extfieldsofstudy: Medicine Chemistry

provenance: AgriculturalAndFoodSciences,2019-2020-Agricultural And Food Sciences-2020.jsonl.gz.gz

text:
Effect of the roasting levels of Coffea arabica L. extracts on their potential antioxidant capacity and antiproliferative activity in human prostate cancer cells

Coffee, besides being one of the most consumed stimulating beverages in the world, has important bioactive activities, which have been attracting increasing attention from researchers. However, the standard process of roasting causes changes in its chemical composition. In the present study, extracts obtained from green and roasted beans (light, medium and dark) of Coffea arabica Linnaeus were submitted to high-power ultrasonic extraction and atomization by spray drying. Colorimetric analysis was used to classify the roasting levels of the dried extract samples. The effects of the roasting process on the bioactivity of the dried extracts were verified through the following assays: caffeine, chlorogenic acid and caffeic acid, by HPLC-PDA; total phenolics by Folin–Ciocalteu; antioxidant activity by DPPH, FRAP, ABTS and ORAC; antiproliferative activity, using the MTT assay; and cell cycle and apoptosis by flow cytometry in metastatic prostate cancer cell lines from bone (PC-3) and brain (DU-145). The results showed that the lowest levels of caffeine, chlorogenic and caffeic acids were observed in dark roasted coffee. In comparison to medium and dark extracts in PC-3 cells, the green and light coffee extracts had higher antioxidant activities and promoted cytotoxicity followed by cell cycle arrest in phase S and apoptosis induction. Thus, the roasting level of the coffee extracts may be related to the potential chemoprotective effects of Coffea arabica L. in prostate cancer cells.

Introduction
Despite the emergence of "energy drinks", coffee is still the main beverage used to start and continue daily activities, 1 due to its stimulant compounds and phytochemicals, 78 which besides improving performance, can also promote wellness. 2 Brazil is the world's leading producer and exporter of coffee, with ten producing states. Minas Gerais is the state with the highest production, followed by Espírito Santo, while Rio de Janeiro occupies the seventh place. The countries that most consume coffee are the United States, followed by Germany, Italy, Japan and Belgium. 3 Coffee belongs to the Rubiaceae family and Coffea genus, which has more than 100 species and subspecies, 25 of which are marketed. 4 Brazil is the main producer of the C. arabica and C. canephora species. 5 The reasons for high consumption of this beverage are due to its palatability and economic accessibility. 6 Arabica coffee accounts for 70% of the world's exports and its sensorial characteristics are considered more appealing than C. canephora. 7 However, the beverage from C. canephora contains higher levels of bioactive compounds, such as chlorogenic acids, trigonelline, caffeic acid and caffeine among others. 8 Coffee displays the highest antioxidant potential compared to other popular beverages. 10 The degree of coffee roasting is controlled by the combination of time and temperature. It is usually qualitatively assessed by colour and classied as light, medium or dark roasted coffee. The roasting process causes changes in the chemical composition and biological activity of coffee: while natural phenolic compounds may be lost, other antioxidant compounds are formed, such some Maillard reaction products. Despite the many studies addressing the antioxidant activity of roasted coffee, little has been reported about the biological activity and variations in the composition of the product in different phases of the roasting process. 11,12 In Brazil, chronic non-communicable diseases (NCDs) are the main cause of death in adults and the elderly. 9 NCDs are associated with endogenous factors such as genetic predisposition and exogenous factors like inadequate eating habits. 13 An analysis conducted by the Brazilian Institute of Geography and Statistics (IBGE) showed that the pattern of food consumption among the Brazilian population has increased the demand for foods with high caloric densities and low nutritional content. 14 Therefore, a good public health strategy would be to encourage consumption of less ultra-processed foods. Although, there is a change in this pattern of eating, natural foods have always been present in the Brazilian diet. Regular coffee consumption, for example, is a habit that has not changed. In fact, there was an increase in this consumer behavior of approximately 5% in 2018, according to studies of the Brazilian research coffee consortium. [15][16][17] This consortium is a strategic arrangement with more than 40 Brazilian institutions, which aims to develop technologies for all stages of the coffee production chain. The consortium is organized by the Brazilian Agricultural Research Corporation -Embrapa, linked to the Ministry of Agriculture. Thus, Brazil is not only the largest coffee producer and exporter worldwide but also a global leader in technical and scientic research on coffee. 80 Prostate Cancer (PCa) is one of the most prevalent cancer types throughout the world, and is one of the most intensely studied problems involving human disease. Several cancer cell lines have been developed and used to determine the mechanisms underlying cancer tumorigenesis and identify markers of therapeutic response. Cancer cell lines have several advantages, including innite proliferation ability and amenability to high-throughput drug screening. Along with LNCaP and PC3 cells lines, DU-145 cells were once considered part of the triad that constituted the gold standard of PCa cell culture lines. DU-145 cells were rst isolated from a brain metastatic prostate tumour. PC3 cells were isolated from a vertebral metastatic prostate tumour and are similar to DU-145 cells in that they are hormone-insensitive and have no androgen receptors. [18][19][20] The objective of the present work was to analyse the antioxidant activities and the effects of green and roasted solid (spray drying) coffee extracts at three gradient levels (light, medium and dark) in prostate adenocarcinoma cell lines (PC-3 and DU-145).

Samples
Coffea arabica (variety 'Catuaí vermelho-amarelo') was grown in Bom Jardim (Brazil). The green coffee bean samples were stored in the Laboratory of Molecular Diagnosis and Mycology of Embrapa Food Technology located in the state of Rio de Janeiro, Brazil.

Raw material and production of extracts
The steps to obtain the coffee extracts started with manual sorting to discard decient beans and dirt, to select those with good quality and to keep the samples homogeneous. 21 In industry, hot air roasting is the most widespread technology used. Medium-dark roasts are the most widely sold in Brazil. 22 The beans were divided into four portions: one for green coffee extracts, and the others according to roast processing, namely light roasting (12 minutes at 230 C), medium (14 minutes at 240 C) and dark (15 minutes at 245 C). The beans were roasted in a Gene Café® roaster (CBF-101, Kyungki-Do, Korea) and were classied according to the Agtron Scale and the manufacturer's manual 23 (Fig. 1).
The green coffee beans were ground in the disc mill (Laboratory Mill 3600) by the grain stiffness criterion. Aer natural cooling, the roasted beans were further ground by brushed steel grinder (Cuisinart®, East Windsor, NJ, USA) to ne consistency. Then, the grain sizes of the samples were standardized using two sets of sieves (Bertel, São Paulo, Brazil) with 0.850 and 0.600 mm mesh.
Aer obtaining granulated green and roasted coffee, infusions were carried out to obtain the extract. In a 250 mL beaker, 25 g of the powder was solubilized in 100 mL of distilled water at 90-95 C for 5 minutes. Aerwards, these infusions were cooled in an ice bath and placed in a high-power ultrasonic processor (Hielscher® UIP1000hdT, Teltow, Germany) (350 W; amplitude ¼ 70% for 10 min). Then the samples were transferred to Falcon tubes and centrifuged at 7000 rpm for 14 min. The liquid part (supernatant) was transferred to a 200 mL beaker and the solid part (precipitate) was discarded. The supernatant was submitted to analysis of total solids (g 100 g À1 ) with a refractometer (Atago PAL-Alpha digital). This measurement was used to delineate the feasibility of spray drying (dehydration at 180 C; vacuum of À60 mbar). Aer drying, the extracts were stored individually in light-protected zipped laminated bags in a cooling chamber at À80 C until analysis.

Colorimetric analysis
The colorimetric analysis of the samples was performed using a Konica Minolta CM-5 benchtop colorimeter (Sensing Americas, Inc.) with prior calibration. Three replications were performed, with direct reading of reectance. The standard colour scale used was CIELAB, with the coordinates L*, a*, b*, where L* is lightness, a* is red/green intensity and b* is yellow/blue intensity. 24 Results were expressed as mean of replicates plus or minus standard deviation.  Samples were submitted to extraction with 1 g to 10 mL of the mobile phase for 10 minutes in a 25 mL volumetric ask with an ultrasonic bath. Each sample was ltered through lter paper (rapid     ltration type) and microltered into disposable hydrophilic Teon lter units with porosity of 0.22 mm. The concentration of the caffeine external standard was 1 mg mL À1 , solubilized with the mobile phase. The wavelength was 280 nm with ow rate of 0.5 mL min À1 . The injection volume was 20 mL and the result was expressed as g of caffeine per 100 g of sample (% w:w). 2.4.2 Determination of chlorogenic acid and caffeic acid. The levels of chlorogenic acid (5-CQA) and caffeic acid were determined by the HPLC method described by Trugo and Macrae (1984), 26,27 using a Waters Alliance 2695 chromatograph, 2996 PDA and Empower® soware (Waters, Milford, MA, USA), along with a Hypersil C 18 BDS column (5 cm Â 4.6 mm and 2.6 mm; Thermo Scientic, Milford, MA, USA). The mobile phase gradient consisted of the initial composition of 5% methanol (phase A) and 95% of 0.1% formic acid solution (phase B), maintained for 6 min. Aer 8 minutes, the composition of the mobile phase reached 80% of phase A and remained there for up to 10 min. Aer 11 min, the composition reached 100% of phase A, and from 12 min to 15 min, the composition returned to the initial conditions. Detection was performed at 325 nm. The ow rate of the mobile phase was 1 mL min À1 and the injection volume was 3 mL. Extraction was performed in an ultrasonic bath for 20 min with 1 g of the sample in 5 mL of acetonitrile (20%) in ultrapure water (v/v), in a 25 mL volumetric ask. The samples were then centrifuged and microltered in disposable hydrophilic Teon® lter units with porosity of 0.22 mm. A pre-calibrated system with chlorogenic acid and caffeic acid external standards (Sigma-Aldrich, New York, NY, USA) at 1.2 mg mL À1 concentration in 0.5% formic acid in ultrapure water was used. Results were expressed as g of the analytes per 100 g of sample (% w/w).

