Chemical Diversity of Volatiles From Parents , Rootstock and Atemoya Hybrid

Hybridization promotes the transfer of genetic material from parental species to the hybrid, which can be closely related to one of its parental species or present new features that provide them higher competitivity concerning other chemical phenotype comparing to its parents. On the other hand, grafting technique, in which occurs the combination between two species, also may lead to changes in the volatile profile of terpenes at the grafted plant. The objective of this research was to characterize the chemical profile of volatile compounds in leaves of atemoya hybrid (Annona × Atemoya) (Mobb.), its male and female parents (Annona squamosa Mill. and Annona cherimola L.), as well as its common rootstock (Annona emarginata (Schltdl.) H. Rainer var. “Terra-fria”). Leaf samples from atemoya, its parents and the rootstock were collected in São Bento do Sapucaí and Santa Fé do Sul, São Paulo state, Brazil. Volatile substances were obtained by microextraction solid phase (SPME). Chemical profile of volatile compounds was determined and identified by gas chromatography coupled to mass spectrometry. Atemoya hybrid presented substances also found in both parents and rootstock, with a variation on the relative percentage of compounds. Such variation allowed to form three clusters, where I was comprised by the hybrid, II comprised by A. cherimola, and the latter, III, grouping the rootstock and the male parent. We concluded that hybridization and grafting influence production of terpenes in atemoya hybrid (Annona × Atemoya).


Introduction
Hybridization allows the transfer of genetic material from parental species to the hybrid, which can be more related to one of its parents or show features completely new.Hybrids can present variable adaptation to the local conditions where they occur, which, added to the inherited features from parents, could provide them higher competitivity comparing to the endemic and rare species (López-Caamal & Tovar-Sánchez, 2014).Though, hybrid phenotype could be typically more robust than that from its parents, resulting in a remarkable importance to agriculture (Greaves et al., 2015).
The Annonaceae family is comprised by the genus Annona, one of its most important, especially due to its edible fruits like those of atemoya (Anonna × atemoya), a hybrid formed by the outcrossing between Annona cherimola Mill.and Annona squamosa L. Atemoya is characterized by intermediate climatic adaptation in relation to its parents, like fruit quality conferred by A. cherimola, as well as the resistance to the climatic conditions conferred by A. squamosa (Tokunaga, 2005).
The theme complexity increases, even more, when hybridization is considered together with grafting process.Apparently, the processes of grafting regulation and control occur due to water and mineral nutrients absorption, hormone synthesis, and because of both protein and mRNA migration (Baron, Bravo, Maia, Pina, & Ferreira, 2016;Davis et al., 2008;Harada, 2010), added to transfer of plastidial DNA from rootstock to the graft (Stegemann & Bock, 2009).The choose of rootstock species is related to the resistance increase against pathogens, faster dissemination and production of fruits, and the ability to impact the graft phenotype, although such mechanisms are not well understood yet (Warschefsky et al., 2016).
Typically, atemoya is grafted in Annona emarginata (Schltdl.)H. Rainer var.'Terra-fria' which confers diseases and pathogens resistance to hybrid, favoring its cultivation (Baron, Amaro, Pina, & Ferreira, 2019).Grafting influences the production and chemical profile of volatiles from hybrid essential oils relatively to its parents by the transfer of genetic material and, subsequently, modification in metabolic routes as well as in the specialized metabolism (Cano & Bermejo, 2011;Harada, 2010).
The expression of specialized metabolites is frequently regulated by different genes, which could explain an intermediate chemical volatile profile in hybrids and its parents (Cheng, Vrieling, & Klinkhamer, 2011;López-Caamal & Tovar-Sánchez, 2014).Variations in genetic expression of specialized metabolites could be linked to changing of its epigenome (Greaves et al., 2015), indicating an inheritance complex pattern from this trait (Cheng et al., 2011;Zito et al., 2018).
Once insects may be the main allies for several species pollination, as well as in the interaction between plants and defense against herbivores and pathogens (Alzogaray et al., 2011;Hommel et al., 2016;Wang, Liu, Wei, & Yan, 2012;Zito et al., 2018), the fluctuations in chemical profile could be important for comprehending plant survival strategies, defense and reproduction.
Species from Annonaceae family, including parents, rootstock and hybrids, present essential oils with different substances that, besides acting in defense strategies and pollinators attraction for reproduction and survival, also may show pharmacological properties with economic importance.
The objective of this research was to characterize and compare chemical profile composition of leaf volatiles in hybrid Annona × atemoya (Mobb.), with its parents Annona cherimola L., Annona squamosa Mill.and the rootstock Annona emarginata (Schltdl.)H. Rainer var."Terra-fria".

