Genotype- and tissue-specific miRNA profiles and their targets in three alfalfa (Medicago sativa L) genotypes

- Genotype- and tissue-specific miRNA profiles and their targets in three alfalfa (Medicago sativa L) genotypes.
- The profiles of the six abundantly expressed miRNA families (miR156, miR159, miR166, miR319, miR396 and miR398) were relatively similar between the whole plants, roots and shoots of these three alfalfa genotypes.
- Further, the generation and analysis of degradome libraries from the three alfalfa genotypes enabled confirmation of 69 genes as targets for 31 miRNA families in alfalfa..
- Full list of author information is available at the end of the article.
- 2018 Open Access This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0.
- In order to identify conserved miRNAs as well as novel miRNAs from alfalfa, we constructed and sequenced small RNA libraries from whole clonally propagated plants, roots and shoots of three alfalfa genotypes (Altet-4, NECS-141 and NF08ALF06).
- Three alfalfa genotypes NECS-141, Altet-4 and NF08ALF06 were evaluated in this study.
- The three alfalfa genotypes (NECS- 141, Altet-4 and NF08ALF06) were clonally propagated and grown in tissue culture.
- An additional 20 clonally propagated plants of these geno- types were grown in a Conviron growth chamber as previ- ously described and used to evaluate the tissue-specific expression of the miRNAs.
- Total RNA was isolated from the whole plants, roots and shoots of three alfalfa genotypes using TRIzol ® Reagent (Invitrogen), according to the manufacturer’ s instructions..
- To identify novel miRNAs, the presence of the miRNA-star (miRNA*) sequences coupled with the predictable hairpin-like struc- ture for the precursor sequences were used..
- After removal of the adapter sequences and low-quality reads, the total reads ranging between 11 to 42 million, and unique reads ranging between 1.8 to 8.5 million reads from these nine libraries were obtained (Table 1).
- How- ever, the quality of the small RNA library generated from the shoots of NF08ALF06 did not meet the threshold criteria, therefore only NECS-141 and Altet-4 were used for the miRNA analyses of shoot tissues..
- Specifically, miR172 levels in roots and shoots of the three genotypes were extremely low and in most cases was below 20 RPTM (Table 2).
- The majority of the miRNA families identified are 21 nt long, although some cases including miR2601 and miR2603 were represented by 22 nucleotides.
- MicroRNA profiles in alfalfa plants, roots and shoots A total of 100 known miRNA families were identified from the small RNA libraries of the three alfalfa geno- types (Table 2).
- The identification of these 79 miRNA families in alfalfa was based on their expression in M..
- Among the highly conserved miRNA families, miR166 was the most highly expressed family in seven of the eight samples that were surveyed in this study.
- Interestingly, miR160 was not sequenced from the roots of three alfalfa genotypes..
- Table 2 Identified miRNA families and their frequencies (reads per ten million [RPTM]) in whole plants, roots and shoots of three alfalfa genotypes (miRNA-stars were marked in bold).
- Table 2 Identified miRNA families and their frequencies (reads per ten million [RPTM]) in whole plants, roots and shoots of three alfalfa genotypes (miRNA-stars were marked in bold) (Continued).
- alfalfa genotypes.
- The miRNA-star sequences corresponding to the 12 of the 21 highly conserved miRNA families were also recov- ered from almost all libraries (Table 2).
- In shoots of NECS-141, miR168 star levels were slightly more than that of miR168, while both in whole plants and roots, the star levels were approximately half of the levels of miR168..
- The sequencing of the small RNAs from multiple tissues of three different alfalfa genotypes would allow us to iden- tify the novel miRNAs more confidently.
- Novel miRNA identification was dependent on sequencing of the miRNA complementary strand (miRNA-star) coupled with the predictable fold back structure for the primary miRNA transcript.
- Because a stable assembly of the tetraploid alfalfa genome sequence is not available, the small RNAs were mapped to the M.
- Mapping of the small RNAs from the three alfalfa genotypes onto the M.
- We have identified a total of 17 novel miRNAs which have been sequenced from all of the three genotypes (Table 3.
- Identification of miRNA targets in alfalfa.
- Although the alfalfa is one of the important legumes ag- ronomically, the genome sequencing and annotations are not available so far.