Determination of total phenolic compounds
An initial aqueous solution (50 mg/10 mL) of each soluble dried Arabica coffee extract was prepared. Then, three dilutions of these solutions, with aliquots of 10, 50 and 100 mL, were added to 2 mL of ultrapure water (these solutions also were used for measurement of antioxidant activity, except ORAC).
The total phenolic content of the extracts was determined according to the Folin-Ciocalteu method, as described by Hudáková et al. 28 From each of the dilutions, in triplicate, 500 mL was retrieved and added to 2.5 mL of Folin-Ciocalteu reagent and 2.0 mL of 4% sodium carbonate solution. The mixture was allowed to rest for 2 h in the dark. Measurements were performed at 750 nm in triplicate, in a Shimadzu UV-VIS 2700 spectrophotometer (Nakagyo-ku, Kyoto, Japan). Gallic acid, in the concentration range of 0-100 mg mL À1 , was used for the calibration curve. The concentration of total phenolic compounds of the extracts was expressed as gallic acid equivalents, which reect the phenolic content as the amount of gallic acid in mg 100 g À1 dry weight of the samples.

Determination of antioxidant activity
The analysis of the antioxidant activities of the soluble extracts of green Arabica coffee and their respective roasted samples were evaluated through four different methods: DPPH scavenging activity or ABTS radical cation scavenging activity; ferric Table 2 Effects of green and roasted Arabica coffee bean extracts (5000 and 10 000 mg mL À1 ) on the cell cycle progression in human prostate cancer PC-3 cells after 24 h of treatment  reducing antioxidant power (FRAP); and oxygen radical absorbance capacity (ORAC). 27 2.6.1 Evaluation of the sequestering activity of DPPH radicals. The diluted extracts were mixed with 2.5 mL of DPPH methanolic solution (0.06 mM) and allowed to react for 1 h in the dark. Measurements were performed at 515 nm with a Shimadzu UV-VIS 2700 spectrophotometer (Nakagyo-ku, Kyoto, Japan). The analysis was performed in triplicate. The decline in the DPPH radical absorbance concentration caused by the extracts was compared to a Trolox standard. The results were expressed as mmol Trolox equivalent (TE) g À1 dry weight of the samples. 29,30 2.6.2 Total antioxidant activity analysis by ABTS*+ radical capture. ABTS cations were prepared by mixing an ABTS stock solution (7 mM in water) with 2.45 mM of potassium persulfate. This mixture was allowed to stand for 24 h at room temperature until the reaction was completed and the absorbance was stable. The antioxidant capacity assay was carried out by the ABTS/TEAC method created in 1993 by Miller et al. 31,32 The ABTS solution (2.5 mL) was added to diluted sample extracts, or commercial antioxidant Trolox to obtain the calibration curve (Trolox equivalent -TEAC), and mixed thoroughly. Absorbance was measured at 734 nm aer 6 min. Results were expressed as mmol TE g À1 dry weight of the samples.
2.6.3 Determination of the total antioxidant activity by the iron reduction method (FRAP). The extracts were measured for antioxidant activity by FRAP according to Runo et al. 27 Aliquots of 2.7 mL of TPTZ reagent (ferric-2,4,6-tripyridyl-s-triazine) were mixed with the diluted extract samples. Aer 30 min at 37 C, the absorbance was read at 595 nm. The antioxidant capacity was expressed as ferrous sulphate equivalents (mmol FeSO 4 g À1 dry weight of the samples). 33 2.6.4 Antioxidant activity test by the ORAC method. The ORAC assay was conducted with an automatic plate reader (Spec-traMax i3x, San Jose, CA, USA) with 96-well plates. Analysis was conducted with eight coffee extract dilutions (0.005, 0.0010, 0.0025, 0.0050, 0.01, 0.02, 0.03 and 0.04 mg mL À1 ), aer which 120 mL of the uorescein solution and 60 mL of 2,2 0 -azobis(2-amidino-propane) dihydrochloride (AAPH) was added. 34 Analysis was conducted in phosphate buffer with pH 7.4 at 37 C. Peroxyl radical was generated using the AAPH reagent, which was freshly prepared for each run. Fluorescein was used as the substrate. Fluorescence conditions were as follows: excitation at 485 nm and emission at 520 nm. The standard curve was linear between 1 mM and 90 mM Trolox. Results were expressed as mmol of TE g À1 dry weight of the samples of coffee extract.  3 Cell viability assay. The viability of the cells was analysed by measuring the enzymatic activity of their mitochondria by the MTT (3-[4,5-dimethyl-thiazol-2-yl]-2,5-diphenyltetrazolium bromide) assay, as described by Mosmann (1983). 35 2.0 Â 10 4 cells per well were plated in 96-well plates. Aer 24 hours, they were incubated with the soluble coffee extracts in concentrations ranging from 78.1 to 10 000 mg mL À1 . Aer 24 hours of treatment, the medium was changed in all wells (150 mL) and 10 mL of MTT (5 g L À1 ) was added to each well. Aer 4 h of incubation with the MTT salt, 80 mL from each well was collected and placed in an oven and 50 mL per well of dimethyl sulfoxide (DMSO) was added to dissolve the generated product. Aer 15 minutes in the oven, the plate was read with a microplate reader (Polaris, CELER®) at 570 nm. The cell proliferation inhibition rate (CPIR) was calculated as a percentage based in on the formula: CPIR ¼ (1 À mean value of the experimental group/mean value of the control group) Â 100.

Cell cycle analysis.
For cell cycle assay, 4.0 Â 10 4 cells per well were plated in 6-well plates and were incubated with extracts for 24 h. Cells were rinsed briey with calcium and magnesium-free phosphate-buffered saline (PBS) and detached with trypsin at room temperature. Aer centrifugation, the cells were washed twice with phosphate-buffered saline; and then resuspended in 500 mL of ice-cold Vindelov solution 36 containing 0.1% Triton X-100, 0.1% citrate buffer, 0.1 mg mL À1 RNase, and 50 mg mL À1 propidium iodide (Sigma Chemical Co., St. Louis, MO, USA). Aer incubation for 15 min, the cell suspension was analysed for DNA content by ow cytometry using a FACSCalibur ow cytometer (Becton Dickinson, Mountain View, CA, USA). The relative proportions of cells with DNA content indicative of apoptosis (<2n), G 0 :G 1 diploid (2n), S (2n < phase < 4n), and G 2 :M phase (4n) were obtained and analysed using the CellQuest WinMDI 2.9 program.
The percentage of cell population at a particular phase was estimated with the FlowJo soware. The cell dissociation procedure did not affect uorescence under the experimental conditions used in this study, or in any other studies of which we are aware. Nuclei of viable cells were gated according to the FL2-width-to-FL2-area ratio.

Detection of apoptosis by annexin V-FITC.
To measure the rate of apoptosis, 4.0 Â 10 4 cells per well were plated in 6-well plates and were incubated with extracts for 24 h. Then, the cells were subjected to staining with annexin V conjugated with FITC. The non-adherent cells were collected, and adherent cells were quickly washed with a calcium/magnesium-free buffered saline solution (BSS) and were detached with 0.125% trypsin/EDTA (Sigma Chemical Co., St. Louis, MO, USA) at room temperature. Subsequently, apoptotic and necrotic cells were stained with annexin V FITC/propidium iodide (PI) (BD PharMingen, Mountain View, CA, USA) according to the manufacturer's instructions, quantied with a ow cytometer (FACSCalibur, BD Bioscience, Mountain View, CA, USA), and analysed using two specic programs, Cell Quest and FlowJo.

Statistical analysis
Results are presented as mean with the corresponding standard deviation of triplicates of two different experiments (n ¼ 6). Data were analysed with the statistical soware GraphPad Prism (version 7.0, GraphPad Soware, San Diego, CA, USA). One-way analysis of variance (ANOVA) and the Tukey post-hoc test at a condence level of 95% were used to test cell viability, cell cycle, and apoptosis.

