Transcriptome profiling reveals the regulatory mechanisms of ascorbic acid and flavonoid synthesis and metabolic processes in fruit development of Ribes nigrum

Transcriptome profiling reveals the regulatory mechanisms of ascorbic acid and flavonoid synthesis and metabolic processes in fruit development of Ribes nigrum | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Research Article Transcriptome profiling reveals the regulatory mechanisms of ascorbic acid and flavonoid synthesis and metabolic processes in fruit development of Ribes nigrum Huixin Gang, Xuelin Zhang, Danni Zhang, Junwei Huo, Dong Qin This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-3330314/v1 This work is licensed under a CC BY 4.0 License Status: Posted Version 1 posted You are reading this latest preprint version Abstract Background The blackcurrant ( Ribes nigrum L.) is rich in nutritional value. It is rich in ascorbic acid and flavonoids with excellent antioxidant properties. Fruit quality is an important factor affecting subsequent processing and production, yet the molecular mechanisms underlying the transcriptional regulation of blackcurrant fruit quality are largely unknown. Results In the current study, comparative transcriptome analysis investigated the similarities and differences between two blackcurrant varieties: 'Adelinia' and 'Heifeng' at four fruit developmental stages (young, expansion, veraison, and ripe fruit). Additionally, we observed variations in a number of physiological indicators during the growth of blackcurrant fruits. The results indicate that, during the development of blackcurrant fruits, ascorbic acid content continued to decrease, whereas flavonoid content was lowest in the veraison period. The fruit size of 'Adelinia' variety was larger than that of 'Heifeng' variety throughout the developmental. Based on the results of the transcriptome sequencing data, a total of 4295 up-regulated genes and 3529 down-regulated genes were obtained between 'Adelinia' and 'Heifeng' varieties; A total of 6,349 up-regulated and 5,770 down-regulated genes were identified between the four developmental periods, respectively. The expression of 4 genes, PMM , APX , GME , and PGI has a strong correlation with AsA content. while two APX , two AO , and one MDHAR genes showed a strong negative correlation with AsA content. A total of 19 genes related to flavonoid biosynthesis were screened. The KEGG pathway enrichment analysis showed that many DEGs were enriched in the plant hormone signal transduction (ko04075) pathway and the photosynthesis-antenna proteins (ko00196) pathway. Conclusions We predicted that the PMM, APX, GME , and PGI genes, which are positively correlated with ascorbic acid, may play an important role in the biosynthesis of AsA; while the negatively correlated APX, AO , and MDHAR genes may be critical for AsA in the recycling pathway. DFR, ANR, CHI, CHS, 4CL , and LAR genes are important regulators of flavonoid synthesis. Also predicted the regulatory influences of various plant hormones on the fruit size of blackcurrants. We provide genetic resources for improving fruit quality and size as well as for rationalizing the use of plant growth regulators to improve fruit quality. Ribes nigrum L. Transcriptional analysis AsA Flavonoids Plant hormone qRT-PCR Expression analysis Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 Figure 6 Figure 7 Figure 8 Figure 9 1. Background Blackcurrant ( Ribes nigrum L.) is a perennial shrub that produces sweet and sour fruit packed with a wide range of nutrients. Biochemical analysis of blackcurrants has revealed the presence of numerous components, including flavonoids, polyunsaturated fatty acids, structural and non-structural carbohydrates, non-volatile organic acids, tannins, and astragals[ 1 ]. Ascorbic acid (AsA), which can enhance antioxidant capacity and health potential[ 2 ], is also found in blackcurrants at higher levels than in raspberries and blueberries[ 3 ]. The high nutritional, medical, health, and cosmetic value of blackcurrant fruits is due to their bioactive components, which are effective in reducing thrombosis, inhibiting tumor growth[ 4 ], lowering cholesterol[ 5 ], inhibiting salmonella enteritidis[ 6 ], and preventing major diseases such as asthma, cardiovascular disease, heart disease, and hypertension[ 7 ]. In addition, blackcurrant is an effective anti-microbial agent[ 8 ] and has the potential to be used as a disinfectant and antiseptic[ 9 ]. The antioxidant properties of blackcurrants are largely due to their phenolic compounds, such as ANC, which can act as hydrogen donors or electron transfer agents depending on their chemical structure. The antioxidant activity of ANC is directly related to its chemical structure, and the differences in the antioxidant activity of flavonoids are due to differences in the type, position, and number of methyl and hydroxyl groups[ 10 ]. Blackcurrant fruits have significant value in nutrition, medicine, health, and beauty. Their bioactive components can effectively reduce blood clot formation, inhibit tumor growth, lower cholesterol levels[ 5 ], suppress Salmonella enteritidis infection[ 6 ], and prevent/treat major diseases such as asthma, cardiovascular disease, heart disease, and hypertension[ 7 ]. Additionally, blackcurrants are effective antimicrobial agents[ 8 ] and hold potential in the production of disinfectants and preservatives[ 9 ]. The rapid development of high-throughput sequencing technology has greatly improved the efficiency of gene discovery[ 11 , 12 ]. The advancement of sequencing technology has provided powerful tools for scientists to study plant transcriptomes. Compared to woody plants, fruit trees have relatively less genomic information and have therefore been less extensively studied. Transcriptomes play a vital role in overcoming the bottleneck that