Introduction {#Sec1}
============

Alfalfa (*Medicago sativa* L.) is an important forage legume species with global adaptation, high forage quality and the capacity for harvesting biomass multiple times during the growing season. Alfalfa is an autotetraploid (2n = 4x = 32), perennial outcrossing species with high levels of genetic diversity in cultivated and non-cultivated populations. Besides its use as a forage, alfalfa also has potential crop for biofuel production \[[@CR1]\]. Alfalfa has the capacity for symbiotic nitrogen fixation and can also contribute to reduce soil erosion \[[@CR2], [@CR3]\].

Endogenous non-coding RNAs of approximately 21--22 nucleotides represent plant miRNAs that silence gene expression by binding to complementary sequences of its target mRNA at the post-transcriptional level. Such targeting results in mRNA cleavage and degradation or repression of translation, with the former being more prevalent in plants \[[@CR4]--[@CR7]\]. The miRNA analyses in different plant species highlight the important regulatory roles of miRNAs in multiple organs (roots, stems, leaves and flowers), differentiation and development, leaf polarity, transition from juvenile to vegetative stages and vegetative to reproductive phases, and regulation of plant responses to biotic and abiotic stresses \[[@CR8]--[@CR10]\].

Several investigations have shown that plant miRNAs can be classified into conserved and novel lineage- or species-specific miRNAs. Conserved miRNAs and their corresponding target genes are commonly found in all or most angiosperms, with some also being described in gymnosperms as well as primitive land plants such as ferns \[[@CR11], [@CR12]\]. However, miRNA analysis in several legumes including *M. truncatula*, soybean (*Glycine max* L), chickpea (*Cicer arietinum* L.), common bean (*Phaseolus vulgaris*), and *Lotus japonicus* indicate the presence of miRNAs that seem to be specific to certain legumes and there could have important gene regulatory roles \[[@CR13]--[@CR19]\]. Although recent attempts were made to report miRNAs from alfalfa (*M. sativa*) \[[@CR20]--[@CR22]\], these do not include the discovery of novel miRNAs, and most importantly, the miRNA target genes have not been confirmed in this legume species. Understanding miRNAs and their target gene regulation in various tissues can provide further insights into the miRNA target networks operating in a tissue-specific manner in alfalfa.

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). The sequenced reads were mapped to known miRNAs in *M. truncatula*, deposited in the miRBase to identify and annotate the miRNAs in alfalfa. Degradome libraries were constructed and sequenced from these three genotypes to characterize the miRNA gene targets.

Materials and methods {#Sec2}
=====================

Plant materials and growth conditions {#Sec3}
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Three alfalfa genotypes NECS-141, Altet-4 and NF08ALF06 were evaluated in this study. NECS-141 is the genotype being used to sequence the tetraploid alfalfa genome \[[@CR23]\]. Altet-4 is an aluminum tolerant genotype used to develop a mapping population \[[@CR24]\]. NF08ALF06 is a commercial breeding line with good agronomic performance (Forage Genetics International). The three alfalfa genotypes (NECS-141, Altet-4 and NF08ALF06) were clonally propagated and grown in tissue culture. After 13 d of growth in rooting media, these were transferred to medium at pH 7 for 96 h as previously described \[[@CR25]\]. The rooting media contains 0.55 g/L Murashige & Skoog Basal Medium with Vitamins (PhytoTechnology \#M519), 1 ml Plant Preservative Mixture, PPM (PhytoTechnology), adjust the pH to 5.8, and add 12 g/L Gelzan. The plants were placed in a Conviron growth chamber (24 °C, 18 h /6 h day/night cycle, 100 μmol light intensity) for root development and growth. An additional 20 clonally propagated plants of these genotypes were grown in a Conviron growth chamber as previously described and used to evaluate the tissue-specific expression of the miRNAs. Tissue samples were harvested and immediately flash frozen in liquid nitrogen and stored at − 80 °C.

Small RNA library construction and sequencing {#Sec4}
---------------------------------------------

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. The quality of total RNA was monitored on 1% agarose gel and their concentrations were measured using Nanodrop spectrophotometer. Small RNA libraries were generated as described previously \[[@CR26]\] by following the protocol described for the Illumina Truseq® Small RNA Preparation kit (Illumina, San Diego, USA). Briefly, 1 μg total RNA per sample was ligated sequentially with 5′ and 3' RNA adaptors. The ligated products were converted into cDNAs and then amplified using PCR. The amplified products were sequenced using an Illumina Hiseq® Analyzer.

Identification of conserved and novel miRNAs {#Sec5}
--------------------------------------------

The raw sequencing reads were processed as follows: adaptor sequences were trimmed off from the raw reads to obtain small RNAs. These reads were then mapped to ribosomal RNA (rRNA), transfer RNA (tRNA), small nuclear RNAs (snRNA), and the aligned and mapped reads were not used for further analysis. The remaining reads were aligned to miRBase v 20 \[[@CR27]\] to identify miRNAs in *M. sativa*. The reads with 100% sequence identity were designated as conserved miRNA homologs. To identify novel miRNAs, the presence of the miRNA-star (miRNA\*) sequences coupled with the predictable hairpin-like structure for the precursor sequences were used.

Degradome library construction and analyses {#Sec6}
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Degradome libraries from the alfalfa genotypes NECS-141, Altet-4 and NF08ALF06 were constructed as previously described to identify potential target mRNAs \[[@CR28]\]. Briefly, the cleaved 5′ monophosphate containing polyadenylated mRNA fragments were ligated to an RNA oligo-nucleotide adapter containing *MmeI* recognition site at its 3′ end. The ligated products were converted into cDNA using reverse transcriptase and the product was amplified using only 5 PCR cycles. The PCR product was eluted, digested with *MmeI* restriction enzyme and then ligated to a double-stranded DNA adapter. The ligated product was again purified and amplified using 15 cycles of PCR. The final PCR product was sequenced. The reads were processed for quality and then aligned to the transcriptome assembly of *M. truncatula* to identify potential miRNA targets using the SeqTar pipeline \[[@CR29]\].

