Limits...
Novel and nodulation-regulated microRNAs in soybean roots.

Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O - BMC Genomics (2008)

Bottom Line: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean.The availability of complete and assembled genome sequence information will enable identification of many other miRNAs.The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA. ssubramanian@danforthcenter.org

ABSTRACT

Background: Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Results: We sequenced approximately 350000 small RNAs from soybean roots inoculated with B. japonicum and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to B. japonicum inoculation were identified. We also identified putative target genes of the identified miRNAs and verified in vivo cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified approximately 50% of miRNAs in soybean roots.

Conclusion: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

Show MeSH
Summary of the filtering process to identify candidate miRNA sequences in the library. See text for details about each filtering process. Numbers in each box represent the read numbers and numbers in parenthesis are frequency calculated as the percentage of reads retained (or discarded) after each step of processing compared to the total number of reads. Enrichment is calculated as the percentage of conserved miRNA reads in the number of library reads retained after each step of processing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC2335117&req=5

Figure 1: Summary of the filtering process to identify candidate miRNA sequences in the library. See text for details about each filtering process. Numbers in each box represent the read numbers and numbers in parenthesis are frequency calculated as the percentage of reads retained (or discarded) after each step of processing compared to the total number of reads. Enrichment is calculated as the percentage of conserved miRNA reads in the number of library reads retained after each step of processing.

Mentions: We cloned and sequenced small RNAs from mock-inoculated (Control library) and B. japonicum-inoculated (3 h) soybean roots (Bj library). We obtained a total of 159145 sequence reads from the control library and 194855 sequence reads from the Bj library using deep pyrosequencing (454 Lifesciences). For computational analysis and filtering of potentially non miRNA reads, we combined reads from both the libraries. For abundance calculations, reads from Control and Bj libraries were differentiated based on ID tags. Only reads with a recognizable adapter (see Methods) sequence were retained for further analysis ('high quality reads' in Figure 1A). Since 454 sequencing is not directional, we used the 5' adapter sequence to determine the direction of each sequence read. Where necessary, the sequences were converted to their corresponding reverse complements to facilitate computational analysis. Adapter sequences were removed and the sequences were cleaned of low quality reads (see Methods). All sequences 17 nt or longer in length were retained ('17nt or longer' in Figure 1A).


Novel and nodulation-regulated microRNAs in soybean roots.

Subramanian S, Fu Y, Sunkar R, Barbazuk WB, Zhu JK, Yu O - BMC Genomics (2008)

Summary of the filtering process to identify candidate miRNA sequences in the library. See text for details about each filtering process. Numbers in each box represent the read numbers and numbers in parenthesis are frequency calculated as the percentage of reads retained (or discarded) after each step of processing compared to the total number of reads. Enrichment is calculated as the percentage of conserved miRNA reads in the number of library reads retained after each step of processing.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC2335117&req=5

Figure 1: Summary of the filtering process to identify candidate miRNA sequences in the library. See text for details about each filtering process. Numbers in each box represent the read numbers and numbers in parenthesis are frequency calculated as the percentage of reads retained (or discarded) after each step of processing compared to the total number of reads. Enrichment is calculated as the percentage of conserved miRNA reads in the number of library reads retained after each step of processing.
Mentions: We cloned and sequenced small RNAs from mock-inoculated (Control library) and B. japonicum-inoculated (3 h) soybean roots (Bj library). We obtained a total of 159145 sequence reads from the control library and 194855 sequence reads from the Bj library using deep pyrosequencing (454 Lifesciences). For computational analysis and filtering of potentially non miRNA reads, we combined reads from both the libraries. For abundance calculations, reads from Control and Bj libraries were differentiated based on ID tags. Only reads with a recognizable adapter (see Methods) sequence were retained for further analysis ('high quality reads' in Figure 1A). Since 454 sequencing is not directional, we used the 5' adapter sequence to determine the direction of each sequence read. Where necessary, the sequences were converted to their corresponding reverse complements to facilitate computational analysis. Adapter sequences were removed and the sequences were cleaned of low quality reads (see Methods). All sequences 17 nt or longer in length were retained ('17nt or longer' in Figure 1A).

Bottom Line: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean.The availability of complete and assembled genome sequence information will enable identification of many other miRNAs.The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

View Article: PubMed Central - HTML - PubMed

Affiliation: Donald Danforth Plant Science Center, 975 N Warson Road, St Louis, MO 63132, USA. ssubramanian@danforthcenter.org

ABSTRACT

Background: Small RNAs regulate a number of developmental processes in plants and animals. However, the role of small RNAs in legume-rhizobial symbiosis is largely unexplored. Symbiosis between legumes (e.g. soybean) and rhizobia bacteria (e.g. Bradyrhizobium japonicum) results in root nodules where the majority of biological nitrogen fixation occurs. We sought to identify microRNAs (miRNAs) regulated during soybean-B. japonicum symbiosis.

Results: We sequenced approximately 350000 small RNAs from soybean roots inoculated with B. japonicum and identified conserved miRNAs based on similarity to miRNAs known in other plant species and new miRNAs based on potential hairpin-forming precursors within soybean EST and shotgun genomic sequences. These bioinformatics analyses identified 55 families of miRNAs of which 35 were novel. A subset of these miRNAs were validated by Northern analysis and miRNAs differentially responding to B. japonicum inoculation were identified. We also identified putative target genes of the identified miRNAs and verified in vivo cleavage of a subset of these targets by 5'-RACE analysis. Using conserved miRNAs as internal control, we estimated that our analysis identified approximately 50% of miRNAs in soybean roots.

Conclusion: Construction and analysis of a small RNA library led to the identification of 20 conserved and 35 novel miRNA families in soybean. The availability of complete and assembled genome sequence information will enable identification of many other miRNAs. The conserved miRNA loci and novel miRNAs identified in this study enable investigation of the role of miRNAs in rhizobial symbiosis.

Show MeSH