Abstract
Insertion/deletion (InDel) markers are second most abundant polymerase chain reaction (PCR)-based molecular markers having enormous applications in genotyping and molecular breeding in different crops. Although standard polymerase chain reaction (PCR) for DNA amplification generally takes ~ 1.5 to 2 h, small amplicons can be effectively generated using dynamic heating and cooling through PCR with “V”-shaped thermal profile (VPCR) in ~ 15 to 20 min. Here, we evaluated the applicability of a partly modified VPCR method for amplifying InDels of tomato genome. Out of the 31 InDel markers tested in 15 diverse tomato genotypes, 29 markers resulted in sharp amplicons, where 26 markers were found to be polymorphic. Using this method, the individual DNA amplification reactions could be completed within ~ 30 min. The method was effective for primers varying in melting temperature (Tm) and GC contents. Furthermore, the need for empirically determining suitable annealing temperature could be bypassed using this generalised thermal profile. Through our results, we advocate the use of this method of DNA amplification in other plants to achieve rapid genotyping using standard molecular biology equipments and procedures.
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All data generated in this research is incorporated in the manuscript and associated supplementary information.
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Acknowledgements
We thank Prof. Pranab Hazra, BCKV for providing seeds of “purple tomato” genotype. ICAR-IIHR, Bengaluru, ICAR-IIVR Varanasi and NBPGR, India are acknowledged for providing other seed materials. The authors thank Arnab Mukherjee and Deepsikha Kumari for their help. Financial support from BAU in terms of project grant (Code: SNP/CI/Rabi/2018-5) is highly acknowledged. This article bears BAU COMMUNICATION NO.1189/220603.
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This research is funded by the project code: SNP/CI/Rabi/2018-5.
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TC conceived the idea. SS and TC performed the experiments. SA, TC and SS analysed the results. All the authors took part in preparation and correction of the manuscript. All the authors read the final manuscript and approved it.
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13205_2023_3499_MOESM2_ESM.tif
Supplementary Figure Sf1 VPCR to generate amplicons of different lengths using plasmid DNA as template. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing efficiency of partly modified VPCR thermal profile in generating amplicons of different lengths using gene-specific primers and recombinant plasmid DNA as template. L = 100 bp DNA ladder (BioLit, SRL) (TIF 1637 KB)
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Supplementary Figure Sf2 Failed and monomorphic InDel markers in 15 tomato lines using VPCR. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing the 2 failed markers (sli2645 and sli2282) and monomorphic amplicons generated by 3 InDel markers (sli394, sli536 and sli572) in 15 tomato lines by the partly modified VPCR method. Lane 1 = H-86, 2 = BRDT-1, 3 = CLN B, 4 = CLN-1621-L, 5 = IIHR 2614, 6 = Kashi Chayan, 7 = VRTOLCV-16, 8 = VRTOLCV-32, 9 = H-88-78-1, 10 = VRKB-08, 11 = VRKB-09, 12 = VRKB-12, 13 = VRCYT-04, 14 = 2018/TOINDVAR-05 and 15 = Purple Tomato. L = 100 bp DNA ladder (BioLit, SRL) (TIF 3888 KB)
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Supplementary Figure Sf3 Standard 3-step PCR using the failed InDel markers. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing the amplicons generated by the 2 failed markers (sli2645 and sli2282) in 4 tomato lines by the standard 3-step PCR method. Lane 1 = H-86, 2 = BRDT-1, 3 = CLN B, 4 = CLN-1621-L. L = 100 bp DNA ladder (BioLit, SRL) (TIF 1522 KB)
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Supplementary Figure Sf4 Dendrogram of 15 tomato genotypes generated on the basis of the polymorphism data generated by the 26 polymorphic InDel markers (TIF 1027 KB)
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Supplementary Figure Sf5 Influence of cycle numbers and ramp rate on VPCR. A. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing the amplicons generated by VPCR for 25, 30 and 35 cycles using 4 dilutions of genomic DNA isolated from the genotype H-86 by rapid method. B. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing the amplicons generated by 35 cycle-VPCR in 75 % and 100 % ramp rate using 4 dilutions of genomic DNA isolated from the genotype H-86 by rapid method. C = crude sample; 2D = 2-fold diluted sample; 5D = 5-fold diluted sample; 10D = 10-fold diluted sample. L = 100 bp DNA ladder (BioLit, SRL) (TIF 1743 KB)
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Supplementary Figure Sf6 Applicability of VPCR in genotyping of other crops. A. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing the amplicons generated by SSR marker emf01J09 (Forward primer: 5´-ATAGCACCCACACTAAACCTTGGG-3´ and Reverse primer: 5´-GTTTCACTTCTTGGTCCATTGTTCAGA-3´) and emb01J19 (Forward primer: 5´-GACAGGGATAGGGGTACGGATAGG-3´ and Reverse primer: 5´-TACCATGTGATGCCTCGATTTTCT-3´) by VPCR using brinjal genomic DNA isolated by rapid method from 4 genotypes. Lane 1 = BRBR-06, 2 = Haritha, 3 = BRBL-02, 4 = BRBL-01. B. Inverse image of ethidium bromide stained 2.5 % (w/v) agarose gels showing the amplicons generated by SSR marker RM413 (Forward primer: 5´-GGCGATTCTTGGATGAAGAG-3´ and Reverse primer: 5´-TCCCCACCAATCTTGTCTTC-3´) using rice genomic DNA isolated by rapid method from 4 genotypes. Lane 1 = Sabour Surbhit, 2 = Rajendra Sweta, 3 = Sabour Deep, 4 = TN-01. L = 100 bp DNA ladder (BioLit, SRL) (TIF 1202 KB)
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Chattopadhyay, T., Sangam, S. & Akhtar, S. Rapid genotyping in tomato by VPCR using agarose gel-resolvable InDel markers. 3 Biotech 13, 85 (2023). https://doi.org/10.1007/s13205-023-03499-x
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DOI: https://doi.org/10.1007/s13205-023-03499-x