Physicochemical results
3.1.1 Colorimetry. The colorimetric results of the samples, as expected, were different from each other and similar to the data obtained in the literature. 23 The CIELAB colour scale has three parameters: L*, for lightness, ranging from zero (black) to 100 (white); the coordinate a*, which goes from positive (+) for the red component to negative (À) for the green component; coordinate b*, which goes from positive (+) for the yellow component and negative (À) for the blue component. The green coffee extract, as expected, 37 presented higher luminosity (L*) value, which was inversely proportional to the degree of roasting, but there were no signicant differences between the soluble extracts of light and medium roasted Arabica coffee. The soluble extract of green coffee showed a greenish-yellow coloration, and the roasted ones showed an initial red-yellow coloration, quickly turning bluish red (Table 1).
According to De Oliveira et al., 38 in the roasting process, the decrease of luminosity is proportional to the roasting intensity due to the darkening of the beans. This occurs due to three biochemical processes: Maillard reactions, Strecker degradation and sugar caramelization. 39,40 These exothermic reactions determine the sensory characteristics of roasted coffee beans, such as avour, aroma and colour.
3.1.2 Bioactive compounds from the extracts of green and roasted Arabica coffee 3.1.2.1 Quantication of caffeine by HPLC. The caffeine content values in the extracts are shown in Fig. 2A. The light roast coffee had the highest concentration (1.8 AE 0.3 g/100 g). The green and medium roasted coffee samples did not present signicant differences (p < 0.05), and the lowest caffeine content was observed in dark roasted coffee extract (1.0 AE 0.1 g/100 g).
Caffeine is a thermoresistant alkaloid that undergoes better release in aqueous media aer light roasting, but may suffer partial loss by the degradation of the coffee bean mass by dehydration in the more intense roasting phase. 39,41 3.1.2.2 Quantication of chlorogenic acid and caffeic acid by HPLC. Caffeic acid is a thermosensitive bioactive of the hydroxycinnamic group of the phenolic acid family. 43 All the extracts analysed showed signicant differences (p < 0.05), and the highest concentration was found in the light roast coffee (0.43 AE 0.01 g/100 g). Green coffee (0.34 AE 0.007 g/100 g), medium roast (0.14 AE 0 g/100 g) and dark roast (0.06 AE 0 g/100 g) extracts presented lower values (Fig. 2B). Higher levels of caffeic acid can be produced from the nal breakdown of chlorogenic acid by thermal reactions. Caffeic acid, however, is also thermally degraded as these reactions continue. 44 Another important bioactive compound in Arabic coffee beans is chlorogenic acid, a thermosensitive phenolic acid composed by ester isomers of quinic and caffeic acid, 48,49 which is associated with high antioxidant activity. Among the chlorogenic acid isomers found in green coffee beans, 5-CQA (neochlorogenic acid) is the most abundant, composing about 80% of chlorogenic acids, followed by 4-CQA (cryptochlorogenic acid) and 3-caffeoylquinic acid (3-CQA). 50 Only 5-CQA was analysed in this study. Chlorogenic acid concentrations were higher in green coffee extracts (2.84 AE 0.03 g/100 g), but the extracts of the medium roasted and dark roasted beans did not present signicant differences (p > 0.05) (Fig. 2C).
According to Manach et al., 45 the bioavailability of caffeic acids is higher than chlorogenic acid because the latter's breakdown by esterase enzymes in the intestine, increasing the content of caffeic acids.
3.1.3 Total phenolic compounds in dry coffee extracts. The quantication of the total phenolic compounds of the extracts of green and roasted Arabica coffee was performed by the Folin-Ciocalteu assay. 51 This analysis demonstrated higher concentration of total phenolics in the extract of light roasted Arabic coffee (4420.58 AE 15.25 mg gallic acid 100 g À1 ), but without signicant differences with the green coffee extract (p > 0.05). The green coffee extract (4140.35 AE 24.53 mg gallic acid 100 g À1 ) also did not present signicant differences with the medium roast, and the lowest concentration of phenolic compounds was observed in extracts of dark roasted coffee, with value of 1780.19 AE 15.52 mg gallic acid 100 g À1 (Fig. 3).
Total phenolic compounds increased from green to light roast, likely due to the breakdown of chlorogenic acid into caffeic acid, which has higher antioxidant capacity. 52 The content of phenolic compounds decreased as roasting degree increased.
3.1.4 Antioxidant activity of extracts of green and roasted Arabic coffee. The analysis of the antioxidant activity of soluble extracts of green coffee and roasted coffee was performed through four different methods: ORAC, ABTS, FRAP and DPPH. Due to the various types of free radicals and their different means of action in living organisms, there is no simple and universal test method that can measure the antioxidant capacity exactly and quantitatively. Hence, a single analysis would be insufficient to express antioxidant activity. 53 The FRAP assay is based on reducing capacity. DPPH, ABTS and ORAC are based in free radical scavenging capacity, where ABTS is more hydrophilic than DPPH. 79 ORAC is a more direct method because it is based in a naturally occurring radical, unlike DPPH and ABTS, which are stable synthetic radicals.
The most sensitive assays were DPPH and ABTS, followed by ORAC and FRAP (all extracts presented concentration dependent activity). Trolox (a vitamin E analogue) was used as the standard for measuring antioxidant strength of the samples for DPPH, ABTS and ORAC. 54,55 The ABTS and FRAP methods showed similar results in all extracts, in accordance with the data from total phenolic analysis, they are also in line with the results of Moreira et al., 56 who correlated chlorogenic acid monomers with higher FRAP activity and the soluble compounds of caffeine and caffeic acid with superior effects in ABTS analysis.
Flavonoids are thermosensitive compounds that can be easily degraded even in the mildest roast. 57 Chlorogenic acids (CGAs) are thermosensitive and also abundant in green coffee extracts. CGAs are directly related to the reduction of the DPPH radical, which is consistent with the results of the present work. 58 Results related to extracts in the DPPH assay showed a roastdependent decrease, which coincided with the chlorogenic acid content found in the samples. 59 In the ORAC method, there were no signicant differences (p > 0.05) between the green coffee and light roasted coffee extracts (1081.5 AE 27.5 and 1150.5 AE 5.16 mmol TE g À1 ) and between extracts of medium roast and dark roast coffee (584.33 AE 14.33 and 511.66 AE 33 mmol TE g À1 sample) (Fig. 4), indicating a negative effect of the roasting process on the antioxidant capacity.

Effects of green and roasted coffee extracts on cell viability, cell cycle and apoptosis of PC-3 and DU-145
By metabolizing the salt of MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) into formazan by the mitochondrial enzymes of PC-3 and DU-145 malignant cells, it was possible to analyse the cytotoxic effects of Arabica coffee extracts. In PC-3 cells, we observed a signicant reduction of cell viability (p < 0.05) with a low dose of green coffee and light roasted extracts (78.1 mg mL À1 ) when compared to medium and dark roasted extracts (p < 0.05). Those samples reduced the viability by almost 50% and remained stable as concentration increased. However, the green Arabica coffee extract at higher concentrations (5000 mg mL À1 and 10 000 mg mL À1 ) decreased the cell viability of PC-3 by around 70%. The medium roasted coffee extract caused a signicant reduction (p < 0.05) of only 33% at the highest concentration (10 000 mg mL À1 ), whereas the soluble extract of dark roasted Arabica coffee caused an average 13% reduction (Fig. 5).
No signicant differences (p > 0.05) were observed in viability of DU-145 cells aer treatment with soluble extracts of light green, medium and dark coffee extracts (p > 0.05). However, extracts of soluble green Arabica coffee induced the most signicant (p < 0.05) reduction (26%) in cell viability, compared with the other extracts aer treatment with the highest concentration (10 000 mg mL À1 ) (Fig. 5B). Soluble extracts of medium and dark Arabica coffee promoted a very small decrease (5%) only at the highest concentration (10 000 mg mL À1 ) (Fig. 5B), without signicant differences (p < 0.05) compared with untreated cells.
PC-3 carcinogenic cells treated at concentrations of 5000 and 10 000 mg mL À1 of soluble green coffee and light roasted coffee extracts showed signicant cell viability reductions compared to control cells (p < 0.05) (Fig. 4C).
DU145 and PC3 prostate cancer cell lines have moderate and high metastatic potential, respectively. It was reported that DU145 and PC3 cells expressed a higher number of the selected inammatory-related genes that were upregulated in hypoxia with a more complete and persistent response in more aggressive PC3 cells. 60,61 Our data indicate that coffee extract acts more effectively against cells with greater metastatic potential. Other researchers incubated extracts rich in chlorogenic acids of green Arabica coffee and green tea in four tumour cell lines and found that their effects did not increase the proliferation of cancer cells, negatively affecting the cellular viability and the proliferation in a dose-dependent form. 62 Differences in extraction involving distinct solutions (organic solvent and water) produced extracts with different chlorogenic acid proles and content, which can explain the different responses of cell lines.
Coffee constituents have been described as having genotoxic and mutagenic properties, but also antimutagenic and antioxidant activities and the capacity to inhibit the action of carcinogens. The meta-analysis of Sang et al. 63 of epidemiological studies showed that coffee consumption reduces the risk of liver cancer. Previous studies have suggested the inhibitory effect of caffeine on the adhesion, motility and proliferation of DU-145 and PC-3 prostate cancer cells. 42 Many bioactive compounds have been shown to be effective at reducing viability of cancer cell lines while not affecting noncancer cells, which is an added advantage to their potential as natural chemopreventive substances against cancer. Several studies have demonstrated no toxic effects of coffee on noncancer cells, such as the broblast CCD-18Co line, normal macrophages cells (RAW 2647) and hepatocytes o(AML-12). 62,64 In such cases, coffee compounds display cell-and tissue-specicity. Priis et al. 65 investigated the toxicity of roasted and green coffee extracts on myoblasts and endothelial cells and found that myoblasts were more sensitive to green coffee extracts, in contrast to endothelial cells.
Based on our data of cell growth inhibition by Arabica coffee extracts in PC-3 cells, we examined the cell cycle and apoptosis by ow cytometry. Signicant changes were observed compared to the control (p < 0.05) in PC-3 cells aer treatment with high concentrations (5000 and 10 000 mg mL À1 ). Green and light Arabic coffee extract promoted a decrease in G 0 /G 1 phases, more pronounced in the soluble extract of green coffee (p < 0.05), in a dose-dependent manner. This effect was followed by S-phase arrest, mainly aer treatment with green and light roasted Arabica coffee extracts (Table 2). Medium roasted extract promoted slight changes in cell cycle prole only in the S phase, while the dark roasted extract caused a reduction in the S phase.
Emerging evidence indicates that epigenetic and nonepigenetic mechanisms cause alteration of DNA methylation and loss of oestrogen receptors during tumour cycle progression, which in turn may be modiable by dietary and lifestyle factors. It has also been shown that exposure to caffeine and caffeic acid signicantly suppressed the proliferation of both ER+ (by 59% and 38%, respectively) and ER-(by 47% and 28%, respectively) human breast cancer cells (MCF-7 and MDA-MB-231) and inhibited DNA methylation. 67,68 Previous studies have reported that caffeine caused an increase in the G 0 /G 1 phase of mouse epithelial cells. 42,66 and some studies have shown that hydroxycinnamic acids cause reduction of cell viability and apoptosis of tumour cells. 46,47 Specically, chlorogenic acids and their derivatives have been shown to be able to decrease the number of cells in phases G 0 / G 1 and increase cells in phase S. 69,70 This may explain the observation that green and light roasts caused greater increase in the S phase than did medium roast, because these compounds are degraded during roasting. Probably in dark roast, the amount of chlorogenic acids was not sufficient to have biological effects on cells. 27,62,65 Many studies have shown the effects of green coffee extracts on normal cells and tumour cells, but few works have demonstrated and tested the effects of roasted coffee extracts on the viability of cancer cell lines. 27 The development of cancer is associated with disorders in the regulation of the cell cycle. An S-phase checkpoint checks the delity of DNA replication and conrms there are no areas of unrepaired DNA damage from previous phases in the cell cycle. The DNA damage checkpoint arrests cells in the S phase depending on the cell cycle phase at the time damage was incurred. Checkpoints can also cause DNA repair and regulate cancer progression in addition to inducing cell cycle arrest. 71 Our ndings showed a cell cycle arrest in S phase by GC and LC. This fact may have promoted repair, or in the case of irreparable damage, stimulated cell death.
To measure the rate of apoptosis, cells treated with the soluble coffee extracts showed a population shi to the upper le quadrant of the graph, as can be seen in Fig. 6. There was also displacement to the lower and upper right quadrants, representing the cell populations in early apoptosis and late apoptosis or non-apoptotic cell death. Medium and dark extracts had the capacity to alter cell populations, but also promoted changes in the non-apoptotic cells that were higher than those observed in the control, suggesting a potential toxicity effect. Apoptosis increased when we used the coffee extract concentration of 5000 and 10 000 mg mL À1 to treat the cells and the light roasted and green bean extracts were better than medium and dark extracts (Fig. 6). Table 3 reports the percentages of cells of the control and the samples treated with the coffee extract concentrations of 5000 and 10 000 mg mL À1 for each phase of cell death.
Cancer can occur when the balance between cell growth and death is disturbed. A cancer therapy that acts solely by induction of apoptosis without intrinsic cytotoxicity would be likely to cause the death of more normal cells than cancer cells. Our data indicate that green and light roasted extracts increased early and late apoptosis, with no signicant changes in nonapoptotic cells.
These results might be directly related to the production of monomeric compounds of chlorogenic acids and caffeic acid, which modulate the cell's epigenetic activities by increasing DNA methylation and silencing some genes involved in proliferation. Even with the presence of acrylamide in its composition, the consumption of roasted coffee has strong effects on the prevention and treatment of chronic NCDs, including cancer, cardiovascular diseases, Parkinson's disease, Alzheimer's disease, among others. 27 Several studies have shown no harmful effects on human health due to coffee consumption and some epidemiological studies have demonstrated signicant positive effects of 3 to 4 cups of coffee per day in combating NCDs. 72 Studies of bioavailability reveal high absorption and high permanence of caffeine in the bloodstream, a supposed passive transport absorbance of monomers of chlorogenic acids and an absorbance by active transport of caffeic acid in the colon. 73 The literature has also reported that coffee extracts protect the living organism from various effects, including antioxidant activity, increased enzymatic activity of glutathione synthesis, and modulated oncogene expression. 65,74,75 Similar to our research, it has been shown that coffee extracts showed cytotoxic activity, induced cell cycle arrest and induced apoptosis in Hela cells. 76 The possible pathways through which coffee mediated biological activity can be correlated with the promotion of programmed cell death (apoptosis), cell cycle regulation and increased pro-apoptotic activity through the pathway involving caspases, bax and p53. 77 Many bioactive compounds in Arabica coffee are well known, but many other compounds have not been described yet. The Maillard reaction by-products and its nal products, melanoidins, are examples of this. Thus, further experiments are needed to elucidate the function of coffee's chemical substances on tumour cells.

