Molecular breeding of a novel orange-brown tomato fruit with enhanced beta-carotene and chlorophyll accumulation
- Ranjith Kumar Manoharan†1,
- Hee-Jeong Jung†1,
- Indeok Hwang1,
- Namhee Jeong1,
- Kang Hee Kho2,
- Mi-Young Chung3 and
- Ill-Sup Nou1, 4Email author
© The Author(s) 2017
Received: 15 July 2016
Accepted: 16 December 2016
Published: 11 January 2017
Tomatoes provide a significant dietary source of the carotenoids, lycopene and β-carotene. During ripening, carotenoid accumulation determines the fruit colors while chlorophyll degradation. These traits have been, and continue to be, a significant focus for plant breeding efforts. Previous work has found strong evidence for a relationship between CYC-B gene expression and the orange color of fleshy fruit. Other work has identified a point mutation in SGR that impedes chlorophyll degradation and causes brown flesh color to be retained in some tomato varieties.
We crossed two inbred lines, KNY2 (orange) and KNB1 (brown) and evaluated the relationship between these genes for their effect on fruit color. Phenotypes of F2 generation plants were analyzed and a novel ‘orange-brown’ fruit color was identified.
We confirm two SNPs, one in CYC-B and another in SGR gene sequence, associated with segregation of ‘orange-brown’ fruit color in F2 generation. The carotenoid and chlorophyll content of a fleshy fruit was assessed across the different phenotypes and showed a strong correlation with expression pattern of carotenoid biosynthesis genes and SGR function. The orange-brown fruit has high β-carotene and chlorophyll. Our results provide valuable information for breeders to develop tomato fruit of a novel color using molecular markers.
Tomatoes are predominantly grown as an agricultural crop and are considered a healthy food due to their high nutritional value . Tomatoes are cholesterol free, rich in fiber and protein, and low in fat and calories. Approximately 80% of the tomatoes produced are used in tomato-based foods that include tomato juice, puree, paste, sauce and salsa . Daily consumption of tomato sauce has been shown to reduce DNA damage in white blood cells and cancerous prostate tissues . In addition, consumption of lycopene-containing foods can reduce the risk of cardiovascular disease and breast cancer . Therefore, many attempts have been made to develop high-lycopene tomatoes using conventional breeding techniques and genetic manipulation. In addition to lycopene content, new color developments also attract consumers in the fresh market. Tomato fruit color is an important indicator of eating quality for consumers and thus considerable research has been directed towards its characterization and measurement . During the ripening stage, tomato color brightens due to carotenoid, lycopene accumulation, independently or in concert with chlorophyll degradation . Subsequent lycopene accumulation during the final stages of fruit ripening affects color development and the health benefits, both important traits for consumers. In green vegetables and leaves, lycopene is concealed by green chlorophyllic pigments. However, in most fruits, lycopene and other carotenoids are responsible for the bright color development during the ripening stage . Lycopene accounts for more than 80% of the accumulated carotenes in ripe tomato fruits. While β-carotene accumulates to a lesser degree, it also constitutes a sizable portion of total carotene accumulation. Both lycopene and β-carotene are essential to fulfill the nutritional requirements of a healthy animal and human diet .
Another important gene, SGR (STAY-GREEN) associated with color has been identified previously in some plant species [18–20]. SGR mutants showed brown color due to carotenoid accumulation and fail to degrade chlorophyll completely at ripening stage [21, 22]. For instance, SGR mutants that have been identified in other plant species (Arabidopsis, pepper, pea, and meadow fescue) displayed green phenotypes due to inhibition of chlorophyll degradation [19, 23–27]. Barry and Pandey  had reported that point mutation in SGR gene causes loss of protein function and leads to inhibit chlorophyll degradation which exhibited green fleshy fruit color in ‘Black cherry’ variety. Flavonoids also play an important role in determining tomato fruit color. Flavonoids primarily accumulate in the tomato fruit peel, and are absent in the flesh, due to a lack of expression of flavonoid biosynthesis genes in flesh tissues [28, 29].
Because of their importance, our study focused on developing a new tomato fruit color that is enriched for β-carotene and chlorophyll content. Thus, our work evaluated the segregation of fruit color and, β-carotene and chlorophyll level, in the F1 and F2 populations, developed by crossing orange and brown fruit.
Tomato seeds of inbred lines KNY2 (orange) and KNB1 (brown) were obtained from Kana Seed Co. Ltd (Korea). Seeds were sown on moist filter paper in petri dishes and germinated at 30 °C in the dark. The germinated seeds were transferred to plastic trays containing soil mix and maintained at growth room conditions. When plants (F1 and F2) had four true leaves, they were transplanted into plastic pots and grown in greenhouse (25 °C day/18 °C night, 70% air humidity and natural light) at Sunchon National University, Korea. Furthermore, three fruits were harvested from individual plant at ripe (57 days after pollination (DAP)) stage for HPLC analysis. Fruit color was confirmed from 6 F1 plants, 192 F2 plants (obtained from 6 F1 plants) and described in results section.
