Comprehensive Analysis of Autophagy-Related Genes in Sweet Orange (Citrus sinensis) Highlights Their Roles in Response to Abiotic Stresses.

Autophagy is a highly conserved intracellular degradation pathway that breaks down damaged macromolecules and/or organelles. It is involved in plant development and senescence, as well as in biotic and abiotic stresses. However, the autophagy process and related genes are largely unknown in citrus. In this study, we identified 35 autophagy-related genes (CsATGs-autophagy-related genes (ATGs) of Citrus sinensis, Cs) in a genome-wide manner from sweet orange (Citrus sinensis). Bioinformatic analysis showed that these CsATGs were highly similar to Arabidopsis ATGs in both sequence and phylogeny. All the CsATGs were randomly distributed on nine known (28 genes) and one unknown (7 genes) chromosomes. Ten CsATGs were predicted to be segmental duplications. Expression patterns suggested that most of the CsATG were significantly up- or down-regulated in response to drought; cold; heat; salt; mannitol; and excess manganese, copper, and cadmium stresses. In addition, two ATG18 members, CsATG18a and CsATG18b, were cloned from sweet orange and ectopically expressed in Arabidopsis. The CsATG18a and CsATG18b transgenic plants showed enhanced tolerance to osmotic stress, salt, as well as drought (CsATG18a) or cold (CsATG18b), compared to wild-type plants. These results highlight the essential roles of CsATG genes in abiotic stresses.

Analysis of phytochemical contributors to antioxidant capacity of the peel of Chinese mandarin and orange varieties.

The phytochemicals in the peel of six oranges and ten mandarins including seven wild varieties and three cultivars were systematically characterised using UHPLC-Q-TOF-MS, and the correlation analysis was performed between phytochemicals and antioxidant capacity in order to investigate the phytochemical contributors to antioxidant capacity. The gradient elution was completed within 16 min and 92 compounds were undoubtedly or tentatively identified. Furthermore, the antioxidant capacities were determined using ABTS, DPPH and FRAP methods. The number of compounds, their contents and the antioxidant capacities were sequenced in the same order of the wild mandarins > cultivated mandarins > oranges. The correlation analysis that showed five compounds were significantly correlated with the antioxidant capacity and can act as main contributors to the citrus varieties with high antioxidant capacities. This study is systematic for the metabolites identification of mandarins and oranges and provides valuable information for effective utilisation of citrus peel and their bioactive compounds.

Phenolic composition and total antioxidant capacity of South African frozen concentrated orange juice as affected by varietal, seasonal and regional differences.

BACKGROUND: There is epidemiological evidence that fruits and vegetables promote general health due to their phenolic composition. The phenolic composition of three commercially important citrus varieties ('Mandarin', 'Navel' orange and 'Valencia' orange), used for frozen concentrated orange juice (FCOJ) production in South Africa, were evaluated based on variety, production season and geographical region (Western Cape (WC) and Eastern Cape (EC)). RESULTS: FCOJ from the WC had significantly (P < 0.05) lower titratable acidity (TA) and higher total soluble solids (TSS):TA ratio compared to FCOJ produced in the EC. The 'Navel' FCOJ, irrespective of season and region, had the highest (P < 0.05) phenolic content (sum of quantified compounds, TP). Regional effects were clear for the 'Mandarin' variety, the EC 'Mandarin' FCOJ had the highest TP and WC 'Mandarin' had the lowest. Seasonal differences were less evident. Variation that could be ascribed to regional differences were also found for individual phenolic compounds. CONCLUSION: Robust data regarding the phenolic profile of FCOJ produced in South Africa, suitable for inclusion in food composition databases, were collected. © 2018 Society of Chemical Industry.

Identification, characterization and expression analysis of lineage-specific genes within sweet orange (Citrus sinensis).

