Fruit Quality Assessment of Novel Hybrid Pummelo × Sweet Orange and Its Molecular Characterization Using Acidity Specific Markers

SUMMARY Research background There is considerable diversity in newly developed pummelo × sweet orange citrus hybrids. Most hybrids showed lower peel thickness and high juice yield but there is a lack of information on fruit quality parameters and molecular characterization. Therefore, the aim of the current study is to determine the content of antioxidants and properties of the fresh juice of 24 new pummelo × sweet orange citrus hybrids (Citrus maxima [Burm. f.] Osbeck × Citrus sinensis [L.] Osbeck) and the parental genotypes along with molecular characteristics determined using acidity specific markers. Experimental approach The correlation and estimate of inheritance of the fruit juice properties: ascorbic acid, total phenol, total flavonoid, total antioxidant, total soluble solid and sugar contents, pH, titratable acidity, along with sensory evaluation was performed. Molecular characterization of these hybrids was carried out using de novo generated acidity specific simple sequence repeat (SSR) markers. Results and conclusions The main constituents of the fruit juice of pummelo × sweet orange hybrids were observed in the range of w(ascorbic acid)=40.00–58.13 mg/100 g, total phenols expressed as gallic acid equivalents w(GAE)=40.67–107.33 mg/100 g, total antioxidants expressed as Trolox equivalents b(Trolox)=2.03–5.49 µmol/g, total flavonoids expressed as quercetin equivalents w(QE)=23.67–59.33 mg/100 g, along with other properties: total soluble solids=7.33–11.33 %, w(total sugar)=2.10–5.76 %, w(reducing sugar)=1.69–2.78 %, w(non-reducing sugar)=0.39–3.17 % and titratable acidity 1.00–2.11 %. The above parameters differed significantly in the fruit juice of the evaluated pummelo × sweet orange hybrids. Considering these parameters, the hybrids SCSH 17-9, SCSH 13-13, SCSH 11-15 and SCSH 3-15 had superior antioxidant properties in terms of these parameters. A higher heritability (≥80 %) was also observed for all juice properties. Molecular characterization of pummelo × sweet orange hybrids showed that >50 % of the hybrids were grouped with medium acidity parents. Both molecular and biochemical parameter-based clustering showed that interspecific hybrids exhibit transgressive segregation with increased antioxidants that help alleviate the health problems. Novelty and scientific contribution These newly developed pummelo × sweet orange citrus hybrids are a valuable source of high-quality antioxidants for a healthy diet. The identification of trait markers that enable selection at the seedling stage is of great benefit to citrus breeders, as the characteristic features of a mature tree are not yet visible at the juvenile stage.


