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Subtractive transcriptomes of fruit and leaf reveal differential representation of transcripts in Azadirachta indica

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Abstract

Azadirachta indica produces a wide array of secondary metabolites of medicinal and agricultural importance in an organ-specific or abundant manner. We used suppression subtractive hybridization strategy to clone and identify the rare and differentially expressed transcripts in fruit and leaf which could be missed out by high-throughput sequencing. Subtractive ESTs were generated, assembled and their comparison showed that around 61.8 % and 33.9 % unigenes were unique to A. indica fruit and leaf, respectively. Around 12.3 % and 16.5 % of our unigenes were not represented in the available A. indica draft genome and transcriptomes sequences, respectively, indicating a significant fraction of novel and rare expressing transcripts. Blast2GO functional annotation could assign 721 Gene Ontology (GO) terms to 50 % of the assembled unigenes, and Kyoto Encyclopedia of Genes and Genomes (KEGG) mapping mapped 66 (27.96 %) unigenes onto 39 different pathways among which 26 (11 %) were metabolism-related. Ten cytochrome p450s were identified, and their KEGG and phylogenetic analysis indicated that five of them were secondary metabolism-related which could be functioning at specific pathway steps to synthesize organ-specific compounds. For reverse transcription-quantitative polymerase chain reaction (RT-qPCR) analysis, we tested three housekeeping genes to be used as internal reference, among which elongation initiation factor 4A was found to be most stable. Expression analysis of secondary metabolism-related candidates showed their variable and tissue-preferential expression indicating their relative enzymatic activities and roles in partitioning of secondary metabolites in different organs. Our small-scale subtractive ESTs being longer in read length provide a useful resource for studying organ-preferential secondary metabolic pathways operating in A. indica and would complement the available draft genome/transcriptome(s).

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Abbreviations

CYP:

Cytochrome p450

EST:

Expressed sequence tag

GO:

Gene Ontology

KEGG:

Kyoto Encyclopedia of Genes and Genomes

NCBI:

National centre for biotechnology information

RT-qPCR:

Reverse transcription quantitative polymerase chain reaction

SRA:

Sequence read archive

SSH:

Suppression subtractive hybridization

TSA:

Transcriptome shotgun assembly

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Acknowledgments

This work was financially supported by Council of Scientific and Industrial Research, Govt. of India, under the grant HCP002. Research fellowship from University Grants Commission, Govt. of India, is also acknowledged. We thank CSIR-National Botanical Research Institute, India for providing A. indica tissues. The authors wish to express their sincere thanks to the Director, CSIR-CIMAP, for encouragement and providing necessary facilities.

Data archiving statement

All the subtractive ESTs were deposited in NCBI GenBank and accessions were obtained. The GenBank ID for the fruit-specific subtractive library is LIBEST_027324 and the accession numbers of all ESTs from fruit library are: JK126220-JK126259, JK341301-JK341454, JK479929-JK480085 and JK623987-JK624163. The GenBank ID for the leaf-specific subtractive library is LIBEST_027962 and all ESTs from this library were submitted under the accession numbers: JK998954-JK999475. A complete list of GenBank accession numbers of our submitted sequences is provided in Tables S1 and S2.

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Authors and Affiliations

  1. Biotechnology Division, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India

    Raja Rajakani & Vikrant Gupta

  2. Department of Metabolic and Structural Biology, CSIR-Central Institute of Medicinal and Aromatic Plants, Lucknow, 226015, India

    Lokesh Narnoliya, Neelam S. Sangwan & Rajender S. Sangwan

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  1. Raja Rajakani
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  2. Lokesh Narnoliya
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  3. Neelam S. Sangwan
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  4. Rajender S. Sangwan
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  5. Vikrant Gupta
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Correspondence to Vikrant Gupta.

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Communicated by W. Ratnam

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Fig. S1

Snapshot representation of SRA BLAST analysis of in-house generated A. indica unigenes with A. indica var. Indica transcriptomes showing partial homology (PPT 1433 kb)

Fig. S2

Blast2GO functional annotation of A. indica unigenes. a GO-level distribution of separately assembled A. indica fruit-specific SSH library. b GO-level distribution of separately assembled A. indica leaf-specific SSH library. c Length distribution, d E-value distribution and e number of GO terms for sequences with length distribution (PPT 280 kb)

Fig. S3

Phylogenetic analysis of A. indica cytochrome p450 unigenes with Arabidopsis CYPs. A neighbor-joining tree was constructed for identified ten A. indica cytochrome p450s along with 482 full-length CYPs from Arabidopsis thaliana. Cytochrome p450 clans and families identified for A. indica CYPs are marked (PPT 2651 kb)

Fig. S4

Quality of total RNA used and specificity of real-time PCR primer pairs. a Total RNA isolated from A. indica fruit, mesocarp, leaf and endocarp (shown in triplicates). b Specific RT-PCR amplification of A. indica candidates selected for expression analysis (PPT 701 kb)

Fig. S5

Melting (dissociation) curve analysis to check specific amplification during RT-qPCR (PPT 2324 kb)

Table S1

GenBank accession numbers for all in-house A. indica subtracted ESTs from fruit-specific library (DOC 625 kb)

Table S2

GenBank accession numbers for all in-house A. indica subtracted ESTs from leaf-specific library (DOC 640 kb)

Table S3

BLAST analysis of in-house unigenes against Azadirachta indica draft genome and transcriptomes at NCBI. (Sheet-1): SRA BLAST against A. indica (var. Indica) draft genome sequence by SRA BLAST analysis. (Sheet-2): BLASTN against A. indica draft genome sequence in TSA database. (Sheet-3): SRA BLAST against A. indica (var. Indica) transcriptomes by SRA BLAST analysis (XLS 55 kb)

Table S4

BLASTX analysis of in-house A. indica unigenes against nr database of NCBI (XLS 103 kb)

Table S5

A. indica unigenes mapped in KEGG pathways with their EC numbers and pathway ID (XLS 27 kb)

Table S6

Blast2GO functional annotation of A. indica unigenes. GO terms and GO IDs associated with the unigenes are given. (Sheet-1): biological process. (Sheet-2): molecular function. (Sheet-3): cellular components (XLS 65 kb)

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Rajakani, R., Narnoliya, L., Sangwan, N.S. et al. Subtractive transcriptomes of fruit and leaf reveal differential representation of transcripts in Azadirachta indica . Tree Genetics & Genomes 10, 1331–1351 (2014). https://doi.org/10.1007/s11295-014-0764-7

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