lncRNAs can be ascribed to cis-regulation [56,57], the investigation of which relies on well-annotated genomes.

The proximal PGs located in a 100 kb genomic window were searched as cis-regulated target genes of lncRNAs [53]. A total of 7765 lncRNAs were found to have potential cisregulatory effects on 5314 PGs in 38,845 gene pairs altogether (Table S5), among which, 89% of lncRNAs targeted more than one gene, 71% targeted two to seven genes, while only two lncRNAs targeted fourteen genes (Figure 3A). Approximately 5% of the PGs corresponded to only one lncRNA, and only six PGs were cis-regulated by up to nineteen lncRNAs (Figure 3A).

**Figure 3.** Numbers of target PGs regulated by lncRNAs and numbers of lncRNAs that have potential cis-regulatory and trans-regulatory effects on PGs in *S. lycopersicum* leaves. (**A**) The numbers of TPGs regulated by lncRNAs and the numbers of lncRNAs that have potential cis-regulatory effects on PGs. (**B**) The numbers of TPGs regulated by lncRNAs and the numbers of lncRNAs that have potential trans-regulatory effects on PGs.

Subsequently, the coexpression relationships between lncRNAs and protein-coding gene pairs were studied using WGCNA. A total of 1116 lncRNAs and 2239 PGs were involved in transcriptome regulation, which formed a complex network (Data S1), including 12 modules (Figure 4A and Table S6). Different modules contained different proportions of lncRNAs, ranging from 16.7% in Module 10 (M10) to 57.6% in M6, with an average of 38.2%. Gene expression patterns for each module were distinguished (Figure 5). Significant GO terms were identified in each module, ranging from 11 in M11 to 133 in M1 (Table S7). GO:0032957, which is associated with the inositol trisphosphate metabolic process, was found in M7 and M11, while GO:0050789 (regulation of biological process) was found in M1 and M6. Among these modules, the highly expressed genes in M2 were involved in photosynthesis and cell wall organization and were annotated as being associated with allelopathy (Figure 6A). The genes in M13 were also predicted to be involved in photosynthesis and cell wall biogenesis (Figure 6B).

**Figure 4.** The weighted gene co-expression network analysis of lncRNAs and TPGs in *S. lycopersicum.* (**A**) Modules of lncRNAs and CTPGs. (**B**) Modules of lncRNAs and TTPGs.

**Figure 5.** Expression patterns of lncRNAs and CTPGs in different modules.

**Figure 6.** GO annotations of CTPGs in Module 2 (M2) (**A**) and Module 13 (M13) (**B**).

In addition, we calculated the Spearman correlations (correlation coefficient cutoff = 0.9) for the expression of each lncRNA and its CTPG pair. The expressions of 458 lncRNAs and their corresponding 975 CTPGs were found to be strongly correlated (Table S8). A total of 1092 gene pairs were positively correlated, and 963 pairs were negatively correlated. Among these target genes, 1348 were upregulated in *S. lycopersicum* leaves in at least one treatment, and 892 were downregulated (Table S9).