Conclusions
Regular roasting (light to medium) causes signicant positive changes in the chemical composition of coffee bean extracts, but darker roasting may negatively affect this composition and potential bioactivity, leading to chlorogenic acid breakdown and the formation of products, which may alter the health benets.
The extraction of coffee solutions in a high-power ultrasonic device and the subsequent spray drying alone can generate a product with health appeal, although further studies are needed for conrmation. Our research demonstrates an important antiproliferative effect of coffee extract in human prostate cancer cell lines that can be contributes to cancer prevention.

Conflicts of interest
There are no conicts to declare.


extfieldsofstudy: Biology Medicine

text:
Fine mapping qGL2H, a major locus controlling grain length in barley (Hordeum vulgare L.)

Key message A major grain length QTL on chromosome 2H was fine mapped to a 140.9 Kb region containing three genes. Abstract Increasing yield is an important target for barley breeding programs. One approach to increase yield is by enhancing individual grain weights through the regulation of grain size. Fine mapping major grain size-related quantitative trait loci is necessary for future marker-assisted selection strategies, yet studies of this nature are limited in barley. In the present study, we utilised a doubled haploid population derived from two Australian malt barley varieties, Vlamingh and Buloke, coupled with extensive genotypic and phenotypic data from three independent environments. A major grain length locus identified on chromosome 2H designated qGL2H was fine mapped to a 140.9 Kb interval. qGL2H was able to account for 25.4% of the phenotypic variation for grain length and 10.2% for grain yield. Underlying qGL2H were three high-confidence predicted genes. One of these genes encodes a MYB transcription factor and represents a promising candidate for further genetic research. Electronic supplementary material The online version of this article (10.1007/s00122-020-03579-z) contains supplementary material, which is available to authorized users.

Introduction
"Yield is king," a sentiment held by many cereal crop breeders across the globe with yield representing the most important driver of grower profitability. Changing climate, rising global population and reduction in arable land area necessitate the need to increase cereal yield potentials beyond what are currently achieved in order to meet future demand, which is expected to more than double by 2050 (Molotoks et al. 2018). Barley is the fourth most important cereal grain crop grown globally in terms of both production and area cultivated (FAOSTAT 2019). It is an important stockfeed and the primary grain used for beer and spirit production (Wendt et al. 2016). Changes to climatic conditions threaten barley production with yield modelling indicating average yield losses of up to 17% as a result of the increased frequency and severity of heat stress events alone. In comparison, growth chamber experiments have indicated this reduction could be as high as 52% when coupled with the increased average global temperature (Ingvordsen et al. 2018;Xie et al. 2018). Like all small grained cereal crops, barley yields are a reflection of two key phenotypic components: number of grain per m 2 and individual grain weight (Fan et al. 2006;Xing and Zhang 2010). Yield improvements through pre-breeding and breeding have largely been driven by the optimisation of flowering time and increases in Communicated by Andreas Graner.

Electronic supplementary material
The online version of this article (https ://doi.org/10.1007/s0012 2-020-03579 -z) contains supplementary material, which is available to authorized users.
1 3 harvest index, enabling lodging resistance and more efficient grain filling (Hill and Li 2016;Prince et al. 2001;Sharma et al. 2018). Despite this, year-on-year yield improvements have begun to stagnate because desirable allelic combinations have largely been captured in current cultivars, and this yield growth stagnation is reflected in increasing numbers of undernourished people globally (FAOSTAT 2019; Ray et al. 2012;Schauberger et al. 2018;Xu et al. 2018a). In some regions, yield growth rates are already decreasing due to recent changes in climatic conditions resulting in poorer cultivar adaptation. Alternative traits to target are therefore necessary in order to improve the yield potential of barley.
Grain size is a major determinant of individual grain weight and has higher heritability than yield per se making it a desirable alternative trait to target. Grain size is not only important from a yield perspective but is also important from a processing standpoint. For example, maltsters prefer a plump grain because it has a more desirable starch/protein ratio, improving the efficiency of alcohol production (Agu et al. 2007). Increasing grain size is therefore an efficient and necessary strategy by which to increase yield and improve grain quality, and has been a recent focus of pre-breeding research (Walker et al. 2013;Watt et al. 2019;Wendt et al. 2016;Xu et al. 2018a).
Grain size characteristics are complex polygenic traits that are also strongly influenced by environment Walker and Panozzo 2016;Xu et al. 2018a). Optimising the most desirable allelic combinations into elite backgrounds can be rapidly achieved through the use of tightly linked molecular markers and marker-assisted selection (MAS) coupled with speed breeding approaches (Watson et al. 2018). Identification and mapping QTL for grain size-related characteristics have been achieved in various genetic backgrounds (Walker et al. 2013;Watt et al. 2019;Xu et al. 2018a;Zhou et al. 2016). In many instances, the identified QTL share close homology to grain size-related genes identified in wheat and rice resulting in promising putative candidates for further research, although research of homologous gene regions in barley is restricted to a small number of publications (Bélanger et al. 2014;Wang et al. 2019;Watt et al. 2019;Xu et al. 2018a). Despite our current understanding of QTL regions contributing to grain size, only one study has fine mapped a major QTL and identified a potential candidate gene (Watt et al. 2019). While analysis of homologous sequence regions between barley and the other widely studied cereal crop species provides a promising starting point for grain size research, independent linkage and association mapping studies are still necessary to validate gene regions and identify desirable alleles underlying these homologs that will improve grain size in barley (Walker and Panozzo 2016;Zhou et al. 2016).
Quantitative trait loci mapping studies have successfully identified significant QTL on all seven barley chromosomes reported to control grain size-related characteristics (Marquez-Cedillo et al. 2000;Mather et al. 1997;Matthies et al. 2012;Walker et al. 2013;Watt et al. 2019;Xu et al. 2018a;Zhou et al. 2016). Although the limiting step is not QTL identification, the process of fine mapping and identifying the underlying candidate gene are responsible for trait variation. This is followed by validation of gene effect and characterisation of desirable alleles in different genetic backgrounds. Development of tightly linked molecular markers to underlying candidate genes and identification of desirable alleles is necessary for effective MAS. Diagnostic molecular markers have been identified for numerous traits such as waterlogging tolerance (Zhang et al. 2016); malting quality (Gong et al. 2013;Xu et al. 2018b) and disease resistance (Dinglasan et al. 2019;Zantinge et al. 2019). Despite numerous studies having mapped significant QTL associated with grain size characteristics on all seven barley chromosomes, limited research fine mapping these regions has impacted the development of diagnostic molecular markers for breeding purposes. A previous study by the authors represents the only research to date with a specific focus on fine mapping a major grain size-related locus, designated qGL5H (Watt et al. 2019). Using the same genetic population as Watt et al. (2019), this study describes the fine mapping result of a major grain length QTL identified on chromosome 2H that was able to explain a high proportion of the phenotypic variation for grain length and yield. An underlying gene identified in the fine-mapped interval represents a promising target for future genetic research, and the development of diagnostic flanking molecular markers in this study has potential use in MAS.

