Different peanut root morphology types caused by root-knot nematodes
Compared with no-infected peanut, the Huayu22 infected by root-knot nematode showed significant at root node number (21.33±4.16 vs 0, p=0.00089) (Fig.1, Fig.2a, Table 1). At the same time, the root-knot nematodes could cause short primary root length (25.06±1.79 vs 28.26±1.38, p=0.0013) (Fig.2c, Table 1), decreased total root surface area (644.94±21.53 vs 478.36±42.96, p=0.0039) (Fig.2d, Table 1), narrower root angle (79.26±7.47 vs 106.58±8.94, p=0.015) (Fig.2e, Table 1), less lateral root density (0.48±0.026 vs 0.69±0.021, p=0.00041) (Fig.2e, Table 1) but more lateral root number (52.33±5.13 vs 41±3, p=0.029) in infected peanut (Fig.1, Fig.2b, Table 1).
The differential expression mRNA in response to peanut root-knot nematode
After investigated the whole genome gene expression profile of peanut in response to root-knot nematodes, a total of 430 differential expression mRNAs (427 up-regulated and 3 down-regulated) were identified in response to peanut root-knot nematode (Fig. S2a, Table S1.). The length of differential expressed mRNA was range from 200bp to 12283 bp (with an average of 1672 bp) (Fig. 3c, Table S1).
Differentially expressed genes were distributed on 20 chromosomes. The number of differential expressed mRNAs (with an average of 21.5) was ranging from 11 on A02 chromosome to 35 on A01 and A03 chromosomes. The number of differential expressed genes was close in sub-A genome (213) and sub-B genome (217) (Fig S1). The functional annotation of differential expressed genes (329 out of 430) showed related to stress adaptation, such as heat shock cognate protein, cytochrome P450, pathogenesis-related protein, peroxidase and WRKY and MYB transcription factor (Table S1). The top GO annotation of differential expressed mRNAs was including defense response, oxidation-reduction process, signal transduction, response to wounding, nucleus, protein binding et al (Table S1).
The differential expression miRNAs and their target genes
To reveal the role of miRNA in response to root-knot nematodes, the expression profiles of miRNAs and target genes were identified. The length of differential expressed miRNA was ranged from 18 to 25 (with an average of 21.75) nucleotide (Fig.3, Table S2). A total of 80 miRNAs showed differential expressed between infected and no-infected by root-knot nematode. In addition, 36 out of 80 showed up-regulated in response to root-knot nematodes (Fig.S2, Table S2).
A total of 1771 mRNAs were the target genes of those 80 miRNAs. There were 111 out of 1771 target genes showed differential expressed in response to root-knot nematodes (Table S3). The 111 target genes annotation were disease resistance-like protein DSC1, peroxidase and WRKY transcription factor et al. According to the GO and KEGG analysis, target genes were involved in a series of adversity response processes, such as, plant-pathogen interaction, MAPK signaling pathway-plant and starch and sucrose metabolism (Table S3). The mRNAs-miRNAs regulatory networks contained miRNA (gma-MIR482c-p5_2ss12GA19CT) and mRNA (CTM7LX, JF37M9, NEIN3W, X5NWFC and Z9NEHU); miRNA (PC-3p-30685_85) and mRNA (AJ79N4, NK4UTA, R13KY7 and XA3BQV); miRNA (PC-3p-14080_193) and mRNA (42IH8X, DQ3LYR and S1GD6Q) were constructed (Fig.4a, Fig.S3).
CircRNAs acts as the sponge of miRNAs in response to peanut root-knot nematode
A total of 123 differential expressed circRNAs (with 60 up-regulated and 63 down-regulated) were detected in response to peanut root-knot nematodes (Fig.S2c, Fig.S4). From 1 on A03 chromosome to 15 on A09 chromosome (with an average of 6.15), the number of circRNAs is unevenly distributed on each chromosome (Fig.S4). A total of 6 (with 2 up-regulated and 4 down-regulated) out of 123 differential expressed circRNAs were predicted bind to 7 miRNAs (with 2 up-regulated and 5 down-regulated). The GO and KEGG analysis showed that the differential expressed circRNAs involving in defense response, response to oxidative stress, response to temperature stimulus et al (Table S4). The circRNAs-miRNAs regulatory networks were constructed by circRNAs (circRNA113 and circRNA442) and miRNA (gma-miR10420_L+1R-1); circRNAs (circRNA226) and miRNA (PC-3p-14080_193); circRNAs (circRNA320) and miRNA (gma-MIR482c-p5_2ss12GA19CT); circRNAs (circRNA43) and miRNA (ptc-miR393a-3p) (Fig.4b, Fig.S3).
The role of lncRNAs played in regulatory ceRNA under root-knot nematode stress
A total of 4453 significantly differentially expressed lncRNAs were identified by false discovery rate (FDR) < 0.05, among which 2909 differentially expressed lncRNAs were up-regulated, and 1544 differentially expressed lncRNAs were down-regulated (Fig.S2d, Table S5). The length of differentially expressed lncRNAs were ranged from 200 to 552581 (most of lncRNAs were 200-300 nucleotide) nucleotide (Fig 3d). There were 13 differential expressed lncRNAs (6 up-regulated and 7 down-regulated) bind to 6 miRNAs (with 3 up-regulated and 3 down-regulated). The lncRNAs-miRNAs regulator interactions were constructed by lncRNAs (MSTRG.12823, MSTRG. 17002, MSTRG.33245 and MSTRG.42738) and miRNA (PC-3p-14080_193 and PC-3p-30685_85); lncRNAs (MSTRG.2115, MSTRG.30601, MSTRG.30599 and MSTRG.31962) and miRNA (gma-MIR482c-p5_2ss12GA19CT); lncRNAs (MSTRG.3150 and MSTRG.37521) and miRNA (mtr-miR319a-3p_R+1) (Fig.4d, Fig S3).
The regulatory ceRNA network of lncRNA/circRNA-miRNA-mRNA in response to root-knot nematode stress
The whole genome of differential expressed lncRNA, circRNA, mRNA and miRNA were detected (Fig.3, Fig.S2, Table S1-S7). The key lncRNA/circRNA-miRNA-mRNA competing endogenous RNA tetraploid sub-network associated with root-knot nematode stress response was reconstruction (Fig.4, Fig. S3, Table S3, Table S6, Table S7). The first sub-network contained 6 lncRNAs (MSTRG.41742, MSTRG.43398, MSTRG.47397, MSTRG.55943, MSTRG.56279 and MSTRG.58883), 2 circRNAs (circRNA113 and circRNA442), 3 miRNAs (gma-miR10420_L+1R-1, vvi-MIR3630-p5_2ss19AG20CT_1 and PC-5p-7845_316) and 4 mRNAs (LMT1MP, J7B49U, 322B5E and K6N5RU). A total of two lncRNAs, one circRNA and two mRNA showed up-regulated in response to peanut root-knot nematode. Meanwhile, the number of lncRNAs, circRNAs, miRNAs and mRNAs showed down-regulated under root-knot nematode stress were four, one three and two, respectively. The second sub-network contains four lncRNAs (all down-regulated), one circRNA (down-regulated), one miRNA (up-regulated) and five mRNAs (three up-regulated and two donw-regulated). The final sub-network was constructed based on one down-regulated circRNA, one up-regulated miRNA and four mRNAs (two up-regulated and two down-regulated) (Fig.5, Fig.S3). Based on the GO and KEGG analysis, the RNAs in ceRNA network was involved in peroxidase activity, lignin biosynthetic process and oxidation-reduction process (Fig.6, Fig.S5).