hinders the development of fruit tree genomes. Currently, RNA-Seq has been widely applied to most fruit trees, such as sweet orange[ 13 , 14 ], apple[ 15 , 16 ], blackberry[ 17 , 18 ], kiwi[ 19 ], blueberry[ 20 ], lychee[ 21 ], Chinese cherry[ 22 ], dragon fruit[ 23 ], pear[ 24 ], strawberry[ 25 ], raspberry[ 26 ], plum[ 24 ], and others. It has been used to investigate the synthesis of carotenoids in yellow-fleshed kiwi[ 27 ], gene expression changes during the maturation and development of blueberry[ 20 ], plum[ 24 ], and kiwi fruits[ 19 ], genetic differences among different varieties of dragon fruit[ 23 ] and strawberry fruits[ 25 ], changes in the cuticle layer during different stages of navel orange fruit[ 13 ], and the production of differentially expressed genes (DEGs) under Huanglongbing bacteria and decline viruses in citrus[ 14 ]. However, the fruit tree genome still faces several challenges, including imperfect bioinformatics studies and the difficulty of identifying key genes responsible for physiological changes. The quality of blackcurrant fruit not only determines its appearance, storage potential, and transportability but also affects subsequent product development and quality. The Northeast region of China has abundant blackcurrant germplasm resources and multiple varieties but lacks comprehensive blackcurrant varieties with superior economic traits. To explore the genetic basis of blackcurrant fruit quality, this study utilized RNA-Seq combined with de novo transcriptome assembly to analyze the differences in fruit quality during ripening between two varieties, 'Adelinia' and 'Heifeng'. The key genes involved in AsA biosynthesis and recycling, flavonoid biosynthesis, and plant hormone signal transduction were identified by integrating physiological experimental results. This study aims to investigate the major candidate genes influencing fruit quality, laying the foundation for future research on gene function and high-quality blackcurrant fruit cultivation. 2. Results 2.1. Changes in fruit weight, transverse and longitudinal diameter during the development of ‘Adelinia’ and ‘Heifeng’ During fruit growth and development, the variation in fruit weight and longitudinal and cross-sectional diameter between ‘Adelinia’ and ‘Heifeng’ was investigated (Fig. 1 ). The single fruit weight of ‘Adelinia’ was consistently greater than that of ‘Heifeng’, and the weight of the ripe fruit of ‘Adelinia’ was three times of ‘Heifeng’. The most significant changes in fruit weight and transverse and longitudinal diameters occurred during the period from expansion to veraison, demonstrating an S-shaped trend of "slow-fast-slow". During fruit development, the longitudinal diameter developed faster, resulting in an oval fruit shape during the young fruit stage. After veraison, the transverse diameter gradually became larger than the longitudinal diameter, resulting in a spherical fruit shape. 2.2. Changes in fruit quality during the development of ‘Adelinia’ and ‘Heifeng’ The contents of soluble solids and titratable acids in blackcurrant fruits increased with fruit development (Table 1 ). Soluble solids increased the slowest from the young fruit stage to the expansion stage and the fastest from the veraison stage to the ripe stage. The contents of soluble solids increased by 4.6% and 3.0% from the veraison stage to the ripe stage in ‘Heifeng’ and ‘Adelinia’ respectively. The contents of soluble solids in ‘Heifeng’ were consistently higher than that in ‘Adelinia’ throughout the fruit development, reaching 1.4 times higher than that of ‘Adelinia’at the ripe fruit stage. Table 1 Changes in quality parameters during fruit development of different cultivars of blackcurrant Varieties Developmental stage The soluble solids (%) Titratable acid (%) Ascorbic acid (mg/g) Total phenol (mg/100g) Flavonoid (mg/100g) Heifeng young fruit 7.6 ± 0.52cd 1.34 ± 0. 02d 16.32 ± 0.05a 17.52 ± 0.04b 11.29 ± 0.93b expansion fruit 8.2 ± 0.64c 1.97 ± 0.11b 15.56 ± 0.32b 13.68 ± 0.07d 3.24 ± 0.69c veraison fruit 11.3 ± 0.97b 2.36 ± 0.21ab 11.33 ± 0.10c 0.13 ± 0.05h 0.26 ± 0.07e ripe fruit 15.9 ± 0.72a 2.55 ± 0.45ab 7.62 ± 0.08f 0.55 ± 0.03f 2.18 ± 0.21d Adelinia young fruit 5.3 ± 0.44d 1.63 ± 0.05c 16.61 ± 0.91a 18.36 ± 0.06a 12.23 ± 0.27a expansion fruit 6.4 ± 0.37d 2.45 ± 0.12ab 15.87 ± 0.99b 15.39 ± 0.07c 3.58 ± 0.34c veraison fruit 8.5 ± 0.62c 3.07 ± 1.02a 10.18 ± 0.07d 0.16 ± 0.01g 0.32 ± 0.06e ripe fruit 11.5 ± 0.43b 3.36 ± 0.58a 8.85 ± 0.03c 1.05 ± 0.01e 2.24 ± 0.28d Note: Data are expressed as mean ± standard deviation. Different letters within the same column indicate significant differences (p < 0.05) based on Duncan's multiple range test. The contents of titratable acid in ‘Adelinia’ and ‘Heifeng’ increased most rapidly from the young fruit stage to the expansion stage, with the content increasing by 0.63% and 0.82%, respectively. The rate of increase in titratable acid gradually slowed down from the expansion stage to the ripe stage. The content of titratable acid in ‘Adelinia’ was consistently higher than that of ‘Heifeng’ throughout fruit development, reaching 1.3 times higher than that of ‘Heifeng’. The AsA content in the fruit showed a continuous decreasing trend during growth and development, with the highest content in the young fruit stage and the lowest in the ripe fruit. The AsA content decreased most from the expansion stage to the veraison stage, with ‘Heifeng’ decreasing by 4.23 mg/g and ‘Adelinia’ decreasing by 5.69 mg/g. The AsA content in the young, expansion, and ripe fruit of ‘Adelinia’ was higher than that of 'Heifeng. The changing trend of total phenols and total flavonoids was similar during fruit growth and development, with the maximum value observed in the young fruit stage and the minimum value in veraison fruit, which slightly increased in ripe fruit. The contents of total phenols and flavonoids from the expansion stage to the veraison stage decreased the most in 'Heifeng' and ‘Adelinia’. The content of total phenols decreased by 13.6 mg/100g and 15.23 mg/100g in 'Heifeng' and ‘Adelinia’, respectively. The content of total flavonoids decreased by 5.11 mg/100g and 7.95 mg/100g in 'Heifeng' and ‘Adelinia’, respectively. The contents of total phenols and flavonoids in ‘Adelinia’ fruit were consistently higher than those in 'Heifeng' fruit throughout the fruit development. The highest total phenolic content was 1.9 times that of 'Heifeng', and the highest total flavonoid content was 1.5 times that of 'Heifeng'. 