BACKGROUND: With the availability of rapidly increasing number of genome and transcriptome sequences, lineage-specific genes (LSGs) can be identified and characterized. Like other conserved functional genes, LSGs play important roles in biological evolution and functions. RESULTS: Two set of citrus LSGs, 296 citrus-specific genes (CSGs) and 1039 orphan genes specific to sweet orange, were identified by comparative analysis between the sweet orange genome sequences and 41 genomes and 273 transcriptomes. With the two sets of genes, gene structure and gene expression pattern were investigated. On average, both the CSGs and orphan genes have fewer exons, shorter gene length and higher GC content when compared with those evolutionarily conserved genes (ECs). Expression profiling indicated that most of the LSGs expressed in various tissues of sweet orange and some of them exhibited distinct temporal and spatial expression patterns. Particularly, the orphan genes were preferentially expressed in callus, which is an important pluripotent tissue of citrus. Besides, part of the CSGs and orphan genes expressed responsive to abiotic stress, indicating their potential functions during interaction with environment. CONCLUSION: This study identified and characterized two sets of LSGs in citrus, dissected their sequence features and expression patterns, and provided valuable clues for future functional analysis of the LSGs in sweet orange.

Genome-wide analysis of the R2R3-MYB transcription factor gene family in sweet orange (Citrus sinensis).

MYB transcription factor represents one of the largest gene families in plant genomes. Sweet orange (Citrus sinensis) is one of the most important fruit crops worldwide, and recently the genome has been sequenced. This provides an opportunity to investigate the organization and evolutionary characteristics of sweet orange MYB genes from whole genome view. In the present study, we identified 100 R2R3-MYB genes in the sweet orange genome. A comprehensive analysis of this gene family was performed, including the phylogeny, gene structure, chromosomal localization and expression pattern analyses. The 100 genes were divided into 29 subfamilies based on the sequence similarity and phylogeny, and the classification was also well supported by the highly conserved exon/intron structures and motif composition. The phylogenomic comparison of MYB gene family among sweet orange and related plant species, Arabidopsis, cacao and papaya suggested the existence of functional divergence during evolution. Expression profiling indicated that sweet orange R2R3-MYB genes exhibited distinct temporal and spatial expression patterns. Our analysis suggested that the sweet orange MYB genes may play important roles in different plant biological processes, some of which may be potentially involved in citrus fruit quality. These results will be useful for future functional analysis of the MYB gene family in sweet orange.

Genome-wide identification and phylogenetic analysis of the AP2/ERF gene superfamily in sweet orange (Citrus sinensis).

Sweet orange (Citrus sinensis) plays an important role in the economy of more than 140 countries, but it is grown in areas with intermittent stressful soil and climatic conditions. The stress tolerance could be addressed by manipulating the ethylene response factor (ERF) transcription factors because they orchestrate plant responses to environmental stress. We performed an in silico study on the ERFs in the expressed sequence tag database of C. sinensis to identify potential genes that regulate plant responses to stress. We identified 108 putative genes encoding protein sequences of the AP2/ERF superfamily distributed within 10 groups of amino acid sequences. Ninety-one genes were assembled from the ERF family containing only one AP2/ERF domain, 13 genes were assembled from the AP2 family containing two AP2/ERF domains, and four other genes were assembled from the RAV family containing one AP2/ERF domain and a B3 domain. Some conserved domains of the ERF family genes were disrupted into a few segments by introns. This irregular distribution of genes in the AP2/ERF superfamily in different plant species could be a result of genomic losses or duplication events in a common ancestor. The in silico gene expression revealed that 67% of AP2/ERF genes are expressed in tissues with usual plant development, and 14% were expressed in stressed tissues. Because the AP2/ERF superfamily is expressed in an orchestrated way, it is possible that the manipulation of only one gene may result in changes in the whole plant function, which could result in more tolerant crops.

A new strategy employed for identification of sweet orange cultivars with RAPD markers.

We optimized RAPD techniques by increasing the length of RAPD primers and performing a strict screening of PCR annealing temperature to distinguish 60 sweet orange cultivars from the Research Institute of Pomology at the Chinese Academy of Agricultural Sciences. A new approach called cultivar identification diagram (CID) was used to improve the efficiency of RAPD markers for cultivar identification. Thirteen effective primers were first screened from 54 RAPD arbitrary 11-mer primers based on their amplification products and amplified polymorphic bands; they were then used for PCR amplification of all 60 cultivars. All cultivars were manually and completely separated by the polymorphic bands appearing in DNA fingerprints from 13 primers; a CID of the 60 sweet orange cultivars was then constructed. This CID separated all the cultivars from each other, based on the polymorphic bands; the corresponding primers were marked in the correct positions on the sweet orange CID. The CID strategy facilitates the identification of fruit cultivars with DNA markers. This CID of sweet orange cultivars will be very useful for the protection of cultivar rights and for early identification of seedlings in the nursery industry.