Results and discussion {#Sec7}
======================

The analyses of small RNA libraries {#Sec8}
-----------------------------------

High-throughput sequencing has been used to identify miRNAs and their target mRNAs in plants \[[@CR15], [@CR30], [@CR31]\]. To catalogue conserved and novel miRNAs in alfalfa, a total of eight small RNA libraries from the whole plants, roots and shoots of Altet-4, NECS-141 and NF08ALF06 genotypes were constructed and sequenced. 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](#Tab1){ref-type="table"}). However, 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.Table 1The mapping of total and unique reads obtained from different small RNA librariesAltet-4 whole plantsNECS-141 whole plantsNF08ALF06 whole plantsAltet-4 RootsNECS-141 RootsNF08ALF06 RootsAltet-4 ShootsNCES-141 ShootsTotal readsUnique readsTotal readsUnique readsTotal readsUnique readsTotal readsUnique readsTotal readsUnique readsTotal readsUnique readsTotal readsUnique readsTotal readsUnique readscdna6,858,719336,2666,197,308430,8198,142,985493,9299,352,477276,27619,665,339732,7388,930,063352,16717,117,689909,49110,943,411669,572ncRNAs6,810,937261,1345,666,067213,3967,722,727284,7859,633,993271,49618,454,661272,5968,834,028237,82714,718,199289,0877,855,629128,514pre-miRBase567,5183182943,97638881,162,2334005147,32624091,102,8795780426,32732132,520,49270513,840,7716449repeats5,451,840162,5524,218,992148,2975,756,349183,4188,267,063158,31015,744,403180,0307,536,403146,04310,855,687192,3773,798,312104,708genome8,951,4301,142,5949,878,8382,398,70511,387,4132,053,58211,557,742784,14029,143,5495,078,32211,588,8321,488,54628,744,2315,246,02729,192,0985,834,731total12,008,8922,343,12011,645,2173,348,18815,733,1023,739,16314,377,3361,860,73633,335,2016,947,62214,378,8592,708,73742,196,8888,564,21834,441,3137,748,996

Quantification of miRNA abundances between the genotypes and tissues was preceded by normalizing the expression levels of miRNA families to reads per ten million (RPTM). The normalized miRNA family read frequencies ranged between 1 to 552,267 RPTM for the whole plants, between 1 to 134,679 RPTM for the root samples, and 1 to 165,310 RPTM for the shoot samples (Table [2](#Tab2){ref-type="table"}). The range of miRNA read frequencies varied slightly between the three genotypes. As expected, the most conserved miRNAs appeared to be the most abundantly expressed in all tissues and genotypes, with the exception of miR169, miR393, miR395 and miR172 which exhibited low abundances. Specifically, miR172 levels in roots and shoots of the three genotypes were extremely low and in most cases was below 20 RPTM (Table [2](#Tab2){ref-type="table"}). The miRNA families with the lowest expression levels, and in some cases as low as 1 RPTM, were largely represented by the non-conserved miRNAs or miRNAs that have been reported exclusively from *M. truncatula* (miRBase) that include miR2601, miR2674, miR5207, miR5241, miR5243, miR5244, miR5255, miR5257, miR5269, miR5282, miR5289, miR5294, miR5296, miR5299, miR5561, miR5744, and miR7701 (Table [2](#Tab2){ref-type="table"}). miR5207 is the only miRNA that was also reported from *Gossypium raimondii* (miRBase). The majority of the miRNA families identified are 21 nt long, although some cases including miR2601 and miR2603 were represented by 22 nucleotides. Further, a total of 23 miRNA families included between miR5267 to miR5299 were 24 nt long. The fact that these small RNAs were initially identified in *M. truncatula* (miRBase), and could be identified in several independent small RNA libraries from three different alfalfa genotypes (Table [2](#Tab2){ref-type="table"}), suggests that these sequences and their associated processing are conserved between alfalfa and its close relative *M. truncatula*. However, their extremely low abundances coupled with their longer read lengths could also indicate that these may be 24-nt long siRNAs. Additional studies are needed to assess the precise nature of these small RNAs, i.e., miRNAs or siRNAs.Table 2Identified 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)Whole plantsRootsShootsAltet-4NECS-141NF08ALF06Altet-4NECS-141NF08ALF06Altet-4NECS-141miR156-5p47127243643610013466314519,80847,306**miR156-3p**32624012299254575554846346420miR159-3p631511,0508484391023,46510,54961,929103,370miR160-5p2254173512027711335058706miR162-3p140229292194454361533517miR164-5p1082753066775748431miR166-3p336,905552,267534,05434,634111,596134,679101,118131,196**miR166-5p**5449606142285084388001216miR167-5p2184707221072403576991389**miR167-3p**21000000miR168-5p1121198016917352960131734605049**miR168-3p**67269176818244319455505638miR169-5p1934354755354659**miR169-3p**7125618722miR171-3p51120232442383166085**miR171e-5p**26394422374276miR172-3p6213824001123**miR172-5p**382011222miR319-3p163136892101160762813323433010,864**miR319-5p**46727432014129559miR390-5p9541031886656234121382miR393-5p11243448102245miR395-3p3871213720miR396-5p12,18521,92622,411283514,549812139,23658,336**miR396-3p**25043743776312188323356miR397-5p5728153716119461**miR398a-5p**19162502143miR398-3p38143223227221014086317635,53826,478miR399-3p1711112526136243miR408-3p26561301109697773757063802866**miR408-5p**17712121485535miR482-3p28274918194541105**miR482-5p**71010111913912miR530-5p27801124miR1507--3963178917018811596123017783349miR1510-5p1959427835205233505142912,49634,705**miR1510-3p**961511675211863256617miR211147201044154227822miR2118560711,94816,13410661030779,977165,310miR219995154221183015613miR2585577742812223910miR2587069010101328miR2590154142235525109177miR25923931350395119161226812241742miR2601-5p00000011miR2603-5p081111524miR2629-5p25413725miR2632-5p010000118miR2634-3p537641596miR2643-3p1502268921063821462948968224,971miR2651-3p27522242174049miR2661-5p345245139miR2666-3p02100140029miR2674-3p00100000miR2678-3p264044412miR4414-3p24401137miR4414-5p13411057miR5037-5p4313382424miR5204-3p410632817610miR5205-5p72214066156miR5207-5p00000101miR5208-3p21100011miR5208d-5p00101011miR5211-5p4328523559714129259miR5213-5p80183688718189182913971379miR5214-3p6315515397414452153201miR5225-5p42831811miR5230-5p12101061miR5231-5p107731114369miR5232-5p67253419565034176023964miR5237-3p22002164miR5238-5p20212100miR5239-5p347269430165272622773miR5241-3p00000001miR5243-3p00000001miR5244-3p01100001miR5248-5p02102103miR5255-3p01100001miR5257-5p10000000miR5261-3p76899322302127283227miR5266-5p00042301miR5267-5p13101102miR5269-3p01110000miR5271-5p11112211miR5272-5p1722121234211818miR5273-3p13113142miR5277-3p60108627599481620miR5279-5p31913116887miR5281-3p294729356918141150miR5282-3p00000010miR5284-3p205250414171023miR5285-5p00111023miR5286-3p20213232miR5287-3p610148941719miR5289-3p01010000miR5290-3p05112126miR5291-3p01103101miR5292-3p163521634215382miR5294-3p00100000miR5295-3p92913315976miR5296-3p10000000miR5297-3p01210111miR5298-3p441401315miR5299-3p01001100miR5558-5p5391938182022041541211031276miR5559-5p73000085miR5561-3p5141805545miR5561-5p00010000miR5743-5p19113601170398miR5744-5p00000100miR5745-3p28394169144171126113miR5752-3p040010811miR5754-5p0619013241**miR7696-5p**01101001miR7696-3p17495253401381841173255miR7701-3p01000000

