Molecular insights to discover the nucleotide variations related to limonin associated delayed bitterness in new interspecific citrus hybrids

In the present study, molecular analysis of limonoid-associated delayed bitterness was carried out in 16 newly developed interspecific 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.