Effect of extraction method on quality of orange juice: hand-squeezed, commercial-fresh squeezed and processed.

BACKGROUND: Fresh orange juice is perceived to be more wholesome than processed juice. Fresh juice may have flavor and nutrients that differ from pasteurized or processed juice. RESULTS: 'Hamlin' and 'Valencia' oranges were extracted using a commercial food service juicer, pasteurized or not, resulting in fresh-commercial juice (FCJ) or pasteurized juice (FCPJ) for comparison with pasteurized processed juice (PPJ) in 2009, and gently hand-squeezed 'Valencia' juice (HSJ) in 2010 for quality attributes. There was higher peel oil, lower pectin content, and less cloud loss in FCJ/FCPJ compared to PPJ and HSJ regardless of pasteurization. Titratable acidity was generally higher and the ratio of solids to acids lower in FCJ/FCPJ or HSJ compared to PPJ. FCJ/FCPJ had generally higher levels of most aroma volatiles than did PPJ and, overall, the highest esters and terpenes, while methanol and ethanol levels were highest in HSJ. For sensory evaluation, FCJ/FCPJ had more peel oil and PPJ more cooked flavor than other samples, while 'Valencia' HSJ was preferred over the other juices. CONCLUSION: High peel oil content and thermo-pasteurization process decreased cloud loss of orange juice. Extraction and finishing processes rather than pasteurization or oil content were major factors in influencing orange juice flavor quality.

[Determination of parameter range in detecting Cu of Gannan navel orange by LIBS setup].

In order to improve the precision and sensitivity of laser-induced breakdown spectroscopy device on detecting heavy metal, parameter optimization should be done. After selecting the initial delay time parameter in detecting navel orange experiment of laser-induced breakdown spectroscopy device, a cell range from 1.10 to 1.30 micros of delay time containing the initial parameter was extracted and experiment on it was conducted. There are more than one peak in the curve of element line intensity and signal-to-background ratio vs the delay time in the cell range, and the peak values are higher than the initial one. Confidence interval of the delay time calculated indirectly by interval estimation in Statistics is (1.13, 1.25). The relative standard deviation decreases from 0. 103 269 548 to 0. 025 322 279 5 when the delay time adjusted from 1.20 to 1.14 micros in the confidence interval. It is suggested that parameters range of LIBS experimental device can be adjusted freely in the range while detecting measured elements so as to improve the precision and sensitivity.

[Identification of different Citrus sinensis (L.) Osbeck trees varieties using Fourier transform infrared spectroscopy and hierarchical cluster analysis].

Researched on diversity of the spring leaf samples of seven different Citrus sinensis (L.) Osbeck varieties by Fourier transform infrared (FTIR) spectroscopy technology, the results showed that the Fourier transform infrared spectra of seven varieties leaves was composited by the absorption band of cellulose and polysaccharide mainly, the wave number of characteristics absorption peaks were similar at their FTIR spectra. However, there were some differences in shape of peaks and relatively absorption intensity. The conspicuous difference was presented at the region between 1 500 and 700 cm(-1) by second derivative spectra. Through the hierarchical cluster analysis (HCA) of second derivative spectra between 1 500 and 700 cm(-1), the results showed that the clustering of the different varieties of Citrus sinensis (L.) Osbeck varieties was classification according to genetic relationship. The results showed that FTIR spectroscopy combined with hierarchical cluster analysis could be used to identify and classify of citrus varieties rapidly, it was an extension method to study on early leaves of varieties orange seedlings.

Limonoid content of sour orange varieties.