MicroRNA profiles in alfalfa plants, roots and shoots {#Sec9}
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A total of 100 known miRNA families were identified from the small RNA libraries of the three alfalfa genotypes (Table [2](#Tab2){ref-type="table"}). Of these, 21 families were represented by the highly conserved miRNAs, whereas the remaining 79 families could be considered as *Medicago*-specific miRNA families. The identification of these 79 miRNA families in alfalfa was based on their expression in *M. truncatula* (miRbase), therefore, these are conserved at least between *M. truncatula* and alfalfa.

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. The only exception to this trend was observed in the shoots of NECS-141 in which the miR2118 family was the most abundant followed by the miR166 family. The miRNA families, miR396 and miR2118 represents the second and third most abundantly expressed in the whole plants, while miR159 and miR396 were the second and third most highly expressed miRNAs in roots. Several additional miRNA families including miR398, miR160, miR168, miR319, miR408, miR1510 and miR2643 were also highly expressed but miR169, miR171, miR393, miR397 and miR395 were expressed at relatively very low levels (Table [2](#Tab2){ref-type="table"}). On the other hand, miR159, miR156, miR319, miR398 miR1507 and miR1510 were highly expressed but miR164, miR169, miR172, miR393, miR397, miR399 and miR482 were expressed at very low levels in roots of these genotypes. Interestingly, miR160 was not sequenced from the roots of three alfalfa genotypes.

Overall, the conserved miRNA families such as the miR156, miR159, miR166, miR168, miR319, miR396, miR398 and miR408 were more highly expressed in the plants, roots and shoots of all three alfalfa genotypes. Among the legume-specific families, miR1507, miR1510, miR2118, miR2592, miR2643, miR5213, miR5232, miR5558 and miR7696 (Table [2](#Tab2){ref-type="table"}) were also abundant in all tissues of alfalfa genotypes. Conversely, some conserved miRNA families represented by miR169 and miR393 recorded very low abundances in all samples. Other notable differences between roots and shoots include relatively low expression levels of miR160, miR167, and miR408 in roots compared to the shoots of alfalfa genotypes (Table [2](#Tab2){ref-type="table"}).

Several miRNA families including miR482, miR1507, miR2118, miR4416 are conserved in *M. truncatula*, soybean, chickpea (miRBase). These miRNA families are known to regulate NBS-LRR genes that are involved in pathogen resistance. The miRNA-guided cleavage on the NBS-LRR genes initiates the generation of phasiRNAs \[[@CR16], [@CR18], [@CR32]\]. In alfalfa, miR482, miR1507 and miR2118 were detected in all three tissues (Table [2](#Tab2){ref-type="table"}), but not miR4416. Both miR2118 and miR1507 families were more abundantly expressed in all tissues and genotypes compared with miR482 family. Remarkably, miR2118 was the top most highly expressed miRNA family in shoots of NECS-141. By contrast, miR2118 levels were very low in roots of three alfalfa genotypes. On the other hand, miR1507 family displayed approximately similar levels in three tissues of alfalfa genotypes.

The miRNA-star sequences corresponding to the 12 of the 21 highly conserved miRNA families were also recovered from almost all libraries (Table [2](#Tab2){ref-type="table"}). Additionally, miRNA-stars for the miR1510, miR4414, miR5208, and miR7696 were also detected. Furthermore, the miRNA-star expression levels for miR156, miR166 and miR168 were very high (Table [2](#Tab2){ref-type="table"}). Intriguingly, like miR168, miR168 star levels differed greatly between different tissue. 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.

miRNA diversity in alfalfa compared with other legumes {#Sec10}
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Several miRNA families are specifically reported from the leguminous plants such as the *M. truncatula, Glycine max*, *Lotus japonicus*, *Phaseolus vulgaris*, *Cicer arietinum*, *Vigna unguiculata* and *Acacia auriculiformis* \[[@CR14], [@CR16], [@CR18], [@CR19], [@CR32], [@CR33]\]. These lineage-specific miRNAs include miR1507, miR1508, miR1509, miR1510, miR1512, miR1514, miR1520, miR1521, miR2118, miR2086, miR2109, miR2199, miR4414, miR5213, miR5232, and miR5234 among others (miRBase). The majority of these were reported from *M. truncatula* and soybean, since these legume species have been the subject of multiple studies exploring small RNAs. Most of these legume-specific miRNAs were also identified in alfalfa and a few of them including miR1507, miR1510, miR2118, miR2592, miR2643, miR5211, miR5213, miR5214, miR5232, miR5239, miR5277, miR5558, and miR7696 were specifically highly expressed in all three genotypes (Table [2](#Tab2){ref-type="table"}).

