Analysis of sRNA population

Altogether 188 million final clean reads were obtained out of 220 million raw reads after quality filtering and adapter trimming. Out of the final clean reads, 84.43% mapped to the genome. Approximately, 12.68% (23,848,593) and 0.94% (1,759,128) were mapped to the known and novel hairpin sequences, respectively (Table 1). The size distribution analysis of these small RNA sequences showed that majority of the reads were 21 to 25 nt in length (Fig. 1). The 24 nt long sequence size class was the most abundant in all the libraries, followed by 23, 21, 22 and 25 nt classes (Fig. 1).

Table 1 Summary of reads from twelve stolon small RNA libraries after adapter removal and filtering. The individual read-based statistics for whole miRNA analysis is also represented Full size table

Fig. 1 Size distribution of unique small RNA sequences identified in all twelve small RNA libraries. Samples LD4, LD7 and LD10 represents 4, 7 and 10 days during long-day induction respectively, whereas samples SD4, SD7 and SD10 represents 4, 7 and 10 days during short-day induction respectively Full size image

Identification of conserved and novel miRNAs

Deep-sequencing analyses using miRPRo showed that among total raw reads, on an average 4.6% of final clean reads were counted as conserved mature miRNAs, whereas 0.76% of final clean reads were counted as novel mature miRNAs. Overall, our study from 12 stolon libraries detected 324 conserved miRNAs belonging to 114 miRNA families, and 311 novel miRNAs (Table 1; Additional file 3: Table S2). Out of 311 novel miRNAs that were identified, star strand was detected for 270 miRNAs. Further, 173 miRNAs had star strand detected in at least two sequencing samples (Additional file 3: Table S2; Novel miRNAs sheet).

Differential expression analyses of miRNAs

Among the conserved miRNAs, only 7 were found to be differentially expressed between LD4 and SD4 conditions (LD4 vs SD4), whereas two miRNAs that were found to be differentially expressed in LD4 vs SD4 conditions, were also differentially expressed between LD10 and SD10 conditions (LD10 vs SD10) (Table 2; Additional file 4: Table S3; Additional file 1: Figure S2). For novel miRNAs, 10 were found to be differentially expressed between LD4 and SD4 comparisons (LD4 vs SD4), whereas two were differentially expressed between LD10 and SD10 conditions (LD10 vs SD10) (Table 2; Additional file 5: Table S4; Additional file 1: Figure S2). No miRNAs showed differential expression for LD7 and SD7 comparisons (LD7 vs SD7) among both conserved and novel miRNAs (Table 2; Additional file 1: Figure S2). Thus, in total, 19 miRNAs (7 conserved miRNAs and 12 novel miRNAs) were differentially expressed at either LD4 vs SD4 or LD10 vs SD10 comparisons.

Table 2 List of differentially expressed conserved as well as novel miRNAs identified from LD4 vs SD4 and LD10 vs SD10 stolon transition time points comparisons. Number of putative targets predicted by psRNA target for each miRNA is also represented with (E < 3.0). None of the miRNAs were differentially expressed at LD7 vs SD7 time point comparison Full size table

Time-course expression analysis of miRNAs

In total, 15 miRNAs (7 conserved and 8 novel), that were differentially expressed at either LD4 vs SD4 or LD10 vs SD10 comparisons, were tested by qRT-PCR for their validation. The expression data for all 15 miRNAs were highly correlated (R2 = 0.8305) between the RNA-seq and qRT-PCR analysis (Fig. 2), suggesting the reliability of the deep-sequencing data.