Plant material
The population consisted of 306 DH lines derived from a cross between Vlamingh and Buloke, two-rowed spring barley varieties with distinctly different grain shapes. This is the same genetic population used by Watt et al. (2019).

Field trials
Phenotypic data from three independent and rainfed field trials (Esperance 2016; South Stirling 2016; Wongan Hills 2017) were used for this study and represented contrasting environments in the Western Australian wheatbelt. Briefly, each genotype was sown in a 1 × 5 m 2 plot, reduced to 3 m in late August of each season. Genotypes were partially replicated with a minimum of one replication and maximum of four in each field trial. Commercial varieties acted as controls. There were two applications of fertiliser, the first at seeding and the second when the crop had begun to tiller at Zadoks growth stage 21 (Zadoks et al. 1974). Biotic stress management varied depending on the severity of infestation and control followed standard agricultural practice for the western region. Growing season rainfall and temperature records were maintained for inclusion as covariates during statistical analysis.

Measurement and analysis of grain size characteristics
Grain size characteristics were measured using an SC6000R digital image analyser (Next Instruments, Condell Park, Australia) using the same protocol as Watt et al. (2019). Yield (t/ha) was determined post-harvest. Grain length, width and thickness were measured on a 20-25 g sub-sample of grain.
Best linear unbiased predictions (BLUPs) for grain size characteristics and yield were calculated using linear mixed models for individual trials and a combined analysis of all three field trials known as a multi-environment trial (MET), the purpose of which was to remove environmental effects. The simplified model is given by where y is the vector of observations for different grain size characteristics; X is a design matrix associated with a vector of fixed effects ; Z is a design matrix associated with a vector of random effects u; and e is the vector of residuals that include residual error variance associated with autoregressive spatial correlations in the row and column directions (Smith et al. 2019). Linear mixed models using advanced restricted maximum likelihood techniques to obtain trait BLUPs were ran using the R software package 'ASReml'. Statistically significant differences between individuals and trials for the phenotypic measurements collected were determined using Students t tests using the R software, which was also used for principle component analysis (PCA).
Genotyping and QTL analysis. Genomic DNA was isolated from grain samples using an adapted method from Ahmed et al. (2009) where instead of a phenol/chloroform/isoamyl alcohol protein degradation step, samples were placed in a 65 °C water bath for 1 h to denature any protein. Following denaturing DNA was precipitated using ethanol, spun at 4000 RPM to pelletise DNA prior to resuspension. Insertion-deletions (InDels) between Vlamingh and Buloke were determined within the QTL region identified from initial whole-genome QTL mapping activities using the BarleyVar database (in-house database) and used for fine mapping on a total of 306 DH lines. Primers were developed using the barley cv. Morex reference genome sequence in the Geneious v10.2.3 software (Kearse et al. 2012;Mascher et al. 2017).
PCR reactions were performed using freshly extracted genomic DNA from leaves in a total volume of 10 µL containing 1 µL of 10 × buffer and GC buffer, 0.25 mM dNTPs, 0.2 µM of each primer, 50 mM MgCl 2 , 50 ng genomic DNA and 0.2 U Taq-polymerase. The PCR protocol for gel markers was as follows: 95 °C for 3 min, 38 cycles of 94 °C for 20 s, 55-57 °C (primer-dependent) for 20 s, 72 °C for 20 s and a final extension at 72 °C for 5 min. Standard InDel markers were separated in 2% agarose gels in 0.5x TBE buffer and visualised under UV light.
Initial whole-genome QTL mapping analyses used a total of 619 DArT, SSR and SNP markers after removal of unmapped markers as outlined in a previous publication by Watt et al. (2019). The genetic map for this initial analysis was developed using JoinMap5 as described by Watt et al. (2019). Composite interval mapping (CIM) algorithms were used to conduct initial whole-genome scans using individual field trial BLUP data in MapQTL v6, culminating in a separate analysis for each field trial. LOD scores were calculated based on 1,000 permutations at a cut-off P-value of 1.0e-08, a minimum walk speed of 1 cM and a threshold LOD value for linkage significance of 3.5. Identified QTL were further investigated using the more sensitive multi-QTL mapping (MQM) algorithm by adding more QTL sequentially until no new loci were detected with similar threshold criteria to CIM. Percentage of phenotypic variation explained by each QTL was estimated as the R 2 (coefficient of determination). The linkage map and identified QTL were drawn using the MapChart software (Voorrips 2002). Detected QTLs that were stable across two or more environments and had similar genetic positions (overlapping) were considered as singular loci. Any QTL that explained > 10% of the phenotypic variation was considered a major locus.
QTL fine mapping and candidate gene annotation. Initial whole-genome QTL analysis using individual trial BLUP data identified three overlapping intervals for qGL2H. Fine mapping of qGL2H was then achieved by saturating the interval delineated by the two outermost flanking markers identified with InDel markers. In total, 95 InDel markers were designed and combined into the genetic map with further QTL analyses using MET-BLUP phenotypic data and then carried out using the same approach described previously. We used the MET-BLUP data during fine mapping to ensure that we captured the stable effect of qGL2H. Following marker development and subsequent QTL analysis with the newly developed InDel marker set were able to narrow down the interval. In the newly identified qGL2H interval, we then identified DH lines that were recombining and used Student's t tests to compare the phenotype of recombinants that fell into two distinct groups to validate allelic effects at this locus. Candidate genes in the fine-mapped interval were identified using the BarleyVar database by inputting the physical positions of the QTL flanking markers. Relative gene expression profiles were investigated using The Barley Genome Explorer (BARLEX) database which is the combination of the barley cv. Morex reference sequence and highand low-confidence gene predictions (Beier et al. 2017;Colmsee et al. 2015;Mascher et al. 2017).

Phenotypic summary
Grain size characteristics were normally distributed in each individual field trial after removal of outliers as expected for polygenic and quantitatively inherited traits. Phenotypic differences between the two parental lines, Vlamingh and Buloke, are shown in Fig. 1a. Transgressive segregation was evident for grain length in all trials (Fig. 1c). Correlation between grain size traits and yield varied significantly, driven by strong environmental effects (Fig. 1b). At the individual trial level, grain length was significantly positively correlated with yield in the Wongan Hills field trial, but there were no significant correlations between grain length and yield in either Esperance or South Stirling trials. Broad sense heritability ranged from 0.27 at Wongan Hills to 0.81 at both Esperance and South Stirling, respectively. The low heritability in Wongan Hills was believed to be driven by heterogeneous field variation that was not properly captured by the model and is reflected in low accuracy of BLUPs for this site despite the high replication of control varieties.
Principal component analysis using combined trial MET-BLUP data indicated that length was the most discriminating grain size trait measured within this population (Fig.  S1). The PCA clearly indicates two distinct groups along the first principle component which is most correlated with grain length, where groups are based on the parental allele present at qGL2H and indicate that lines with the Buloke allele tend to be longer on average. Predicted values from combined trial MET-BLUP analysis indicated that all traits were significantly correlated apart from width and yield, which is inconsistent with the correlations detected in the raw field trial data (Fig. 1b). There were significant negative correlations between length and the other three traits compared. Grain thickness was significantly positively correlated with width and yield. Grain length was significantly positively correlated with yield in the Wongan Hills trial. Wongan Hills representing a harsher environment than South Stirling and Esperance, with grain length likely contributing more to yield than grain width and thickness, were more important contributors to yield in the more favourable growing environments of Esperance and South Stirling.

Identification and mapping of QTL
Using the two-stage linkage mapping procedure in MapQTL (CIM followed by MQM), 15 significant QTLs were detected for the grain size traits only, and none for yield using wholegenome marker data and individual trial BLUPs. Of these QTL, nine were associated with grain length, a conclusion support by PCA where width and thickness are not large drivers of grain size diversity within this population (Fig.  S1). Significant QTL were detected on all chromosomes aside from 3 and 7H. Chromosomes 2H and 5H were hotspots with a total of 11 QTL detected. Major loci that were detected in two or more environments (consensus) were only identified on chromosomes 2H and 5H using the individual trial BLUP data (Table 1). In this study, we were only able to detect two consensus major QTL regions, both of which regulated grain length, one being qGL5H and the other qGL2H (Table 1). These loci were identified in each individual trial QTL analysis reflecting the heritability and stability of this trait in this population.