INTRODUCTION
Globally, malnutrition affects over two billion people, with 924 million experiencing undernourishment.This condition contributes to approximately 45% of deaths among children under the age of five (1).Besides around 828 million are being suffered from hunger worldwide and the outbreak of the covid-19 pandemic has further worsened the situation (2).The highest number of under-nourished people (15.2 %) live in India, and about 38.4 % of the children (<5 years) in India are stunted, whereas more than 35.7 % are underweight (3).Zero hunger and good health are the major goals of United Nations.The Sustainable Development Goals Report 2023 has shown the wide gaps to achieve the targeted goals.Citrus fruits are widely cultivated fruit crops across the globe and are consider the most commonly consumed fruits for enhanced health properties worldwide (4).For their pharmaceutical attributes Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
including anti-inflammatory, antisclerotic, antiviral, antibacterial, and anticancer properties, citrus fruits have long been esteemed as integral to a healthy diet (5).Despite being nutritionally rich, the nutritional properties of citrus fruits have often gone unnoticed (6,7).
Various species of citrus fruits exhibit distinct chemical compositions.The principal constituents of the sweet orange include sugars (such as glucose, fructose and sucrose) and acids, primarily citric acid with a smaller amount of malic acid.Citrus fruits contain considerable number of ascorbic acids (vitamin C) (8).In most citrus fruits, the total soluble solids (TSS) in the fruit juice typically range from 8-12 °Bx, while the titratable acidity commonly falls within the range of 0.5-1.5 %.Generally, orange and mandarins have TSS:acid ratio of 12-14:1, but in pummelo it is 6:1.The vitamin C in the fruit juices ranged between 25 and 85 mg per 100 mL of juice.Furthermore, citrus fruits are abundant in nutrients such as flavonoids and fiber, which play a role in safeguarding arteries, reducing inflammation, improving gastrointestinal health, and potentially aiding in the prevention of conditions like diabetes, cancer, and neurological disorders.Vitamin C is essential for the development and maintenance of strong bones, skin, connective tissues, and blood vessels.The immune system is strongly aided by vitamin C, which also functions as an antioxidant that may help shield your cells from the damaging effects of free radicals, and helps to reduce inflammation (9).The non-heme iron found in plant foods, which is absorbed by the body with the aid of vitamin C. Thus, consuming citrus fruits coupled with plant foods like nuts, seeds, and legumes helps to improve the body's absorption of iron.The preventive properties of citrus fruits extend to a wide range of nutrients, including a sizable plant chemical family known as the flavonoids (10).The bioactive chemical compounds present in citrus, such as flavonoids, carotenoids, terpenes, and limonoids, hold promising potential in combating obesity, inflammatory conditions, atherosclerosis, neurological disorders, and cancer owing to their remarkable antioxidant properties.By reducing fat absorption, pancreatic lipase (PL), an essential enzyme involved in the hydrolysis of triglycerides in the digestive system, can reduce obesity.There is a continuous demand for developing the new improved cultivars fortified with mineral nutrients.
Hybridization stands as a pivotal method in crop improvement for perennial fruit crops, notably in citrus, facilitating advancements in tree growth attributes, extended harvesting of high-quality fruits, and imparting resistance against both biotic and abiotic stresses (11).In contemporary citrus crop improvement programs, the focus lies on developing high-quality fruits enriched with health-promoting bioactive compounds as the targeted traits (11).
Pummelo (Citrus maxima (Burm.f.) Osbeck) is a significant citrus species renowned for its health-promoting properties.Nevertheless, it faces challenges in fruit quality, characterized by Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.a thick peel, lower juice percentage, and a higher seed count.So, a systematic citrus improvement programme was launched at ICAR-Indian Agricultural Research Institute, New Delhi (India), in the early 21st century.The primary goal of this program was to enhance the quality of pummelo through strategic crossbreeding with sweet orange.Currently, there are no records of any citrus variety specifically developed for high fruit juice chemical nutrients.
Considering the widespread issue of malnutrition, cultivating nutrient-rich citrus varieties holds promise in significantly reducing disorders related to mineral nutrition in humans.A crucial prerequisite for this endeavour is understanding the biochemical composition of fruits to identify hybrids with superior nutritional values.The characterization of various hybrids and parentages plays a critical role in the breeding of elite cultivars.Considering the mentioned circumstances, the current study was undertaken to evaluate the concentration of phytochemical nutrients in interspecific citrus hybrids, particularly orangelo, encompassing both parental genotypes.The study also aimed to determine correlations among juice nutrients, estimate genetic variability in citrus fruit juice, and characterize these hybrids using recently developed acidity-specific SSR markers.

Experimental site and plant materials
The current study was carried out during 2022-2023, on a group of 24 orangelo hybrids, aged between 7 and 10 years, which were developed at ICAR-Indian Agricultural Research Institute, New Delhi.These hybrids were derived from four parent genotypes: pummelo (C.maxima [Burm.f.] Osbeck) and sweet orange (Citrus sinensis [L.] Osbeck), which were evaluated for biochemical nutrient in fruit juices.The specific hybrid genotypes and their parentage information can be found in Table S1.The hybrid seedlings were planted with a spacing of 3 m×3 m, whereas the parental genotypes were budded onto Citrus jambhiri Lush.rootstock.The experimental site experienced typical sub-tropical climatic conditions characterized by hot and dry summers followed by cold winters.All plants were uniformly subjected to irrigation and fertilization management practices, adhering to the recommended package of practices for citrus crops within the same agro-climatic zone.Recommended production technology was adopted to manage all hybrids including parents.
Mature fruits were harvested from different direction of the tree at proper maturity as adjudged by standardized TSS/TA ratio for each genotype from main orchard of Fruits and Horticultural Technology division, ICAR-IARI, New Delhi.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
After harvesting, each fruit of Pummelo × Sweet orange hybrids and their parents was washed thoroughly under tap water and dried with a paper towel in order to remove surface impurities.Each fruit was cut in half and the juice was extracted with the citrus press juicer, filtered, juice yields, juice recovery were calculated and stored at -20 °C until analysis.