Fig. 2 Validation of deep-sequencing data by qRT-PCR analysis. Differentially expressed selective miRNAs were chosen for validation. a Validation of conserved miRNAs. b Validation of novel miRNAs. U6 was used as a reference gene for normalization of qRT-PCR data. The expression level of miRNAs under LD at corresponding time point was considered as 1 for measurement of relative expression at respective SD time-points. Data is represented as means from two biological replicates and three technical replicates. c A correlation analysis shows the relationship of miRNAs expression between the RNA-seq and qRT-PCR analysis Full size image

Two conserved miRNAs (miR477a-5p and miR477b-5p) showed significantly higher expression levels at SD4 and SD10 d time-points compared to the LD4 time-point, whereas miR477b-5p exhibited significant reduction in its expression at LD10 compared to LD4 (Fig. 3). MiR319-3p expression was significantly higher at SD4 compared to LD4, however its expression remained unchanged at other time points tested (Fig. 3). One novel miRNA (n-miR-147) showed a significant increase in its expression level under SD conditions compared to LD conditions at 4 and 7 d time-points (Fig. 3). The expression of a novel miRNA (n-miR-206) was significantly high under SD conditions than LD conditions at all three time points tested, but the difference was more distinct at 7 and 10 d than the 4 d time-point (Fig. 3).

Fig. 3 Expression analysis of novel and conserved miRNAs at 4, 7 and 10 d time-points under LD vs SD photoperiod conditions. For qRT-PCR analysis, U6 was used as a reference gene for normalization and the expression level of miRNAs at LD4 was considered as 1 for measurement of relative expression at other time-points. Error bars represent standard error of means from two biological replicates. For graphs, a dotted line represents LD condition, whereas a thick line represents SD condition. Asterisks (one and two) indicate significant differences at p < 0.05 and p < 0.01, respectively, using a Student’s t-test. ns = not significant at p < 0.05 Full size image

Novel miRNA, n-miR-139, exhibited significantly low expression at SD4 compared to LD4, but its expression significantly increased towards SD10 d time point (Fig. 3). MiRNA (miR8006-5p) showed a significant increase in its expression under SD conditions compared to LD conditions at 4 and 10 d time point compared to LD4; however, the expression level remained unchanged at 7 d time-point (Fig. 3). A conserved miRNA (miR482d-3p) and two novel miRNAs (n-miR-302 and n-miR-93) exhibited a distinct reduction in their expression levels at 4 d time-point under SD compared to LD conditions (Fig. 3). Moreover, miR482d-3p and n-miR-302 also showed reduced expression at SD7 and SD10 d time points, respectively (Fig. 3). Although miR479 showed a significant increase in its expression level under SD conditions at 4 d time-point, its relative expression levels remained significantly low under both LD and SD conditions at 10 d time-point (Fig. 3). A novel miRNA, n-miR276, exhibited a reduction in its expression level at 7 d time-point under SD conditions compared to LD4 (Fig. 3). Another novel miRNA (n-miR-40) had significantly high expression at SD4 as well as LD7 and SD7 time points when compared with LD4 (Fig. 3). In case of miR399g-3p, a significant reduction in its expression was observed under SD4 conditions than LD4 conditions (Fig. 3). A novel miRNA, n-miR221, showed a significant reduction in its expression at 4 d time-point under SD conditions; however, the expression level was significantly high at LD7 compared to LD4 time-point (Fig. 3).

GO analysis for predicted targets of miRNAs

Altogether, 1414 putative targets were predicted for 653 conserved/novel miRNAs (Additional file 6: Table S5). For differentially expressed conserved and novel miRNAs, several interesting genes were identified as putative targets, such as TEOSINTE BRANCHED 1, cycloidea and PCF transcription factors TCP2 and TCP4 (both targeted by miR319-3p), GRAS family transcription factors DELLA and SCARECROW (targeted by miR477a/b-5p and miR479, respectively), protein phosphatase 2c (targeted by miR8006-5p), cytochrome P450 (targeted by novel-miR-147) and methyl-transferase ASHR3 (targeted by novel-miR-221) (Additional file 6 Table S5; Targets of DE miRNAs sheet). GO analysis for 1414 putative targets categorised them into a total of 1970 GO terms. Of which, 1235 GO terms belongs to biological processes, 236 GO terms were included under cellular components, whereas 499 GO terms were categorised to molecular functions (Additional file 7: Table S6; Individual GO types sheet). In the biological process category, cellular, metabolic, response to stimulus, biological regulation, development, localization and signalling were most enriched, whereas several functions such as nucleic-acid binding TF activity, catalytic activity, regulation and electron carrier activity were greatly enriched in molecular functions category (Fig. 4). Moreover, in cellular component category, cell and cell part, membrane and organelle categories were highly enriched (Fig. 4).