Identification of novel miRNAs from alfalfa {#Sec11}
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The sequencing of the small RNAs from multiple tissues of three different alfalfa genotypes would allow us to identify 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. truncatula genome*. Mapping of the small RNAs from the three alfalfa genotypes onto the *M. truncatula* genome enabled the identification of novel miRNAs more confidently because they have been sequenced from *M. sativa* and mapped on to the *M. truncatula*, suggesting their conservation between *M. sativa* and *M. truncatula*. Moreover, the novel miRNA identification in this study is more robust as it includes sequencing of these small RNAs from three different genotypes. We have identified a total of 17 novel miRNAs which have been sequenced from all of the three genotypes (Table [3](#Tab3){ref-type="table"} and Fig. [1](#Fig1){ref-type="fig"}). Among these, t50582913 was highly expressed followed by t50063038. In roots, t50582913 was highly expressed in NECS-141 and Altet-4 but not in NF08ALF06. In shoots, t50063038 was highly expressed followed by the t50582913 and t51235783.Table 3Identified novel miRNAs based on sequencing both 5′ and 3′ reads and the most abundant ones that is marked in bold denotes potential novel miRNA based on their greater abundancesmiR-5pmiR-5p_seqmiR-3pmiR-3p_seqAltet-4 PlantsNECS-141 PlantsNF08ALF06 PlantsAltet-4 RootsNECS-141 RootsNF08ALF06 RootsAltet-4 ShootsNECS-141 Shootst61680599UUUCUUUGACUGGUUUUUGAAU**t21108041**CAAAAGCCUGUCAAUGAAAAUG031003120032t46402976UAGCAUCAAGCGUCGCGUCGAU**t28372577**CGACCCGAGGCUUAUGCGAUC1159714581479229335315t59820880UUGGCAGAAUCACGGUGUGCC**t29809748**CGGUGGCAUCGUGAUUUUGAC0625168147t21870702CAACUCGGUCCUUCUGUUAAC**t44359413**UAACAGAAGGACUGAGUUGCC01131411224103t62603216UUUUCAAGUUGGUCCCUUACG**t44814359**UAAGGGACCAACUUGAAAACU771781967240107529899t8901469ACCUGGAGACAGAGAUGCAAU**t45832108**UACGUCUCUGUCUUUCGGGUUG155282222286247t12927907AGGAUAACAAUGUUGCAUAAG**t47767430**UAUGUAGCACUGUUUUUCUGA1343851427314783262**t63076572**UUUUUAGAUACAUUGAAUAAUt47960370UAUUCAAUGUAUCUAAAAAG1010144402208177t53501433UGAUUAUUCUACUACCCGGACC**t50063038**UCCGGGUAGCAGAAUAAUCAUC35037178453711817,05720,494t12458129AGCGGUUGGUACAAUGCAAUAu**t50582913**UCGCCUUGUACCAACCUACUGC5449150123114808811453t40560414GGUCCUGAUGUUUUUUAGAGC**t51235783**UCUCAAAGACAUAAGGAACCUC1928102476202691655t55270980UGUCUUUAGCUUCCGAAACAa**t55621674**UGUUCCGGUAGAUGAAGUCAC44022302440t14211567AGUUAAUUGUGUUGCAUGAGUU**t57726911**UUCAGCAACAUGAGUUAACUCA172660348224250**t8194733**ACAUUUUAGAUUGUUGAGGAAt27568341CCUCAAUUAUCUAUUAUGUUU00303660**t62313817**UUUGUUAAACAUUUGUUUCCt311560AAAACAAAUGUUUAGCUAAG0600151012t55268921UGUCUUGGUUUCAAAAAGAAGu**t52170136**UCUUUUUGCAAACCAACUCAAU419131294956**t51870988**UCUUAUUUUCGACAUUGCAAAGt59475847UUGCAGGUCGAGAAUAAAAUG1999711913531072Fig. 1The predicted foldback structures using the novel miRNA precursor sequences. **a** The fold-back structures for six novel miRNAs. **b** The distribution of small RNA reads on the precursors of the novel miRNAs depicted in Fig. 1a

Identification of miRNA targets in alfalfa {#Sec12}
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Although the alfalfa is one of the important legumes agronomically, the genome sequencing and annotations are not available so far. Due to this, studies have utilized the well-studied and closely related *M. truncatula* genome annotations as a model for alfalfa studies. The nucleotide identity for some genes was greater than 97% between *M. sativa* and *M. truncatula* \[[@CR34]\]). Thus, using *M. truncatula* transcript annotations can facilitate identification of miRNA targets in alfalfa. We used SeqTar algorithm (Zheng et al., 2012) to identify miRNA targets by allowing a maximum of 4 mismatches between miRNAs and their potential target transcripts.

