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Assessment of resistance to rice tungro disease in popular rice varieties in India by introgression of a transgene against Rice tungro bacilliform virus

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Abstract

Rice crops in South and Southeast Asian countries suffer critical yield losses due to rice tungro disease caused by joint infection with rice tungro bacilliform virus (RTBV) and rice tungro spherical virus (RTSV). Previously, for generating RNA interference-based transgenic resistance against tungro viruses, RTBV ORF IV was used as a transgene to develop RTBV resistance in a popular high-yielding scented rice variety. The transgene from this line was then introgressed into five popular high-yielding but tungro-susceptible rice varieties by marker-assisted backcross breeding with a view to combine the resistant trait with the agronomic traits. The present work includes a resistance assay of the BC3F5 lines of these varieties under glasshouse conditions. Out of a total of 28 lines tested, each consisting of 12 individual plants, eight lines showed significant amelioration in height reduction and 100- to 1000-fold reduction in RTBV titers. The RNAi-mediated resistance was clearly manifested by the presence of virus-derived small RNA (vsRNA) specific for RTBV ORF IV in the transgenic backcrossed lines.

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Acknowledgements

This work was funded by Department of Biotechnology, Government of India (Grant No. BT/PR-15033/AGR/02/773/2011). GK is indebted to University Grants Commission, New Delhi, for research fellowships during this work. Funds received from R&D Grant of University of Delhi, DST-PURSE Grant, and the departmental infrastructure grant from DST-FIST are also gratefully acknowledged.

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Correspondence to I. Dasgupta.

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Suppl. Figure S1

Schematic representation of the orientation of forward and reverse primers in the two strategies (a and b) used for validation of pRTBV-Inf infection efficiency

Suppl. Figure S2

Confirmation of RTBV in rice plants upon agroinoculation. M, marker; lane 1, negative control; lanes 2 to 13, 12 plants selected for PCR; lane 14, RTBV-positive genomic DNA (1/10th dilution); lane 15, positive control (pRTBV-Inf plasmid)

Suppl. Figure S3

Validation of infection efficiency of the pRTBV-Inf agroinfectious clone. (a) With primer ISEPCIRCU FP/RP: lanes 1-6 and 9-14, plant samples; lane 7, negative control; lane 8, pRTBV-Inf plasmid. (b) With primer IINSVEC FP/RP: lane 1, negative control; lanes 2-13, plant samples; lane 14, positive control (pRTBV-Inf plasmid)

Suppl. Figure S4

Comparison of virus titer of the test TN1 plants at two time points

Suppl. Figure S5

Confirmation of RTBV (amplified as approximately 1-kb bands) in ASD 16 parent and backcrossed lines along with susceptible TN1 and resistant Utri Merah variety. –ve is the mock-inoculated DNA, while +ve is the 1/10th-diluted plasmid. ‘M’ corresponds to the 1-kb DNA marker

Suppl. Figure S6

Confirmation of RTBV (amplified as approximately 1-kb bands) in BPT 5204 parent and backcrossed lines. –ve is the mock-inoculated DNA, while +ve is the 1/10th-diluted plasmid. ‘M’ corresponds to the 1-kb DNA marker

Suppl. Figure S7

Confirmation of RTBV (amplified as approximately 1-kb bands) in CR 1009 parent and backcrossed lines. –ve is the mock-inoculated DNA, while +ve is the 1/10th-diluted plasmid. ‘M’ corresponds to the 1-kb DNA marker

Suppl. Figure S8

Confirmation of RTBV (amplified as approximately 1-kb bands) in Shatabdi and Khitish parent and backcrossed lines. –ve is the mock-inoculated DNA, while +ve is the 1/10th-diluted plasmid. ‘M’ corresponds to the 1-kb DNA marker

Suppl. Figure S9

Standard curve for estimation of RTBV titers

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Kumar, G., Jyothsna, M., Valarmathi, P. et al. Assessment of resistance to rice tungro disease in popular rice varieties in India by introgression of a transgene against Rice tungro bacilliform virus. Arch Virol 164, 1005–1013 (2019). https://doi.org/10.1007/s00705-019-04159-3

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