Measurement of juice recovery, juice pH, TSS, titratable acidity and TSS:acid ratio of juice
The juice yield from each type of the selected citrus fruits was measured in mL/g.The overall yield was calculated by using the following formula: Juice percentage (%) = Average juice mass (g) Average fruit mass (g) × 100 /1/ The juice was analyzed for the pH using digital pH meter (pH meter 700; Eutech Instruments, NY, USA).The total soluble solids (TSS) were measured using digital refractometer (Milwaukee MA871; Milwaukee Instruments, Inc., Rocky Mount, NC, USA).The values were expressed in °Bx.The machine was standardized using purified water before taking readings.
Titratable acidity (TA) was determined as percentage using a previously described AOAC method (12).A volume of 10 mL of each juice was measured into a volumetric flask and then made up to 100 mL mark using distilled water.Then 10 mL of each diluted juice were titrated against 0.1 N NaOH using phenolphthalein indicator while the end-point was noted (the colour changed from colourless to pale pink).The total acidity (g/100 mL) was then calculated in terms of citric acid as follows: Titratable Acidity (% citric acid) = Titre value × acid factor × 100 10(mL juice) ⁄ /2/ where acid factor for citric acid=0.0064.
The TSS:acid ratio was calculated by dividing TSS with the acidity.

Determination of ascorbic acid, total flavonoid content, total phenolics, total antioxidant activity and sugar fractions
The ascorbic acid content was determined using AOAC method (12).About 10 mL of citrus juice were taken and volume was made with 3 % metaphosphoric acid.Then, 10 mL aliquot of metaphosphoric extract were taken and titrated with the standard 2, 6dichlorophenol-indophenol dye to pink end point.The pink end point should persist for 15 s.
The ascorbic acid content was computed by using the following formula given by Rangana (13).The ascorbic acid content was measured as mg/100 mL.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
The total flavonoid content of citrus was estimated by a calorimetric method using the aluminium chloride reagent and quercetin as standard (14).A volume of 1 mL of citrus juice was extracted in 10 mL of 80 % ethanol.Subsequently, the mixture was centrifuged at 10 000×g for 20 min at 4 °C.A volume od 1 mL of properly diluted extract (concentration 1 mg/mL) was mixed with 1.4 mL of distilled water and 0.3 mL NaNO2 (5 % m/V).Additionally, 0.3 mL of AlCl3 (10 % m/V) was added 5 min later and allowed to react for another 6 min.Following this, 2 mL of NaOH solution (1 M) were added.The resultant mixture is then made up to a total volume (V) of 5 mL using distilled water.The solution is meticulously mixed, and its absorbance (A) was measured using a UV-Vis spectrophotometer (GENESYS; Thermo Fisher Scientific, Japan) at a wavelength of 510 nm.The results were expressed in mg of quercetin equivalents per mL of juice extract (mg QE/100 g).
Total flavonoids (mg QE/100 g) =  ×  × DF × 0.1 Factor for QE (0.001) ×  × Aliquot /4/ Total phenolic content of each citrus juice extract was determined using the Folin-Ciocalteu calorometric method suggested by Singlton et al. (15).One-gram citrus pulp was crushed in mortar and pestle using 80 % ethanol and centrifuged for 20 min at 10 000 rpm.To 100 µL supernatant, 2.9 mL distilled water were added along with 0.5 mL 0.2 N Folin-Ciocalteu reagent (FCR).After standing for 10 min at room temperature, 2 mL of sodium carbonate solution (20 % m/V) were added.The solutions were mixed and allowed to stand for 30 min at room temperature for incubation.The absorbance (A) was measured with a UV-Vis spectrophotometer (GENESYS; Thermo Fisher Scientific) at 760 nm for developed dark blue colour complex.Results were expressed in mg of gallic acid equivalents per gram of dry extract (mg GAE/100 g dry extract).
Total phenolic content (mg GAE 100 g) =  ×  × DF × 100 Factor for GAE (0.02) × Aliquot ×  × 1000 /5/ The DPPH radical scavenging capacity of each citrus juice extract was determined as suggested by (16) with some modifications.A solution of 2,2-diphenyl-2-picryl-hydrazyl (DPPH; 6•10 -2 mM) in methanol was prepared, and 2.9 mL of this solution were mixed with 0.1 mL of each extract previously dissolved in methanol.The mixture was kept in the dark at room temperature for 30 min.The absorbance (A) was measured using a UV-Vis spectrophotometer (GENESYS; Thermo Fisher Scientific) at 517 nm.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Sensory evaluation of Pummelo × Sweet orange hybrids
The sensory evaluation plays a pivotal role in defining product quality.Judges were tasked with rating various evaluation parameters using a nine-point hedonic scale, following the guidelines established by Jellinick (18).These parameters included attributes like appearance, color, flavor, taste, consistency, and overall acceptability.A semi-trained panel Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
of ten judges conducted the analysis.Initially, an organoleptic assessment was carried out on the juice immediately after extraction.The judges documented the sensory characteristics of both the fruit and the juice on a dedicated sensory evaluation sheet.Subsequently, statistical analysis was employed to decode and interpret the scores obtained, facilitating the derivation of meaningful conclusions regarding the sensory attributes of the products.

