Rapid Differentiation of Closely Related <i>Citrus Genotypes</i> by Fluorescence Spectroscopy

Abstract

The differentiation of closely related Citrus genotypes is a meticulous, laborious, and time-consuming task that involves the assessment of complex traits such as growth, tolerance to stress, photosynthetic efficiency, yield, and many others. Such a task is generally accomplished either by analyzing specific features of adult plants or by applying molecular markers to young trees. On one hand, only after plants start yielding can distinct genotypes be differentiated by comparing their fruit sizes, shapes, taste, and the number of seeds. On the other hand, molecular markers are expensive, and demand expertise and time for the analysis of a larger number of plants. For these reasons, the development of techniques that could assist in an early, quick and accurate differentiation of closely related Citrus varieties is of utmost importance. In this context, laser-induced fluorescence spectroscopy (LIFS) is a promising technique, since it is rapid, highly sensitive, and inexpensive. Previous studies showed that LIFS can differentiate a variety of sweet orange. However, this new study aimed to determine LIFS accuracy in the differentiation and grouping of very closely varieties of four Sunki mandarin selections: Comum, Florida, Tropical, and Maravilha. Furthermore we compared the results with ISSR and SSR molecular markers for the same varieties. LIFS technique distinguished the four selections with accuracy greater than 70%. Only with molecular markers was possible distinguishing clearly Tropical from Maravilha, but not Comum from Florida selections. In this way the results suggest that LIFS may be a sound tool for helping the identification of closely Citrus varieties.

Laser-induced Fluorescence Spectroscopy (LIFS) for Discrimination of Genetically Close Sweet Orange Accessions (Citrus sinensis L. Osbeck)

“…For the measurements performed with the LIFS-560 nm, the spectrometer parameters adopted were: 2 ms for integration time, 20 for the spectral averages, and 2 for boxcar. 18,19 The experimental procedure consists of positioning the optical fiber probe perpendicularly on the ventral portion of the leave in natura near the midrib, making sure that the light does not focus directly on the veins of the leaf. Before the measurement, each sample was exposed to excitation source for about 2 s, which is enough to reach fluorescence steady state.…”

“…Recently, another technique called laser-induced fluorescence spectroscopy (LIFS) was employed as a tool to identify citrus varieties. 18,19 The results showed that this technique was able to discriminate the different citrus varieties in a nursery. Milori et al.…”

“…Recently, another technique called laser-induced fluorescence spectroscopy (LIFS) was employed as a tool to identify citrus varieties. 18,19 The results showed that this technique was able to discriminate the different citrus varieties in a nursery. Milori et al 18 used an excitation laser of 561 nm and showed that LIFS combined with statistical methods is able to discriminate different citrus varieties.…”

“…These changes are successfully identified by optical techniques because it alters the characteristic optical signature of the leaves from each accession and discriminate them among its genetics. 9,18,19 Two different LIFS systems, one with excitation wavelength at 405 nm and the other one with excitation wavelength at 561 nm, combined with chemometric tools, were used to discriminate these sweet oranges varieties. The evaluation was performed in two different sets of leaves, from ten different citrus varieties in each set collected in two different seasons: the first one in winter time and the second one in summer time.…”

“…However, FDA using Mahalanobis distance showed the best success rate for classification of the Natal variety (around 81%). In previous work described in Santana-Vieira et al., 19 two different excitation lasers, 405 nm and 561 nm, aimed to determine LIFS accuracy in the differentiation and grouping very closely varieties of four Sunki mandarin selections: Comum, Florida, Tropical, and Maravilha. The results obtained with LIFS was compared with the ones obtained with ISSR and SSR molecular markers for the same varieties.…”