Fig. 4 Gene Ontology (GO) categorization for predicted targets of novel and conserved miRNAs identified. GO terms were categorized into biological process, cellular components and molecular functions. GO terms with > 10 sequences were considered for preparing a graph in each category Full size image

Our GO analysis of putative miRNA targets clearly demonstrated that auxin, cytokinin (CK) and gibberellin (GA) metabolism related genes were identified as putative targets of conserved and novel miRNAs (Additional file 7: Table S6; Target gene annotation sheet). For example, auxin biosynthesis gene (indole-3-pyruvate monooxygenase YUCCA8), auxin signaling related genes (TRANSPORT INHIBITOR RESPONSE 1-like), auxin-responsive factors (SAUR71-like, SAUR32-like, ARF10/16) and auxin inducible gene (ARGOS) were identified as miRNA targets (Additional file 7: Table S6; Target gene annotation sheet; yellow highlighted). From CK biosynthesis genes, we identified cytokinin dehydrogenase, LOG10, zeatin O-glucosyltransferase, UDP-glycosyltransferase 708c1 as miRNA targets (Additional file 7: Table S6; Target gene annotation sheet; green highlighted). Additionally, potential CK transporters, such as equilibrative nucleotide transporter 3-like (ENT) and purine permease 1-like (PUP); one CK response regulator (ARR1-like) were also identified (Additional file 7: Table S6; Target gene annotation sheet; green highlighted). GA precursor (GGPP) biosynthesis enzyme such as farnesyl pyrophosphate synthase, and GA signaling regulator (DELLA RGL1-like) (Additional file 7: Table S6; Target gene annotation sheet; blue highlighted) were also identified as miRNA targets. miRNA targets were also from ethylene and abscisic acid (ABA) biosynthesis pathway (Additional file 7: Table S6; Target gene annotation sheet; brown highlighted). Several ABA response related genes, i.e. E3 ubiquitin- ligase LOG2, phosphatase 2C 55, EARLY RESPONSIVE TO DEHYDRATION 15-like and DEHYDRATION-INDUCED 19 homolog 5-like isoform X2, were also identified as targets (Additional file 7: Table S6; Target gene annotation sheet; brown highlighted). Epigenetic modifiers class (methyl- and acetyl-transferases) was also enriched as miRNA targets (Additional file 7: Table S6; Target gene annotation sheet; grey highlighted). Light-mediated response related genes were also found to be the targets of miRNAs e.g. cryptochrome 1, cryptochrome-1-like isoform X1, ultraviolet-B receptor UVR8, transcription factor PHYTOCHROME INTERACTING FACTOR 1 (PIF1-like), B-box zinc finger 19-like (Additional file 7: Table S6; Target gene annotation sheet; red highlighted). Number of genes associated to flowering, growth regulation, Cytochrome P450 like, Argonaut proteins, variety of transcription factors and kinase/phosphatases were also identified as miRNA targets (Additional file 7: Table S6; Target gene annotation sheet).

MiRNAs and their putative targets relationship

To investigate the correlation between the expressions of conserved or novel miRNAs with their putative targets, the expression levels of five miRNAs (chosen from differential expression analysis; Table 2), and their corresponding target genes were studied by real-time analysis at 4 d time-point under SD and LD conditions (Fig. 5). All five miRNAs showed an inverse correlation (R2 = − 0.2739) with their respective putative target mRNA levels (Fig. 5f), suggesting the reliability of the miRNA target prediction software used in the analysis. MiRNAs, such as miR477a-5p, miR319-3p, miR479 and n-miR-206 were up-regulated under SD conditions compared to LD photoperiod, whereas their respective targets (replication factor C [miR377a-5p], StTCP2 [miR319-3p], StGRAS [miR479], exostosin family protein [n-miR-206]) were downregulated (Fig. 5a-c; e). In case of n-miR-302, the expression was reduced, whereas its predicted target (metal dependent phopsphohydrolase HD domain containing protein) was up-regulated under SD conditions compared to LD photoperiod (Fig. 5d).