Previous studies have revealed that conserved miRNAs are strongly associated with the regulation of genes that encode transcription factors \[[@CR35]\]. These transcription factors in turn regulate key developmental processes and pathways in plants. Degradome sequencing has been very effective in identifying plant miRNA targets. Besides identifying the conserved targets, this approach can also identify non-conserved targets for the conserved miRNAs \[[@CR28], [@CR36], [@CR37]\]. Degradome sequencing was used in this study to identify the cleaved mRNA fragments corresponding 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](#Tab4){ref-type="table"}) and these reads were analysed using SeqTar program. In total, we have identified 69 targets for 31 miRNA families that included 16 highly conserved families (Table [5](#Tab5){ref-type="table"}). With respect to the conserved miRNAs, 33 targets for 16 conserved miRNA families were identified (Table [5](#Tab5){ref-type="table"}). The known targets for miR162, miR165/166, miR398 and miR399 families were not identified in this study. Although miR165/166 family is the most abundantly expressed as scored from their read frequencies in almost all libraries but the cleaved fragments from the HD-Zip target transcripts were not recovered from degradome libraries of alfalfa genotypes.Table 4Mapping of the reads obtained from the degradome librariesDatabaseAltet-4NECS-141NF08ALF06Total readsUnique readsTotal readsUnique readsTotal readsUnique reads*M. truncatula* genome852,790487,5821,541,055791,2943,091,8321,021230*M. sativa* genome1,488,681957,8662,691,7631,435,6594,591,1301,877,041Cds770,970426,2781,436,059727,3302,928,098933,425ncRNA231,07622,907186,81318,0141,305,68136,585Repeats171,35816,804116,74116,759636,67523,958Pre-miRBase34,63183735,979104550,1361192Total28,674,6782,286,69330,573,2703,137,32730,812,6063,885,547Table 5miRNA targets identified in the degradome libraries generated from three alfalfa genotypes. \#Mis. is number of mismatches on the miRNA complementary site; Valid reads is Reads corresponding to the expected cleavage site; Total reads is Total reads mapped to the cDNA of the gene; Percent is Percent reads at the expected cleavage sitegenotypesmiRNA id\#Target gene\#Mis.Valid readsTotal readsPercentTarget gene annotationAltet-4miR156eMedtr7g028740.2042317.4squamosa promoter-binding-like proteinAltet-4miR156aMedtr7g444860.102287.1squamosa promoter-binding-like proteinAltet-4miR156aMedtr3g099080.101333.3squamosa promoter-binding 13A-like proteinAltet-4miR159bMedtr8g042410.12.51166.3MYB transcription factorAltet-4miR160cMedtr2g094570.3142119.1auxin response factor 1Altet-4miR164dMedtr2g064470.112345.9NAC transcription factor-like proteinAltet-4miR164dMedtr8g058330.1254910.2protein transporter Sec61 subunit alpha-like proteinAltet-4miR167b-5pMedtr8g079492.344626.5auxin response factor 2Altet-4miR169e-5pMedtr2g099490.221205CCAAT-binding transcription factorAltet-4miR171fMedtr0092s0100.21.552420.8GRAS family transcription regulatorAltet-4miR172aMedtr4g094868.311137.7AP2 domain transcription factorAltet-4miR172aMedtr5g016810.211185.6AP2 domain transcription factorAltet-4miR172aMedtr2g093060.3041723.5AP2-like ethylene-responsive transcription factorAltet-4miR319d-3pMedtr2g078200.132345.9TCP family transcription factorAltet-4miR319d-3pMedtr8g463380.132728.6TCP family transcription factorAltet-4miR393aMedtr1g088950.11118313.3transport inhibitor response-like proteinAltet-4miR393aMedtr7g083610.123813428.4transport inhibitor response 1 proteinAltet-4miR395jMedtr1g102550.111761.3ATP sulfurylaseAltet-4miR396b-5pMedtr1g017490.234710047growth-regulating factorAltet-4miR396b-5pMedtr2g041430.3351241.7growth-regulating factor-like proteinAltet-4miR396b-5pMedtr5g027030.1351533.3growth-regulating factorAltet-4miR396a-5pMedtr3g052060.1211100hypothetical proteinAltet-4miR398cMedtr4g114870.1382334.8plastocyanin-like domain proteinAltet-4miR398a-3pMedtr8g064810.1353613.9protein disulfide isomerase (PDI)-like proteinAltet-4miR408-3pMedtr8g089110.133933.3basic blue-like proteinAltet-4miR408-3pMedtr8g007020.13.55736.9plastocyanin-like domain proteinAltet-4miR408-3pMedtr8g007035.13.551234.1plastocyanin-like domain proteinAltet-4miR408-5pMedtr3g074830.13.524420.5phosphate-responsive 1 family proteinAltet-4miR1510a-5pMedtr2g012770.111520disease resistance protein (TIR-NBS-LRR class)Altet-4miR2199Medtr7g080780.222825helix loop helix DNA-binding domain proteinAltet-4miR2643aMedtr3g010590.111156.7F-box protein interaction domain proteinAltet-4miR2643aMedtr6g053240.132450F-box