Molecular insights to discover the nucleotide variations related to limonin-associated delayed bitterness in new interspecific citrus hybrids
Main Article Content
Abstract
In the present study, molecular analysis of limonoid-associated delayed bitterness was carried out in 16 newly developed inter-specific citrus scion hybrids (Citrus maxima Merr. × Citrus sinensis (L.) Osbeck) along with their parental genotypes. The Limonoid UDP-glucosyltransferase, a key enzyme responsible for debittering through glucosylation, was targeted for de novo primer design. However, PCR amplification using these primers could not decipher polymorphism based on the amplicon size among the 16 citrus hybrids and their parental genotypes. Therefore, five genotypes having contrasting bitterness properties (Low bitterness: SCSH-9-11/12, SCSH-11-9/13; high bitterness: SCSH-17-8/14 and parents White Fleshed Pummelo, and Mosambi) were selected for Sanger sequencing of PCR amplified products to decipher the variation at the nucleotide level. The analysis of variants and their annotation in the genomic region of the reference genome (Citrus sinensis (L) Osbeck) indicated a total of 19 missense variants corresponding to high limonin content and 12 missense variants for low limonin content. The transition to transversion ratio in the studied genotypes was found to be 0.83 and 0.29 for the high and low limonin groups, respectively. The changes in amino acids with respect to nucleotide variants in low limonin were identified. The identified nucleotide variations were exploited to design bitterness-specific primers in citrus sp., which serve as a reference SNV dataset. Further, this resource could be utilized to develop bitterness-specific markers for marker-assisted breeding in perennial citrus fruits.
Downloads
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
References
Anonymous 2023. Area and production of horticultural crops for the year 2021–22. Ministry of Agriculture & Farmers Welfare, Govt of India, New Delhi.
Anticona, M., Fayos, M. C., Esteve, M. J., Frigola, A., Blesa, J. and Lopez-Malo, D. 2022. Differentiation of juice of mandarin-like hybrids based on physicochemical characteristics, bioactive compounds, and antioxidant capacity. Eur. Food Res. Technol., 248(9): 2253–2262.
Baldwin, E., Plotto, A., Manthey, J., McCollum, G., Bai, J., Irey, M., Cameron, R. and Luzio, G. 2010. Effect of liberibacter infection (huanglongbing disease) of citrus on orange fruit physiology and fruit/fruit juice quality: Chemical and physical analyses. J. Agric. Food Chem., 58: 1247–1262.
Chandler, B. V. and Kefford, J. F. 1966. The chemical assay of limonin, the bitter principle of oranges. J. Sci. Food Agric., 17: 193–197.
De Mori, G. and Cipriani, G. 2023. Marker-assisted selection in breeding for fruit trait improvement: A review. Int. J. Mol. Sci., 24(10): 8984.
Deterre, S. C., McCollum, G., Leclair, C., Manthey, J. A., Bai, J., Baldwin, E. A., Raithore, S., Stover, E. and Plotto, A. 2021. Effect of Poncirus trifoliata on the chemical composition of fruits in pedigrees of citrus scion hybrids. Sci. Hortic., 277: 109816.
Doyle, J. and Doyle, J. 1987. A rapid procedure for DNA purification from small quantities of fresh leaf tissue. Phytochem. Bull., 19: 11–15.
FAO 2020. Citrus Fruit, Fresh and Processed: Statistical Bulletin 2020. Food and Agriculture Organization of the United Nations (FAO), Rome, Italy, 2020.
Fong, C. H., Hasegawa, S., Herman, Z. and Ou, P. 1989. Limonoid glucosides in commercial citrus juices. J. Food Sci., 54(6): 1505–1506.
Guadagni, D. G., Maier, V. P. and Turnbaugh, J. G. 1973. Effect of some citrus juice constituents on taste thresholds for limonin and naringin bitterness. J. Sci. Food Agric., 24(10): 1277–1288.
Gupta, A. K., Dhua, S., Kumar, V., Naik, B., Magwaza, L. S., Ncama, K. and Mishra, P. 2023. Current and emerging applications in detection and removal of bitter compounds in citrus fruit juice: A critical review. Food Biosci., 102995.
Huang, S., Dong, T., Xiong, B., Qiu, X., Sun, G., Liao, L. and Wang, Z. 2021. Variation in the content and composition of limonoids in fruits of four pummelo varieties during fruit development: The natural debittering process in pummelo fruits. J. Food Compos. Anal., 100: 103928.
Karim, M. R. and Hashinaga, F. 2001. Screening and some properties of limonoid glucosyltransferase from selected citrus varieties. Pak. J. Biol. Sci., 4: 486–483.
Kita, M., Hirata, Y., Moriguchi, T., Endo-Inagaki, T., Matsumoto, R., Hasegawa, S., Suhayda, C. G. and Omura, M. 2000. Molecular cloning and characterization of a novel gene encoding limonoid UDP-glucosyltransferase in citrus. FEBS Lett., 469(2–3): 173–178.