Fine mapping of 2H major grain length QTL region
Two significant major grain length QTL were identified using combined trial MET-BLUP data in this population, one each on chromosome 2H and 5H. The interval identified on chromosome 2H overlapped with the major consensus region qGL2H identified through individual trial QTL analyses (Table 1). Using MET-BLUP data, the LOD scores for qGL2H and qGL5H were 22.7 and 20.9, respectively. The percentage of phenotypic variation was 25.4% and 21.6% for qGL2H and qGL5H, respectively. Furthermore, a previous study found a strong QTL for grain length in a similar location to qGL2H making it a suitable candidate for fine mapping . Whole-genome QTL analyses using individual trial BLUP data indicated that qGL2H was flanked by SNP markers 2651-1774 and 6117-1507, which based on the barley cv. Morex reference sequence is a large interval spanning 530.3 Mb (Table 1). Analysis using MET-BLUP data found qGL2H accounted for 25.4% of phenotypic variation for grain length with a LOD score of 22.7. No other grain size characteristic was influenced by qGL2H which was consistent with initial whole-genome mapping results. Interestingly, qGL2H was also able to explain 10.2% of the phenotypic variation for grain yield when using the MET-BLUP data, a relationship not observed during individual trial QTL analyses. To fine map qGL2H, we designed 95 polymorphic InDel markers saturating this 530.3 Mb interval and used the combined trial MET-BLUP data to undertake successive QTL analyses and marker development to continuously narrow down the target region. Using this dataset which consisted of the entire DH population being genotyped, we were able to fine map qGL2H to a 140.9 Kb interval between markers 2H638,235,731 and 2H638,376,721 representing a substantial reduction in the size of this interval (Figs. 2 and 3,  S2). At a population level, DH lines that have the Buloke allele at both of these flanking markers have an average grain length of 8.78 mm compared to 8.47 mm for those with Vlamingh alleles which are significantly different at p = 0.001. Of the population, four recombinant DH lines in total were identified within the qGL2H interval (Fig. 3), three of which had Buloke alleles between the flanking markers (Rec061, Rec068 and Rec081) and in each individual trial had grains that were significantly longer than the recombinant line with Vlamingh alleles, Rec160. This is supported by an effect at this locus indicating that the Vlamingh allele reduces grain length upwards of 0.22 mm in this population (Table 1).
Within this newly mapped interval, three highconfidence predicted genes were identified, HOR-VU2Hr1G089310, a predicted MYB transcription factor of subgroup 15, which is a promising candidate as it has been reported to be involved in cell cycle control and cell division in the longitudinal direction (Qi et al. 2018;Tombuloglu et al. 2013;Wu et al. 2017). The other predicted genes were HORVU2Hr1G089320 (hexosyltransferase) and HORVU2Hr1G089330 (root UV-B sensitive 2 protein). Relative gene expression indicates that of the three candidate genes, the MYB transcription factor has the highest expression of the three genes in tissues associated with the developing inflorescence (Fig. S3). Table 1 Consensus genomic regions harbouring significant QTL for grain length using whole-genome marker data Chr, chromosome ID; Env, Number of environments detected; Pos, genetic position; LOD, logarithm of the odds; R 2 , % trait variation explained by the QTL; Effect, mean locus effect; Higher, parent contributing discriminating allele; Marker, QTL discriminating flanking markers; Physical position, based on barley reference genome in Mb; and Name, of locus * Consensus; an overlapping chromosomal region detected in two or more field trials and considered to be the same locus  (Table S1) and associated fine-mapped QTL region (black) Fig. 3 Genotypes and phenotypes of parents and recombinant DH lines using BLUP data for grain length. Genetic structure depicted as white (homozygous Buloke) and black (homozygous Vlamingh) in qGL2H (dash) using a subset of the 95 interval markers. All recombi-nant lines had Buloke alleles between qGL5H flanking markers. Table on the right indicates variation in grain length; numbers in brackets indicate number of plots of each genotype and letters depict significant differences at p = 0.05

Discussion
Linkage mapping is an efficient method to identify the genetic control of polygenic traits such as grain length. In the present study, Vlamingh and Buloke, two elite Australian two-rowed malting varieties with contrasting grain lengths were used to generate a DH population to fine map a major grain length locus that overlapped with one previously identified on chromosome 2H (Watt et al. 2019).
Grain length was normally distributed in each environment as expected for a polygenic and quantitatively inherited trait (Lai et al. 2017;Sadeghzadeh et al. 2010;Vafadar Shamasbi et al. 2017). Correlation between the three grain size characteristics varied from weakly positive to negative in each trial, indicating that there is a significant genotype by environment interaction occurring. Within individual field trials, grain length was only significantly correlated with grain width in the Esperance environment (Fig. 1b).
For the most part, grain plumpness characteristics, width and thickness tended to be significantly positively correlated with yield in individual environments apart from grain width at Wongan Hills which was negatively correlated with yield (p = 0.05). A possible reason for this distinction is Esperance and South Stirling are milder environments compared to Wongan Hills, enabling a genotypes maximum grain width and thickness to fully express, whereas in the harsher Wongan Hills environment grain length would become a larger contributing factor for grain yield as supported by the significant positive correlation between the two (Fig. 1b). Watt et al. (2019) previously identified 23 significant QTLs for grain yield and three grain size characteristics of which two represented major loci controlling grain length in this population. One designated qGL5H was previously fine mapped to a 1.7 Mb interval that was able to explain 21.6% of the phenotypic variation for grain length. In this present study, we fine mapped the second major grain length QTL originally identified, designated qGL2H to a 140.9 Kb interval containing three high-confidence predicted genes. Consistent with studies in wheat and rice, grain length QTL tend not to coincide with other grain size-related characteristics such as width and thickness. This is due to the fact that grain length is to a large extent controlled at the cellular level by cell elongation and proliferation in the longitudinal direction in developing endosperm and husk tissues (Rabiei et al. 2004;Yu et al. 2017). In contrast, grain width and thickness correlate primarily with endosperm cell proliferation in the transverse direction (Segami et al. 2016;Wang et al. 2015). In the present study, qGL2H did not coincide with other grain size-related characteristics; however, it did represent a major locus for grain yield explaining 10.2% of the phenotypic variation for this trait using MET-BLUP data but not individual trial BLUPs. This result is likely to be driven by the reduced shrinkage of BLUPs towards the mean when running a MET compared to individual trial analyses. Interestingly, a previous study identified significant QTL for grain length, a similar region to qGL2H, although they also led to variation in other grain sizerelated characteristics such as grain width . The fine mapping result of qGL2H indicates there are two QTL regions near one another on chromosome 2H contributing to grain length variation; however, it is evident that due to the genetic background of each population, neither were able to be identified simultaneously.
Three high-confidence predicted genes are located within qGL2H. A promising candidate for the control of grain length is HORVU2Hr1G089310 which encodes a MYB transcription factor protein of subgroup 15. Previous research in other cereal species has indicated that transcription factors are heavily involved in the control of grain size through regulation of cell proliferation and differentiation (Arora et al. 2017;Gong et al. 2018;Ji et al. 2019;Qi et al. 2018). Specifically, a MYB transcription factor designated GL4 was shown to regulate cell elongation in the outer and inner glumes of African rice which directly regulated grain length (Wu et al. 2017). In the present study, comparisons between the three putative candidate genes indicated that the MYB transcription factor has the highest relative expression in developing inflorescence tissues of the lemma and palea compared to the other two genes (Fig. S3). Wu et al. (2017) found that in rice, a premature stop codon in the coding region of this MYB transcription factor was responsible for the significant differences in grain length observed. Interestingly, the MYB transcription factor gene located within qGL2H shows approximately 90.37% DNA sequence homology to SH4, a MYB-like protein encoding gene associated with non-shattering in Asian rice that is orthologous to GL4. A review of the current literature did not find any clear link between the hexosyltransferase and root UV-B sensitive protein encoding genes and any grain size-related characteristic, further reinforcing that the MYB transcription factor is the likely candidate gene underlying qGL2H. Watt et al. (2019) previously conducted whole-genome QTL analysis to identify loci controlling yield and three components contributing to grain size (length, width and thickness). The present study fine mapped qGL2H, which was found to significantly associate with grain length and yield. We identified one promising candidate gene that based on current research is likely to regulate grain size, annotated as HORVU2Hr1G089310 and encoding a MYB transcription factor. MYB-like proteins represent one of the largest families of transcription factors and have been linked to numerous biological functions, including abiotic stress tolerance and control of grain size characteristics through 1 3 regulation of cell division and differentiation within certain developmental tissues (Qi et al. 2018;Wu et al. 2017;Xiong et al. 2014). While the other two candidate genes cannot be ruled out, the MYB transcription factor represents the most promising avenue on which to focus further research. In lieu of further research into these genes, the two flanking InDel markers of qGL2H could be highly useful for MAS as they are diagnostic for grain length and yield within this population. As it stands, qGL2H represents a promising genetic region with which yield and grain length can be manipulated simultaneously.
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Agronomic yield and quality of industrial tomatoes under NPK doses

: High doses of nitrogen, phosphorus and potassium in irrigated systems may lead to an increase in the productivity of industrial tomatoes, but may also alter the final quality of the fruits. The objective of this was to evaluate the agronomic yield and post-harvest quality of industrial tomatoes, under NPK doses for climatic conditions of Northern Minas Gerais state, Brazil. The study was conducted in the city of Jaíba, MG, Brazil, in randomized block design with four repetitions in a 3 x 3 factorial scheme, consisting of three doses of N (90, 135 and 180 kg ha -1 ), of P (270, 405 and 540 kg ha -1 ) and of K (225, 337 and 450 kg ha -1 ), corresponding to 50, 75 and 100% of the fertilization recommendation; and three tomato hybrids: BRS Sena, Heinz 9553 and BHN 0574. The following variables were evaluated: number of bunches, number and weight of fruits, productivity and fertilizer efficiency index; besides the physico-chemical characteristics: firmness, soluble solids concentration, pH and pulp industrial yield. The hybrid Heinz 9553 showed higher weight, number of fruits, productivity and industrial yield at doses of 75 and 100% of NPK recommendation. The fertilizer doses did not interfere on the soluble solids, pH and fruit firmness of the three hybrids. The hybrid BRS Sena was not influenced by the increase of NPK and there was lower weight, number of fruits, productivity and fertilization efficiency. The hybrid BHN and Heinz hybrids can be indicated for cultivation at doses of 100 and 75% of NPK recommendation, because they promote higher agronomic yield and do not alter the post-harvest quality of the tomatoes.