2.3. Transcriptome sequencing quality of blackcurrant fruit and denovo-assemble of unigenes In order to explore the difference in gene expression in the two varieties during fruit ripening, transcriptome sequencing was performed on young, expansion, veraison, and ripe fruit stages of ‘Adelinia’ and ‘Heifeng’. After removing low-quality reads, a total of 186.71 Gb reads were obtained from 24 samples, with an average of 6.74 Gb per sample (Table S2). The Q30 base percentage of all samples was greater than 94.08%, and GC content ranged from 44%-47%, indicating good sequencing quality and credible results. Then the clean reads of all the samples were used for de novo assembly, and a total of 220,757 transcripts and 116,793 unigene were obtained. The average length was 1,129 bp and 729 bp respectively. The N50 was 1,998 and 1,342 respectively (Table S3), indicating that the assembly results were credible. Unigene length ranged from 150 to 16,047 bp, with most unigenes being 300 to 2,000 bp in length. 2.4. Gene function annotation, GO, and KOG functional classification All assembled unigenes were annotated with databases. A total of 23,788, 41,786, 22,044, 35,830, 41,872, 29,652, 64,522, and 66,902 unigenes were matched with COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG and NR databases, respectively. A total of 70,224 unigene were identified. There were 8,257, 13, 7,701, 12,554, 16,740, 13,041, 19,607, and 19,977 unigene genes with length ≥ 1,000 bp (Table S4). GO annotation was performed on 41,786 unigenes, which are included in 54 branches. Twenty branches of biological processes involve 110,234 annotated information, accounting for 45.82% of all annotated information. The branches with the most unigenes are the "metabolic process" with 30,074 annotated information and the "cellular process" with 24,290 annotated information. In the cellular component category, 17 branches involve 78,115 annotated information, accounting for 32.47% of all annotated information. The branches with the most annotated information are "cell" and "cell part," both with 17,483 annotated information. In the molecular function category, 17 branches involve 52,216 unigenes, accounting for 21.71% of all annotated information. The branches with the most molecular function are "catalytic activity" with 23,645 unigenes and "binding" with 20,421 unigenes (Fig. 2 ). The KOG database has a wide range of functions related to life activities, with a total of 35,830 genes annotated and divided into 26 categories represented by letters A to Z. The "General function prediction only" accounts for 20.56% of all gene sequences, with 7,368 genes in this category. The next most abundant category is "Posttranslational modification, protein turnover, chaperones", which accounts for 10.48% with 3,755 genes. Additionally, there are 3,202 genes involved in "Translation, ribosome structure, and biogenesis", accounting for 8.94% of the total sequence. Furthermore, there are 2,762 genes involved in "Signal transduction mechanisms", 2,488 genes involved in "Amino acid transport and metabolism", and 2,102 genes involved in "Carbohydrate transport and metabolism". The category with the fewest number of genes is "cell motility", with only 21 genes, followed by "extracellular structures" and "Nuclear structure" with 123 and 181 genes, respectively. The number of genes in each category is shown in Fig. 3 . 2.5. DEGs analysis of ‘Adelinia’ and ‘Heifeng’ fruits at the same stage The expression levels of unigenes in the fruits of ‘Adelinia’ and ‘Heifeng’ were compared at four developmental stages, namely, young fruit, expansion fruit, veraison fruit, and ripe fruit stages. A total of 483 DEGs were detected in the young fruit stage, including 241 up-regulated genes and 242 down-regulated genes. In the expansion stage, 1,909 DEGs were detected, including 1,020 up-regulated genes and 889 down-regulated genes. In the veraison fruit stage, 1,548 genes were differentially expressed, including 752 up-regulated genes and 796 down-regulated genes. In the ripe fruit stage, 2,822 genes were differentially expressed, including 1,602 up-regulated genes and 1,220 down-regulated genes (Fig. 4 , A). The KEGG enrichment analysis of the DEGs in the fruits of ‘Adelinia’ and ‘Heifeng’ at the same stage is shown in Fig. 4 , B. Specifically, DEGs in the young fruits of ‘Adelinia’ and ‘Heifeng’ were significantly enriched in monoterpenoid biosynthesis (ko00902). DEGs in the expansion fruits of ‘Adelinia’ and ‘Heifeng’ were significantly enriched in plant hormone signal transduction (ko04075), DNA replication (ko03030), plant-pathogen interaction (ko04626), and mismatch repair (ko03430) pathway. DEGs in the veraison fruits of ‘Adelinia’ and ‘Heifeng’ were significantly enriched in photosynthesis-antenna proteins (ko00196), flavonoid biosynthesis (ko00941), and plant-pathogen interaction (ko04626) pathway. DEGs in the ripe fruits of ‘Adelinia’ and ‘Heifeng’ were significantly enriched in glyoxylate and dicarboxylate metabolism (ko00630), carbon metabolism (ko01200), citrate cycle (TCA cycle, ko00020), flavonoid biosynthesis (ko00941), photosynthesis-antenna proteins (ko00196), alanine, aspartate and glutamate metabolism alanine (ko00250), cutin, suberine and wax biosynthesis (ko00073), pyruvate metabolism (ko00620) pathway. 