Sequence, Secondary Structure, and Phylogenetic Conservation of MicroRNAs in Arabidopsis thaliana

Abstract

MicroRNAs are small non-coding RNA molecules that are produced in a cell endogenously. They are made up of 18 to 26 nucleotides in strength. Due to their evolutionary conserved nature, most of the miRNAs provide a logical basis for the prediction of novel miRNAs and their clusters in plants such as sunflowers related to the Asteraceae family. In addition, they participate in different biological processes of plants, including cell signaling and metabolism, development, growth, and tolerance to (biotic and abiotic) stresses. In this study profiling, conservation and characterization of novel miRNA possessing conserved nature in various plants and their targets annotation in sunflower (Asteraceae) were obtained by using various computational tools and software. As a result, we looked at 152 microRNAs in Arabidopsis thaliana that had already been predicted. Drought tolerance stress is mediated by these 152 non-coding RNAs. Following that, we used local alignment to predict novel microRNAs that were specific to Helianthus annuus. We used BLAST to do a local alignment, and we chose sequences with an identity of 80% to 100%. MIR156a, MIR164a, MIR165a, MIR170, MIR172a, MIR172b, MIR319a, MIR393a, MIR394a, MIR399a, MIR156h, and MIR414 are the new anticipated miRNAs. We used MFold to predict the secondary structure of new microRNAs. We used conservation analysis and phylogenetic analysis against a variety of organisms, including Gossypium hirsutum, H. annuus, A. thaliana, Triticum aestivum, Saccharum officinarum, Zea mays, Brassica napus, Solanum tuberosum, Solanum lycopersicum, and Oryza sativa, to determine the evolutionary history of these novel non-coding RNAs. Clustal W was used to analyze the evolutionary history of discovered miRNAs.

Introduction

Sunflower (Helianthus annuus) belongs to the Asteraceae family. By cloning method, 700 types of miRNA were identified in plants; in 2012, miRNA was identified in Arabidopsis thaliana. All the processes of miRNA targets are based on coding and non-coding sequence.¹ Previous studies show that RNA polymerase play important role in the transcription of the miRNA gene.²

In recent years, for the identification of miRNA scientists have used high throughput sequencing and computational analysis techniques.³ Almost all scientists have concluded that microRNAs involved in the regulatory function of flowering and non-flowing plants are conserved.⁴ Plants are damaged by 2 types of environmental stresses categorized as biotic and abiotic stress. Damage to living organisms by living organisms like parasites, bacteria, viruses, and fungi is known as abiotic stress as well as damage to a living organism by the source of nonliving factors called abiotic stress.⁵ To describe abiotic stress, we should study the function of different organisms that survived in different environments. Stress always affects the plant’s tissues. plants need enough water for their sufficient growth. Up and down movement of water expand plant cells, which causes plant growth. Enough amount of water expands the plant’s cells and transfers the minerals from the soil to the tip of the leaves but stress causes an imbalance in the plant’s routine processes.

According to research every year, we lost 50% of our food production due to abiotic stress. Abiotic stress affects plants’ fruits, crops, metabolism, respiration processes of plants, and at the end plant’s seeds. Seeds are used for next generation; hence, unhealthy seeds affect further production.⁶ H. annuus typically refer to annual species that tend to spread rapidly and can become aggressive.⁷ The plant family improvement depends on the genetically resistant varieties, seed productivity, modern cultivation, and biotic-abiotic stress tolerance. Plant improvement can be assessed by studying its genetic makeup and sowing in a different location.⁸ For the human, it acts as an important source of nutrients. Nutritionally, it is the main source of vital nutrients inkling carbohydrates, proteins, and dietary fibers, and provides almost 20% of the dietary energy supply. According to the miRNA database, H. annuus contains a total of 6 precursors and 7 mature microRNA that are compared with A. thaliana for the identification of novel microRNA.⁹ A. thaliana contained 205 precursors and 384 mature non-coding RNA. All data regarding miRNA are present in miRBase.