- Degradome sequencing was used in this study to identify the cleaved mRNA fragments cor- responding to the miRNA recognition sites in all three alfalfa genotypes.
- Approximately 30 million degradome reads were obtained from the transcripts of each of the alfalfa genotypes (Table 4) and these reads were analysed using SeqTar program.
- Specifically, five mem- bers of the squamosa promoter-binding-like protein (SPL) targeted by the miR156 family, five members of the auxin response factors targeted by both miR160 and miR167 families, five members of the apetala2 (AP2)-domain containing transcription factors, four members of the growth-regulating factor (GRFs) family targeted by miR396, two members of the TCP family transcription fac- tors targeted by miR319, and, a NAC domain-containing.
- transcription factor-like protein (NAC) targeted by miR164 [35].
- On the other hand, we have identified three potentially non-conserved targets (plastocyanin, protein disulphide isomerase and a hypothetical protein) for miR398 in three alfalfa genotypes.
- b The distribution of small RNA reads on the precursors of the novel miRNAs depicted in Fig.
- To increase the confidence in identification of targets for the non-conserved miRNAs that are usually expressed at low abundances and the cleavage frequencies on those targets are relatively low, we considered as ‘targets’ only those for which the cleavages were detected at least in two of the three alfalfa genotypes..
- Additionally, two other F-box pro- tein interaction domain protein genes were also identified as targets for miR2643 in alfalfa genotypes (Table 5)..
- Because some of the miRNA-stars are also highly expressed, we scrutinized the degradome reads for poten- tial cleavages on the transcripts that are complementary to the miRNA-stars.
- Auxin re- ceptors (TIR1 proteins) and ARFs targeted by miR393 and miR160, miR167, are components of the auxin signalling pathway that regulates several aspects of plant growth and development.
- Yet another interesting target include the F-box protein interaction domain proteins that are regulated by miR2643, one of the very abundantly expressed miRNA in alfalfa..
- Table 4 Mapping of the reads obtained from the degradome libraries.
- Table 5 miRNA targets identified in the degradome libraries generated from three alfalfa genotypes.
- Total reads is Total reads mapped to the cDNA of the gene.
- Altet-4 miR156e Medtr7g squamosa promoter-binding-like protein Altet-4 miR156a Medtr7g squamosa promoter-binding-like protein Altet-4 miR156a Medtr3g squamosa promoter-binding 13A-like protein Altet-4 miR159b Medtr8g MYB transcription factor.
- Altet-4 miR164d Medtr2g NAC transcription factor-like protein.
- Altet-4 miR164d Medtr8g protein transporter Sec61 subunit alpha-like protein Altet-4 miR167b-5p Medtr8g auxin response factor 2.
- Altet-4 miR169e-5p Medtr2g CCAAT-binding transcription factor Altet-4 miR171f Medtr0092s GRAS family transcription regulator Altet-4 miR172a Medtr4g AP2 domain transcription factor Altet-4 miR172a Medtr5g AP2 domain transcription factor.
- Altet-4 miR172a Medtr2g AP2-like ethylene-responsive transcription factor Altet-4 miR319d-3p Medtr2g TCP family transcription factor.
- Altet-4 miR319d-3p Medtr8g TCP family transcription factor Altet-4 miR393a Medtr1g transport inhibitor response-like protein Altet-4 miR393a Medtr7g transport inhibitor response 1 protein.
- Altet-4 miR396b-5p Medtr2g growth-regulating factor-like protein Altet-4 miR396b-5p Medtr5g growth-regulating factor.
- Altet-4 miR398a-3p Medtr8g protein disulfide isomerase (PDI)-like protein Altet-4 miR408-3p Medtr8g basic blue-like protein.
- Altet-4 miR408-3p Medtr8g plastocyanin-like domain protein Altet-4 miR408-3p Medtr8g plastocyanin-like domain protein Altet-4 miR408-5p Medtr3g phosphate-responsive 1 family protein Altet-4 miR1510a-5p Medtr2g disease resistance protein (TIR-NBS-LRR class) Altet-4 miR2199 Medtr7g helix loop helix DNA-binding domain protein Altet-4 miR2643a Medtr3g F-box protein interaction domain protein Altet-4 miR2643a Medtr6g F-box protein interaction domain protein Altet-4 miR4414a-5p Medtr3g BZIP transcription factor bZIP124.