Molecular characterization of Pummelo × Sweet orange citrus hybrids using acidity specific markers
Twenty-eight Pummelo × Sweet orange citrus hybrids and their parents listed were used for molecular profiling specific to acidity.Genomic DNA isolation from leaf tissues was carried out using CTAB (cetyltrimethylammonium bromide) method with minor modifications (19) Thermal cycler, Bio-Rad, USA) was used for thermo cycling.PCR amplified products were run in 3 % high resolution agarose gels.Electrophoresis was carried out at 100 V for 3 to 4 h.DNA profiles were visualized on UV trans-illuminator and photographed on gel documentation system (Alphaimager Mini Gel Documentation, USA).The genetic similarity between individual pairs of genotypes was analyzed by using the NTSYS pc 2.1 software (22).

Statistical analysis
The experimental setup followed a Randomized Block Design (RBD) in a factorial arrangement.Each treatment consisted of three replications, with one tree per replication.The collected data underwent statistical analysis of variance (ANOVA) using SAS 9.

version
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
software (SAS, USA INC) (23).Duncan's Multiple Range Test (DMRT), were employed to compare significant differences at p ≤ 0.05.Principal component analysis (PCA) was performed using the average concentration of physico-chemical quantitative trait, to highlight the distances between genotypes, using R Studio (R Studio, PBC, Version 2022.07.1-554, (24).Correlation analysis was done for biochemical parameters using Pearson's correlation was carried out using same software.The assessment of genetic similarity among pairs of genotypes was conducted using the NTSYS pc 2.1 software (22).The average similarity across all genotype pairs served as a threshold value for delineating clusters.For the estimation of genetic similarity, the Dice coefficient (25) was employed.Dendrograms were then generated by employing clustering analysis via the unweighted pair group method with arithmetic means (UPGMA).Phenotypic co-efficient of variation (PCV) and genotypic coefficient of variation (GCV), heritability and genetic advance were estimated.Broad sense heritability was calculated as genotypic variance/phenotypic variance (26).