2.6. DEGs analysis of ‘Adelinia’ and ‘Heifeng’ fruits during ripening To investigate the DEGs during fruit ripening, we compared young fruit with expansion fruit, expansion fruit with veraison fruit, and veraison fruit with ripe fruit in both ‘Adelinia’ and ‘Heifeng’. The number of DEGs was 1499 (810 up-regulated DEGs and 689 down-regulated DEGs) and 2311 (1163 up-regulated DEGs and 1148 down-regulated DEGs) in the comparison group of young fruit with expansion fruit in ‘Adelinia’ and ‘Heifeng’, respectively. The number of DEGs was 2250 (850 up-regulated DEGs and 1400 down-regulated DEGs) and 3746 (1767 up-regulated DEGs and 1979 down-regulated DEGs) in the comparison group of expansion fruit with veraison fruit in ‘Adelinia’ and ‘Heifeng’, respectively. The number of DEGs was 1356 (1258 up-regulated DEGs and 98 down-regulated DEGs) and 960 (501 up-regulated DEGs and 456 down-regulated DEGs) in the comparison group of veraison fruit with ripe fruit in ‘Adelinia’ and ‘Heifeng’, respectively (Table 2 ). Table 2 Up-regulated and down-regulated DEGs in the young fruit, expansion fruit, veraison fruit, and ripe fruit stage Compared group Total DEGs Up-regulated Down-regulated Y1 vs. Y2 1499 810 689 Y2 vs. Y3 2250 850 1400 Y3 vs. Y4 1356 1258 98 H1 vs. H2 2311 1163 1148 H2 vs. H3 3746 1767 1979 H3 vs. H4 960 501 456 All DEGs in the comparison groups were mapped to KEGG pathway enrichment to analyze the enriched pathways. The DEGs in the comparison groups of Y1 vs Y2 and H1 vs H2 were both significantly enriched in plant hormone signal transduction (ko04075), Phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), Stilbenoid, diarylheptanoid and gingerol biosynthesis (ko00945), Starch and sucrose metabolism (ko00500), Phenylalanine metabolism (ko00360), Cutin, suberine, and wax biosynthesis (ko00073) pathway. The DEGs in the comparison groups of Y2 vs Y3 and H2 vs H3 were both significantly enriched in plant hormone signal transduction (ko04075), starch and sucrose metabolism (ko00500), phenylpropanoid biosynthesis (ko00360), homologous recombination (ko03440), phenylalanine metabolism (ko00360), flavonoid biosynthesis (ko00941), DNA replication (ko03030), Cutin, suberine, and wax biosynthesis (ko00073) pathway. While the DEGs in the comparison groups of Y3 vs Y4 and H3 vs H4 did not share the same significantly enriched pathway (Fig. 5 ). 2.7. Identification of genes involved in AsA biosynthesis and recycling pathway during fruit ripening According to the gene expression level of transcriptome sequencing, crucial genes involved in AsA biosynthesis and recycling pathway were identified. Including 3 genes of glucose-6-phosphate isomerase ( PGI ), 1 gene of mannose-6-phosphate isomerase ( PMM ), 1 gene of GDP-mannose-3’,5’-epimerase ( GME ), 2 genes of GDP-L-galactose phosphorylase ( GGP ), 1 gene of Myo-inositol oxygenase ( MIOX ) and 3 genes of L-galactono-1,4-lactone dehydrogenase ( GalLDH ) associated with AsA biosynthesis. And 6 genes of ascorbate peroxidase ( APX ), 6 genes of ascorbate oxidase ( AO ), 3 genes of monodehydroascorbate reductase ( MDHAR ) and 1 gene of glutathione reductase ( GR ) associated with AsA recycling. As shown in Fig. 6 , most of these genes were highly expressed in the young fruits of both ‘Adelinia’ and ‘Heifeng’. Such as the expression of the PGI gene (c179793.graph_c1 and c181113.graph_c0) and MDHAR (c160843.graph_c0) were highest in the young fruit and lowest in the veraison fruit in both ‘Adelinia’ and ‘Heifeng’. The expression of the PMM gene (c184228.graph_c0), GME gene (c181107.graph_c0), GalLDH gene (c178565.graph_c2), and AO gene (c185688.graph_c1) was the highest in the young fruits and lowest in the ripe fruit. In addition, we calculate the correlation coefficient (r 2 ) of all the genes identified in AsA biosynthesis and recycling pathway with AsA content in the four fruit developmental stages of ‘Adelinia’ and ‘Heifeng’. The expression of a PMM gene (c184228.graph_c0) has a strong correlation (r 2 = 0.84) with AsA content. Similarly, the expression of an APX (c176242.graph_c0), a GME (c181107.graph_c0), and a PGI (c181113.graph_c0) gene were strongly correlated with AsA content, with r 2 of 0.74, 0.71 and 0.71, respectively. These results revealed that the above genes may be crucial for AsA biosynthesis in blackcurrant. The expression of 2 APX (c176953.graph_c0, c172886.graph_c0) gene showed a strong negative correlation with AsA content, with − 0.93 and − 0.77, respectively. The expression of 2 AO (c184540.graph_c1, c185556.graph_c0) genes has a strong negative correlation with AsA content, with r 2 of -0.83 and − 0.74, respectively. An MDHAR (c172058.graph_c0) gene also showed a strong negative correlation (r 2 = -0.72) with AsA content. These genes may be crucial for the AsA recycling pathway in blackcurrant. 2.8. Identification of genes involved in flavonoid biosynthesis pathway during fruit ripening As the flavonoid contents were variational during fruit ripening, we then identified the key genes involved in the flavonoid biosynthesis pathway. A total of 6 genes of 4-coumarate-CoA ( 4CL ), 3 genes of chalcone synthase ( CHS ), 2 genes of chalcone isomerase ( CHI ), 1 gene of Flavanone-3-hydroxylase ( F3H ), 2 genes of dihydro flavonol reductase ( DFR ), and 1 gene of leucoanthocyanidin dioxygenase ( LDOX ), anthocyanin synthase ( ANS ), anthocyanidin reductase ( ANR ), flavonoid 3-O-glucosyl transferase ( UFGT ) and leucoanthocyanidin reductase ( LAR ) were identified that associated with flavonoid biosynthesis. Most of these genes were expressed at their highest levels in the young fruits, by the flavonoid contents during fruit ripening. The expressions of the 4CL gene (c183421.graph_c1), CHS genes (c115942.graph_c0, c183294.graph_c1), CHI genes (c157264.graph_c0, c163054.graph_c0), DFR gene (c183820.graph_c4), ANS gene (c187123.graph_c2), ANR gene (c183720.graph_c1) and LAR (c162573.graph_c1) were highest in the young fruit of both ‘Adelinia’ and ‘Heifeng’, and were significantly down-regulated in the expansion fruits. Similarly, the expression of a DFR (c183820.graph_c4), an ANR gene showed a strong correlation (both r 2 = 0.88) with flavonoid content. The expression of 2 CHI (c157264.graph_c0, c163054.graph_c0) with r 2 of 0.87and 0.83, and 2 CHS (c115942.graph_c0, c183294.graph_c1) genes with r 2 of 0.82 and 0.80 showed strong correlation with flavonoid content. A 4CL and a LAR gene were also strongly correlated with flavonoid content, with r 2 of 0.73 and 0.71, respectively. These results indicated that the above genes may be crucial for flavonoid biosynthesis in blackcurrant (Fig. 7 ). 