Introduction
Brazil is the eighth largest producer of tomato for industrialization in the world (WPTC, 2016), producing 1.2 million tons of tomato for industrial processing in 2016. The use of hybrids with higher productivity, phenotypic adaptability and resistance to diseases allowed greater profitability of this crop in the country (Peixoto et al., 2017).
The northern part of Minas Gerais state, Brazil, which is a semiarid region, has potential for the production of commercial plantations of irrigated industrial tomato, due to low air relative humidity and high insolation, which guarantee higher fruit quality. However, studies demonstrating the productive behavior of hybrid genotypes in the semiarid region of Minas Gerais state, Brazil, are necessary (Figueiredo et al., 2016).
Balanced supply of fertilizers, in the correct quantity and time, is a primordial factor for maximum expression of the production potential of tomato hybrids. The nutrient supply can also influence the nutritional quality of the fruits as well as their yield, but these parameters do not increase at the same rate as the increment of nutrients (Marschner, 2012). According to Silva et al. (2012), fertilization with doses of 120 kg ha -1 of nitrogen (N), 600 kg ha -1 of phosphorus (P) and 250 kg ha -1 of potassium (K) is indicated for the cultivation of tomato for processing in the Brazilian Cerrado region.
The nitrogen is cited as one of the main nutrients for tomato, because it constitutes organic compounds that are vital to plants and contributes to fruiting, productivity and economic profitability, but can decrease fruit firmness (Marschner, 2012). Despite being absorbed in smaller quantity and having lower concentrations in plant tissue, the phosphorus is extremely important in root formation and growth, besides increasing the commercial quality of the fruit (Mueller et al., 2013;Nowaki et al., 2017). The potassium is the most required nutrient by tomato (Prado et al., 2011) and contributes to the processes of photosynthesis, enzymatic activation and protein synthesis (Rebouças Neto et al., 2016). Balance between nutrient availability, fertilizer dose and application time should be recommended in order to reduce the risk of environmental contamination and increase the efficiency of fertilizer use by cultivated plants.
Thus, the adoption of balanced NPK doses, besides increasing productivity and soluble solids concentration, can increase pulp yield, fruit firmness of industrial tomatoes, among others. However, studies related to the adoption of balanced doses of nutrients in irrigated industrial tomato hybrids in the semiarid region are scarce. Therefore, the objective of this study was to evaluate the agronomic yield and postharvest quality of industrial tomato hybrids after fertilization with NPK doses.

Material and Methods
The study was conducted in 2015, in the Irrigation District of Jaíba (IDJ), municipality of Jaíba, MG, Brazil (15º 12' S, 44º 01' W and 456 m of altitude). The climate of the region is Aw, according to Köppen's climatic classification, tropical with dry winter and the soil of the experimental area was characterized as Inceptisol. Before the experiment began, the area was cultivated in rotation with maize for grain production and plant residues were incorporated into the soil by conventional tillage, twice with a heavy harrow and once with a light harrow.
The experimental design consisted of randomized blocks , with four repetitions, in a 3 x 3 factorial scheme, corresponding to three tomato hybrids for industrial processing: BRS Sena (Embrapa Hortaliças), Heinz 9553 (Heinz Seeds) and BHN 0574 (Blueseed), and three doses of N: 90, 135 and 180 kg ha -1 , of P: 270, 405 and 540 kg ha -1 and of K: 225, 337 and 450 kg ha -1 . These doses corresponded, respectively, to 50, 75 and 100% of the fertilizer recommendation for industrial tomato, as described by Filgueira et al. (1999) with adaptations in doses and managements for irrigated systems.
The treatments with NPK fertilizer in the tomato hybrids were managed as follows: at planting (basal fertilization) 20, 100 and 40% of nutrients N, P and K, respectively, were applied by means of the NPK 2-30-10 formulation; as topdressing, N and K were split into six times and applied by sprinkler fertigation at 18, 35, 40, 46, 51 and 59 days after transplanting (DAT). The following fertilizers were used as sources: ammonium sulfate, potassium nitrate and potassium chloride and superphosphate (simple).
The hybrids used in the study have the following characteristics: BRS Sena was developed by EMBRAPA, has great performance in mechanical harvesting, cycle of 110 days, soluble solids concentrations varying from 5.8 to 6.7 °Brix; the hybrid Heinz 9553 has a cycle of 110 to 120 days and soluble solids concentration of 4.9 to 5.1 °Brix; and the hybrid BHN 0574, developed by the company Blueseed, has concentrated maturity and high soluble solids concentration.
Each experimental plot consisted of four planting rows and the two central rows were considered for the observations, with 30 m in length and 5.2 m in width, in a total area of 5,616 m², totaling 240 plants per plot. The seedlings were produced on polypropylene trays of 400 cells, with the commercial substrate Carolina Soli and grown in greenhouse. Transplanting was performed on May 20, 2015, when the seedlings had three to four true leaves. Planting in the experimental area was carried out mechanically, in a single-row system, with spacing of 1.3 m between rows and 0.25 m between plants.
Phytosanitary treatments were performed with the application of herbicides (glyphosate, metribuzin), systemic insecticides (Actara) and contact insecticides (pyrethroid), applied preventively, and fungicides (chlorothalonil, copper hydroxide, famoxadone and mancozeb) applied according to the schedule of the farm. Irrigation was carried out according to the water need of the crop, using the center-pivot sprinkler irrigation system. The total water depth applied during the crop cycle was 414 mm.
At the end of the crop cycle (September 5, 2015), the four central plants were evaluated for the agronomic characteristics: number of bunches (NB), number of fruits per plant (NF), fruit weight per plant (kg) (FW), productivity (PRODUCT) estimated in t ha -1 , and fertilization efficiency index (FEI), obtained by dividing the productivity (kg ha -1 ) by the dose of each nutrient used.
In addition, 3 kg of fruits with uniform maturation from the four central plants were separated and sent to the Postharvest Laboratory to determine the physico-chemical characteristics: firmness -FIRM, expressed in Newtons (N), determined using a hand-held penetrometer, with a thin tip (8 mm), in the median region of the fruit; soluble solids concentrationo Brix, by refractometry, using a digital refractometer (Atago), with values expressed in ºBrix; pH, determined directly in the processed juice of the fruits, using a pH meter; and industrial pulp yield, using the formula: Yield -PY (t ha -1 of pulp) = [(Productivity (t ha -1 ) x 0.95) x ºBrix of the Juice]/28 (Clemente & Boiteux, 2012) .
The obtained data were subjected to normality (Shapiro-Wilk) and homogeneity tests and then analysis of variance was performed. When significant, the data were subjected to Tukey's means comparison test at p < 0.05. Statistical analysis was carried out using the statistical software R.

Results and Discussion
There was interaction between the doses of NPK fertilizer and the hybrids for the characteristics fruit weight per plant (FW), number of fruits per plant (NF) and productivity (PRODUCT). For the number of bunches (NB), difference was only caused by the hybrids (Table 1).
For NB, the hybrid Heinz 9553 was superior, with 23 bunches per plant, followed by the BRS Sena (19 bunches) and BHN 0574 (17 bunches). On the contrary, the NPK doses did not influence the NB (Table 1), indicating that this is a genetic trait not influenced by fertilization. This same aspect was reported by Blanco & Folegatti (2008), who attributed the lack of difference to genetic characteristics of the genotypes.
The doses of 100 and 75% of NPK recommendation promoted increase of FW in the hybrids BHN 0574 and Heinz 9553, with weights of 4.93 and 5.18 kg per plant, respectively (Table 1). These results suggest that higher doses of NPK favor the growth and formation of tomato fruits, especially for the hybrids BHN 0574 and Heinz 9553. The increase in fruit weight can be explained by the increase in photoassimilates transported in the plant from source organs to sink organs (Singh et al., 2010). For the industry, it is desirable to produce larger fruits in size and weight, because this usually leads to greater pulp production (Silva et al., 2012). For the hybrid BRS Sena, there was no difference in FW with the NPK doses (Table 1), probably due to the high natural fertility of the soil used in this experiment.
The hybrid Heinz 9553 under 75% NPK dose obtained higher FW compared to the other tomato genotypes studied (Table 1). It is known that this hybrid is widely used in Brazilian production and experimental fields, mainly due to its characteristics of production and adaptability (Figueiredo et al., 2016;Luz et al., 2016). Carvalho et al. (2003), evaluating tomato hybrids under three doses of NPK fertilizer, reported higher weight in the fruit of the Heinz 9553, which indicates higher genetic potential of production. The lower FW obtained in BRS Sena for the doses of 100 and 75% NPK was attributed to its production characteristic, because it has lower average weights (70 g) compared to the hybrids Heinz 9553 (80 g) and BHN 0574 (95 g) (Quezado-Duval et al., 2014).
The highest NPK dose (100%) increased NF only for the hybrid Heinz, compared to the 50% NPK dose (Table 1). On the other hand, Singh et al. (2010), working with NPK doses in tomato, found higher number of fruits (37) under the dose of NPK (120:60:60 kg ha -1 ). However, for the hybrid BRS Sena, the highest NF was obtained with the dose of 50% NPK, and there was no effect of fertilization on the hybrid BHN 0574 (Table 1).
Means followed by the same lowercase letter in the column and uppercase letter in the row do not differ by Tukey test (p ≤ 0.05). *Refers to the fertilization efficiency of the nutrients nitrogen, phosphorus and potassium (FEI = PRODUCT/Dose of the nutrient), respectively