Methodology

Many tools are used as comparative genomics approaches to achieve novel and interesting information about miRNAs in plants and animals. In the initial step, identify sequences and reference sequences, and download them from the microRNA Registry Database. This miRBase Pre-miRNA database was available at https://www.mirbase.org/ freely. Pre-miRNA potential candidates were predicted by subjecting the downloaded mature and precursor miRNAs sequence through the Basic Local Alignment Tool. For this purpose, nucleotide BLAST available freely at Genbank of the National Center for Biotechnology Information (https://blast.ncbi.nlm.nih.gov/Blast.cgi?PAGE_TYPE=BlastSearch) was used. The miRNA* sequences, both mature and precursor, were subjected to BLAST against H. annuus expressed sequence tags (ESTs) sequentially using the BLASTn program following a maximum of up to 4 mismatches with miRNAs*.

EST single-tone selection

BLASTn program was used and the setting of the parameters was adjusted as expect values, 1000; low complexity, the sequence filter, database, others; organism, H. annuus; program selection, somewhat similar sequences; and all other parameters, by default. To identify the coding part of miRNA, we used BLASTx. BLASTx highlights coding regions.¹⁰

Prediction of miRNAs secondary structure

MFOLD, a secondary structure prediction tool was used to produce a stem-loop structure for the initially identified potential H. annuus. All the initial candidate sequences that failed to develop stable secondary structures were discarded. MFOLD software updated as UNAfold http://www.unafold.org/ and then clicked on MFOLD and then selected application (RNA fold form Version 2.3).¹¹

UNAfold → MFOLD → Application → RNA fold form version

The setting of MFOLD parameters was adjusted as RNA sequence, linear; folding temperature, 37°C, ionic concentration, 1 mol/L of National Center for Biotechnology Information (NCBI) having no divalent ions; percent sub-optimality number, 5; maximum interior loop size 30.

Conservation and phylogenetic analysis

Clustal W was selected for phylogenetic analysis. Clustal W is used for multiple sequence alignment or the alignment of more than one sequence available at https://www.genome.jp/tools-bin/clustalw.

Results

New potential miRNAs in sunflower

In all research, we predicted 152 miRNAs that performed regulatory process against drought-tolerant stress in A. thaliana. miRNAs that respond to drought stress are 156a, 156b, 156c, 156d, 156e, 156f, 158a, 159a, 164a, 164b, 165a, 165b, 166a, 166b, 166c, 166d, 166e, 166f, 166g, 168a, 168b, 169a, 170, 171a, 172a, 172b, 173, 156b, 319a, 319b, 169b, 169c, 169d, 169e, 169f, 169g, 169h, 169i, 169j, 169k, 169l, 169m, 169n, 171b, 171c, 172c, 172d, 339a, 339b, 394a, 394b, 397a, 397b, 398a, 398b, 398c, 399a, 399b, 399c, 399d, 399e, 399f, 400, 401, 402, 403, 404, 408, 159g, 156h, 158b, 159c, 319c, 164c, 172e, 417, 418, 414, 415, 416, 419, 420, 426, 427a, 427b, 427c, 827, 830, 833a, 835, 836, 837, 839, 841a, 842b, 844, 845a, 846, 848, 850, 851, 847, 855, 854a, 854b, 854c, 856, 857, 858, 859, 860, 861, 865, 845b, 870, 1886, 1888a, 2933a, 2933b, 2936, 3434, 774b, 4221, 854e, 5021, 5024, 5025, 5026, 5029, 5641, 5642a, 858b, 833b, 156i, 156j, 5652, 5653, 5654, 5655, 5656, 5658, 5188, 1888b, 5666, 5996, 8121, 8165, 8170, 8178, 8180, and 8181. All these miRNA sequences are reported in A. thaliana.