Capture and Analysis of Volatile Compounds
Leaves were dried in a forced air oven with a temperature held at 30 °C to achieve its constant dry mass.Volatile substances were captured by headspace-solid phase microextraction (HS-SPME).So, 50 mL of leaf dry mass grinded were stored in a 20 mL glass vial with a cover provided of a septum, and subsequently added 10 mL of distilled water.The vial glass was heated for 1 hour in a water bath at 90 °C.Afterward, the volatile compounds were captured by headspace for 15 minutes using a DVB/PDMS fiber (Manual Holder-SUPELCO).The extraction of volatile compounds was executed in triplicate for each sample.Chemical analysis of volatile compounds was determined by gas chromatograph coupled to a mass spectrometer (GC-MS-Shimadzu, QP-5000), operating at 70 eV, equipped with a DB-5 fused silica capillary column (30 m × 0.25 mm × 0.25 μm), helium as carrier gas (1.0 mL/min), injector at 240 °C, detector at 230 °C, split: 1/20, using the temperature program of 60-240 °C, 3 °C/min.Substances were identified by comparing its mass spectrum with data bank from CG-EM (Nist 62.lib) system; Retention indexes (IR) and literature data (Adams, 2017).Retention indexes (IR) of the substances were obtained by analysis of the mixture of n-alkanes (C 9 -C 24 Sigma Aldrich 99%), at the same operational conditions of samples, in which it was applied the equation of Van den Dool & Kratz , 1963(Van Den Dool & Dec. Kratz, 1963).
The dendrogram presents the formation of three clusters, in which cluster I is comprised by atemoya hybrid, cluster II by A. cherimola and, III groups the rootstock A. emarginata and male parent A. squamosa (Figure 2).t (A.emale as a t the compatibility and success in hybrid grafting frequently occur due to similar features between rootstock and at least one its parents, according to the already recorded by Baron et al. (2019), condition that may be suggested by this present work about chemical profile of volatiles.
According to the interpretation of Cheng et al. (2011), the studied chemical profiles of volatiles reveal that atemoya hybrid (Annona × atemoya) expressed intermediate phenotype in relation to its parents.However, our results reveal that atemoya also present a chemical profile intermediate to the rootstock.These results are in agreement with studies in literature, which report that the majority of specialized metabolites can be expressed in similar concentrations or even intermediate to those ones from its parents (Zito et al., 2018).Besides, in hybrid, substances may have its relative percentages raised or even suppressed (Cheng et al., 2011;López-Caamal & Tovar-Sánchez, 2014), condition that can contribute with the intermediate chemical profile.
Although it was not possible to identify and confirm the origin of substances presented in hybrid from its parents and rootstock, hybridization and grafting associated to both environment and intraspecific conditions, may contribute to variations in the expression of the volatile profile (Selmar & Kleinwächter, 2013;Zito et al., 2018).This variation can provide greater resistance to pathogens, allowing a better environment adaptation in different climatic conditions (Baron et al., 2019;Chezem & Clay, 2016).
Despite the variation in relative percentage and substances predominance of volatile profile from both parents and rootstock, these plants show similar substances, which suggest that hybridization and grafting influence terpene production in atemoya hybrid.