Fig. 5 Relationship between miRNAs and their predicted targets by qRT-PCR at 4 d time-point under LD vs SD photoperiod conditions. Conserved miRNAs and their targets - (a) Stu-miR477a-5p and its target replication factor C, (b) Stu-miR319-3p and its target StTCP2, (c) Stu-miR479 and its target StGRAS. Novel miRNAs and their targets – (d) Stu-novel-miR302 and its target metal dependent phophohydrolase HD domain containing protein, and (e) Stu-novel-miR206 and its target exostosin family protein. f A correlation analysis shows the inverse relationship between miRNA expression and their putative target genes. U6 and EIF3e were used for normalization of miRNAs and target genes, respectively. The expression level of respective miRNA or target gene at LD4 time point was considered as 1 to measure relative expression at SD4 time point. Error bars represent standard error of means from two biological replicates. Asterisks (one, two and three) indicate significant differences at p < 0.05, p < 0.01, p < 0.001, respectively, using a Student’s t-test. ns = not significant at p < 0.05 Full size image

Cleavage site mapping for targets of conserved miRNAs

A modified 5′ RLM-RACE analysis validated StARF10 as a target of miR160 with high cleavage frequency (7 of 7) at 10th/11th nucleotides position (Fig. 6a). Similarly, StGRAS (10 of 10) and StGAMYB (12 of 12) were found to be true targets of miR479 and miR319b, respectively (Fig. 6b-c). Moreover, it was found that StTm2 transcript was cleaved by miR6026-3p with a very low frequency (1/11) (Fig. 6d). Sequencing results for RACE cloning are shown in Additional file 1: Figure S6.

Fig. 6 5′ RLM-RACE for cleavage site mapping. Arrows show frequency of 5’ RACE clones showing cleavage sites and numbers represent fractions with proportions of clones showing these cleavage sites. a StARF10, which is a target of Stu-miR160 was used a positive control [35]. Two conserved miRNAs (stu-miR479 and stu-miR319b) were chosen to map cleavage site on StGRAS (b) and StGAMYB (c) transcription factors, respectively. d Cleavage site mapping for miR6026-3p is shown on StTm2 TAS-like locus. Alignment between mature miRNA (bold) and target gene sequences is shown. The transcript accession IDs are- StARF10: PGSC0003DMT400020874; StGRAS: PGSC0003DMT400031475; StGAMYB: PGSC0003DMT400058426, and StTm2: PGSC0003DMT400051269 Full size image

MiRNA target gene expression analysis

The expression profiling of nine interesting target genes was studied at 4, 7 and 10 d under LD/SD photoperiodic conditions. Some of these genes were selected based on the available literature about tuberization pathway and their potential involvement in this process. StASHR3, StSWI3 and StHT showed a significant reduction in their transcript level under SD at both 4 and 7 d time point, whereas their transcript levels (except StASHR3) were significantly high at SD10 time point compared to LD4 (Fig. 7). The mRNA levels of TCP transcription factors (StTCP2 and StTCP4) were significantly reduced at all three points under SD conditions compared to LD4 (Fig. 7). Another miRNA target, StPTB6 exhibited a significant increase in its expression under SD at all three time points tested, however, the effect was more enhanced at SD10 time point. The transcript abundance of a GA signaling components (StDELLA and StGAMYB) remained unchanged at all time points tested compared to LD4, except SD7 time point for StGAMYB; where its expression was significantly low at SD7. The mRNA level of StGRAS was significantly low at SD4 and SD10 time points, whereas it was significantly high under LD10 time point when compared to LD4 (Fig. 7).