Prediction of novel miRNA

In A. thaliana, after identifying the 152 microRNA sequences, we performed local alignment of these sequences by BLASTn with the parameters of somewhat similarities and H. annuus in organisms and selected those BLAST sequences that were 80% to 100% similar. So in local alignment, we got 19 sequences of A. thaliana against H. annuus. The accession numbers of these 19 sequences are XR_002550943, XR_002552183, XR_002552875, XR_002554296, XR_002556375, XR_002562643, XR_002570335, XR_002574508, XR_2575503, XR_002579567, XR_002582121, XR_002587840, XR_002549854, XR_002592285, XR_004862999, XR_004863001, XR_004865012, XR_004869948, and XR_004890935 (see in Table 1).

Table 1.

Novel microRNA in Helianthus annuus that is predicted with the reference of Arabidopsis thaliana sequences.

	H. annuus miRNA	Accession no by BLASTn	Novel miRNA sequences
1	MIR156	ACCESSION	TTTCGTTATTATCATCTATTCTTGTGGCACGAG
	a	XR_002550943	AAAGAGAAGTTTGGTTGAGAACTGACAGAAG
			AGAGTGAGCACAGGCC
		ACCESSION	CTCAAACTTTATGCACTCTTTTACTTCTGTTGA
		XR_004869948	TTATGTTCTTTGATGGGATGATATATATGGTT
			ACAAAGATGAAGAAAGCTGACAGAAGAGAGT
			GAGCATATGCAACCAGTTGTATATAGAGTATG
			CATTTATTGGGA
2	MIR164	ACCESSION	GACATGTAAAGCGAGTGGGTGGGTTTATAAG
	a	XR_002562643	ATTACTAAGGTGGGTGTTGAGCAAGATGGAG
			AAGCAGGGCACGTGCATTACAAACTCATCAT
			GCAAAACTTCATTCAAATTTCAACAAAACACC
			CTTTCTCAGGTTTGA
3	MIR165	ACCESSION	5p
	a	XR_002587840	TCTCTTTCCCACCTATACCATCACCATTTACAC
			CCATCTTTCTCTCACTAAAACTGTACCCAAAA
			GATGA
			ACAAGAAGAAAAGAACTGAAGCTGAAAGCTG
			TTTTCTTTTGAGGGGAATGTTGTCTGGCTCGA
			GGCCACT
			3p
			AAATGATGTTTGTGAAATGTTTGATTACTGCG
			AGAAGTTACTGATCTGGTGTCGTCGGACCAGG
			CTTCATTCCCCCCAATTGTGGCTTCCTGTGTTC
			TAAAAGAATTGTTTTTTCTTGAATCTACATGTT
			TCCA
		ACCESSION	5p
		XR_002549854	GAGGTCTTGGGTTCAAGTCCCACTGACGACAG
			GAATAAAAGAAATTTGCCGTTAAAAAAAAAT
			GAAGAAGAAGATTGGGGTAATGATGAGTTCT
			GAGAATCTAACAAAATTCCAGAAACAGGATG
			3p
			TTCATAATCACATAATTGATCAATCTTTGTTG
			ATCAATGATTAAAGATTAGAATCTTGTGTTGT
			CGGACCAGGCTTCATTCCCCTC
		ACCESSION	5p
		XR_002552183	TTCTTTTGAGGGGAATGTTGTTTGGCTCGAGG
			TCATTAGAAACCATGGATCTTTATCTCTCTCTA
			TATAT
			ATATGTATGTATATATATTGATGTATGTA
			3p
			ATCCATCATCTATGGTCTTTGTTGATCAATGA
			GTTTTAGATTAATTATATAAAGTGTTAAAGAG
			AGTATC
			TTGATCATTGGATCTGGTGTGCTCGGACCAGG
			CTTCATTC
		ACCESSION	5p
		XR_002582121	GAAGAGAAGAAGAAGCTATATCTTTTGAGGG
			GAATGTTGTTTGGCTCGAGGTCAATAGAAACC
			AAAGATCTATATCTCTTTTTCTCTAACTGTTAA
			TAAAGATGTACATATATATGTATGTATATGAT
			GTTATAGTCAAC
			3p
			GGTTATTCATTTAGAGTGTTGAATGAGAGAAA
			CTTGATCATTGGATGATCTGCTGTACTCGGAC
			CAGGCTTCATTCCCCCCAATTGTTGCTTCA
		ACCESSION	5p
		XR_002570335	CAATCTTTCTCCCTCTCTCTAAGATCACACCTA
