Small RNAs have emerged as one of the most important regulators of gene expression in both plants and animals. DCL(Dicer-like), RDR (RNA-dependent RNA polymerase) and AGO (Argonaute) gene families are known to be essential for the biogenesis andfunction of small RNAs. It is therefore imperative to investigate the role of protein/gene families involved in regulation of expression of smallRNAs in important legume crops like chickpea and pigeonpea. HMM (Hidden Markov Model), blastp searches followed by domain scanningidentified a total of 4 DCL, 5 RDR, 13 AGO proteins in chickpea and 5 DCL, 5 RDR, 13 AGO proteins in pigeonpea. Phylogenetic analysisprovided insights into the evolutionary aspects and resulted in clustering of these members into different groups based on their orthologsfrom other species. Motif analysis, promoter prediction, functional annotation and gene structure analysis of these families in both chickpeaand pigeonpea were carried out. These results were in concurrence with the previous reports in arabidopsis, rice, maize and soybeanindicating the conservation of small RNA function and structure in plants. Our study unraveled 17 out of 22 and 20 out of 23 identifiedproteins as potential targets for the microRNAs (miRNAs) in chickpea and pigeonpea, respectively. This study has not only identified thegenes involved in regulation of gene expression in these two crops, but also would result in identification of probable candidate genesimparting resistance against different stresses.

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Genome wide identification of gene families involved in the regulation of small RNA expression in chickpea and pigeonpea

Summary

Vanika Garg1,2, Gaurav Agarwal1, Aamir W Khan1, Dadakhalandar Doddamani1, Himabindu Kudapa1, Lekha Pazhamala1,PB Kavi Kishor 2, Rajeev K Varshney1,3*

Department of Science and TechnologyGovernment of India

Comparative analysis of orthologusrelationship of DCL, RDR and AGO genesamong chickpea, pigeonpea and soybean.The syntenic chromosomes arerepresented by similar colors. Red, blueand green colored links correspond toDCL, RDR and AGO proteins respectively.

Phylogenetic analysis of DCL, AGO and RDR genes

Identified and characterized three classes of proteins (DCL, RDR and AGO) involvedin biogenesis of small RNAs in chickpea and pigeonpea

A total of 20 orthologs of chickpea DCL, RDR and AGO were found in pigeonpeaDuplication studies identified 3, 6 paralogs in chickpea and pigeonpea respectivelyDCLs were localized in nucleus and AGOs were found both in nucleus and cytoplasmInteraction studies revealed a strong association between DCL, RDR and AGO

proteins

Identification and orthologous relationship

Chromosomal localization and duplication

Chickpea Pigeonpea Arabidopsis RiceGene Motif Gene Motif Gene Motif Gene Motif

CaAGO3b DDD/F CcAGO4a DDH/P AGO2 DDD/H OsAGO1 DDH/PCaAGO4a DDH/A CcAGO4c DDH/A AGO3 DDD/H OsAGO2 DDD/HCaAGO10c DDH/Q CcAGO9 DDH/S AGO4 DDH/S OsAGO3 DDD/HCaAGO4c DDH/A CcAGO6 DDH/S AGO6 DDH/P OsAGO4a DDH/PCaAGO6b DDH/P CcAGO3b DDD/N AGO9 DDH/R OsAGO4b DDH/PCaAGO6a DDH/P CcAGO3a DDD/H OsAGO11 GDH/HCaAGO3a DDD/H OsAGO13 -DH/H

OsAGO15 DDH/POsAGO16 DDH/POsAGO17 HDR/COsAGO18 DDH/S

Abundance of genes

Comparison of conserved motifs

Comparison among AGO proteins with missing catalyticresidue(s) in Piwi domains of chickpea, pigeonpea, arabidopsisand rice

Distribution of DCL, RDR and AGO genes across chickpea(C1) and pigeonpea (C2) chromosomes. Lines linking thegenes represent the duplicated genes. Tandem blocks arehighlighted in colored background.

A1 A2 A3

C1

C2

B

Unrooted phylogenetic tree of proteins involved in biogenesis of sRNA in chickpea, pigeonpea,soybean and arabidopsis. MEGA 6.0 was used to construct the tree with NJ method and 5000 bootstrapreplicates. Phylogenetic relationship of dicer like proteins (A1), RNA dependent RNA polymerases (A2),Argonautes (A3). B represents phylogenetic relationship, domain composition and intron–exonstructure of Argonautes. Green boxes represent exons and black lines represent introns.

1 International Crops Research Institute for the Semi-Arid Tropics (ICRISAT), Hyderabad, India2 Department of Genetics, Osmania University, Hyderabad, India3 School of Plant Biology and Institute of Agriculture, The University of Western Australia, Crawley, Australia* Address for correspondence: r.k.varshney@cgiar.org

Abstract