Juice recovery, juice pH, TSS, titratable acidity and TSS:acid ratio of juice
Citrus juices, such as pummelo juice, grapefruit juice, orange juice, and lemon juice, have distinctive flavors, aromas and nutritional profiles due to a variety of biochemical properties.The high juice volume is an important factor determining the suitability of a variety for processing as well for fresh consumption (Fig. S1).Data on juice content in Table S2 shows significant variations among hybrids.Among 24 hybrids, the juice volume was the highest in SCSH 5-5 (232.33 mL) and the lowest in SCSH 11-15 (56.67 mL) which is non-significant to SCSH 9-2.Among parents, sweet orange cv.Mosambi recorded maximum fruit juice content (133.33 mL), which is non-significant compared with the other two parents, and the lowest in PS-10 (Pummelo white) (65.67 mL).Data also suggested that many hybrids yielded juice either higher or lower than their parents.
Juice recovery percentage is an important parameter in citrus fruits.Hybrids and parents had significantly different juice recovery (Table S2).Significantly the highest juice recovery was recorded in SCSH13-14 (45.64 %), followed by CRH 20-11, SCSH 15-19, SCSH 11-19, SCSH 5-5 and SCSH 3-15.However, the lowest juice recovery was found in SCSH 3-10 (25.08 %), which exhibited statistical parity with SCSH 17-9.Furthermore, those two parents showed statistical similarity in juice recovery while significantly higher recovery of juice in Sweet orange cv.Mosambi was measured than in the other three parents.In comparison to parents, hybrids exhibited intermediate values of juice recovery between both female and Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
male parents.This is well explained by Fiévet et al. (27), stating that heterosis is a result of multiple component traits, even when these traits can be entirely explained by an additive genetic model.When the component traits show phenotypic divergence in parents, the manifestation of heterosis in the complex trait is frequently observed in progeny, even if the component traits themselves remain close to the mid-parent values.The hybrids showed significant variation in pH of fruit juice (Table S2).It was evident that the maximum pH was shown by hybrid SCSH 3-10 (4.00), followed by SCSH 3-14, while minimum pH was found in hybrid SCSH 15-18 (3.45), which was non-significant compared to SCSH 13-13.Among parents Mosambi, a male parent recorded highest fruit juice pH (4.73), while PS- (1.00 %).Furthermore, the three parents showed statistical similarity for acidity, while significantly higher acidity (1.50 %) was estimated in PS-2 (Pummelo red) than the rest of the all parents.Compared to parents, hybrids exhibited higher and lower titratable acidity values than parents (Table S2).The total acidity of the citrus juices essentially showed an inverse relationship with the corresponding pH values (28).The content of total soluble solids (TSS) and acids as well as the fine balance between them holds considerable importance in defining the flavor and internal quality of citrus fruits.In the present study, SCSH 13-14 (9.57) recorded the highest TSS/acid ratio, followed by SCSH 13-17, while it was the lowest in hybrid SCSH 21-10 (4.09).In comparison to parents, two parents showed statistical similarity for TSS acid ratio, while significantly higher ratio was recorded in female parent, i.e.PS-10 (Pummelo white) than in other three parents.The hybrids exhibited higher and lower values than the parents (Table S2).Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Ascorbic acid, total flavonoid, total phenolics, total antioxidant activity and sugar fractions content Ascorbic acid content in fruit juice of citrus hybrid and their parent showed notable differences (Table S2).Total phenolic content (TPC) in fruit juice showed notable differences among both the hybrids and their parent varieties (Table S2).The fruit juice of the hybrid SCSH 13-13 (107.33 mg GAE/100 g) had significantly highest TPC.On the other hand, it was the lowest in SCSH 3-10 (40.67 mg GAE/100 g), which exhibited statistical parity with SCSH 5-5 and SCSH 3-15.
Among the parents, the highest TPC was found in PS-2 (Pummelo red) (168.17mg GAE/100 g), while the lowest was found in PS-10 (Pummelo white) (96.83 mg GAE/100 g).The hybrids showed higher and lower values than the parents.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Data presented in
The data on sugar content clearly indicated substantial differences between the hybrids and their parent varieties (Table S2).Significantly highest total sugar content was measured in the fruit juice of hybrid SCSH 13-14 (5.76 %).However, hybrid SCSH 11-15 recorded the lowest total sugar content (2.05 %).Among the parents, the highest total sugar content was found in PS-5 (Pummelo white) and the lowest was in PS-2 (Pummelo red).Some hybrids yielded higher while other showed lower sugar profile as of either parent.

Sensory acceptability of the Pummelo × Sweet orange hybrids
The ultimate criterion for the desirability of any food product to consumers is its flavor.
According to Sharma et al. (30), quality is as a crucial parameter in evaluating the suitability of any food product for human consumption.Quality of the product can be determined in terms of sensory quality, shelf life and microbial growth using standard techniques.According to Thakkar and Shah (31) sensory analysis of food is now becoming increasingly significant in evaluating the acceptability of a particular food item.Sensory analysis is a technique which uses human testers as a measuring tool.Numerical scoring test is used to analyze the characteristics of one or more samples (32).The sensory property serves as a significant parameter in assessing the quality of a product.Currently, sensory evaluation has become an indispensable tool in the food industry, playing a crucial role in interactions with key sectors of food production (33).
The surface characteristics of food item contribute to the appearance.The mean score for the appearance were recorded, with the highest mean score of 9.0 for hybrids SCSH 15-3, CRH 20-11 and Mosambi, and the lowest mean score of 6. 3 for parent PS-10 (Pummelo white) (Table 1).Colour is very important attribute to judge the overall quality of fruit and juice.
The mean scores for fruit colour ranged from 6.3 to 9.0.Hybrid SCSH 9-20 and parent PS-2 Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
(Pummelo red) had the highest mean score (9.0), while the hybrid SCSH 13-17 and parent PS-10 (Pummelo white) obtained 6.The lowest mean score (6.7) was obtained for SCSH 13-13.Taste is an aesthetic appreciation in the mouth and it is the most important sensory characteristic which determines the acceptability of fruit and juice.Selected Pummelo × Sweet orange hybrids were tested by a sensory panel, who found that hybrids SCSH 3-10, SCSH 15-3, CRH 20-11 and male parent Mosambi had the highest taste mean score of 9.0, followed by SCSH 19-2 and PS-2 (Pummelo red) with the mean scores of 8.7 (Table 1).The overall acceptability of the fruits was analyzed by sensory analysis and maximum mean score was noticed in SCSH 3-10 and