			AAAGATGAACACATTGAAAGCCTTTTTATCTT
			TTGAGGGGATTGTTGTCTGGCTCGAGGCCACT
			AGAAAGCCTAGATCTTTCTCTCTCTATATACA
			TATCTATATCA
			3p
			GGGATTGTTGTCTGGCTCGAGGCCACTAGAAA
			GCCTAGATCTTTCTCTCTCTATATACATATCTA
			TATCACAGTCAACGCAACCATTGATTACTGAG
			AGAGATTCTTGATCTGGTGTCGTCGGACCAGG
			CTTCATTCCCC
4	MIR170	ACCESSION	GATGTTGGTTCGGTTCAATAAGAACTCAATGT
		XR_004890935	TCAAATGATGCATTGAACGTTGCTTTTTGATT
			GAGCCGTGCCAATATCACGTGCATGTTGCTTC
			TAAATTTCCACAAGTCTTTTGTAAACTTCTGTG
			AAAAGGACCTA
5	MIR172	ACCESSION	AGAATCTTGATGATGCTGCATCGGAAATCAAT
	a	XR_002592285	TGACCACTTTAAAAATCAACGCATCAAATACG
			ATTTTGTTGGATCTCATACAAACAAAGGTAAT
			CTTT
		ACCESSION	TATAGATTGGCTGGAATCTAAGCACAAGGGT
		XR_002556375	GTGTTTTTTCTGTAGATTTAATAATGAAAATA
			CTGATCACACACCAGAAAAGAGAAATAGGGC
			TTTGCTGTTATTGTATTTTTTTATTGTATTTAGT
			TTGAAGATTTTG
		ACCESSION	AGAAAGAAAAAGAAAGTGGTTTATGGGCCCC
		XR_004865012	TTGATGGTTTGAGAATCTTGATGATGCTGCAG
			CGGCAATTGCTGGCTAATTATGATCTTTAAAA
			CTGGATTATGGTAAGTTGTGACCATGTAGATT
			TAATCTTAAAATA
6	MIR172	ACCESSION	5p
	b	XR_004862999	TCAAACAACATCAGCAGGTAGCAGCTCCACCT
			CCTCAGTCACCAGCGCCGACTGCCGCTGCTCC
			GCCTCT
			GCCCTCCGGCTGCTCCGCCTTCGACGATTGCT
			GCCGAAACTTTGTTT
			3P
			ACTAAGTAGCTATTAGCTAATTTTTGTTCTTGT
			TTAATTTTGAATTCAGGCTGTTTTGAGTTGTGG
			GATC
			AAGCTAGAACAAGATTT
7	MIR319	ACCESSION	AATTAGCTGCCGACTCATTCATTTAACCACTC
	a	XR_004863001	AGTAGAAAAGGGTTCACTTTATGCTACTTTGA
			TTGAGTGAATGATGCGGGAGATAGTTTTCATC
			CCTTGCTAATCTGTACTTGGACTGAAGGGAGC
			TCCCTCGTTCTT
8	MIR393	ACCESSION	5P
	a	XR_002552875	TCCCTCTCTCTCTCTCTCTCTCTCTCTCTCTCTC
			TCTCTCTCTTACACACACATCTCTCTCCCCGTC
			TCTATATGTGCGAAGATTACAACGGTAGCTAA
			AGGACGCATCCAAAGGGATCGCATTGATCCT
			AAATCCCATA
			3P
			CGCATAGTATATGGGTATGATATACCGAGTTG
			GGATCATGCTATCCTTTTGGATTCCTTCTTCGG
			TTGCTTCTAACTTTATAAATATCACACGCACA
			AGCCAAGAACAATCAAAGATGGAAGAGAATG
			TGATGTTTGATT
9	MIR394	ACCESSION	CTTTAGGGTTTTTAGGGTTTCTTTCATGGTGGT
	a	XR_002554296	TTAATAAAGAGTTTCCAGCAGATTTCTTTGGC
			ATTCTGTCCACCTCCATATTCATTGATCTATGT
			ATCTCACTGTTGTGTAAATGTGTAATTAGGGT
			TTATTGGTTT
10	MIR399	ACCESSION	GGTCGGAAGAAAAGGAGCTGAGACAGCTGGA
	c	XR_002574508	TGTTTTGAAGCAGGCAATTGTGTTTGGGCTGG
			GTGAAGACAACTGGCTCTGGGCACGATAACG
			CAAGGGGTTTTTCCATGCGCCTGCCAATAGAA
			ATATGCGGTATAGT
11	MIR156h	ACCESSION	ATGTTGATGAAACGGGTTGAAGTGTCTCGATG
		XR_002579567	ATGTTGTTGACAGAAGATAGAGAGCACAGAT
			GACGAAGTTGCAGCTAATATTTGGCATCTTTT
			TTCTTTGTGCCCTCTATTGTTCTGTCATCATCA
			CATATCTTCTTC
12	MIR414	ACCESSION	ATATTCATTGAGTTATTATTATTTTATGAAAAT
		XR_002575503	TTTCATGTTCTTTGAAACTTATCATGTCTTATG
			AAATCGTTTCAGTCCTCAGTCTTAGGGGCTGT
			TTGGTTGCCTCTTAATGGCTCCATTAAGAAGC
			TTGGCCTCTGAATCGGTCAGACATGGGGTGT