Kore, V. T. and Chakraborty, I. 2015. Efficacy of various techniques on biochemical characteristics and bitterness of pummelo juice. J. Food Sci. Technol., 52: 6073–6077.
Kruger, M. J., Davies, N., Myburgh, K. H. and Lecour, S. 2014. Proanthocyanidins, anthocyanins and cardiovascular diseases. Food Res. Int., 59: 41–52.
Lamo, K., Bhat, D. J., Kour, K. and Solanki, S. P. S. 2017. Mutation studies in fruit crops: A review. Int. J. Curr. Microbiol. Appl. Sci., 6(12): 3620–3633.
Liu, Y., Heying, E. and Tanumihardjo, S. A. 2012. History, global distribution, and nutritional importance of citrus fruits. Compr. Rev. Food Sci. Food Saf., 11(6): 530–545.
Maier, V. P., Hasegawa, S. and Hera, E. 1969. Limonin D-ring-lactone hydrolase. A new enzyme from citrus seeds. Phytochemistry, 8: 405–407.
Nishad, J., Singh, S. P., Singh, S., Saha, S., Dubey, A. K., Varghese, E. and Kaur, C. 2018. Bioactive compounds and antioxidant activity of selected Indian pummelo (Citrus grandis L. Osbeck) germplasm. Sci. Hortic., 233: 446–454.
Pardo, H., Abiola Owoyemi, O. B., Goldenberg, L., Yaniv, Y., Doron-Faigenboim, A., Carmi, N. and Porat, R. 2021. Sensory analysis of a new citrus juice made from 'Aliza' fruit: A new pomelo x mandarin hybrid. J. Food Sci. Nutr. Res., 4(1): 1–11.
Peterson, J. J., Beecher, G. R., Bhagwat, S. A., Dwyer, J. T., Gebhardt, S. E., Haytowitz, D. B. and Holden, J. M. 2006. Flavanones in grapefruit, lemons, and limes: A compilation and review of the data from the analytical literature. J. Food Compos. Anal., 19: S74–S80.
Puri, M., Marwaha, S. S., Kothari, R. M. and Kennedy, J. F. 1996. Biochemical basis of bitterness in citrus fruit juices and biotech approaches for debittering. Crit. Rev. Biotechnol., 16: 145–155.
Raghavan, S. and Gurunathan, J. 2021. Citrus species – a golden treasure box of metabolites that is beneficial against disorders. J. Herb. Med., 28: 100438.
Raithore, S., Dea, S., McCollum, G., Manthey, J. A., Bai, J., Leclair, C., Hijaz, F., Narciso, J. A., Baldwin, E. A. and Plotto, A. 2016. Development of delayed bitterness and effect of harvest date in stored juice from two complex citrus hybrids. J. Sci. Food Agric., 96(2): 422–429.
Rouseff, R. L. 1982. Nomilin, a new bitter component in grapefruit juice. J. Agric. Food Chem., 30: 504–507.
Saini, M. K., Capalash, N., Kaur, C. and Singh, S. P. 2019. Comprehensive metabolic profiling to decipher the influence of rootstocks on fruit juice metabolome of Kinnow (C. nobilis × C. deliciosa). Sci. Hortic., 257: 108673.
Salonia, F., Ciacciulli, A., Poles, L., Pappalardo, H. D., La Malfa, S. and Licciardello, C. 2020. New plant breeding techniques in citrus for the improvement of important agronomic traits: A review. Front. Plant Sci., 11: 1234.
Singh, N., Sharma, R. M., Dubey, A. K., Awasthi, O. P., Saha, S., Bharadwaj, C., Sharma, V. K., Sevanthi, A. M., Kumar, A. and Deepak. 2023. Citrus improvement for enhanced mineral nutrients in fruit juice through interspecific hybridization. J. Food Compos. Anal., 119: 105259.
Wang, F., Yu, X., Liu, X., Shen, W., Zhu, S. and Zhao, X. 2016. Temporal and spatial variations on accumulation of nomilin and limonin in pummelos. Plant Physiol. Biochem., 106: 23–29.
Wu, G. A., Terol, J., Ibanez, V., López-García, A., Pérez-Román, E., Borreda, C., Domingo, C., Tadeo, F. R., Carbonell-Caballero, J., Alonso, R. and Curk, F. 2018. Genomics of the origin and evolution of Citrus. Nature, 554(7692): 311–316.
Zaare-Nahandi, F., Hosseinkhani, S., Zamani, Z., Asadi-Abkenar, A. and Omidbaigi, R. 2008. Delay expression of limonoid UDP-glucosyltransferase makes delayed bitterness in citrus. Biochem. Biophys. Res. Commun., 371(1): 59–62.
Zhang, P., Liu, X., Yu, X., Wang, F., Long, J., Shen, W., Jiang, D. and Zhao, X. 2020. The MYB transcription factor CiMYB42 regulates limonoids biosynthesis in citrus. BMC Plant Biol., 20: 1–12.