RNA extraction and PCR amplification
Total RNA was extracted from fleshy fruit tissue at early (E, 17 DAP), mature (M, 39 DAP), turning (T, 45 DAP), and ripe (R, 57 DAP) stages using an RNA extraction kit (Qiagen, USA) according to manufacturer’s instructions. High-quality RNA was eluted in RNase-free water and treated with RNase-free DNase I (Qiagen) before cDNA synthesis. Quantitative RT-PCR (RT-qPCR) was conducted using cDNA synthesized from the RNA of each stage. PCR was performed with the following conditions: initial denaturation at 95 °C for 10 min, followed by 40 cycles of 95 °C for 20 s, 60 °C for 20 s, 72 °C for 40 s and final extension at 72 °C for 2 min.
Single nucleotide polymorphisms (SNPs) were detected using 3’-blocked and unlabeled oligonucleotide probes (HybProbe). PCR was performed using LightCycler® 480 Resolight saturating dye (Roche, Germany) to generate melting curves characteristic of the genotype under the probe. Melting curves were generated and analyzed using the LightCycler®96 Instrument System (Roche, Germany). PCR reactions were performed with a 95 °C pre-denaturation for 5 min, followed by 45 cycles of denaturation at 95 °C for 20 s, annealing at 60 °C for 20 s, and extension at 72 °C for 30 s, with a final extension s at 72 °C for 40 s. Primer and probe sets used for SNP detection are described in Additional file 2 Table S1.
Standards for β-carotene, lycopene, phytoene, and chlorophyll were purchased from Sigma- Aldrich (Sigma Co., USA). The carotenoids and chlorophyll were separated using reverse phase columns (Kinetex 206 μm, C18 100A, 100 × 4.60 mm, Phenomenex, USA). The whole fruit extracts were filtered with a 0.2 μm PTFE filter prior to injection. Mobile phase A was 78% methanol and B was 100% ethyl acetate. Release conditions were 0–5 min, 0% B; 5–15 min, 10% B; 15–20 min, 100% B; 20–30 min, 0% B at a flow rate of 1 mL/min. The phytoene, carotenoids and chlorophyll were identified and quantified based on the retention time and the absorbance between 280 nm, 450 nm and 660 nm of standards. The values represent the mean of three biological replicates.
The EF1α gene was used as reference for normalization. The relative gene expression was calculated based on ΔΔCt method (LightCycler®96, Roche Diagnostics, Mannheim, Germany). Data are presented as the mean of three biological replicates. The data were analyzed using a Tukey Pairwise Comparisons test (P < 0.05) in the Minitab 17 Statistical Software (State College, Pennsylvania, USA). Chi-square analysis to test goodness-of-fit was performed using Graphpad Prism 7.02 (Graphpad software, California, USA).
Results and discussion
Phenotypic and genotypic segregation of F2 generation plants from KNB1 x KNY2
SGR 371 position
CYC-B -77 position
Segregation ratio of fruit color in F2 generation plants from KNB1 x KNY2
Expression of CYC-B and SGR
Barry and Pandey  reported that a SNP (C → T) at nucleotide 371 in the tomato SGR gene results in a mutation that truncates the protein at glutamine 91. Characterized in the variety ‘Black Cherry,’ the truncation introduces a premature stop codon and is predicated to carry null alleles that cause complete loss of protein function. Varieties with this SNP could thus inhibit chlorophyll degradation and retain green flesh. Consistent with these results, the new orange-brown phenotype reported herein retains brown coloration may be due to loss of SGR protein function while concurrent high expression levels of CYC-B result in orange fruit color. This combination could explain the orange-brown phenotype we observed.
HPLC analysis of carotenoid and chlorophyll content
Phytoene, lycopene, β-carotene, and chlorophyll content in F2 generation plants. The carotenoid pigments were quantified using HPLC (μg/g fresh weight) (n = 3, ± s.e.m) during mature (M), turning (T), and ripe (R) stages of ripening
Selected plant in F2 generation
The present study developed a new tomato fruit color with an orange-brown phenotype. This fruit has high β-carotene content and retains chlorophyll through ripening. The expression of CYC-B mRNA coincided with the accumulation of β-carotene. The point mutation in SGR gene causes loss of protein function and leads to inhibit chlorophyll degradation. Present work provides insight into development of genotypes with enhanced β-carotene accumulation and chlorophyll retention in tomato fruits. Combination of these two SNPs would be suitable for breeding ‘orange-brown’ color tomato cultivars.
This research was supported by the Golden Seed Project (Center for Horticultural Seed Development), Ministry of Agriculture, Food and Rural Affairs (MAFRA), Ministry of Oceans and Fisheries (MOF), Rural Development Administration (RDA) and Korea Forest Service (KFS).
Availability of data and materials
Plant materials and cDNA samples are available from the authors.
ISN, KHK and MC conceived and designed the study. HJJ managed the experimental plants, collected samples, prepared cDNA and performed qPCR analysis. NJ and IH prepared samples for HPLC. RKM wrote the manuscript. All authors read and approved the final manuscript.
The authors declare that there is no conflict of interest regarding the publication of this paper.
Consent for publication
Ethics approval and consent to participate
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