BACKGROUND: Modern Citrus cultivars are thought to have arisen from three parents: the pummelo, the mandarin, and citron. Taxological and genetic data support that sweet and sour oranges share a common parentage. However, as their name suggests, the organoleptic properties of the fruit from these two families is distinctly different. Analysis of the limonoid content of sour orange varieties has been limited. RESULTS: Juice samples prepared from a selection of sour orange cultivars were evaluated for their limonoid A-ring lactone, aglycone, and glucoside contents. Limonoate A-ring lactone concentrations ranged from 11.1 to 44. 9 mg L⁻¹, whereas nomilinoate A-ring lactone levels were found not to exceed 1.2 mg L⁻¹. Total limonoid aglycone and total limonoid glucoside concentrations varied from 2.4 to 18.4 mg L⁻¹ and from 149.0 to 612.3 mg L⁻¹, respectively. Limonoid glucoside profiling by liquid chromatography-mass spectrometry suggest that the sour oranges are distinctly different from sweet oranges and other citrus species. CONCLUSION: Limonoid aglycone and A-ring contents across sweet and sour oranges are similar, whereas limonoid glucoside profiles are distinctly different. Juice prepared from Citrus maderaspatana had the highest limonoid concentrations among the samples tested and could potentially be used for the isolation of limonoid A-ring lactones and glucosides.

[The characteristic analysis of several mineral contents in Chinese orange juice].

A study was carried out on the contents of mineral elements such as K, Ca, Na, and Mg in seven different orange varieties, namely Pineapple orange, Hamlin, Trovita, Jincheng, 1,232 Tangor, Olinda Valencia and Delta Valencia, by flame atomic absorption spectrometry. The results indicated that the content sequence of different nutritional elements was K > Mg > Ca > Na, with a range of 1,233.75-1,866.23, 77.51-167.15, 49.32-125.29 and 1.22-9.26 mg x L(-1) respectively. The range scale of the four elements was largely consistent with the reference value of AIJN (Association of the Industry of Juices and Nectars from Fruits and Vegetables of the European Union). The samples can be clustered into 2 groups by factor analysis, and lower Na content would be the characteristic of the Valencia varieties. All these data would offer important information for orange juice adulteration determination and quality evaluation.

Daily Consumption of Orange Juice from Citrus sinensis L. Osbeck cv. Cara Cara and cv. Bahia Differently Affects Gut Microbiota Profiling as Unveiled by an Integrated Meta-Omics Approach.

We have investigated the effect of intake of two different orange juices from Citrus sinensis cv. "Cara Cara" and cv. "Bahia" on faecal microbiota and metabolome using an integrated meta-omics approach. Following a randomized crossover design, healthy subjects daily consumed 500 mL of orange juice from Cara Cara or Bahia juices or an isocaloric control drink. Stools were collected at baseline (T0) and after a week (T7) of intervention. Operational taxonomic units (OTUs) were pyrosequenced targeting 16S rRNA, and faecal metabolites were analyzed by an untargeted metabolomics approach based on 1H NMR spectroscopy. The major shift observed in microbiota composition after orange juice intake was the increased abundance of a network of Clostridia OTUs from Mogibacteriaceae, Tissierellaceae, Veillonellaceae, Odoribacteraceae, and Ruminococcaceae families, whose members were differently affected by Cara Cara or Bahia juice consumption. A core of six metabolites such as inositol, choline, lysine, arginine, urocanic acid, and formate significantly increased in Cara Cara compared to the Bahia group.

Carotenoid biosynthesis and quality characteristics of new hybrids between tangor (Citrus reticulata x C. sinensis) cv. 'Murcott' and sweet orange (C. sinensis) cv. 'Pêra'.

Phenotypic characteristics, as well as the relation between carotenoid accumulation and gene expression during ripening were determined in fruits of five new hybrids between tangor cv. 'Murcott' and sweet orange cv. 'Pêra'. The genotypes were classified into the orange-like group, showing mainly epoxycarotenoids, oval fruit shape and yellowish color, or in the mandarin-like group, showing mainly ß-cryptoxanthin, flattened shape and deep-orange coloration; although some hybrids presented intermediate characteristics. The diversity in carotenoid composition of hybrids and genitors were mostly explained by patterns of gene expression. High carotenoid (250-426 µg/g dry weight [dw]) and ß-cryptoxanthin (81-125 µg/g dw) contents, observed in the mandarin-like group, were generally associated with high expression of upstream genes (GGPPS1, PSY, PDS). On the other hand, low expression/repression of these genes and high expression of downstream genes (BCHX and ZEP) were associated with low carotenoid (~158 µg/g dw) and ß-cryptoxanthin (5-22 µg/g dw) contents and epoxycarotenoid accumulation, as occurred in the orange-like group. Breeding experiments resulted in hybrids with outstanding higher carotenoid contents than both genitors (up to 426 µg/g dw versus 158-250 µg/g dw in genitors), which was attributed to transgressive segregation. Differences among genotypes have great impact on commercial fruit quality and potential health benefits, such as the provitamin A content.

