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Genome wide identification, characterization and validation of novel miRNA-based SSR markers in pomegranate (Punica granatum L.)

Abstract

A total of 17,439 mature miRNAs (~ 21 nt) earlier generated through RNA seq in the pomegranate were used for in silico analysis. After complexity reduction, a total of 1922 representative mature miRNAs were selected and used as query sequences against pomegranate genome to retrieve 2540 homologous contigs with flanking regions (~ 800). By using pre-miRNA prediction web server, a total of 1028 true contigs harbouring pri-miRNAs encoding 1162 pre-miRNAs were identified. Survey of these sequences for SSRs yielded a total of 1358 and 238 SSRs specific to pri-miRNA and pre-miRNAs, respectively. Of these, primer pairs were designed for 897 pri-miRNA and 168 pre-miRNA SSRs. In pri-miRNA sequences, hexa-nucleotides repeats were found to be most abundant (44.18%) followed by mono- (18.41%) and di-nucleotide (17.01%), which is also observed in pre-miRNA sequences. Further, a set of 51 randomly selected pre-miRNA-SSRs was examined for marker polymorphism. The experimental validation of these markers on eight pomegranate genotypes demonstrated 92.15% polymorphism. Utility of these functional markers was confirmed via examination of genetic diversity of 18 pomegranate genotypes using 15 miRNA-SSRs. Further, potential application of miRNA-SSRs for discovery of trait specific candidate genes was showed by validating 51 mature miRNA against publically available 2047 EST sequences of pomegranate by target and network analysis. In summary, the current study offers novel functional molecular markers for pomegranate genetic improvement.

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Abbreviations

pri-miRNA:

Primary micro RNA

pre-miRNA:

Precursor micro RNA

SSRs:

Simple sequence repeats

CTAB:

Cetyl trimethyl ammonium bromide

NCBI:

National Centre for Biotechnology Information

UPGMA:

Unweighted pair group method of arithmetic averages

NJ:

Neighbor joining

PIC:

Polymorphic information content

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Acknowledgements

The authors are highly grateful to the Indian Council of Agricultural Research (ICAR), New Delhi for extending financial support through ICAR-National Research Centre on Pomegranate, Solapur to carry out this work.

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Correspondence to Prakash G. Patil.

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Patil, P.G., Singh, N.V., Parashuram, S. et al. Genome wide identification, characterization and validation of novel miRNA-based SSR markers in pomegranate (Punica granatum L.). Physiol Mol Biol Plants 26, 683–696 (2020). https://doi.org/10.1007/s12298-020-00790-6

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Keywords

  • MicroRNA-SSRs
  • Functional markers
  • Genetic diversity
  • Pomegranate