protein interaction domain proteinAltet-4miR4414a-5pMedtr3g117120.143843.6BZIP transcription factor bZIP124Altet-4miR5213-5pMedtr6g084370.121250disease resistance protein (TIR-NBS-LRR class)Altet-4miR5213-5pMedtr6g088245.131520disease resistance protein (TIR-NBS-LRR class)Altet-4miR5239Medtr3g018680.131520F-box/RNI superfamily protein, putativeAltet-4miR5561-3pMedtr2g045295.131425hypothetical proteinAltet-4miR5752bMedtr8g066820.1494232.1PLATZ transcription factor family protein \|Altet-4miR7696a-5pMedtr1g072130.132277.4PHD finger protein, putativeAltet-4miR7696c-3pMedtr3g081480.132219.5endoplasmic reticulum vesicle transporterAltet-4miR7696d-5pMedtr3g112250.13.583622.2hypothetical proteinAltet-4miR7696c-3pMedtr4g011600.23.51263.9sulfate transporter-like proteinAltet-4miR7696c-3pMedtr7g085650.43.51616.7sulfate adenylyltransferase subunit 1/adenylylsulfate kinaseAltet-4miR7701-3pMedtr6g011380.2211370.7SPFH/band 7/PHB domain membrane-associated family proteinNF08ALF06miR156eMedtr7g028740.20143638.9squamosa promoter-binding-like proteinNF08ALF06miR156aMedtr7g444860.1011440.7squamosa promoter-binding-like proteinNF08ALF06miR156h-3pMedtr7g091370.131119.1heat shock transcription factorNF08ALF06miR159bMedtr8g042410.12.543013.3MYB transcription factorNF08ALF06miR160cMedtr2g094570.3184617.4auxin response factor 1NF08ALF06miR160dMedtr1g064430.20.532412.5auxin response factor 1NF08ALF06miR160dMedtr3g073420.10.521711.8auxin response factor, putativeNF08ALF06miR164dMedtr2g064470.114115127.2NAC transcription factor-like proteinNF08ALF06miR164dMedtr8g058330.1251154.4protein transporter Sec61 subunit alpha-like proteinNF08ALF06miR167b-5pMedtr8g079492.3491336.8auxin response factor 2NF08ALF06miR167aMedtr4g076020.13.55776.5GRAS family transcription factorNF08ALF06miR171fMedtr0092s0100.21.56011552.2GRAS family transcription regulatorNF08ALF06miR172aMedtr4g094868.311452.2AP2 domain transcription factorNF08ALF06miR172aMedtr5g016810.211841.2AP2 domain transcription factorNF08ALF06miR172aMedtr2g093060.3043511.4AP2-like ethylene-responsive transcription factorNF08ALF06miR172aMedtr4g061200.411283.6AP2-like ethylene-responsive transcription factorNF08ALF06miR172aMedtr7g100590.1121711.8AP2 domain transcription factorNF08ALF06miR319d-3pMedtr2g078200.1321261.6TCP family transcription factorNF08ALF06miR319d-3pMedtr8g463380.132484.2TCP family transcription factorNF08ALF06miR393aMedtr1g088950.115426820.2transport inhibitor response-like proteinNF08ALF06miR393aMedtr7g083610.1247277161.2transport inhibitor response 1 proteinNF08ALF06miR393aMedtr8g098695.241462.2transport inhibitor response 1 proteinNF08ALF06miR396b-5pMedtr1g017490.2342374257growth-regulating factorNF08ALF06miR396b-5pMedtr2g041430.33307540growth-regulating factor-like proteinNF08ALF06miR396b-5pMedtr5g027030.13104223.8growth-regulating factorNF08ALF06miR396a-5pMedtr3g011560.131333.3TNP1NF08ALF06miR396a-5pMedtr3g052060.1231127.3hypothetical proteinNF08ALF06miR396a-5pMedtr8g017000.131250Ulp1 protease family, carboxy-terminal domain proteinNF08ALF06miR398cMedtr4g114870.13144928.6plastocyanin-like domain proteinNF08ALF06miR398a-3pMedtr8g064810.1384418.2protein disulfide isomerase (PDI)-like proteinNF08ALF06miR408-3pMedtr8g089110.1383423.5basic blue-like proteinNF08ALF06miR408-3pMedtr8g007020.13.5103752.7plastocyanin-like domain proteinNF08ALF06miR408-3pMedtr8g007035.13.5106751.5plastocyanin-like domain proteinNF08ALF06miR408-5pMedtr3g074830.13.5279482.9phosphate-responsive 1 family proteinNF08ALF06miR482-5pMedtr1g064430.23.51244.2auxin response factor 1NF08ALF06miR530Medtr3g072110.12.531022.9transmembrane amino acid transporter family proteinNF08ALF06miR1507--3pMedtr8g036195.124944.4NBS-LRR type disease resistance proteinNF08ALF06miR1510a-5pMedtr7g108860.43.52110612CS domain proteinNF08ALF06miR2199Medtr7g080780.221263.9helix loop helix DNA-binding domain proteinNF08ALF06miR2643aMedtr6g053240.13253375.8F-box protein interaction domain proteinNF08ALF06miR4414a-5pMedtr3g117120.1482603.1BZIP transcription factor bZIP124NF08ALF06miR5037cMedtr4g070550.132444.6F-box protein interaction domain proteinNF08ALF06miR5213-5pMedtr4g014580.11.53319.7TIR-NBS-LRR class disease resistance proteinNF08ALF06miR5238Medtr3g077740.22.512590.4pantothenate kinaseNF08ALF06miR5239Medtr3g018680.134439.3F-box/RNI superfamily protein, putativeNF08ALF06miR5561-3pMedtr2g045295.131128.3hypothetical proteinNF08ALF06miR5752aMedtr8g066820.14139361.4PLATZ transcription factor family proteinNF08ALF06miR7696a-5pMedtr1g072130.1342591.5PHD finger