Table 1. Number of bunches per plant (NB), fruit weight (FW), number of fruits (NF), productivity (PRODUCT) and fertilization efficiency index (FEI) of industrial tomato hybrids under NPK fertilizer doses
The classification of tomato genotypes destined for processing considers as the best the ones which have lower fruit weight and higher number of fruits per plant (Carvalho et al., 2003;Silva et al., 2012). Santos et al. (2001) working with three doses of NPK in tomato hybrids, observed that the highest number of fruits was obtained at the highest NPK doses, 3.5 and 5 t ha -1 . Salam et al. (2010) also found that, at the highest dose of NPK (2.2:1.8:1), there was higher number of tomato fruits. Higher nutrient availability for tomato plants prolongs the vegetative stage, increasing the production of inflorescences and, consequently, the number of fruits (Santos et al., 2001;Marschner, 2012).
At the dose of 50% NPK, there was a similar productivity among the hybrids, corresponding to 126.67 t ha -1 (Table 1). However, the productivity obtained is within the production pattern of commercial plantations of tomato for processing, demonstrating the high production capacity of these hybrids for the northern region of Minas Gerais. Brazil. At the dose of 75% NPK, the hybrid Heinz 9553 was superior (191.39 t ha -1 ), differing from the other hybrids. At the highest fertilizer dose (100%), there was average productivity of 150.26 t ha -1 and the hybrids BHN (182.15 kg ha -1 ) and Heinz (155.38 t ha -1 ) outperformed BRS Sena (113.23 t ha -1 ) ( Table 1). These productivities were considered higher than those obtained by Carvalho et al. (2003), which corresponded to 124 t ha -1 for the hybrid Heinz under drip irrigation system.
In general, the productions and productivities obtained in this study were considered superior to those described in the literature (Silva et al., 2012). The expected productivity for the Brazilian Cerrado region is approximately 100 t ha -1 in plantations with high technological dose, because the use of highly productive genetic materials associated with the application of modern technologies results in high productivity (Peixoto et al., 2017).
For the hybrid BRS Sena, the NPK doses did not influence productivity (111.59 t ha -1 ) ( Table 1). This result is of great importance, since 50% of the recommended dose (90 kg ha -1 of N, 270 kg ha -1 of P and 225 kg ha -1 of K) statistically led to the same gain in productivity as the doses of 75 and 100% NPK. The productivity obtained by this hybrid was considered higher than the average of 87.6 t ha -1 obtained for the Jaíba region, MG, Brazil (Quezado-Duval et al., 2014). Fratoni et al. (2016) also observed no difference in the productivity of fertigated Italian tomatoes with the increase of K doses.
For the hybrid Heinz 9553, the addition of 75% NPK led to a productivity of 191.39 t ha -1 (Table 1), not differing from the dose of 100% NPK. These results indicate that the application of 75% of NPK (N: 135 kg ha -1 , P: 405 kg ha -1 and K: 337 kg ha -1 ) promotes adequate productivity for the hybrid Heinz, cultivated under irrigated system in the northern region of Minas Gerais, Brazil.
The lowest productivity was observed in the hybrid BRS Sena, compared to the others (Table 1), indicating its lower adaptation to the climatic conditions of the semiarid region. Quezado-Duval et al. (2014) reported that there is a reduction in the productivity of BRS Sena when it is cultivated in the hottest periods of the Cerrado in Goiás, Brazil. In turn, the hybrid BHN 0574 obtained higher productivity at the 100% NPK dose (Table 1), corresponding to 182.15 t ha -1 , expressing its maximum genetic potential under this condition of fertilization.
The hybrid Heinz 9553 obtained higher fertilization efficiency index (FEI) at all NPK doses (Table 1), whereas the opposite was observed for BRS Sena. Higher indices suggest that there is efficiency in the amount of tomato produced per unit of nutrient applied. BRS Sena had higher FEI at the dose of 50% NPK, for the nutrients nitrogen, phosphorus and potassium (Table 1), confirming that this hybrid has greater efficiency in the use of NPK fertilizers at the lowest fertilization dose, which was also verified for the hybrid BHN 0574. Badr et al. (2016) found that the lowest dose of N (120 kg ha -1 ) led to higher efficiency in the use of the fertilizer and, on the other hand, at the highest dose of N (300 kg ha -1 ), there was lower agronomic efficiency in low-growing tomato. For Heinz 9553, there was higher FEI at the doses of 50 and 75% NPK, corroborating its maximum productivity, which was obtained at the dose of 75% NPK.
In the evaluation of fruit postharvest characteristics, it was observed that firmness and pulp industrial yield were influenced by the interaction between doses of NPK fertilizer and the hybrids studied (Table 2). However, the soluble solids concentration was not influenced by the studied factors, with an average of 5.65 °Brix (Table 2), a value considered higher than that recommended in the industry of tomato products, of 5 °Brix (Clemente & Boiteux, 2012). The soluble solids concentration is directly linked to pulp yield; for every increase of one ºBrix, there is a 20% increment in the industrial yield of tomato, leading to lower energy expenditure to obtain the pulp (Rocco & Morobito, 2016).
The characteristic pulp pH was influenced only by the cultivated hybrids ( Table 2). The BRS Sena showed higher pulp pH than the others (4.71), while the hybrids Heinz 9553 (4.53) and BHN 0574 (4.49) did not differ from each other ( Table  2). For the industry of tomato products, the desirable pH is less than 4.5, because high pH promotes greater proliferation of microorganisms in the final product, besides increasing the heating time in the process of obtaining the pulp. On the Means followed by the same lowercase letter in the column and uppercase letter in the row do not differ by Tukey test at p ≤ 0.05 Table 2. Soluble solids concentration (°Brix), hydrogen potential (pH), fruit firmness (FIRM) and pulp yield (PY) of industrial tomato hybrids under doses of NPK fertilizer other hand, pH less than 4 results in excessively acidic product (Clemente & Boiteux, 2012). Thus, the pH was within the range desired by the industry (4.49) only for the hybrid BHN 0574.
The NPK doses did not influence fruit firmness in the evaluated hybrids (Table 2), with mean values varying from 24.32 to 25.69 N, which are considered adequate for the resistance to transport (Clemente & Boiteux, 2012). However, among the hybrids studied, at the lowest dose of NPK (50%), BHN 0574 obtained higher fruit firmness (28.34 N), differing from Heinz 9553 (21.37 N), which in turn had firmness similar to that of BRS Sena (25.88 N). Luz et al. (2016), evaluating 53 genotypes of industrial tomato in the field, reinforce that this is one of the main characteristic to select hybrids with resistance to transport. Among the 53, 13 genotypes were selected as firm and concentrated maturity, including the hybrid Heinz 9553, which was used as a comparative among the genotypes.
The pulp yield of the fruits was influenced by the NPK doses only in the BHN 0574. The dose of 100% NPK resulted in a yield of 27.41 t ha -1 , higher than those obtained at doses of 75% (17.35 t ha -1 ) and 50% (20.83 t ha -1 ) of NPK, which did not differ from each other ( Table 2). The greater availability of N, P and K in the treatment with 100% of the fertilization possibly contributed to the higher plant growth, favoring the production and accumulation of photoassimilates and productivity (Santos et al., 2001;Badr et al., 2010;Singh et al., 2010), and these factors contribute to the highest pulp industrial yield.
Among the hybrids evaluated, higher pulp yield (29.11 t ha -1 ) was obtained in the Heinz 9553, at the dose of 75% NPK compared to the others (Table 2). This hybrid also had the highest pulp yields at the doses of 100 and 50% NPK, but at the dose of 100% NPK, the pulp yield between the hybrids Heinz 9553 and BHN 0574 did not differ and were superior to the results obtained with the BRS Sena. The higher pulp yield of Heinz 9553 was attributed to its higher productivity, since the soluble solids concentration did not vary between the studied hybrids. Rocco & Morabito (2016) report that the amount of pulp after processing is the combined result of productivity and soluble solids concentration. This result suggests that the hybrid Heinz has greater efficiency in the use of NPK fertilizers. However, Nowaki et al. (2017) found no difference between the yields of tomato plants working with P in areas of high and low fertility in São Paulo state, Brazil, and these authors point out the need for balanced management of nutrients during tomato cultivation.

Conclusions
1. The hybrid Heinz 9553 has higher fruit weight, number of fruits, productivity and pulp industrial yield at the dose of 75% of the NPK recommendation.
2. The dose of 100% of the NPK recommendation promotes greater fruit weight, number of fruits, productivity, and pulp industrial yield for the hybrid BHN 0574.
3. The fertilizer doses did not interfere with the fruit weight, number of fruits, productivity and pulp industrial yield of the hybrid BRS Sena.
4. Highest fertilization efficiency index was obtained at the 50% of the NPK recommended dose for all hybrids. The Heinz 9553 obtained higher fertilization efficiency index. 5. Soluble solids concentration, pH and firmness are not altered by NPK doses. The pulp industrial yield is higher at the 75% of the NPK recommendation dose for the hybrid Heinz 9553 and, at the 100% NPK dose, there was higher pulp yield for the hybrid BHN 0574.