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

Although there is substantial diversity among cultivated sweet oranges genotypes with respect to morphological, physiological, and agronomic traits, very little variation at DNA level has been observed. It is possible that this low DNA molecular variability is due to a narrow genetic basis commonly observed in this citrus group. The most different morphological characters observed were originated through mutations, which are maintained by vegetative propagation. Despite all molecular tools available for discrimination between these different accessions, in general, low polymorphism has been observed in all groups of sweet oranges and they may not be identified by molecular markers. In this context, this paper describes the results obtained by using laser-induced fluorescent spectroscopy (LIFS) as a tool to discriminate sweet orange accessions ( Citrus sinensis L. Osbeck) including common, low acidity, pigmented, and navel orange groups, with very little variation at DNA level. The findings showed that LIFS combined with statistical methods is capable to discriminate different accessions. The basic idea is that citrus leaves have multiple fluorophores and concentration depends on their genetics and metabolism. Thus, we consider that the optical properties of citrus leaves may be different, depending on variety. The results have shown that the developed method, for the best classification rate, reaches an average sensitivity and specificity of 95% and 97.5%, respectively. An interesting application of this study is the development of an economically viable tool for early identification in seedling certification, in citrus breeding programs, in cultivar protection, or in germplasm core collection.

Curcumin solid dispersion based on three model acrylic polymers: formulation and release properties

Abstract To investigate structure-property relationship of polymer-based curcumin solid dispersion (SD), three acrylic polymers were used to formulate curcumin SD by solvent evaporation method. Curcumin Eudragit EPO SD (cur@EPO), curcumin Eudragit RS PO SD (cur@RSPO) and curcumin Eudragit RL PO SD (cur@RLPO) showed deep red, golden orange and reddish orange color, respectively. Cur@RSPO entrapped 15.42 wt% of curcumin followed by cur@RL PO and cur@EPO. FTIR spectra indicated that in cur@EPO, curcumin may transfer hydrogen to the dimethylaminoethyl methacrylate group and thus change its color to red. In contrast, curcumin may form hydrogen bonding with Eudragit RS PO and Eudragit RL. Curcumin exists in amorphous state in three SDs as proved by differential scanning calorimetry and X-Ray diffraction measurement. In vitro digestion presented that lower pH value in simulated gastric fluid (SGF) stimulates the curcumin release from cur@EPO while permeability influences the release profile in other two SDs. When in simulated intestinal fluid (SIF), first order release model governs the release behaviors of all three SDs which showed sustained release pattern. Our results are helpful to elucidate how structure of polymer may impact on the major properties of curcumin contained SD and will be promising to broaden its therapeutic applications.

Differentiation Using Microwave Plasma Torch Desorption Mass Spectrometry of Navel Oranges Cultivated in Neighboring Habitats.

The molecular fingerprinting of intact fruit samples combined with statistical data analysis can allow the assessment of fruit quality and location of origin. Herein, microwave plasma torch desorption ionization mass spectrometry (MPT-MS) was applied to produce molecular fingerprints for the juice sac and exocarp of navel oranges cultivated in three closely located habitats, and the mass spectrometric fingerprints were differentiated by principal component analysis (PCA). Because of the relatively high temperature and high ionization efficiency of MPT, the volatile aroma compounds and semivolatile chemicals in the navel oranges were sensitively detected and confidently identified by collision induced dissociation (CID). The limit of detection (LOD) of MPT-MS for vanillin was 0.119 µg/L, with the relative standard deviation (RSD, n = 10) of 1.7%. The results showed that MPT-MS could be a powerful analytical platform for the sensitive molecular analysis of fruits at molecular level with high chemical specificity, allowing differentiation between the same sorts grown in neighboring habitats.

Characterization of the Key Aroma Compounds in Five Varieties of Mandarins by Gas Chromatography-Olfactometry, Odor Activity Values, Aroma Recombination, and Omission Analysis.