Fig. 7 Expression analysis for selected target genes of novel and conserved miRNAs at 4, 7 and 10 d time-points under LD vs SD photoperiod conditions. EIF3e was used as a reference gene for normalization. The expression level of each target gene at LD4 was considered as 1 for measurement of relative expression at other time-points. Error bars represent standard error of means from two biological replicates. For graphs, a dotted line represents LD condition, whereas a thick line represents SD condition. Asterisks (one, two and three) indicating significant differences at p < 0.05, p < 0.01, p < 0.001, respectively, using a Student’s t-test. ns = not significant at p < 0.05 Full size image

Identification of potential TAS-like loci

Our data analysis (at p < 0.0001) resulted in identification of 830 putative TAS-like loci from all 12 stolon sample libraries. Out of these loci, 275 were found to be present in genic (Additional file 8: Table S7; line 2–276), whereas 555 in intergenic (Additional file 8: Table S7; line 276–831) regions of the potato genome. We observed that many putative TAS-like loci reside in genes associated with plant growth and development, such as PHO2, cytochrome P450 like TBP, auxin related genes (auxin:hydrogen symporter, AUX/IAA 2, TIR1 receptor), serine/threonine protein kinase pk23, P450 mono-oxygenase, F-box family proteins, AGO1–1, squamosa promoter binding protein, WD-repeat protein and COP1 homolog (Additional file 8: Table S7; yellow highlighted). Off 830 TAS-like loci, 24 were found to be targeted by either conserved (11) or novel (13) miRNAs, respectively (Table 3). From which, 16 were present in the intergenic regions, whereas 8 loci were present in the genic regions.

Table 3 List of TAS-like loci predicted to be cleaved by conserved or novel miRNAs. Twenty-four TAS-like loci with their genomic locations and miRNA cleaving them are listed. PGSC transcript IDs and annotation for TAS-like loci that are present in genic regions is also mentioned. Two TAS-like loci present within StTm2 and StPHO2 genes (captured in bold) were used for further experimental validations Full size table

Identification of phased siRNAs and their target analysis

We identified 59 phased siRNAs generated from 11 TAS-like loci triggered by conserved miRNAs (i.e. miR399a-f, miR482c, miR5303a-g, miR6026-3p, miR7983-5p, miR8005a-c, miR8008a, miR8009), whereas 131 phased siRNAs from 13 TAS-like loci targeted by novel miRNAs (i.e. novel-miR-13, novel-miR-41, novel-miR-52, novel-miR-53, novel-miR-71, novel-miR-72, novel-miR-124, novel-miR-171, novel-miR-204, novel-miR-213, novel-miR-230, novel-miR-263, novel-miR-272, novel-miR-276) (Table 3; Additional file 9: Table S8). To gain further insights, we identified targets of these predicted siRNAs (Additional file 9: Table S8; siRNA targets sheet). Altogether, 3441 targets were predicted using psRNA target finder (Additional file 10: Table S9; B2G analysis sheet). Further, GO analysis of targets showed that off 5918 GO terms, 1780 belongs to biological processes, 2210 terms included in molecular function category, whereas 1919 GO terms categorised in cellular components (Additional file 10: Table S9). All GO terms were further subjected to KEGG pathway analysis for functional reconstruction of targets. Altogether, we obtained 103 enriched pathways from our KEGG analysis (Additional file 10: Table S9; KEGG pathways sheet). It was also observed that siRNA target genes were enriched in starch and sucrose, fructose and mannose, purine and pyrimidine as well as in several amino acid metabolism (Additional file 10: Table S9; KEGG analysis sheet; grey highlighted).

Interestingly, two of the phased siRNAs from StTm2 and StRGA4 found to target key GA metabolic genes StGA2ox1 (Additional file 9: Table S8, line 862 of siRNA target sheet and Additional file 10: Table S9, line 2330 of B2G analysis sheet; yellow highlighted) and StGA3ox1 (Additional file 9: Table S8, line 4443 of siRNA target sheet and Additional file 10: Table S9, line 443 of B2G analysis sheet; yellow highlighted), respectively. Apart from this, many genes involved in various other hormones (auxin, CK, ABA and ethylene) transport, metabolism and signalling were also identified as targets of siRNAs (Additional file 10: Table S9). Different target genes of siRNAs also included calcium signalling related genes (CDPKs and calmodulin- and calcineurin- binding proteins) (Additional file 10: Table S9, B2G analysis sheet; green highlighted), and cell-cycle and cell-division associated genes (cyclin C5, cyclin D4/D6 and cyclin-dependent kinases/inhibitors) (Additional file 10: Table S9, B2G analysis sheet; brown highlighted). Additionally, genes encoding for different homeobox TFs, F-box proteins, early flowering 3, Dof zinc finger protein (StCDF4), POTH1, phloem mobile RNA binding protein (StPTB1), and zinc/ring finger proteins were found to be siRNA targets (Additional file 10: Table S9, B2G analysis sheet; blue highlighted).

