Unraveling phenotypic ambiguities of kagzi and ornamental lime accessions: a comprehensive exploration through morpho-biochemical and DNA marker profiling

The complexity of citrus phylogeny and taxonomy, influenced by polyembryony, genetic diversity, and extended generation cycles, necessitates assessing genetic relationships for improved production and disease resilience. DNA polymorphism markers are pivotal in genotyping and resolving phenotypic varietal ambiguities through accurate and objective genetic relatedness and distinctiveness assessments. In this study, 27 accessions of kagzi and ornamental lime were evaluated at morphological (based on IPGRI citrus descriptor), biochemical, and molecular levels using DNA-based markers, namely, random amplified polymorphic DNA (RAPD), start codon targeted (SCoT) polymorphism, and simple sequence repeats (SSR). Morphological analysis unveiled significant variation in leaf lamina attachment, shape, margins, fruit characteristics, and texture across all parameters. Biochemical research noted minimal differences, with total soluble solids (TSS) ranging from 5.7° to 8.0° Brix and titratable acidity (TA) from 0.36 to 0.50%. Notably, ornamental lime accessions exhibited higher TSS:TA ratios of 16.00. Molecular markers (RAPD, SCoT, and SSR) corroborated these findings, yielding 115, 74, and 64 amplicons, respectively, with polymorphic percentages varying from 33.33 to 100%. Polymorphic information content ranged from 0.20 to 0.90 for RAPD, 0.41 to 0.99 for SCoT, and 0.07 to 0.67 for SSR markers. Analysis of molecular variance (AMOVA) substantiated significant genetic differences among the kagzi and ornamental lime accessions (RAPD PhiPT = 0.267, SCoT PhiPT = 0.351, SSR PhiPT = 0.107). Furthermore, STRUCTURE software 2.3.4 revealed a ΔK value of two (K = 2), indicating two distinct genetic pools within the accessions, possibly attributable to bud mutations generating varying morphological traits within the same plant. Molecular marker-based germplasm characterization can mitigate confusion linked to morphological characteristics and multiple cultivar registrations.