Heritability studies
In this study, a broad spectrum of variability was noted across all biochemical properties in fruit juice.The observed phenotypic coefficient of variation (PCV) for all 13 properties exceeded their respective genotypic coefficient of variation (GCV), suggesting that environmental factors significantly contribute to the observed variations alongside genetic factors.This aligns with earlier findings reported by Mishra et al. (37).Moreover, the minor difference between these two estimates (GCV and PCV) suggests a reduced impact of the environment on the observed variability.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
high (>30 %) for non-reducing sugar, juice volume, total antioxidants and total phenolic content; moderate (20-30 %) for total flavonoids, TSS:acid and juice recovery, while it was low (<20 %) for total sugars, acidity, reducing sugars, TSS, ascorbic acid and juice pH (Table 2).Citrus species exhibit high levels of heterozygosity, leading to extensive genotypic and phenotypic diversity in their first-generation progeny (38,39).The current study has revealed a higher heritability (≥80 %) for all the biochemical properties in the juice, which strengthens the indication of a limited impact of environmental factors on these traits.The elevated values of both genotypic coefficient of variation (GCV) and heritability (>80 %) suggest the potential for successful selection to enhance these traits.Additionally, a high genetic advance as a percentage of the mean (GA as % mean) was estimated for non-reducing sugar, juice volume, total phenolic content, and total antioxidants, further indicating the prospects for improvement through selection in these specific traits (Table 2).The juice properties such as pH, total sugars, non-reducing sugar, reducing sugars, and total phenolic content, exhibiting high heritability and genetic advance as a percentage of the mean, suggest that these attributes are primarily governed by additive genes with less influence from the environment.This indicates their suitability for selecting hybrid genotypes based on these traits (40).Whereas non-additive gene action might be responsible for these biochemical properties (Total flavonoids, TSS:acid, juice recovery, total sugars, acidity, reducing sugars, TSS, ascorbic acid and juice pH) as revealed by low to moderate GA (20-30 %) along with high heritability (>80 %), which could be enhanced in the fruit juice through heterosis breeding as reported for nutrient content in banana (26).Previously, the role of both additive gene action and nonadditive gene action for mineral content in banana fruit pulp and ridge gourd reported by .At the biochemical level, complex phenotypes result from the interplay of multiple component metabolites over time (27).The levels or concentrations of metabolites represent a complex phenotype in themselves, as they are influenced by enzyme quantities and activities within pathways, along with the flux, which denotes the rate of turnover through the pathway (27,(48)(49).To the best of our knowledge, this is the initial study on citrus interspecific Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
(Pummelo x Sweet Orange) hybrids, focusing on the estimation of heritability for juice properties.
PCA was performed using the mean of different biochemical properties in the juice of 24 citrus hybrids and four parental genotypes.Considering a minimum threshold eigen value of one the five PCs (juice volume, juice recovery (JR), TSS, juice pH, titratable acidity (TA)) were considered, explaining 71.16 % of the total variation among the studied citrus hybrids hybrids with their parents (Fig. 2).PC1 contributed 24.43 % of the total variation, followed by PC2 (17.40 %).Similarly, these parameters were found important in study of Dubey et al. (50).
Cluster analysis clearly showed that cluster A1 consist mainly the hybrids which are good in juice volume however, outstanding performing hybrids SCSH 13-15 and SCSH 11-15 were presented as an outgroup (Table 3).
In order to find out the biochemical level relationship and per cent similarities, a dendrogram was constructed from the pair wise distance matrices.A dendrogram generated based on biochemical data grouped all the 24 interspecific hybrids along their parents into two major cluster A and cluster B at similarity value of 0.56.Cluster A comprised most of the studied genotypes and further subdivided into two clusters A1 and A2 at 60.71 % similarity value and cluster B comprised remaining genotypes and further dived into two clusters as B1 and B2 at 39.29% similarity value.Citrus hybrids SCSH 3-15 and SCSH 11-15 were presented as an out-group based on biochemical traits.Biochemical parameters-based grouping showed that most of the hybrids had close association with female parent (Pummelo red).In the Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
present study, some of the Pummelo × Sweet orange hybrids proved superior for antioxidants, vitamin C, phenolics and flavonoids of juice over their parental genotypes.The heterotic effect for nutrients in edible tissue has been previously reported in several food crops (41,51-52).
Nevertheless, the antioxidants, vitamin C, and other aspects of the biochemical nutrition profile in several hybrids were found to be intermediate compared to their parents.This phenomenon is attributed to non-transgressive segregation, wherein alleles at multiple loci from different parental populations recombine in hybrids, resulting in intermediate trait expression (53).The cluster analysis effectively categorized the citrus genotypes into distinct groups based on their juice nutrient concentrations.Interestingly, the results indicated that identifying genotype groups belonging to the same genomic or parentage group was unfeasible, suggesting that hybrids displayed a blend of characteristics concerning their juice mineral contents.These findings align with prior studies on minerals and fruit quality traits observed in banana (26), citrus (54), and apricot (55).

