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Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures.

ABSTRACT: MicroRNAs (miRNAs) are endogenous, noncoding, short RNAs directly involved in regulating gene expression at the post-transcriptional level. In spite of immense importance, limited information of P. vulgaris miRNAs and their expression patterns prompted us to identify new miRNAs in P. vulgaris by computational methods. Besides conventional approaches, we have used the simple sequence repeat (SSR) signatures as one of the prediction parameter. Moreover, for all other parameters including normalized Shannon entropy, normalized base pairing index and normalized base-pair distance, instead of taking a fixed cut-off value, we have used 99% probability range derived from the available data.We have identified 208 mature miRNAs in P. vulgaris belonging to 118 families, of which 201 are novel. 97 of the predicted miRNAs in P. vulgaris were validated with the sequencing data obtained from the small RNA sequencing of P. vulgaris. Randomly selected predicted miRNAs were also validated using qRT-PCR. A total of 1305 target sequences were identified for 130 predicted miRNAs. Using 80% sequence identity cut-off, proteins coded by 563 targets were identified. The computational method developed in this study was also validated by predicting 229 miRNAs of A. thaliana and 462 miRNAs of G. max, of which 213 for A. thaliana and 397 for G. max are existing in miRBase 20.There is no universal SSR that is conserved among all precursors of Viridiplantae, but conserved SSR exists within a miRNA family and is used as a signature in our prediction method. Prediction of known miRNAs of A. thaliana and G. max validates the accuracy of our method. Our findings will contribute to the present knowledge of miRNAs and their targets in P. vulgaris. This computational method can be applied to any species of Viridiplantae for the successful prediction of miRNAs and their targets.

SUBMITTER: Nithin C

PROVIDER: S-EPMC4464996 | biostudies-literature | 2015

REPOSITORIES: biostudies-literature

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Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures.

Nithin Chandran C Patwa Nisha N Thomas Amal A Bahadur Ranjit Prasad RP Basak Jolly J

BMC plant biology 20150612

<h4>Background</h4>MicroRNAs (miRNAs) are endogenous, noncoding, short RNAs directly involved in regulating gene expression at the post-transcriptional level. In spite of immense importance, limited information of P. vulgaris miRNAs and their expression patterns prompted us to identify new miRNAs in P. vulgaris by computational methods. Besides conventional approaches, we have used the simple sequence repeat (SSR) signatures as one of the prediction parameter. Moreover, for all other parameters ...[more]

PMID: 26067253

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Project description:BackgroundOver recent years, a growing effort has been made to develop microsatellite markers for the genomic analysis of the common bean (Phaseolus vulgaris) to broaden the knowledge of the molecular genetic basis of this species. The availability of large sets of expressed sequence tags (ESTs) in public databases has given rise to an expedient approach for the identification of SSRs (Simple Sequence Repeats), specifically EST-derived SSRs. In the present work, a battery of new microsatellite markers was obtained from a search of the Phaseolus vulgaris EST database. The diversity, degree of transferability and polymorphism of these markers were tested.ResultsFrom 9,583 valid ESTs, 4,764 had microsatellite motifs, from which 377 were used to design primers, and 302 (80.11%) showed good amplification quality. To analyze transferability, a group of 167 SSRs were tested, and the results showed that they were 82% transferable across at least one species. The highest amplification rates were observed between the species from the Phaseolus (63.7%), Vigna (25.9%), Glycine (19.8%), Medicago (10.2%), Dipterix (6%) and Arachis (1.8%) genera. The average PIC (Polymorphism Information Content) varied from 0.53 for genomic SSRs to 0.47 for EST-SSRs, and the average number of alleles per locus was 4 and 3, respectively. Among the 315 newly tested SSRs in the BJ (BAT93 X Jalo EEP558) population, 24% (76) were polymorphic. The integration of these segregant loci into a framework map composed of 123 previously obtained SSR markers yielded a total of 199 segregant loci, of which 182 (91.5%) were mapped to 14 linkage groups, resulting in a map length of 1,157 cM.ConclusionsA total of 302 newly developed EST-SSR markers, showing good amplification quality, are available for the genetic analysis of Phaseolus vulgaris. These markers showed satisfactory rates of transferability, especially between species that have great economic and genomic values. Their diversity was comparable to genomic SSRs, and they were incorporated in the common bean reference genetic map, which constitutes an important contribution to and advance in Phaseolus vulgaris genomic research.

