Rapid identification of columnar apples based on chlorophyll a/b ratio

【Objective】Columnar apple is a spontaneous bud mutant characterized by compact growth, shortened internodes, and easy formation of spurs. This unique architecture, coupled with high photosynthetic efficiency, would eliminate the need for extensive training and pruning. Consequently, the columnar phenotype would be well- suited for standardized, mechanized orchard management systems. Therefore, the columnar apple would be a highly valuable germplasm resource for breeding new apple varieties for simplified cultivation. The columnar growth habit is a qualitative trait governed by a single dominant gene, designated Co. Although molecular markers developed through the identification and characterization of this Co gene are available to facilitate the precise genetic identification of columnar apples, these techniques have considerable practical limitations. A previouse method including tissue sectioning and staininghas been developed in our laboratory, provided an alternative identification strategy for columnar apples. The present study was tried to develop another optional method for easy selection of columnar apples from hybrid population.【Methods】This investigation employed an ImagingPAM (Pulse Amplitude Modulated) chlorophyll fluorescence imaging system for the non-destructive assessment of photosynthetic parameters. A standardized leaf sampling protocol was instituted: commencing from the apex of a normally growing shoot, the first leaf exceeding 0.5 cm in length was designated as position one, and the eighth leaf from this point was systematically selected for all subsequent analyses. Leaf samples, collected from both confirmed columnar and non- columnar apple genotypes, were subjected to a dark adaptation period of 30 minutes to fully relax photosynthetic reaction centers. Following this adaptation, a comprehensive chlorophyll fluorescence analysis was conducted. Key parameters included the minimal fluorescence level (F ) and the maximum fluorescence yield (Fm) in the darkadapted state were recorded. These values were utilized to compute the maximum photochemical efficiency of Photosystem Ⅱ (PSⅡ), expressed as Fv/Fm, through the standardized formula: Fv/Fm=(Fm-F )/ Fm. In parallel, chlorophyll extraction and quantification were performed on leaf tissue obtained from the identical, standardized nodal position. The central midrib was meticulously excised and discarded from each sample leaf. The remaining lamina tissue was then finely fragmented, accurately weighed, and subsequently homogenized using a mechanical grinder in the presence of 2.5 mL of 95% ethanol. The grinding process continued after the addition of a further 10 mL of 95% ethanol until the plant material was completely bleached. The resulting homogenate was allowed to settle for 3 minutes before being quantitatively transferred, including insoluble residues, into a 15 mL volumetric flask, which was then brought to its final volume with 95% ethanol. For spectrophotometric analysis, a 1 mL aliquot of the primary extract was diluted with 2 mL of 95% ethanol. This diluted solution was transferred into a spectrophotometric cuvette with a 1 cm optical path length. Using a pure 95% ethanol solution as the blank reference, the absorbance of the sample was measured at two specific wavelengths: 665 nm and 649 nm. The respective concentrations of chlorophyll a and chlorophyll b were calculated using established arnon equations, from which the chlorophyll a to chlorophyll b ratio (Chla/b) was precisely determined. A comparative analysis of chlorophyll content and, more specifically, the Chla/b ratio was conducted between the columnar and non-columnar apple groups. The observed differential in the Chla/b ratio served as the foundation for proposing a new, physiology-based method for the rapid identification of columnar apple types. To rigorously validate the reliability and accuracy of this proposed physiological marker, an extensive trial was undertaken utilizing 62 individual plant samples sourced from a diverse panel of 20 distinct apple germplasms. All samples underwent the standardized chlorophyll extraction and Chla/b analytical procedure described above. The results derived from this physiological assay were systematically compared against those obtained from a established molecular marker analysis for the Co gene, which served as the definitive control or“gold standard”for genotypic identification, thereby allowing for a critical assessment of the new method's diagnostic precision.【Results】The experimental results indicated that under normal growth conditions, the maximum photochemical quantum yield of PSⅡ (Fv/Fm) was higher in the columnar apples compared with the non-columnar apples. The maximum fluorescence (Fm) values of the non-columnar apples were generally lower than those of the columnar apples. The minimum fluorescence (F ) values showed no significant difference between the columnar and the non-columnar apples, with columnar apples exhibiting slightly higher overall values. The overall identification accuracy of the method based on chlorophyll extraction and Chla/b analysis reached 97.6%. Specifically, the identification accuracy for columnar apples was as high as 100%. 【Conclusion】In conclusion, measuring the Chla/b ratio would provide a rapid, cost-effective, and reliable method for early screening of columnar apple progenies, with 100% accuracy in identifying columnar types.