Molecular characterization using newly generated acidity specific markers
The use of conventional breeding methods in citrus is limited by its long generation time, usually from 3 to 7 years.Evaluation period longer when breeding for quality traits such as fruit acidity.Therefore, identification of trait-linked markers is highly beneficial to citrus breeders.A total of eight primers were designed and synthesized for the molecular profiling of important citrus genotypes specific to acidity.Out of 8 primers seven primers viz., NRCH2, NRCH3, NRCH4, NRCH5, NRCH6, NRCH7 and NRCH8 were polymorphic showing a total of 87.5% polymorphism and NRCH1 primer did not amplified shown in Table S3.The amplicon  CRH 20-11 and all four parents.In the present study it has been observed that there is substantial variation in acidity status.Molecular characterization of Pummelo × Sweet orange hybrids showed that >50 % hybrids grouped with parents with medium acidity level (56).
Sometimes complementation of genes and alleles happens (from different parents) and due to which there is chance of a hybrid better than the parents.Also, there is a phenomenon of overdominance wherein F1 becomes better than the parents.Also, in case of quantitative trait there is a chance of additive effect (accumulation of different QTLs) of genes/QTLs in hybrids (57).

CONCLUSIONS
The findings from this study highlight that newly developed orangelo hybrids represent a valuable source of antioxidant properties, potentially fulfilling additional nutritional requirements for a healthy body.Moreover, the study revealed significant variations in nutritional value among both the parental varieties and the resulting hybrid genotypes.The overall acceptability of the fruits was noticed in SCSH 3-

FUNDING
This research did not receive any specific grant from funding agencies in the public, commercial, or not-for-profit sectors.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

SUPPLEMENTARY MATERIALS
All supplementary materials are available at: www.ftb.com.hr.

AUTHORS' CONTRIBUTION
Raushan Kumar performed the experiment, collected, processed the data, prepared Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Available from:
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

06) fg
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.(1.12±0.

01) jk
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.The results are presented as mean value± standard deviation (N=3).Mean values of different letters in superscript in the column are statistically different (p≤0.05).JV-Juice Volume, JR-Juice Recovery, TSS-Total Soluble Solids, TA-Titratable Acidity, T:A ratio -TSS: Acid ratio, AA-Ascorbic Acid, TPC-Total Phenolic Content, TFC-Total Flavonoid Content, TA ox-Total Anti-Oxidant Content, TS-Total Sugars, RS-Reducing Sugars, NRS-Non -Reducing Sugars Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