These 19 sequences code for different miRNAs that were novel. In our research, we predicted 12 novel miRNAs in sunflowers against A. thaliana. These novel predicted miRNAs are MIR156a, MIR164a, MIR165a, MIR170, MIR172a, MIR172b, MIR319a, MIR393a, MIR394a, MIR399a, MIR156h, and MIR414 (see in Table 2).

Table 2.

Conservation analysis of Helianthus annuus with other organisms such as Gossypium hirsutum, H. annuus, Arabidopsis thaliana, Triticum aestivum, Saccharum officinarum, Zea mays, Brassica napus, Solanum tuberosum, Solanum lycopersicum, and Oryza sativa.

	G. hirsutum	H. annus	A. thaliana	T. aestivum	S. officinarum	Z. mays	B. napus	S. tuberosum	S. lycopersicum	O. sativa
MIR156a	present	present	present	present	present	present	present	present	present	present
MIR164a	present	Non	present	present	Non	present	present	present	present	present
MIR165a	Non	non	present	non	non	Non	non	non	non	non
MIR170	non	non	present	non	non	Non	non	non	non	non
MIR172a	present	non	present	non	non	present	present	present	present	present
MIR172b	present	non	present	non	non	present	present	present	present	present
MIR319a	non	non	present	present	non	present	non	present	present	present
MIR393a	present	non	present	Non	non	present	present	present	present	present
MIR394a	present	non	present	non	non	present	present	non	present	present
MIR399c	present	non	present	present	non	present	present	present	present	present
MIR156h	non	non	present	present	present	present	non	present	non	present
MIR414	non	non	present	non	non	non	non	non	non	present

Novel sunflower miRNAs structures predicted from A. thaliana

In our research, we predicted 12 novel microRNAs in the sunflower that respond against drought stress (MIR156a, MIR164a, MIR165a, MIR170, MIR172a, MIR172b, MIR319a, MIR393a, MIR394a, MIR399a, MIR156a, and MIR414). Here, we explain the miRNAs structures on the base of both 5 and 3 prime by MFOLD.

Ancestral conservation of H. annuus

In addition, novel sunflower precursor miRNAs were selected for conservation studies. For this process, all novel miRNAs selected to evaluate their conservation in other plants. For conservation studies, we selected 10 different types of plants like Gossypium hirsutum, H. annuus, A. thaliana, Triticum aestivum, Saccharum officinarum, Zea mays, Brassica napus, Solanum tuberosum, Solanum lycopersicum, and Oryza sativa. Using the same process, many researchers have determined precursor conservation and phylogenetic analysis in different plants.