- Altet-4 miR5752b Medtr8g PLATZ transcription factor family protein | Altet-4 miR7696a-5p Medtr1g PHD finger protein, putative.
- Altet-4 miR7696c-3p Medtr4g sulfate transporter-like protein.
- NF08ALF06 miR156e Medtr7g squamosa promoter-binding-like protein NF08ALF06 miR156a Medtr7g squamosa promoter-binding-like protein NF08ALF06 miR156h-3p Medtr7g heat shock transcription factor NF08ALF06 miR159b Medtr8g MYB transcription factor NF08ALF06 miR160c Medtr2g auxin response factor 1 NF08ALF06 miR160d Medtr1g auxin response factor 1 NF08ALF06 miR160d Medtr3g auxin response factor, putative NF08ALF06 miR164d Medtr2g NAC transcription factor-like protein.
- NF08ALF06 miR164d Medtr8g protein transporter Sec61 subunit alpha-like protein NF08ALF06 miR167b-5p Medtr8g auxin response factor 2.
- NF08ALF06 miR167a Medtr4g GRAS family transcription factor NF08ALF06 miR171f Medtr0092s GRAS family transcription regulator NF08ALF06 miR172a Medtr4g AP2 domain transcription factor NF08ALF06 miR172a Medtr5g AP2 domain transcription factor.
- NF08ALF06 miR172a Medtr2g AP2-like ethylene-responsive transcription factor NF08ALF06 miR172a Medtr4g AP2-like ethylene-responsive transcription factor NF08ALF06 miR172a Medtr7g AP2 domain transcription factor.
- NF08ALF06 miR319d-3p Medtr2g TCP family transcription factor NF08ALF06 miR319d-3p Medtr8g TCP family transcription factor NF08ALF06 miR393a Medtr1g transport inhibitor response-like protein NF08ALF06 miR393a Medtr7g transport inhibitor response 1 protein NF08ALF06 miR393a Medtr8g transport inhibitor response 1 protein NF08ALF06 miR396b-5p Medtr1g growth-regulating factor.
- NF08ALF06 miR396b-5p Medtr2g growth-regulating factor-like protein NF08ALF06 miR396b-5p Medtr5g growth-regulating factor.
- NF08ALF06 miR398a-3p Medtr8g protein disulfide isomerase (PDI)-like protein NF08ALF06 miR408-3p Medtr8g basic blue-like protein.
- NF08ALF06 miR2643a Medtr6g F-box protein interaction domain protein NF08ALF06 miR4414a-5p Medtr3g BZIP transcription factor bZIP124 NF08ALF06 miR5037c Medtr4g F-box protein interaction domain protein NF08ALF06 miR5213-5p Medtr4g TIR-NBS-LRR class disease resistance protein NF08ALF06 miR5238 Medtr3g pantothenate kinase.
- NF08ALF06 miR5752a Medtr8g PLATZ transcription factor family protein NF08ALF06 miR7696a-5p Medtr1g PHD finger protein, putative.
- NF08ALF06 miR7696c-3p Medtr3g endoplasmic reticulum vesicle transporter NF08ALF06 miR7696c-5p Medtr7g DEAD-box ATP-dependent RNA helicase-like protein NF08ALF06 miR7696d-5p Medtr3g hypothetical protein.
- NF08ALF06 miR7696c-3p Medtr4g sulfate transporter-like protein.
- NCES-141 miR156e Medtr7g squamosa promoter-binding-like protein NCES-141 miR156a Medtr7g squamosa promoter-binding-like protein NCES-141 miR156a Medtr8g squamosa promoter-binding 13A-like protein NCES-141 miR156a Medtr3g squamosa promoter-binding-like protein NCES-141 miR156h-3p Medtr7g heat shock transcription factor NCES-141 miR159b Medtr8g MYB transcription factor NCES-141 miR160c Medtr2g auxin response factor 1.
- NCES-141 miR164d Medtr2g NAC transcription factor-like protein.
- NCES-141 miR164d Medtr8g protein transporter Sec61 subunit alpha-like protein NCES-141 miR167b-5p Medtr8g auxin response factor 2.