3 .
According to Bajaj et al.(34), flavour is one of the central attributes which imparts identifiable character to the food item.Regarding the flavour of the hybrids, mean score ranged from 6.7 to 9.0.Hybrid SCSH 3-10 and male parent Mosambi, female parent PS-2 (Pummelo red) had the highest mean score (9.0), followed by SCSH 3-10, SCSH 9-2, SCSH 9-20, SCSH 15-3, and the lowest score was obtained for PS-10 (Pummelo white) female parent.Texture is a physical property detained by the skin, eye and muscle senses of the mouth.With regard to the texture, hybrids SCSH 15-2 and CRH 20-11 obtained the highest mean score of 8.7.
size ranged from 100 to 190 bp.Detailed study based on molecular data grouped all 28 citrus genotypes on the basis of acidity specific primers into one major cluster A (96.43 %) and one out-group B (3.57 %) (Fig. S2).Cluster A comprised 23 hybrids and all four parents.Cluster A further subdivided into one out group A1 comprised only one hybrids NRCH 3-10 and A2 comprised most of the hybrids and all parents.In out-group B only one hybrid SCSH 19-8 was present.The sweet orange originated as a natural hybrid of mandarin and pummelo.Most of the cultivars are obtained by somatic mutation from one ancestor tree.The acidity values of progeny of all three cross combinations [PS-2 (P Red) × SO cv.Mosambi, PS-5 (P white) × SO cv.Mosambi, PS-10 (P white) × SO cv.Mosambi] shown in 10 and Mosambi followed by SCSH 9-2, SCSH 9-20, SCSH 15-2, SCSH 15-3, CRH 20-11 and PS-2 (Pummelo red) parent.In the present study, a higher heritability (≥80 %), also estimated for all the biochemical properties in the juices further confirms the weak influence of environmental factors.On the basis of titratable acidity, juice recovery, TSS and vitamin C content in citrus hybrid SCSH 11-15 found superior and hybrid SCSH 3-15 best in total antioxidants and total flavonoid content.ACKNOWLEDGMENTS Authors are thankful to Director, IARI for providing the research facilities and Deepak and Arvind (Technical's, FHT, ICAR-IARI, New Delhi) for sample collection and photographs, respectively.

Fig. 1 .
Fig. 1.Pearson's correlation coefficients for the biochemical properties of Pummelo × Sweet orange hybrids fruit juice

Fig S2 .
Fig S2.Genetic tree based on acidity specific marker between 24 citrus hybrids and their four parents . A total of four gene sequences of Citrus sinensis were retrieved from National Center for designing primers were as follows: primer length 20-23 bp, GC content 30-58 %, and optimum primer Tm 55-62 °C.Eight novel expressed sequence tag-derived simple sequence repeat marker (EST-SSRs) designed from nucleotide sequences of these acidity specific sequences.These primers located at Chromosome 3, 4 and 6.SSR motif GAC and GCTT were predominant for these primers.The PCR was carried out in 12 μL reaction mixture containing 1 μL each primer (forward and reverse), 4 μL of 25 ng/μL genomic DNA as template, 2 μL of PCR buffer, 1 μL of dNTPs mix and 0.15 μL Taq polymerase.The volume was made up to 12 μL with sterile distilled water.Applied Biosystems-Thermocycler (Veriti 96 Paithankar et al. (29) reported TSS 8.39 °Bx, acidity 7.18 %, mass 54.31 g, juice 52.41 % and ascorbic acid 31.47 mg/100 mL in Sarbati lime.
The highest content of ascorbic acid was observed in SCSH 17-9 (58.13 mg/100 mL), followed bywhile hybrid SCSH 11-19 (40.00 mg/100 mL) had the lowest ascorbic acid content.Moreover, among the parents, significantly highest ascorbic acid content was noted in the fruit juice of sweet orange cultivar Mosambi, a male parent, while all three female parents showed lower values than Mosambi, but without having significant differences from each other.Furthermore, none of the hybrids showed higher values of ascorbic acid than either of the parents, and exhibited intermediate values of ascorbic acid.

Table S4
Please note that this is an unedited version of the manuscript that has been accepted for publication.This version will undergo copyediting and typesetting before its final form for publication.We are providing this version as a service to our readers.The published version will differ from this one as a result of linguistic and technical corrections and layout editing.

Table 1 .
Mean scores obtained for sensory analysis of fresh fruits

Table 2 .
Estimation of descriptive statistics, heritability and genetic advance of Pummelo ×Sweet orange hybrids fruit juices biochemical

Table S2 .
The physico-chemical traits of citrus hybrids (Pummelo × Sweet orange) with their parents

Table S4 .
Grouping of citrus hybrids based on acidity specific markers (Three cross combinations)