Validation of exonic region TAS-like loci and detection of putative phased siRNAs

Two putative TAS-like loci (Chr09:15393324..15393575 and Chr02:33977738..33977989), that were predicted to be present within the genic regions of StTm2 (predicted target of miR6026-3p) and StPHO2 (predicted target of miR399a), respectively, were chosen for further analysis based on the targets of phased siRNAs they produce (Table S9). The partial transcript sequences of these two genes were detected in stolon samples through real-time PCR using primers on either side of predicted cleavage site (Fig. 8b). Subsequently, two phased siRNAs generated each from StTm2 (2 off 18 siRNAs) and StPHO2 (2 off 8 siRNAs) transcripts were also detected in stolon samples (Fig. 8b; Additional file 1: Figure S5) and were sequence confirmed. Further, a modified 5′ RLM-RACE confirmed StTm2 transcript as a target of miR6026-3p with a very low frequency (1/11) (Fig. 6d). Interestingly, we observed that one of the siRNAs generated from StTm2 locus, i.e. (−) siR8, cleaved its own locus with 10 of 11 cleavage frequency (Fig. 8c; Additional file 1: Figure S5), which might generate secondary siRNAs from StTm2 locus. Since predicted cleavage site of miR6026-3p on StTm2 locus is only 65 bp upstream to (−) siR8 generation/cleavage site, this could be the reason why the frequency of cleavage by miR6026 was very low (1/11) on this locus. From these results, it appeared that the efficiency of cleavage by (−) siR8 is higher (10/11) than that of miR6026-3p (1/11). Sequencing results for RACE cloning are shown in Additional file 1: Figure S6.

Fig. 8 a Graphical representation of StTm2 and StPHO2 TAS-like loci and the most abundant siRNAs generated from these two loci are shown. Phased siRNAs predicted to be generated from StTm2 and StPHO2. Eight siRNAs off 18, whereas 4 siRNAs off 8, with higher abundance values are shown for both loci, respectively. Black arrows pointing downwards represent predicted miRNA cleavage site in each locus. b Detection of two phased siRNAs (highlightd in bold in panel (a) on sense strand of StTm2 ([+]siR1 and [+]siR3) and two from antisense strand of StPHO2 ([−]siR3 and [−]siR4) by RT-PCR. c 5′ RLM-RACE assay confirms the cleavage of StTm2 by one of the siRNAs generated from itself i.e. (−)siR15. The siRNA cleavage frequency is also shown by downward arrows in black (10/11). Alignment between mature miRNA (bold) and target gene sequence is shown. d Expression analysis of StTm2 and StPHO2 at 4, 7 and 10 d time-points under LD vs SD photoperiod conditions. EIF3e was used as a reference gene for normalization. The expression level of respective target gene at LD4 was considered as 1 for measurement of relative expression at other time-points. Error bars represent standard error of means from two biological replicates. e Expression analysis for two phased siRNAs generated from StTm2 ([+]siR1 and [+]siR3) and two from StPHO2 ([−]siR3 and [−]siR4) loci at 4, 7 and 10 d time-points under LD vs SD photoperiod conditions. U6 was used as a reference gene for normalization. The expression level of respective siRNA at LD4 was considered as 1 for measurement of relative expression at other time-points. Error bars represent standard error of means from two biological replicates. For graphs, a dotted line represents LD condition, whereas a thick line represents SD condition. Asterisks (one, two and three) indicate significant differences at p < 0.05, p < 0.01, p < 0.001, respectively, using a Student’s t-test. ns = not significant at p < 0.05 Full size image