2.9. Identification of DEGs involved in plant hormone signal transduction pathway To explore changes in gene expression related to plant hormone signal transduction, we analyzed the DEGs involved in auxin, cytokinine, gibberellin, ethylene, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid signal transduction pathways. A total of 31 DEGs were detected involved in the auxin signal transduction pathway, and 18 genes were expressed highest in the young fruit stage of both ‘Adelinia’ and ‘Heifeng’ (Fig. 8 ). These genes included 2 ARG7 genes(c171898.graph_c0, c167577.graph_c0), 4 AUX10A genes (c177750.graph_c0, c179267.graph_c0, c173102.graph_c0, c178278.graph_c0), AUX28 (c182867.graph_c3), AUX6B (c163920.graph_c0), 2 GH3.1 genes (c179273.graph_c0, c187153.graph_c1), GH3.5 (c175332.graph_c0), GH3.9 (c175298.graph_c0), IAA7 (c159328.graph_c0), IAA9 (c177889.graph_c1), IAA14 (c184998.graph_c1), 2 IAA26 genes (, c176546.graph_c1, c176147.graph_c3), IAA27 gene (c179588.graph_c0). This indicates that the auxin signal transduction pathway plays an important role in the young fruit stage. We also found that the expression of ARG7 (c174082.graph_c0), IAA7 (c159328.graph_c0), and IAA11 (c164719.graph_c0) was significantly higher in ‘Adelinia’ than ‘Heifeng’ in the expansion fruit stage. The expression of AGR7 (c167451.graph_c0) was significantly higher in ‘Adelinia’ than ‘Heifeng’ in the expansion and veraison fruit stages. As the fruit grows rapidly in the expansion stage, these genes may be responsible for the difference in volume and weight between ‘Adelinia’ and ‘Heifeng’ fruits. A total of 10 DEGs were detected as involved in the cytokinine signal transduction pathway. Three genes including ARR3 (c178666.graph_c0), ARR9 (c173285.graph_c1), and AHP gene (c181299.graph_c0) were expressed at their highest level in the young fruit stage of both ‘Adelinia’ and ‘Heifeng’. Two genes including ARR1 (c179842.graph_c0) and ARR12 (c183630.graph_c1) were expressed highest in the expansion fruit stage of both ‘Adelinia’ and ‘Heifeng’. The HK2 (c185911.graph_c0) gene was expressed at its highest level in the ripe fruit stage of both ‘Adelinia’ and ‘Heifeng’. A total of 4 DEGs were detected involved in the gibberellin signal transduction pathway. The expression of the PIF3 (c182315.graph_c1) and GAI (c151333.graph_c0) gene was higher in the young and expansion fruit than veraison and ripe fruit of both ‘Adelinia’ and ‘Heifeng’. While the expression of the GID1B (c184501.graph_c3) gene was expressed higher in the veraison and ripe fruit than in young and expansion fruit of both ‘Adelinia’ and ‘Heifeng’. A total of 5 DEGs were detected involved in the jasmonic acid signal transduction pathway. Four genes including TIFY3B (c179099.graph_c0), TIFY11B (c183995.graph_c4), MYC2 (c182574.graph_c0), and bHLH14 (c167287.graph_c0) were expressed highest in the young fruit of both ‘Adelinia’ and ‘Heifeng’. A total of 4 genes were detected involved in the ethylene signal transduction pathway. Two genes of EBF1 (c168698.graph_c0, c168698.graph_c1) were expressed higher in the young and ripe fruit stage than expansion and veraison fruit stage of both ‘Adelinia’ and ‘Heifeng’. In addition, the expression of EBF1 (c168698.graph_c0, c168698.graph_c1) and EIN3 (c180971.graph_c4) genes was higher in the expansion fruit of ‘Heifeng’ than ‘Adelinia’. A total of 11 DEGs were detected involved in abscisic acid signal transduction. The PYL8 (c169965.graph_c0) gene was expressed highest in the young fruit stage. One of the ABI5 (c186178.graph_c0) genes was expressed highest in the expansion fruit stage, while another ABI5 (c172267.graph_c0) gene was expressed highest in the veraison fruit stage. The PP2C (c177710.graph_c0) gene was expressed highest in the ripe fruit stage. In addition, the expression of PP2C (c176253.graph_c0) was higher in the expansion fruit of ‘Adelinia’ than ‘Heifeng’. The expression of another PP2C (c177479.graph_c0) and PYR1 (c141701.graph_c0) gene was higher in the ripe fruit of ‘Heifeng’ than in ‘Adelinia’. A total of 8 DEGs were detected involved in the brassinosteroid signal transduction pathway. Three genes including SIK (c169178.graph_c0, c186784.graph_c1) and THX (c156073.graph_c0) genes were expressed higher in the veraison and ripe fruit stages than in young and expansion fruit stages of both ‘Adelinia’ and ‘Heifeng’. In addition, CYCD3 (c173925.graph_c0, c178067.graph_c0) gene was expressed highest in the young fruit stage of both ‘Adelinia’ and ‘Heifeng’. Three DEGs were detected as involved in the salicylic acid signal transduction pathway. PR1 (c181784.graph_c1) gene was expressed higher in the ripe fruit, while TGA3 (c174510.graph_c1) gene was expressed higher in the expansion fruit stage of both ‘Adelinia’ and ‘Heifeng. The result indicated that these DEGs involved in the plant hormone signal transduction pathway may play an important role in the fruit growth and development of blackcurrant. 