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Development of EST-based methylation specific PCR (MSP) markers in Crocus sativus

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Abstract

Background

Saffron (Crocus sativus) is high valued spice crop, but due to its sterile nature, the crop is propagated exclusively through corms. Thus, the genetic base of this crop is very narrow, however, frequency of phenotypic variability is observed; and suggested the potential role of epigenetics in saffron crop growth and development.

Methods and results

To facilitate epigenetic studies in saffron, we developed 1525 methylation-specific PCR (MSP) markers using MethPrimer. For this purpose, we used 6767 EST sequences of saffron available on the NCBI database. We also mine CpG islands (2555) and found that 32.7% of EST sequences had CpG islands. Out of 1525 MSP markers developed during the present study, 725 covered the CpG islands and 800 were without CpG islands. PCR amplification was found successful for 82% of MSP markers. A preliminary analysis suggested that 53.7% of genomic sites were methylated and more prominent (60% methylations) in non-CpG island regions, although, more comprehensive studies are required to validate it further.

Conclusions

The epigenetic resource developed during the present study will strengthen the epigenetic studies like epiQTL mapping, epiGWAS to explore the molecular mechanisms and genomic/epigenomic regions associated with phenotype; and further may be utilized for saffron improvement programs through epibreeding.

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Acknowledgements

This study was supported by the Council of Scientific and Industrial Research (CSIR) for providing funds (MLP-201), V.J. also thanks to the Science and Engineering Research Board (SERB) for the Early Career Research Award and Department of Science and Technology (DST) for the INSPIRE faculty award. VC thanks CSIR-UGC for Junior Research Fellowship. This manuscript represents CSIR-IHBT communication number 5100.

Funding

This study was supported by the Council of Scientific and Industrial Research (CSIR) for providing funds (MLP-201) to Vandana Jaiswal.

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Authors and Affiliations

  1. CSIR-Institute of Himalayan Bioresource Technology, Palampur, Himachal Pradesh, 176061, India

    Vishek Choudhary, Deepika Shekhawat, Anita Choudhary & Vandana Jaiswal

  2. Academy of Scientific and Innovative Research (AcSIR), Ghaziabad-201002, India

    Vishek Choudhary, Anita Choudhary & Vandana Jaiswal

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  1. Vishek Choudhary
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  2. Deepika Shekhawat
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  3. Anita Choudhary
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  4. Vandana Jaiswal
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Contributions

VC- data acquisition, wet-lab experiments, analysis, writing; DS- data acquisition, analysis; AC- data acquisition, analysis; VJ- conceptualization, supervision, writing and editing, fund acquisition.

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Correspondence to Vandana Jaiswal.

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Supplementary Information

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11033_2022_7967_MOESM1_ESM.xlsx

Supplementary file1 (XLSX 202 KB)Table S1 Details of methylation-specific PCR (MSP) markers developed during present study.

11033_2022_7967_MOESM2_ESM.xlsx

Supplementary file2 (XLSX 13 KB)Table S2 Conserved domain, pfam ID and putative functions of expressed sequence tags (ESTs) from where methylation-specific PCR (MSP) markers were developed and used for PCR amplification in saffron.

11033_2022_7967_MOESM3_ESM.pptx

Supplementary file3 (PPTX 35 KB)Fig. S1 The workflow of MSP marker development and its profiling in saffron plants for detection of methylated and non-methylated sites.

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Choudhary, V., Shekhawat, D., Choudhary, A. et al. Development of EST-based methylation specific PCR (MSP) markers in Crocus sativus. Mol Biol Rep 49, 11695–11703 (2022). https://doi.org/10.1007/s11033-022-07967-0

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