Project description:BackgroundCommon beans (Phaseolus vulgaris L.) are widely consumed as a source of proteins and natural products. However, its yield needs to be increased. In line with the agenda of Phaseomics (an international consortium), work of expressed sequence tags (ESTs) generation from bean pods was initiated. Altogether, 5972 ESTs have been isolated. Alcohol dehydrogenase (AD) encoding gene cDNA was a noticeable transcript among the generated ESTs. This AD is an important enzyme; therefore, to understand more about it this study was undertaken.ObjectiveThe objective of this study was to elucidate P. vulgaris L. AD (PvAD) gene cDNA sequence and to predict the three-dimensional (3D) structure of deduced protein.Materials and methodspositive and negative strands of the PvAD cDNA clone were sequenced using M13 forward and M13 reverse primers to elucidate the nucleotide sequence. Deduced PvAD cDNA and protein sequence was analyzed for their basic features using online bioinformatics tools. Sequence comparison was carried out using bl2seq program, and tree-view program was used to construct a phylogenetic tree. The secondary structures and 3D structure of PvAD protein were predicted by using the PHYRE automatic fold recognition server.ResultsThe sequencing results analysis showed that PvAD cDNA is 1294 bp in length. It's open reading frame encodes for a protein that contains 371 amino acids. Deduced protein sequence analysis showed the presence of putative substrate binding, catalytic Zn binding, and NAD binding sites. Results indicate that the predicted 3D structure of PvAD protein is analogous to the experimentally determined crystal structure of s-nitrosoglutathione reductase from an Arabidopsis species.ConclusionsThe 1294 bp long PvAD cDNA encodes for 371 amino acid long protein that contains conserved domains required for biological functions of AD. The predicted deduced PvAD protein's 3D structure reflects the analogy with the crystal structure of Arabidopsis thaliana s-nitrosoglutathione reductase. Further study is required to validate the predicted structure.

Project description:BackgroundMicrosatellites, or simple sequence repeats (SSRs), are tandemly repeated DNA sequences, including tandem copies of specific sequences no longer than six bases, that are distributed in the genome. SSR has been used as a molecular marker because it is easy to detect and is used in a range of applications, including genetic diversity, genome mapping, and marker assisted selection. It is also very mutable because of slipping in the DNA polymerase during DNA replication. This unique mutation increases the insertion/deletion (INDELs) mutation frequency to a high ratio - more than other types of molecular markers such as single nucleotide polymorphism (SNPs).SNPs are more frequent than INDELs. Therefore, all designed algorithms for sequence alignment fit the vast majority of the genomic sequence without considering microsatellite regions, as unique sequences that require special consideration. The old algorithm is limited in its application because there are many overlaps between different repeat units which result in false evolutionary relationships.FindingsTo overcome the limitation of the aligning algorithm when dealing with SSR loci, a new algorithm was developed using PERL script with a Tk graphical interface. This program is based on aligning sequences after determining the repeated units first, and the last SSR nucleotides positions. This results in a shifting process according to the inserted repeated unit type.When studying the phylogenic relations before and after applying the new algorithm, many differences in the trees were obtained by increasing the SSR length and complexity. However, less distance between different linage had been observed after applying the new algorithm.ConclusionsThe new algorithm produces better estimates for aligning SSR loci because it reflects more reliable evolutionary relations between different linages. It reduces overlapping during SSR alignment, which results in a more realistic phylogenic relationship.

Dataset Information

Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures.

Publications

Computational prediction of miRNAs and their targets in Phaseolus vulgaris using simple sequence repeat signatures.

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