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Transcript profiling leads to biomarker identification for agarwood resin-loaded Aquilaria malaccensis

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Abstract

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The expression of terpenoid and defense response genes elevated during agarwood formation. TDFs obtained from these genes can serve as biomarkers to distinguish resin-loaded Aquilaria plant from healthy plants.

Abstract

Agarwood develops in a fraction of the wild Aquilaria population as a result of natural infections and wounding in the live vascular tissues of the Aquilaria malaccensis tree. Only an expert agarwood farmer or trader can identify resin-loaded trees by detecting the hole in the trunk, but this approach is less reliable and non-scientific. This must be replaced with a scientific approach to remove ambiguity and establish a standard protocol to assess the quality and hence the economic value of the specific Aquilaria tree. Each year, a significant number of trees are harvested due to high market demand and indiscriminate felling of Aquilaria tree. This study involves a cDNA-AFLP approach for transcript profiling using 64 selective primer combinations. This produced 2760 transcript-derived fragments (TDFs), of which 50 differentially expressed TDFs (DE-TDFs) were sequenced and identified. Amongst these seven were linked to terpenoid biosynthesis, one found in each LOX, abscisic acid, jasmonic acid, and defense response pathway. TDF-1–3, 5, and 6 were identified as sesquiterpene synthase, TDF-4 as a branch point enzyme, TDF-48 as a MAP kinase, TDF-47, and TDF-49 as transcription factors (TFs). To validate the cDNA-AFLP results qRT-PCR and semi-quantitative PCR was performed for 11 DE-TDFs. These DE-TDFs were expressed only in infected plants and thus can be used as a biomarker to identify and delineate economically valuable resin-containing plants.

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Acknowledgements

The authors would like to thank Dr. Mojibur Rahman Khan, IASST, Guwahati for providing access to the laboratory facilities and Dr. Mohammad Ghaznavi Idris, Assistant Professor, Department of Bioengineering and Technology, GUIST, Gauhati University, Guwahati for hel** in qualitative improvement of the manuscript along with the two anonymous reviewers for their valuable inputs. Authors would like to thank all the homestead growers of Aquilaria who provided assess to their plantations for sampling.

Funding

Authors acknowledge the Department of Biotechnology (DBT), Ministry of Science and Technology, Goverment of India, for the Research Grant (Grant no-BT/PR6346/GBD/27/405/2012). MRI acknowledges the Ministry of Minority Affairs, Goverment of India, for the Maulana Azad National Fellowship for Minority Students Grant, under Grant no—F1-17.1/201718-MANF-2017-18-ASS-78710/(SA-III/website).

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  1. Department of Bioengineering and Technology, GUIST, Gauhati University, Guwahati, Assam, 781014, India

    Md. Rofiqul Islam & Sofia Banu

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Correspondence to Sofia Banu.

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Data archiving statement

The differentially expressed TDF sequences were submitted to the NCBI GenBank database with accession number MK751302–MK751351. The full list of accession numbers and details of the sequences are included in supplementary Table 1.

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468_2021_2180_MOESM1_ESM.tif

Supplementary Fig.1 Representative image of molecular cloning. A). Blue-white screening of the recombinant clones. B). Colony PCR of the selective recombinant clones, C1-C7 are selective positive clones containing the TDF of interest. C). Recombinant plasmids (~ 3.5 kb) containing TDF of interest were isolated from C1 and C3 clones. D). PCR product of C1 and C3 plasmids amplified with TDF-5 (SS4) primers. M is a 100 bp molecular size marker in images B and D and a 1 kb molecular size marker in image C (TIF 9576 kb)

468_2021_2180_MOESM2_ESM.tif

Supplementary Fig.2 Fold change (gene expression level) values of the 11 identified DE-TDFs in five different geographical locations (TIF 191 kb)

468_2021_2180_MOESM3_ESM.tif

Supplementary Fig.3 Representative image of validation of 11 biomarkers a). DSS, DGS, MK, DXPS, DGS1, and SS4. b). FPS, LOX, MAPK3, MYC2, WRKY1, and GAPDH using semi-quantitative PCR analysis. The housekee** gene GAPDH is used as an internal control for this experiment. Sample coding: Nt1, Nt2, Nt3, Nt4, Nt5, Nt6, and Nt7 are infected plants of Namti and Ntn1, Ntn2, Ntn3, Ntn4, Ntn5, Ntn6, and Ntn7 are non-infected plants of Namti (TIF 8820 kb)

Supplementary file4 (TIF 7962 kb)

468_2021_2180_MOESM5_ESM.tif

Supplementary Fig.4 Artificial induction and natural infection of A. malaccensis A). Site of mechanical wounding with an electrical drilling machine B). Sealing the drilled hole with a bamboo stick C). Naturally infected A. malaccensis tree (TIF 3428 kb)

468_2021_2180_MOESM6_ESM.docx

Supplementary Table 1: List of identified DE-TDFs with their Genbank accession numbers and functional annotations (DOCX 20 kb)

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Islam, M.R., Banu, S. Transcript profiling leads to biomarker identification for agarwood resin-loaded Aquilaria malaccensis. Trees 35, 2119–2132 (2021). https://doi.org/10.1007/s00468-021-02180-1

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