Time-course expression analysis of genic TAS-like loci and their siRNAs

To investigate if photoperiod has any influence on the expression of StTm2 and StPHO2, qRT-PCR assays were performed. StTm2 showed a significant reduction in transcript abundance at SD7, whereas its transcript level was significantly enhanced at SD10 time point, when compared to LD4 (Fig. 8d). A significant increase in expression level was observed for StPHO2 at LD7 and SD10 time points, whereas its transcript level remained significantly low at SD7 and LD10 time points compared to LD4 (Fig. 8d). Two siRNAs generated from StTm2 (+siR1 and + siR3) and two from StPHO2 (−siR3 and -siR4) TAS-like loci were selected for qRT-PCR analysis (Fig. 8e). From StTm2, both siRNAs (+siR1 and + siR3) showed a significant reduction under LD10 and SD10 time points compared to LD4. However, +siR3 levels were significantly high under SD4, LD7 and SD7 time points when compared to LD4 (Fig. 8e). From StPHO2, −siR3 exhibited a significant reduction in its expression under SD at 4 and 10 d time points, whereas a significant increase in its expression was observed at LD7 and LD10 compared to LD4 (Fig. 8e). -siR4 exhibited a significantly high expression at LD7 time point, but its expression was found to be significantly low at both LD and SD 10d time points compared to LD4 (Fig. 8e).

Conserved TAS loci in potato

In our analysis, one TAS locus (Chr01:37276576..37276827, hereafter referred as StTAS3) fulfilled the TAS3 criteria as described in Xia et al. [4] (Additional file 8: Table S7; line 21; red highlighted). This locus was present in the genic region of a hypothetical protein (PGSC0003DMT400034044) and contains two miR390 cleavage sites. When StTAS3 transcript was aligned to Arabidopsis thaliana AtTAS3b, Nicotiana tabacum NtTAS3a (1) and NtTAS3a (2), and Solanum lycopersicum SlTAS3, a close conservation across the plant species was observed. Moreover, two siRNAs regions StTAS3-siR1 (abundance value 134) and StTAS3-siR2 (abundance value 148) showed a high conservation in all the above mentioned plant species. These siRNAs were predicted to cleave StARF3 and StARF2 transcripts with expectancy values of 0.5 and 1.0, respectively (Fig. 9). Thus, TAS identified in our analysis appears to be a potential StTAS3 locus.

Fig. 9 a Alignment of TAS3 transcripts from Arabidopsis thaliana AtTAS3b, Nicotiana tabacum NtTAS3a (1) and NtTAS3a (2), Solanum tuberosum StTAS3 and Solanum lycopersicum SlTAS3. miR390 target sites (5′ and 3′) on all TAS3 transcripts are highlighted with red boxes. b Magnified view of the alignment of TAS3 transcripts from above mentioned plant species. StTAS3-siR1, with an abundance value of 134 and StTAS3-siR2, with an abundance value of 148, showed a close conservation across chosen plant species. c psRNATarget prediction shows that StTAS3-siR1 cleaves Auxin Response Factor StARF3, whereas StTAS3-siR2 cleaves StARF2 with a cleavage expectancy of E = 0.5 and E = 1.0, respectively. Accessions: StARF3, PGSC0003DMT400081282; StARF2, PGSC0003DMT400036765. d Alignment of Solanum tuberosum StTAS5 transcript with that of Solanum lycopersicum SlTAS5 transcript. miR482c/miR482a-3p target site is highlighted in red box. e Magnified view of TAS5 transcripts alignment from potato and tomato. StTAS5-siRNA (−), with an abundance value of 1734, showed a high conservation with tomato SlTAS5–3′ D12(−) Full size image

Apart from StTAS3, we also found TAS5 locus in potato (Chr06:5518510..5518761, hereafter referred as StTAS5), which reside in the NBS-coding resistance gene (PGSC0003DMT400022440), and this locus shared a close conservation with SlTAS5 from tomato (Additional file 8: Table S7; line 133; red highlighted). Moreover, the target site of miR482c/miR482a-3p on these loci was conserved. Similarly, StTAS5-siRNA[−], with abundance value of 1734, displayed a high conservation with a tomato SlTAS5–3′ D12(−) (Fig. 9).