Phylogenetic analysis of non-coding microRNAs

The study of the evolutionary history of a species or a group of organisms or a particular characteristic of an organism. Here we have done phylogenetic analysis by clustal W.

Phylogenetic analysis of MIR156a

The phylogenetic analysis of mir156a is described in Figures 1 and 2. According to the MIR156a phylogenetic analysis, H. annuus with the Accession number “XR_002550943” and “XR_4869948” shows close relation with T. aestivum, B. napus, A. thaliana, G. hirsutum, O. sativa, Z. mays, and S. officinarum.

Figure 1.

MIR156a.

XR_002550943.

Figure 2.

MIR 156.

XR_004869948.

Phylogenetic analysis of MIR164a

The phylogenetic analysis of mir164a is described in Figure 3. According to the phylogenetic analysis of MIR164a, H. annuus with the Accession number “XR_002562643” shows close relation with T. aestivum, B. napus, A. thaliana, O. sativa, Z. mays, S. lycopersicum, and G. hirsutum and shows a distance relationship with S. tuberosum.

Figure 3.

MIR164a.

XR_002562643.

Phylogenetic analysis of MIR172b

The phylogenetic analysis of mir172b is described in Figure 4. According to the phylogenetic analysis of MIR172b, H. annuus with the Accession number “XR_004862999” shows close relation with A. thaliana, Z. mays, S. lycopersicum, S. tuberosum and shows a distance relationship with G. hirsutum.

Figure 4.

MIR172b.

XR_004862999.

Phylogenetic analysis of MIR172a

Phylogenetic analysis of mir172a is described in Figures 5 to 7. According to the phylogenetic analysis of MIR172a, H. annuus with the Accession number “XR_002592285” and “XR_002556375” shows close relation with A. thaliana, Z. mays, S. lycopersicum, O. sativa, B. napus and with “XR_004865012” shows close relationship with G. hirsutum.

Figure 5.

MIR 172a (XR_002592285).

Figure 7.

MIR172a (004865012).

Figure 6.

MIR172a (002556375).

Phylogenetic analysis of MIR319

The phylogenetic analysis of mir319 is described in Figure 8. According to the phylogenetic analysis of MIR319, H. annuus with the Accession number “XR_004863001” shows close relation with A. thaliana, S. lycopersicum, and T. aestivum.

Figure 8.

MIR319(XR_004863001).

Phylogenetic analysis of MIR393a

The phylogenetic analysis of mir393 is described in Figure 9. According to the phylogenetic analysis of MIR93a, H. annuus with the Accession number “XR_002552875” shows close relation with A. thaliana, G. hirsutum, O. sativa, S. tuberosum, and Z. mays.

Figure 9.

MIR393a(XR_002552875).

Phylogenetic analysis of MIR394

The phylogenetic analysis of mir393 is described in Figure 10. According to the phylogenetic analysis of MIR94, H. annuus with the Accession number “XR_002554296” shows a distance relation with A. thaliana, G. hirsutum, O. sativa, B. napus, S. lycopersicum, and Z. mays.

Figure 10.

XR_002554296.

Phylogenetic analysis of MIR399

The phylogenetic analysis of mir399 is described in Figure 11. According to the phylogenetic analysis of MIR94, H. annuus with the Accession number “XR_002574508” shows a distance relation with A. thaliana, G. hirsutum, O. sativa, B. napus, S. lycopersicum, and Z. mays.

Figure 11.

MIR399(XR_002574508).

