Stem trichome small RNA-Seq from the 20 accessions

Total and Small RNA Isolation

Total RNA from stem trichomes (n = 1) were isolated using concentrated TRIzol reagent (Life Technologies). Total RNA was isolated using the E.Z.N.A.^® MicroElute RNA Clean Up Kit (Omega Bio-Tek). Briefly, TRIzol Reagent (Life Technologies) and chloroform was added according to the manufacturer's instructions. After centrifugation, the RNA-containing aqueous phase was collected, mixed with 1.5 volume of 100% ethanol and applied to a MicroElute spin column (Omega Bio-Tek). The column was washed according to the manufacturers's instructions: once with RWT buffer (Qiagen), once with RPE washing buffer (Qiagen) and finally with 80% ethanol. The RNA concentration was measured on a NanoDrop ND-2000 (Thermo Scientific) and RNA integrity was examined using the 2200 TapeStation System with Agilent RNA ScreenTapes (Agilent Technologies).

Total RNA was spiked with ERCCs spike-in mix 1 (Life Technologies) as well as a synthetic spike-in set for Size Range Quality Control (SRQC) together with an External Reference for Data Normalization (ERDN; Locati et al., 2015).  The total RNA was divided in a large and a small fraction. The large RNA fraction was bound to a mirVana™ spin column (mirVana™ miRNA Isolation Kit, Life Technologies) according to the manufacturer's instructions. Small RNAs (<200 nts) were purified from the flow-through by adding ethanol to a final concentration of 65% (v/v) and bound to an E.Z.N.A.^® MicroElute spin column. The column was washed once with RWT buffer, once with RPE buffer and once with 80% ethanol (Qiagen). The concentration and integrity of small RNA was examined as described above.

Next-Generation Sequencing

Bar-coded small RNA libraries were generated according to the manufacturer's protocols using the Ion Total RNA-Seq Kit v2 and the Ion Xpress™ RNA-Seq bar-coding kit (Life Technologies). The size distribution and yield of the bar-coded libraries were assessed using the 2200 TapeStation System with Agilent D1K ScreenTapes (Agilent Technologies). Sequencing templates were prepared on the Ion Chef™ System using the Ion PI Hi-Q Chef Kit (Life Technologies). Sequencing was performed on an Ion Proton™ System using Ion PI v3 chips (Life Technologies) according to the manufacturer's instructions.

References

Locati et al. 2015. Improving small RNA-seq by using a synthetic spike-in set for size-range quality control together with a set for data normalization. Nucleic Acids Res. (2015). 43(14):e89. doi: 10.1093/nar/gkv303. 

Table of genotypes used

| accession  | species                        | accession nr | synonym | origin      |
|------------|--------------------------------|--------------|---------|-------------|
| LA2172     | S. arcanum                     | TR0009       | -       | Peru        |
| LA1401*    | S. cheesmaniae f. minor        | EA00652      | -       | Ecuador     |
| LA1840     | S. chmielewskii                | -            | -       | unknown     |
| LA2695     | S. chmielewskii                | EA00759      | -       | Peru        |
| LA0407     | S. habrochaites f. glabratum   | EA00558      | -       | Ecuador     |
| LA1777     | S. habrochaites f. hirsutum    | EA00703      | -       | Peru        |
| PI134418   | S. habrochaites f. glabratum   | TR00015      | LYC38   | unknown     |
| LYC4       | S. habrochaites f. hirsutum    | TR00017      | -       | unknown     |
| LA1718     | S. habrochaites f. glabratum   | EA00699      | LYC4934 | Peru        |
| PI127826   | S. habrochaites f. hirsutum    | -            | -       | Peru        |
| LA1364     | S. huaylasense                 | TR00030      | -       | Peru        |
| Moneymaker | S. lycopersicum                | -            | C32     | Netherlands |
| LA4024     | S. lycopersicum                | TA209        | -       | unknown     |
| LA2386     | S. lycopersicoides             | -            | -       | Peru
| LA2133     | S. neorickii/L. parviflorum    | EA00729      | -       | Peru        |
| LA0735     | S. neorickii                   | TR00025      | LYC140  | unknown     |
| LA0716     | S. pennellii                   | EA00585      | -       | Peru        |
| LA1278     | S. peruvianum/pimpinellifolium | TR00005      | -       | unknown     |
| LA1954     | S. peruvianum                  | EA00713      | -       | Peru        |
| LA1578     | S. pimpinellifolium            | EA00674      | -       | Peru        |