2.10. Validation of DEGs by qRT-PCR To confirm the accuracy and reliability of the transcriptome analysis results, three ascorbic acid biosynthesis and recycling-related genes, five flavonoid biosynthesis genes, and one hormone signal transduction-related gene were selected. The gene expression levels at different developmental stages were further examined by qRT-PCR, as shown in Fig. 9 . For all nine genes, the qRT-PCR analysis results had the same expression trend as the FPKM data from RNA-seq. 3. Discussion 3.1. AsA biosynthesis and recycling in ‘Adelinia’ and ‘Heifeng’ during fruit ripening Several factors can influence the content of ascorbic acid (AsA) in plants, including variety, climate, growing conditions, cultivation practices, maturity at harvest, and storage conditions[ 28 ]. For example, blackcurrant grown in high-latitude regions of Finland tend to have higher AsA content compared to those grown in lower latitudes[ 28 ]. The AsA content in blackcurrant fruits also varies under different light intensities, with shading leading to a decrease in AsA content[ 29 ]. Additionally, the AsA content changes during the ripening process of blackcurrant fruits. Under natural growth conditions, the accumulation of AsA in blackcurrant begins in the early stages of berry growth and reaches a stable period at fruit maturity[ 29 ]. This study also found that the AsA levels were highest during the early fruiting stage in the two varieties, 'Adelinia' and 'Heifeng', and gradually decreased as the fruits matured, with the lowest content observed at the ripe stage. In addition, the synthesis of AsA in blackcurrant occurs through the L-galactose pathway[ 30 ], which is consistent with apple ( Malus domestica ), kiwifruit ( Actinidia ), tomato ( Solanum lycopersicum ), and citrus fruits[ 31 ]. In the AsA recycling pathway of blackcurrant, ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and L-galactono-1,4-lactone dehydrogenase (GalLDH) collectively regulate the total AsA content in blackcurrant fruits[ 29 ]. In this study, a total of 26 genes were identified from the AsA biosynthesis and recycling pathway based on transcriptome sequencing results. Among them, the expression of a PMM (c184228.graph_c0), an APX (c176242.graph_c0), a GME (c181107.graph_c0), and a PGI (c181113.graph_c0) gene have a strong correlation with AsA content. These genes may be crucial for the AsA biosynthesis pathway in blackcurrant. Similarly, the suppressed expression of the PMM gene caused a substantial decrease in AsA content in tobacco[ 32 ]. In tomato, the overexpression of the SlGMEs gene enhanced AsA accumulation[ 33 ]. However, the expression of 2 APX (c176953.graph_c0, c172886.graph_c0) gene, 2 AO (c184540.graph_c1, c185556.graph_c0) gene, and an MDHAR (c172058.graph_c0) gene showed strong negative correlation with AsA content. These genes may be crucial for the AsA recycling pathway in blackcurrant. Consist with our result, Shu et al. identified an AcAO1 and an AcAPX2 gene from comprehensive metabolic analysis of kiwifruit during ripening, and the overexpression of the two genes led to reduced L-AsA and total AsA content in tobacco[ 34 ]. The study on AsA indicated that the overexpression of chloroplastic monodehydroascorbate reductase ( MDHAR ) led to an increase in AsA content, a decrease in DHA content, and an approximately twofold increase in the AsA/DHA ratio in tomato[ 35 ]. The DEGs identified in this work will be a valuable gene resource for exploring the molecular mechanism of AsA biosynthesis and breeding of blackcurrant with high AsA levels. 3.2. Flavonoids biosynthesis in ‘Adelinia’ and ‘Heifeng’ during fruit ripening Flavonoid compounds, as a type of phenolic substance, possess powerful antioxidant and free radical scavenging activities. There is a wide variety of flavonoids, including anthocyanins, flavanols, flavones, flavanones, and flavonols[ 36 ]. Flavonoids play important roles in plant defense against microbial stress[ 37 ] as well as in promoting human health[ 38 ]. Among all the components of flavonoids, myricetin has the highest content in blackcurrants, followed by quercetin[ 39 ]. The content of flavonoids shows significant variations at different stages of fruit development. The flavonoid content in Rubus fruits[ 40 ] and navel oranges [ 41 ] has been observed to decline with fruit maturation. In this study, the peak flavonoid content was observed in young fruit, while the minimum was observed in fruits during the coloration stage. Similarly, most of the genes involved in flavonoid biosynthesis showed their maximum expression levels in young fruit, consistent with the changes in flavonoid contents during fruit ripening. The DEGs in the comparison groups Y1 vs Y2, H1 vs H2, Y2 vs Y3, and H2 vs H3 were significantly enriched in both phenylpropanoid biosynthesis and flavonoid biosynthesis pathway. These findings may account for the pronounced variations in flavonoid synthesis and accumulation at different stages of fruit development. The molecular mechanisms regulating flavonoid synthesis have been elucidated in many plants. In recent years, significant progress has also been made in research on flavonoid synthesis in fruit trees, including grapes, blueberries, apricots[ 42 ], blackberries[ 42 ], and apples. Flavonoid synthesis begins with phenylalanine. The health benefits of blackcurrants are partly attributed to their high flavonoid content, so it is important to elucidate the molecular mechanism of flavonoid accumulation in blackcurrants. In melon, flavonoid accumulation is regulated by the up-regulation of 4CL , F3H , F3'H , IFS , FNS , and FLS genes and the down-regulation of ANS , and UFGT genes[ 43 ]. A study on apricot fruits has demonstrated the 4CL gene transcript level has a strong positive correlation with flavonoid contents[ 42 ]. CHS was identified as the backbone gene for flavonoid biosynthesis