In this study, volatile compounds of five varieties of mandarin juices [Tankan, Miyagawa, Mashui (MS), Skiranui, and Ponkan (PG)] were investigated by gas chromatography-olfactometry (GC-O) and gas chromatography-mass spectrometry (GC-MS). A total of 47 volatile compounds were identified by GC-MS. Partial least-squares regression was used to process the mean scores from sensory evaluation by panelists of volatile compounds and samples. The sample PG was associated with "fruity", "floral", and "sweet" notes, while MS was correlated with "green" and "peely" notes. In addition, 36 aroma-active compounds, including esters, alcohols, aldehydes, ketones, and monoterpenes, were detected by GC-O. According to the quantitative results, 29 aroma compounds were important, which indicated that their odor activity values (OAVs) were ≥1. On the basis of the GC-O results and OAVs of these volatile compounds, 22 odor-active compounds were mixed to simulate successfully the overall aroma of PG mandarin juice. Furthermore, omission experiments confirmed that nonanal, hexanal, linalool, and (R)-(+)-limonene were the key odorants for the overall aroma of PG juice sample and that ß-ionone, decanal, γ-terpinene, and methyl butyrate were also important odor-active compounds.

Fresh-Squeezed Orange Juice Properties Before and During In Vitro Digestion as Influenced by Orange Variety and Processing Method.

This study aims to analyze the influence of processing and orange variety on initial quality, antioxidant activity, total polyphenol content (TPC), and ascorbic acid content of fresh-squeezed orange juice during in vitro digestion. Fresh-squeezed orange juice was made from Fukumoto Navel, Lane Late Navel, Olinda Nucellar Valencia, and Campbell Valencia oranges, and was processed thermally and nonthermally. Antioxidant activity (FRAP and ABTS assays), TPC (Folin-Ciocalteu method), and ascorbic acid were analyzed before and after gastrointestinal digestion. Bioaccessibility was calculated by comparing the measured values after digestion with the initial value for each juice. Orange variety significantly influenced pH, acidity, and total soluble solids (P < 0.0001). Antioxidant activity by FRAP was significantly higher (P < 0.0001) in Fukumoto Navel orange juice (16.0 ± 0.4 mM Trolox) than the other juices (range: 9.1 to 10 mM Trolox). TPC was significantly influenced by orange variety (P < 0.0001) and ranged from 521 ± 6 (Campbell Valencia) to 800 ± 11 mg gallic acid/L (Lane Late Navel). Processing method did not influence antioxidant and polyphenol bioaccessibility (P > 0.05). However, antioxidant activity by ABTS and ascorbic acid bioaccessibility were significantly influenced by orange variety (P < 0.0001). These results indicate that fruit variety and nutrient bioaccessibility should be considered to optimize processing and formulation parameters. PRACTICAL APPLICATION: Processing method and variety of fruit during beverage manufacturing may influence its nutrient bioaccessibility. We present a detailed examination of the influence of orange variety, harvest time, and processing method on initial quality parameters and antioxidant bioaccessibility. It is recommended that nutritional analysis of beverages take into consideration both the bioaccessibility of nutrients and the initial nutrient content of the beverage. These results can be used for juice processors to take into consideration the harvest season and produce variety to manufacture beverages with higher nutritional quality.

The high content of ß-carotene present in orange-pulp fruits of Carica papaya L. is not correlated with a high expression of the CpLCY-ß2 gene.

We investigated the transcriptional regulation of six genes involved in carotenoid biosynthesis, together with the carotenoid accumulation during postharvest ripening of three different papaya genotypes of contrasting pulp color. Red-pulp genotype (RPG) showed the lowest content of yellow pigments (YP), such as ß-cryptoxanthin, zeaxanthin, and violaxanthin, together with the lowest relative expression levels (REL) of CpLCY-ß2 and CpCHX-ß genes. On the contrary, the yellow-pulp genotype (YPG) showed the highest content of YP and the highest REL of CpLCY-ß2 and CpCHX-ß genes. Interestingly, the orange-pulp genotype (OPG) showed intermediate content of YP and intermediate REL of CpLCY-ß2 and CpCHX-ß genes. The highest content of ß-carotene shown by OPG despite having an intermediate REL of the CpLCY-ß2 genes, suggests a post-transcriptional regulation. Thus, the transcriptional level of the genes, directing the carotenoid biosynthesis pathway, can partially explain the accumulation of carotenoids during the postharvest ripening in C. papaya genotypes of contrasting pulp color.