protein, putativeNF08ALF06miR7696c-3pMedtr3g081480.132464.4endoplasmic reticulum vesicle transporterNF08ALF06miR7696c-5pMedtr7g076830.1331032.9DEAD-box ATP-dependent RNA helicase-like proteinNF08ALF06miR7696d-5pMedtr3g112250.13.553016.7hypothetical proteinNF08ALF06miR7696c-3pMedtr4g011600.23.511031sulfate transporter-like proteinNF08ALF06miR7696c-3pMedtr7g085650.43.521020sulfate adenylyltransferase subunit 1/adenylylsulfate kinaseNF08ALF06miR7701-3pMedtr3g108910.12.523750.5hypothetical proteinNF08ALF06miR7701-3pMedtr6g011380.222862.3SPFH/band 7/PHB domain membrane-associated family proteinNCES-141miR156eMedtr7g028740.20184639.1squamosa promoter-binding-like proteinNCES-141miR156aMedtr7g444860.1041014squamosa promoter-binding-like proteinNCES-141miR156aMedtr8g096780.101119.1squamosa promoter-binding 13A-like proteinNCES-141miR156aMedtr3g085180.111250squamosa promoter-binding-like proteinNCES-141miR156h-3pMedtr7g091370.132540heat shock transcription factorNCES-141miR159bMedtr8g042410.12.53368.3MYB transcription factorNCES-141miR160cMedtr2g094570.31123732.4auxin response factor 1NCES-141miR164dMedtr2g064470.113310033NAC transcription factor-like proteinNCES-141miR164dMedtr8g058330.121411911.8protein transporter Sec61 subunit alpha-like proteinNCES-141miR167b-5pMedtr8g079492.34101019.9auxin response factor 2NCES-141miR167aMedtr4g076020.13.54458.9GRAS family transcription factorNCES-141miR167b-3pMedtr4g124900.23.511540.7auxin response factor 2NCES-141miR168aMedtr6g477980.2422450.8argonaute protein 1ANCES-141miR171fMedtr0092s0100.21.5367051.4GRAS family transcription regulatorNCES-141miR172aMedtr4g094868.312504AP2 domain transcription factorNCES-141miR172aMedtr5g016810.212563.6AP2 domain transcription factorNCES-141miR172aMedtr2g093060.301195.3AP2-like ethylene-responsive transcription factorNCES-141miR172aMedtr4g061200.413329.4AP2-like ethylene-responsive transcription factorNCES-141miR319d-3pMedtr2g078200.131551.8TCP family transcription factorNCES-141miR319d-3pMedtr8g463380.131263.9TCP family transcription factorNCES-141miR393aMedtr1g088950.113822217.1transport inhibitor response-like proteinNCES-141miR393aMedtr7g083610.1233753962.5transport inhibitor response 1 proteinNCES-141miR395jMedtr1g102550.1111630.6ATP sulfurylaseNCES-141miR396b-5pMedtr1g017490.2320135257.1growth-regulating factorNCES-141miR396b-5pMedtr5g027030.1361637.5growth-regulating factorNCES-141miR396b-5pMedtr8g020560.131714.3growth-regulating factor-like proteinNCES-141miR396a-5pMedtr3g011560.1311100TNP1NCES-141miR396a-5pMedtr8g017000.1311100Ulp1 protease family, carboxy-terminal domain proteinNCES-141miR397-5pMedtr7g062310.11.52450laccase/diphenol oxidase family proteinNCES-141miR398cMedtr4g114870.1382138.1plastocyanin-like domain proteinNCES-141miR398a-3pMedtr8g064810.13478952.8protein disulfide isomerase (PDI)-like proteinNCES-141miR398cMedtr5g089180.1341921.1hypothetical proteinNCES-141miR408-3pMedtr8g089110.1391850basic blue-like proteinNCES-141miR408-3pMedtr8g007020.13.572093.4plastocyanin-like domain proteinNCES-141miR408-3pMedtr8g007035.13.583812.1plastocyanin-like domain proteinNCES-141miR408-5pMedtr3g074830.13.5147032phosphate-responsive 1 family proteinNCES-141miR482-3pMedtr5g027900.12.51195.3disease resistance protein (CC-NBS-LRR class) family proteinNCES-141miR530Medtr3g072110.12.511190.8transmembrane amino acid transporter family proteinNCES-141miR1510a-5pMedtr7g108860.43.5177462.3CS domain proteinNCES-141miR2643aMedtr3g010620.112722.8F-box protein interaction domain proteinNCES-141miR4414a-5pMedtr3g117120.1421341.5BZIP transcription factor bZIP124NCES-141miR5037cMedtr4g070550.131362.8F-box protein interaction domain proteinNCES-141miR5213-5pMedtr6g084370.121520disease resistance protein (TIR-NBS-LRR class)NCES-141miR5213-5pMedtr4g014580.11.51185.6TIR-NBS-LRR class disease resistance proteinNCES-141miR5213-5pMedtr6g088245.131714.3disease resistance protein (TIR-NBS-LRR class)NCES-141miR5238Medtr3g077740.22.511510.7pantothenate kinaseNCES-141miR5561-3pMedtr2g045295.131911.1hypothetical proteinNCES-141miR5752bMedtr8g066820.1487651.1PLATZ transcription factor family proteinNCES-141miR7696a-5pMedtr1g072130.1321351.5PHD finger protein, putativeNCES-141miR7696c-5pMedtr7g076830.135786.4DEAD-box ATP-dependent RNA helicase-like proteinNCES-141miR7696d-5pMedtr3g112250.13.594420.5hypothetical proteinNCES-141miR7696c-3pMedtr4g011600.23.511240.8sulfate transporter-like proteinNCES-141miR7696c-3pMedtr7g085650.43.511010sulfate adenylyltransferase subunit 1/adenylylsulfate kinaseNCES-141miR7701-3pMedtr3g108910.12.524440.5hypothetical protein