- NCES-141 miR167a Medtr4g GRAS family transcription factor NCES-141 miR167b-3p Medtr4g auxin response factor 2.
- NCES-141 miR168a Medtr6g argonaute protein 1A.
- NCES-141 miR171f Medtr0092s GRAS family transcription regulator NCES-141 miR172a Medtr4g AP2 domain transcription factor NCES-141 miR172a Medtr5g AP2 domain transcription factor.
- NCES-141 miR172a Medtr2g AP2-like ethylene-responsive transcription factor NCES-141 miR172a Medtr4g AP2-like ethylene-responsive transcription factor NCES-141 miR319d-3p Medtr2g TCP family transcription factor.
- NCES-141 miR319d-3p Medtr8g TCP family transcription factor NCES-141 miR393a Medtr1g transport inhibitor response-like protein NCES-141 miR393a Medtr7g transport inhibitor response 1 protein.
- NCES-141 miR395j Medtr1g ATP sulfurylase.
- NCES-141 miR396b-5p Medtr1g growth-regulating factor NCES-141 miR396b-5p Medtr5g growth-regulating factor.
- The analyses of small RNA libraries from the whole plants, shoots and roots resulted in the identification of 100 miRNA families that included highly conserved miRNAs as well as miRNAs that are at least conserved between M..
- NCES-141 miR396b-5p Medtr8g growth-regulating factor-like protein.
- NCES-141 miR396a-5p Medtr3g TNP1.
- NCES-141 miR396a-5p Medtr8g Ulp1 protease family, carboxy-terminal domain protein NCES-141 miR397-5p Medtr7g laccase/diphenol oxidase family protein.
- NCES-141 miR398c Medtr4g plastocyanin-like domain protein.
- NCES-141 miR398a-3p Medtr8g protein disulfide isomerase (PDI)-like protein.
- NCES-141 miR398c Medtr5g hypothetical protein.
- NCES-141 miR408-3p Medtr8g basic blue-like protein.
- NCES-141 miR408-3p Medtr8g plastocyanin-like domain protein NCES-141 miR408-3p Medtr8g plastocyanin-like domain protein NCES-141 miR408-5p Medtr3g phosphate-responsive 1 family protein.
- NCES-141 miR482-3p Medtr5g disease resistance protein (CC-NBS-LRR class) family protein NCES-141 miR530 Medtr3g transmembrane amino acid transporter family protein NCES-141 miR1510a-5p Medtr7g CS domain protein.
- NCES-141 miR2643a Medtr3g F-box protein interaction domain protein NCES-141 miR4414a-5p Medtr3g BZIP transcription factor bZIP124 NCES-141 miR5037c Medtr4g F-box protein interaction domain protein NCES-141 miR5213-5p Medtr6g disease resistance protein (TIR-NBS-LRR class) NCES-141 miR5213-5p Medtr4g TIR-NBS-LRR class disease resistance protein NCES-141 miR5213-5p Medtr6g disease resistance protein (TIR-NBS-LRR class) NCES-141 miR5238 Medtr3g pantothenate kinase.
- NCES-141 miR5561-3p Medtr2g hypothetical protein.
- NCES-141 miR5752b Medtr8g PLATZ transcription factor family protein NCES-141 miR7696a-5p Medtr1g PHD finger protein, putative.
- NCES-141 miR7696c-5p Medtr7g DEAD-box ATP-dependent RNA helicase-like protein NCES-141 miR7696d-5p Medtr3g hypothetical protein.
- NCES-141 miR7696c-3p Medtr4g sulfate transporter-like protein.
- NCES-141 miR7696c-3p Medtr7g sulfate adenylyltransferase subunit 1/adenylylsulfate kinase NCES-141 miR7701-3p Medtr3g hypothetical protein.
- This article has been published as part of BMC Genomics Volume 19 Supplement 10, 2018: Proceedings of the 29th International Conference on Genome Informatics (GIW 2018): genomics.
- The full contents of the.
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- MicroRNA directs mRNA cleavage of the transcription factor NAC1 to downregulate auxin signals for Arabidopsis lateral root development.
- MicroRNA regulation of the CUC genes is required for boundary size control in Arabidopsis meristems.

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