in sweet orange[ 44 ]. Here, we identified 19 candidate genes associated with flavonoid biosynthesis. Among them, the expressions of 4CL gene (c183421.graph_c1), CHS genes (c115942.graph_c0, c183294.graph_c1), CHI genes (c157264.graph_c0, c163054.graph_c0), DFR gene (c183820.graph_c4), ANS gene (c187123.graph_c2), ANR gene (c183720.graph_c1) and LAR gene (c162573.graph_c1) followed the same trend as that of flavonoid content in fruits. These genes may be crucial for flavonoid biosynthesis in blackcurrants. Consistent with our results, Zhou et al. reported that the expression of CHS , DFR , and ANS genes peaked at the early stages, decreased when fruits turned white from green, and then increased during anthocyanin biosynthesis in strawberry[ 45 ]. Our results provide valuable information on the dynamic changes in flavonoid content and the molecular mechanism of flavonoid accumulation during the maturation of blackcurrants in the future. 3.3. The difference between ‘Adelinia’ and ‘Heifeng’ in fruit weight Fruit size is one of the most important indicators of fruit appearance and quality, and it is one of the breeding objectives that breeders cannot ignore. Fruit size is affected by a variety of factors such as varietal characteristics, genetic factors, plant hormones, cultivation, and management levels, and is a quantitative trait with multi-gene control. The fruit weight of different varieties of blackcurrant varies greatly[ 46 ], and our data also show that ‘Adelinia’ fruit weight at maturity can be up to three times that of ‘Heifeng’. The influence of genetic factors on fruit size is mainly determined by cell division and expansion, and are the most fundamental factors in determining fruit size. Plant hormones play an important role in fruit development and fruit size. For example, BRASSINOSTEROID INSENSITIVE1 ( BRI1 ) Enlarges Cell Size by Regulating the Cooperation of BR-GA Signaling in Tomato[ 47 ]. Hence, the genes involved in the regulation of cell development and plant hormone signaling are important regulators of fruit size. When studying the influencing factors of fruit size in apricot, Huang et al. identified a series of DEGs involved in plant hormone signal transduction, biosynthesis, and metabolism based on the KEGG enrichment results[ 48 ]. Genes associated with fruit size in the early stage of fruit development in Pyrus pyrifolia significant enrichment in the plant hormone signal transduction passway of zeatin and gibberellin[ 49 ]. The main purpose of plant photosynthesis is to produce carbohydrates, an important energy material for plants to carry out various life activities. An adequate supply of carbohydrates plays an important role in plant growth[ 50 ]. Autotetraploid and diploid mulberry trees, with large fruit size differences, DEGs significant enrichment in the plant hormone signal transduction pathways, and many DEGs were involved in photosynthesis[ 51 ]. In our study, the difference in fruit size and weight between the two varieties increased significantly after the expansion stage, at the same time, KEGG pathway enrichment analysis revealed that DEGs were significantly enriched in the plant hormone signal transduction (ko04075) pathway in the expansion fruit stage of ‘Adelinia’ and ‘Heifeng’, in photosynthesis-antenna proteins (ko00196) pathway in the veraison fruit stage and ripe fruit stage. Combined with the above results, we believe that the fruit size of blackcurrant is also affected by the regulation of hormone signals and photosynthesis. To clarify the specific regulation mechanism of different kinds of hormones on fruit size, we analyzed the DEGs involved in auxin, cytokinine, gibberellin, ethylene, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid signal transduction pathway. Among them, a total of 9 genes had different gene expression levels between the ‘Adelinia’ and ‘Heifeng’ varieties after the expansion stage. Such as ARG7 (c174082.graph_c0), IAA7 (c159328.graph_c0), and IAA11 (c164719.graph_c0) in the expansion fruit stage. AGR7 (c167451.graph_c0) in the expansion and veraison fruit stage. EBF1 (c168698.graph_c0, c168698.graph_c1) and EIN3 (c180971.graph_c4) gene in the expansion fruit. PP2C (c176253.graph_c0) in the expansion fruit. PP2C (c177479.graph_c0) and PYR1 (c141701.graph_c0) genes in the ripe fruit. SlARF9 affects cell division in early tomato fruit development, SlARF9-OE fruit cells were on average bigger than in wild-type fruits, inversely the SlARF9 -RNAi lines, cells were smaller[ 52 ]. Silencing of Sl-IAA17 resulted in the production of larger fruit than the wild type[ 53 ]. MdKNOX19 binds directly to the MdABI5 promoter to activate expression-enhanced ABA sensitivity-affected organ development and fruit size[ 54 ]. genome-edited mutants of tomato ETHYLENE INSENSITIVE 2( SlEIN2 ) decreased auxin biosynthesis to reduce fruit size[ 55 ]. In brief, the above genes may be important influencing factors in cell enlargement and fruit enlargement, and they regulate plant growth and development by regulating the synthesis and metabolism of various hormones. The results provide new insights into the molecular mechanisms underlying fruit development and may be useful for improving fruit yield and quality. 4. Conclusions In this study, we generated a high-quality, functionally annotated reference transcriptome for blackcurrant. Based on the transcriptome, a comparison of different developmental stages and different varieties was performed and DEGs were detected. Detection of differentially expressed genes led to the identification of regulatory genes associated with the AsA biosynthesis and recycling, flavonoids biosynthesis pathways. A large number of differentially expressed genes were also found to be involved in plant hormone signal transduction. In addition, the accuracy of the RNA-Seq results was further verified by qRT-PCR. Our study provides insights into AsA biosynthesis and recycling, flavonoid biosynthesis, and fruit size regulation in blackcurrant. 