Development and Application of Tetra/Penta-Nucleotide SSR Markers for Paternal Identification in the Tea Plant...

In this study, twenty-seven novel SSR markers derived from tetranucleotide or pentanucleotide repeat sequences were developed for tea plants (Camellia sinensis). These markers, along with three previously reported pentanucleotide SSR markers, were assessed for their polymorphisms and capabilities for parental analysis. Analysis of 48 tea cultivars revealed 142 alleles with an average polymorphic information content (PIC) of 0.44, confirming the high polymorphism of these markers. Meanwhile, the capability of these markers for paternal identification in tea plants was also validated. Theoretical calculations yielded a cumulative exclusion probability (CPE) over 99.9999%. In the analyses of real samples using the exclusion method, among eight samples with confirmed parent-offspring relationships, both pair- and trio-mismatch counts were ≤1, whereas non-paternal samples exhibited average pair- and trio-mismatches of 5.13 and 7.96, respectively. When assessed by the combined parentage index (CPI), all trio-CPI values for confirmed parents-offspring exceeded 10⁴ (average: 1.18 × 10⁷), while the average trio-CPI for combinations with correct maternal but incorrect paternal parents remained below 10^-2. Finally, using this set of markers, we successfully identified 41 offspring derived from reciprocal crosses with open pollination between 'Fuding Dabaicha' and 'Chuancha Erhao'. Their trio-mismatches with the parent pair were ≤1, while trio-CPI exceeded 10⁴, meeting the established criteria for parentage relationships.