Discussion

Sunflower is the fourth biggest oil-seed crop in the world. The seeds of sunflowers are used in food as well as their dried stalk is used as fuel. It has previously been used as an ornamental plant and was also used in ancient ceremonies.¹² Moreover, different parts of sunflowers are used in body painting, decorations, and making dyes for the textile industry. Its oil is used in the manufacturing of margarine and salad dressings, and cooking. With roasted seeds, a coffee type could be made. In industry, it is used in cosmetics and paints. Due to its lack of anti-nutritional factors and high nutritional values, it is a potential source of protein for human consumption. Due to its metabolic, physiological, and morphological adaptation strategies, the sunflower is one of the most important oil-seed crops and is resistant to various abiotic stresses. This crop is of special interest for its adaptation to limited water availability, high temperatures, high salinity, and heavy-metal concentrations in soil. The dried stems which are used for fuel contain potassium and phosphorous which can be composed and returned to the soil as fertilizer.¹³

MiRNAs arise from primary longer RNA transcripts that include a self-complementary fold-back, from which the mature miRNAs are excised. They are short RNA molecules containing 19-24 nucleotides in size.^14,15 They are familiar as regulators of gene expression by binding to open reading frames (ORF) or untranslated regions (UTR) of specific mRNAs, targeting them for directing or cleavage translation inhibition at the mRNA level. It has been demonstrated that around 60% of protein-coding genes are targets of miRNAs and are modulated by these small RNAs.

miRNAs are derived from hairpin pre-miRNA from which both miRNA and the imperfectly complementary miRNA* strands are released. Their sequences are not conserved between plants and animals, and even not have been seen in fungi. Many miRNAs within the kingdom have an ancient origin, some being completely conserved among sunflower, Arabidopsis, rice, and even liverworts, mosses, and hornworts.¹⁶

By regulating gene expression, miRNA plays a vital role to regulate the developmental processes of organisms.¹⁷ The negative regulation of miRNAs in gene expression in both plants and animals has been demonstrated.¹⁸ MicroRNAs have been revealed to modulate diverse developmental processes, including polarity, identity, and organ separation, and to regulate their function and biogenesis.¹⁸ In our study, we used the miRBase database to find miRNAs from A. thaliana that were tolerant against drought stress that was 152 in strength and then performed local alignment of these miRNAs against sunflower and found 12 novel miRNAs (MIR156a, MIR164a, mir165a, mir170, mir172a, mir172b, mir319a, mir393a, mir394a, mir399c, mir156h, and mir414). The secondary structure of these 12 novel miRNAs, including forward and reverse strands, was forecasted by MFOLD software by using default parameters. Later, conservation and phylogenetic analysis were done by selecting 10 different organism type such as G. hirsutum, H. annuus, A. thaliana, T. aestivum, S. officinarum, Z. mays, B. napus, S. tuberosum, S. lycopersicum, and O. sativa. In our study, all novel miRNAs are present in only A. thaliana.

In our study, mir-156a is present in all species; mir-164 is present in all except H. annus and S. officin;¹⁹ mir-165a and mir-170 are present in only A. thaliana; mir-172a and mir172-b are present in all except H. annus, T. aestiyum, and S. officin;²⁰ mir-319a is present in all except G. hirsutum, H. annus, S. officin, and Z. mays; mir-393a is present in all except H. annus, T. aestiyum, and S. officin; mir-394a is present in all except H. annus, T. aestiyum, S. officin, and B. napus; mir-399c is present in all except H. annus and T. aestiyum; mir-156h is present in all except G. hirsutum, H. annus, B. napus, and S. lycopersicum; and mir-414 is absent in all except A. thaliana, and O. sativa.

Conclusions

Twelve novel miRNAs (MIR156a, MIR164a, mir165a, mir170, mir172a, mir172b, mir319a, mir393a, mir394a, mir399c, mir156h, and mir414) were identified against drought stress in A. thaliana. We targeted these miRNAs to cope with the drought tolerance in sunflowers. In our study, all novel miRNAs are present in only A. thaliana. Moreover, different parts of sunflowers are used in body painting, decorations, and making dyes for the textile industry. Its oil is used in the manufacturing of margarine and salad dressings, and cooking. With roasted seeds, a coffee type could be made. In industry, it is used in cosmetics and paints. The improvement method also increases the production of sunflowers and benefits economically.