The identified miRNA targets in all three genotypes include mainly transcription factors. Specifically, five members 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 factors targeted by miR319, and, a NAC domain-containing transcription factor-like protein (NAC) targeted by miR164 \[[@CR35]\]. Additionally, transcripts encoding Argonaute targeted by miR168, laccase targeted by miR397, and three plantacyanin containing proteins targeted by miR408 were also identified. Although evidence indicates that that miR398 targets Cu/Zn superoxide dismutases and a copper chaperone for the superoxide dismutases (CCS) in plants \[[@CR28], [@CR38]\] these relationships were not apparent in the data from this study. 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. In addition to the GRFs, our analyses revealed potential non-conserved targets for miR396 including TNP1, Ulp1 protease and hypothetical proteins (Table [5](#Tab5){ref-type="table"}).

The analyses of legume-specific miRNAs and their targets have revealed an interesting miRNA: target networks between the miRNAs and the NBS-LRR genes \[[@CR14], [@CR16], [@CR18], [@CR32]\]. In this study, we identified NBS-LRR disease resistance genes as targets for four different miRNA families including miR482, miR1507, miR1510 and miR5213 in alfalfa (Table [5](#Tab5){ref-type="table"}).

Degradome analyses has also been utilized to identify potential targets for several non-conserved miRNAs or miRNAs that are present only in closely related species such as the *M. truncatula*. 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. The transcripts for Medtr6g053240.1 (F-box protein interaction domain protein) had a cleavage frequency of approximately 75% and were targeted by the miR2643 in NF08ALF06 genotype. Additionally, two other F-box protein interaction domain protein genes were also identified as targets for miR2643 in alfalfa genotypes (Table [5](#Tab5){ref-type="table"}). These results suggest that the F-box protein interaction domain protein family are regulated by this potential legume-specific miRNA. Another notable observation is that 6 different genes identified as potential targets for miR7696, and the cleavage frequency of a particular target gene (hypothetical protein, Medtr3g112250.1) was more abundant in all three alfalfa genotypes (Table [5](#Tab5){ref-type="table"}).

Because some of the miRNA-stars are also highly expressed, we scrutinized the degradome reads for potential cleavages on the transcripts that are complementary to the miRNA-stars. This analysis has identified potential targets for at least four conserved miRNAs. Specifically, miR156-star targets a heat shock transcription factor, miR164-star targets a protein transporter Sec61 subunit alpha-like protein, miR167-star targets a GRAS family transcription factor, and, miR482-star targets an auxin response factor 1 in alfalfa (Table [5](#Tab5){ref-type="table"}).

The confirmed targets of conserved miRNAs are known to regulate diverse developmental processes in the lifecycle of plants. For example, the SPL transcription factors (target of miR156) which regulate the transition from juvenile to adult phase of the life cycle in land plants \[[@CR39]\]. Auxin receptors (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. The roles of NAC factors (targeted by miR164) include shoot meristem initiation and later root formation in Arabidopsis \[[@CR40], [@CR41]\]. Similarly, TCP family transcription factors have several different roles including regulating leaf morphogenesis \[[@CR42], [@CR43]\]. In Arabidopsis, seven out of nine GRFs are known targets for miR396 \[[@CR44]\], and we have identified four GRFs as targets for miR396 in alfalfa (Table [5](#Tab5){ref-type="table"}). By interacting with its coactivators called GRF-interacting factors (GIFs), this regulatory network (miR396-GRFs-GIFs) regulate leaf size, leaf growth and senescence in Arabidopsis \[[@CR44]\]. The known targets for miR397 include laccase, which is involved in oxidative polymerization of lignin in plants \[[@CR45]\]. Similarly, miR408 is targeting a family of plantacyanins, which could function in shuttling electron-transfer between proteins \[[@CR46], [@CR47]\].

The miR398 family is known to target CSDs and a copper chaperone for superoxide dismutase (CCS) genes in plants \[[@CR28], [@CR38]\]. In this study, we have identified plastocyanin-domain like proteins (plastocyanin is an essential electron carrier which shuttles the electrons between cytochrome *b*~6~*f* and PS I) represents a novel target for miR398. Protein disulphide isomerase (PDI) is a member of a family of dithiol/disulfide oxidoreductases, the thioredoxin superfamily, which functions in the formation of disulphide linkage between the cysteine residues for proper protein folding \[[@CR48]\]. Our degradome analyses confirms that PDI represents a novel target for miR398 in alfalfa (Table [5](#Tab5){ref-type="table"}). The other confirmed miRNA target transcripts include Leucine rich repeat resistance (LRR) proteins (TIR-NBS-LRR and CC-NBS-LRR) that play important roles in plant pathogen recognition and activation of plant innate immune responses \[[@CR14], [@CR16], [@CR18], [@CR32]\]. 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.

Conclusions {#Sec13}
===========

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. truncatula* and alfalfa. The conserved miRNA profiles share some similarities and a few differences between genotypes and types of tissues (roots and shoots). The tissue-specific profiles were used to identify miRNAs that are highly abundant as well as those miRNAs that are expressed at low levels. Additionally, 17 novel miRNAs with varying levels of expression were also identified in alfalfa. The present study also reports identification of 69 targets for 31 miRNA families. In addition to the conserved targets for conserved miRNAs, a few non-conserved targets such as the PDI for miR398 were confirmed. Similarly, miR2643 is targeting three transcripts encoding F-box protein interaction domain containing proteins in alfalfa. In summary, the results from this study have increased our understanding of miRNAs and miRNA-mediated gene regulation in alfalfa that could result in potential tangible targets for practical applications in alfalfa and related legume species to increase biomass yield and address abiotic and biotic limitations to agricultural productivity.

Not applicable.

Funding {#FPar1}
=======

This research was funded by the Noble Research Institute and Forage Genetics International, a hatch grant from NIFA-0229360 (OKL02844) to RS and MM, and the National Natural Science Foundation of China (numbers 31460295 and 31760314) to YZ. This work was also partially supported by the Neustadt-Sarkeys Distinguished Professorship to RS. Publication costs are funded by the Oklahoma Agricultural Experiment Station, Oklahoma State University, Stillwater.

Availability of data and materials {#FPar2}
==================================

The small RNA and degradome datasets generated and analyzed in the present study are available in the National Center for Biotechnology Information Gene Expression Omnibus (NCBI GEO) under accession number GSE119460 available at: <https://www.ncbi.nlm.nih.gov/geo/query/511acc.cgi?acc=GSE119460>.

About this supplement {#FPar3}
=====================

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 supplement are available online at <https://bmcgenomics.biomedcentral.com/articles/supplements/volume-19-supplement-10>.

RS and MM conceived the idea and designed the study. CM and TH cultured the plants used in this study; RP isolated the RNA from samples and generated the small RNA libraries; YZ, SR, QW, SA and RS analyzed the small RNA and degradome libraries; RP, SA and RS wrote the manuscript; MM edited the manuscript. All authors reviewed and approved the final manuscript.

Ethics approval and consent to participate {#FPar4}
==========================================

Not applicable.

Consent for publication {#FPar5}
=======================

Not applicable.

Competing interests {#FPar6}
===================

The authors declare that they have no competing interests.

Publisher's Note {#FPar7}
================

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