Unravelling rutin content of tartary buckwheat of north western Himalayas and insights into nucleotide polymorphisms in PAL gene to infer the associations with rutin biosynthesis

Tak, Aatif Mateen; Hami, Ammarah; Bhat, Basharat; Bhat, Sajad Ahmad; Masoodi, Khalid Z.; Bhat, M. Ashraf; Shah, M. D.; Khan, Mohd. Kamran; Zargar, Sajad Majeed

doi:10.1007/s13205-022-03218-y

Original Article
Published: 02 July 2022

Unravelling rutin content of tartary buckwheat of north western Himalayas and insights into nucleotide polymorphisms in PAL gene to infer the associations with rutin biosynthesis

Aatif Mateen Tak¹,
Ammarah Hami¹,
Basharat Bhat²,
Sajad Ahmad Bhat³,
Khalid Z. Masoodi⁶,
M. Ashraf Bhat⁶,
M. D. Shah⁴,
Mohd. Kamran Khan⁵ &
Sajad Majeed Zargar ORCID: orcid.org/0000-0001-6649-9447¹

3 Biotech volume 12, Article number: 156 (2022) Cite this article

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Abstract

Buckwheat (Fagopyrum spp.) has immense nutritional and nutraceutical potential. All the plant parts of buckwheat possess various metabolites, such as rutin, quercetin, vitexin etc. The high content of rutin in this pseudo cereal crop strongly adapts it to grow under adverse environments. In the present study 50 germplasm lines of Fagopyrum tataricum were used for estimation of seed endosperm rutin content through HPLC. Furthermore, molecular analysis of PAL gene (Phenylalanine Ammonia Lyase), an upstream gene in rutin biosynthesis pathway was targeted for detection of SNPs to understand the variations in the concentrations of seed endosperm rutin content, among tartary buckwheat genotypes with highest and lowest seed endosperm rutin content. Three primer pairs were employed for amplification of PAL gene for F. tartaricum (covering whole gene) followed by sequencing. Rutin concentration in seed endosperm of F. tartaricum ranged from 194.86 to 1403.22 ppm with an average of 617.06 ppm. Highest rutin concentration was found in genotype BWZ90 and lowest in BWZ16. Significant variations were observed in the seed endosperm rutin content among the genotypes of tartary buckwheat. Furthermore, alignment of PAL gene sequences of genotypes with high seed endosperm rutin content and low seed endosperm rutin content revealed variations at 21 polymorphic sites. The amino acid sequences obtained from the nucleotide sequences were also aligned and the variations were detected at 19 positions. The putative protein structure showed conformational changes among predicted proteins from two contrasting genotypes for endosperm rutin content. We here established an inventory of seed endosperm rutin content of tartary buckwheat. This study also provided insights about role of these SNPs in rutin biosynthesis. Furthermore, this information can be used for breeding buckwheat for high metabolite contents.

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Abbreviations

HPLC:: High-performance liquid chromatography
PAL:: Phenylalanine Ammonia Lyase
SNP:: Single nucleotide polymorphism
UV-B:: Type-B ultraviolet
NBPGR:: National Bureau of Plant Genetic Resources
DCM:: Dichloromethane
CTAB:: Cetyl-trimethyl ammonium bromide
dNTP:: Deoxynucleotide triphosphate
MgCl₂ :: Magnesium chloride
DMSO:: Dimethylsulfoxide
QTL:: Quantitative trait locus

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Acknowledgements

SMZ acknowledges support of National Mission on Himalayan Studies [NMHS] implemented by Ministry of Environment, Forest & Climate Change [MoEF&CC] vide project sanction No.: GBPNI/NMHS-2017-18/SG24/622. SMZ acknowledges the support of Prof. R K Salgotra (SKUAST-Jammu) for helping in HPLC analysis.

Funding

The work was partly funded by National Mission on Himalayan Studies [NMHS] implemented by Ministry of Environment, Forest & Climate Change [MoEF&CC] vide project sanction No.: GBPNI/NMHS-2017–18/SG24/622.

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Authors and Affiliations

Proteomics Laboratory, Division of Plant Biotechnology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Jammu and Kashmir, 190025, Srinagar, India
Aatif Mateen Tak, Ammarah Hami & Sajad Majeed Zargar
NAIP, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Jammu and Kashmir, 190025, Srinagar, India
Basharat Bhat
Division of Basic Sciences, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Jammu and Kashmir, 190025, Srinagar, India
Sajad Ahmad Bhat
Division of Plant Pathology, Sher-e-Kashmir University of Agricultural Sciences and Technology of Kashmir, Shalimar, Jammu and Kashmir, 190025, Srinagar, India
M. D. Shah
Department of Soil Science and Plant Nutrition, Faculty of Agriculture, Selcuk University, Konya, 42079, Turkey
Mohd. Kamran Khan
Division of Plant Biotechnology, Sher-e-Kashmir University of Agricultural Sciences & Technology of Kashmir, Shalimar, Srinagar, India
Khalid Z. Masoodi & M. Ashraf Bhat

Authors

Aatif Mateen Tak
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Ammarah Hami
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Basharat Bhat
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Sajad Ahmad Bhat
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Khalid Z. Masoodi
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M. Ashraf Bhat
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M. D. Shah
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Mohd. Kamran Khan
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Sajad Majeed Zargar
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Contributions

AMT did biochemical and molecular research; AH and SAB helped in biochemical analysis; BB, MDS and MKK helped in bioinformatics analysis, SMZ conceived research idea, guided all laboratory and bioinformatics work, finalized the manuscript. AMT and AH wrote the first draft of manuscript which was further edited by KZM, MAB, MKK and SMZ. AH, MKK, BB and SMZ did revision of the manuscript.

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Correspondence to Sajad Majeed Zargar.

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Supplementary Information

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Supplementary file 1: BLASTX against NCBI non-redundant protein sequence database (Fagopyrum–taxid:3616) (DOCX 13 kb)

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Tak, A.M., Hami, A., Bhat, B. et al. Unravelling rutin content of tartary buckwheat of north western Himalayas and insights into nucleotide polymorphisms in PAL gene to infer the associations with rutin biosynthesis. 3 Biotech 12, 156 (2022). https://doi.org/10.1007/s13205-022-03218-y

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Received: 01 April 2022
Accepted: 14 June 2022
Published: 02 July 2022
DOI: https://doi.org/10.1007/s13205-022-03218-y

Keywords

Buckwheat
Rutin
PAL
Nutraceutical
Protein structure