The majority of 21-PHAS loci correspond to disease resistance proteins triggered by miR482

3. ARTICLE 3 - THE FLORAL DEVELOPMENT OF THE ALLOTETRAPLOID Coffea

3.3. Results

3.3.2 The majority of 21-PHAS loci correspond to disease resistance proteins triggered by miR482

Plant phasiRNAs can be processed from long noncoding RNAs (lncRNAs) or protein coding transcripts that, after a precise cleavage guided by a miRNA, generate secondary siRNAs by the activity of DICER-like enzymes. We used small RNA libraries from vegetative and reproductive organs to identify loci producing phasiRNAs in Coffea arabica.

We identified 173 21-PHAS loci, most of them from disease resistance (R) genes (Figure 1 - second internal cycle, green dots; Supplemental Table 2). We found miRNA triggering at least 51 21-PHAS, including 23 triggered by miR482 family members. In addition, 15 21-PHAS loci were found to be triggered by a putative novel MIR family, the candidate miR245889. Similarity based analysis suggests that this candidate diverged from the miR482 superfamily (Supplemental Figure 2).

Figure 1 Genome-wide distribution of MIRs, PHAS and PHAS-like loci in the allopolyploid Coffea arabica. The outermost cycle represents chromosomes from each parental ancestor;

from C. eugenioides (ceu) or C. canephora (ccp). First inner cycle; Chromosomal coordinates of miRNA precursors, black dots are conserved families while violet dots are putative novel.

Second inner cycle; green dots point out the chromosomal coordinates of 21-PHAS loci. Third inner cycle; point out the chromosomal coordinates of 24-PHAS loci. Innermost cycle; red dots point out the chromosomal coordinates of 24-PHAS-like loci

Our prediction of loci with phasing patterns allowed us to identify two candidate tasiRNA loci. Their miRNA target sites were similar to miR390 and miR828. Further manual analysis of those loci revealed that they are the respective orthologs of TAS3 and TAS4 (Supplemental figure 3). We did not find any orthologs for TAS1 or TAS2 in accordance with the lack of its conserved trigger, miR173. We also identified two DICER-like 2 (DCL2), one in chromosome 6e and another in 9c, as loci generating phasiRNAs. This phasing of DCL2 has been described before in Fabaceae ⁴⁴, and Solanaceae ⁴⁵. We were not able to identify a

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MIR 390 M Ir390

MIR 159 MIR 167 MIR1 67 MIR 167 MIR 27 1876

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2592 MIR1037 MIR

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6283 MIR

11479 MIR36740 MIR166 MIR10411 MIR

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IR26 19 M IR53

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IR4237 MIR MIR396 1446 MIR MIR171 M156 IR162 M IR162 M IR333194 M IR159 M IR169 M IR169 M IR169 MIR

169 MIR

11515 M IR2

629 M IR34

6958 MIR

9386 M IR3 5713

6 M IR 171 M IR53

76 M IR 39 7 M IR1 85 8 M IR 209 0 M IR 56 48 M IR 10 59 82 M IR 2662 M IR 9386 M IR 7509 M IR 3629 M IR 1075 M IR 171 M IR8 60 M IR

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M IR6143

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IR1 1178 MIR 7701

M IR4

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MIR 394 M IR18

85 M IR9568

M IR 29 26 M IR5

30 M IR8 30 M IR 109

88 M IR1 510 M IR6

15 6 M IR 977 3 M IR 111 08 M IR60

24 M IR 10 982 M IR7 82 2 M IR3 19 M IR2 454 91 M IR 482 M IR 48 2 M IR4 82 M IR 482 M IR 48 2 M IR 24 57 47 M IR 482 M IR 245 88 9 M IR3 98 M IR2 47400 M IR 39

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specific trigger of DCL2 phasing. It is possible that another small RNA is performing this task.

3.3.4 24-PHAS loci are not triggered by any expressed miRNA

The presence of 24-nt phasiRNAs was recently reported in many eudicots, and in those reports, these small RNAsare highly enriched in reproductive organs, specifically anthers ^46–48. Because several phased siRNA annotation methods can frequently mistake heterochromatic siRNAs with 24-PHAS loci ⁴⁹ we manually evaluated all putative PHAS loci with the IGV genome browser. Doing so, we were able to identify 189 24-PHAS (Figure 1 - third internal cycle with yellow dots; Supplemental Table 3). Of those loci, 56 overlap with annotated protein coding genes (PCG) that were mostly identified as ―uncharacterized proteins‖ by the similarity based local alignment tool BLASTP ⁵⁰. That way, these putative protein-coding loci could be lncRNAs that were misannotated. Meanwhile, other loci seem to be bonafide PCGs that, due to their proximity to the 24-PHAS loci, may be transcribed and processed as such. In addition, 58 of those phasiRNAs overlapped with transposable elements (TEs) such as Gypsy (34%) and hAT (22%).

Unexpectedly, we were unable to find any apparent miRNA triggers of the loci with the 24-nt phasing pattern. miR2275, broadly present in eudicots, is the usual trigger of 24-nt phasiRNA biogenesis ⁴⁷. However, it is known to be absent in some lineages such Brassicales, Caryophyllales, Cucurbitaceas and Lamiales. In Coffea arabica, we found four putative miR2275 precursor loci in the genome, but without evidence of expression in the analyzed organs. Something similar was previously reported for the Solanales tomato and petunia in which 24-nt reproductive phasiRNAs were found to be abundant in meiotic anthers, nevertheless, no apparent miRNA target site was present ⁴⁷. MEME analysis of the 189 Cofea arabica 24-PHAS loci showed that 181 of those loci contain 24-nt rich-A motifs, with statistical significance ⁵¹ (exemplified in the Supplemental Figure 4). However, no expressed miRNA (or any of the evaluated small RNA) has complementarity to those putative target sites once miRNAs tend to be poor in A-U stretches. It is also possible that due to the relatively weak pairing of adenine and thymine, with two hydrogen bonds instead of three from the guanine and cytosine, the double strand of DNA can be easily separated allowing transcription and further processed into double-stranded RNA. Then, further degradation in the precise 24-nt pattern by a yet unknown mechanism can take place.

3.3.5 Hundreds of loci present a 24 nt phasing pattern but could not be included in the

No documento GENE REGULATORY NETWORKS: CO-EXPRESSION MODULES OF PROTEIN CODING GENES AND SMALL RNAS GOVERNING ESSENTIAL BIOLOGICAL PROCESSES IN Coffea arabica L. (páginas 89-92)