5. Materials and methods 5.1. Plant materials and growth condition Two varieties of blackcurrants, 'Adelinia' and 'Heifeng', were used as samples in this study. These plants were grown in the germplasm nursery of Northeast Agricultural University, Harbin, China, at coordinates 44°04’ N, 125°42’ E, under natural conditions. Adelinia is an early-maturing variety, while Heifeng is a late-maturing variety. Four stages of fruit development were collected, including young fruit, expansion fruit, veraison fruit, and ripe fruit. Samples were immediately frozen in liquid nitrogen and stored at -80°C for RNA extraction. 5.2. Determination of Soluble solids, titratable acid, Total phenol, flavonoids, and ascorbic acid The soluble solids were measured using a refractometer, according to the GB/T 12295 − 1990 method. Titratable acid content was measured using acid-base titration, according to the GB/T 12295 − 1990 method. Total phenol was measured using the method reported by John[ 56 ] with some modifications. Flavonoid content was determined using the method reported by Zou[ 57 ]. According to the manufacturer's instructions, AsA content was determined using a Solarbio detection kit. Three fruits from different trees were used for each measurement. 5.3. Library construction, and sequencing Total RNAs were extracted from the collected fruit samples using the CTAB method. The RNA quality was assessed using agarose gel electrophoresis and RNA quantity was determined by an Agilent 2100 Bioanalyzer (Palo Alto, USA). Strand-specific RNA-Seq libraries were constructed and sequenced on the Illumina HiSeq 2000 platform by Biomarker Technologies (Beijing, China). Three biological replicates of each fruit sample were used. 5.4. RNA-Seq data processing, and transcriptome assembly Raw RNA-Seq reads were processed using in-house Perl scripts to trim adapters and low-quality sequences. The transcriptome was assembled based on the left.fq and right.fq using Trinity[ 58 ] with the minimum Kmer coverage set to 2. Gene function was annotated based on NR (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence database), KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) databases. GO functional enrichment analysis of genes in major KEGG enriched pathways was performed using BMKCloud ( www.biocloud.net ). For convenience, the young fruits of ‘Adelinia’ and ‘Heifeng’ were labeled as Y1 and H1. The expansion fruits of ‘Adelinia’ and ‘Heifeng’ were labeled as Y2 and H2, the veraison fruits of ‘Adelinia’ and ‘Heifeng’ were labeled as Y3 and H3, and the ripe fruits of ‘Adelinia’ and ‘Heifeng’ were labeled as Y4 and H4, respectively. 5.5. Identification of differentially expressed genes (DEGs) Differential expression analysis was performed using the DESeq R package to identify DEGs between the two groups of samples collected at different stages of fruit development, including young fruit, expansion fruit, veraison fruit, and ripe fruit, for Adelinia and Heifeng varieties. For identifying differentially expressed genes (DEGs), a fold change (FC) ≥ 2 and false discovery rate (FDR) < 0.01 were used as the screening criteria. FC represents the ratio of gene expression values in two groups. Genes with an adjusted P-value < 0.05 determined by DESeq were assigned as differentially expressed. 5.6. Quantitative Reverse Transcription PCR (qRT-PCR) Gene expression analysis was performed for each sample using RT-qPCR. Total RNA from ‘Adelinia’ and ‘Heifeng’ fruit was extracted using the Biomarker Plant Total RNA Isolation Kit (Polysaccharides & Polyphenolics–rich) according to the manufacturer’s instructions. The RNA concentration and quality were measured using an ultraviolet spectrophotometer (NanoPhotometer NP80, IMPLEN) and 1% agarose gel electrophoresis. cDNA was synthesized from 500 ng of the total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO) according to the manufacturer’s instructions. Specific primers for qRT-PCR are listed in Table S1 , which were designed using Premier 5.0. The qRT-PCR reaction was performed in a 20 µL reaction volume containing 10 µL of SYBR®Green Realtime PCR Master Mix (TOYOBO), 2 µL of cDNA, 0.8µL of each forward and reverse primer, and 6.4 µL ddH 2 O. The analysis was carried out on three biological replicates on a qTOWER3 fluorescence quantitative PCR instrument (Analytik Jena). qRT-PCR was carried out under the following conditions: denaturation at 95 ◦C for 3 min prior to 45 amplification cycles (95°C for 5 s, 60°C for 30 s), followed by melt-curve analysis (60–95°C, 1°Cincrement for 15 s per step) to test the amplicon specificity. Relative expression was assessed by the 2 −∆∆CT method using Actin (c178043.graph_c0) as the internal control gene[ 59 ]. Abbreviations DEGs Differentially expressed genes qRT-PCR Quantitative real-time PCR GO Gene ontology KOG Clusters of orthologous groups KEGG Kyoto encyclopedia of genes and genomes AO Ascorbate oxidas APX Ascorbate peroxidase GalDH L-galactose dehydrogenase GGP GDP-L-galactose-phosphorylase GalLDH L-galactono-1,4-lactone dehydrogenase GME GDP-D-mannose3′, 5′-epimerase GMP GDP-mannose pyrophosphorylase GPP L-galactose-1-phosphate phosphatase GSH Glutathione GSSH Oxidized glutathione MDHAR Monodehydroascorbate reductase MIOX Myoinositol oxygenase PGI Phosphoglucose isomerase PMI Phosphomannose isomerase PMM Phosphomannomutase GR Glutathione reductase 4CL 4-coumarate-CoA CHS Chalcone synthase CHI Chalcone isomerase F3H Flavanone-3-hydroxylase DFR Dihydro flavonol reductase LDOX Leucoanthocyanidin dioxygenase ANS Anthocyanin synthase ANR Anthocyanidin reductase UFGT Flavonoid 3-O-glucosyl transferase LAR Leucoanthocyanidin reductase ARG Argentina AUX Auxin IAA Indole acetic acid. Declarations Acknowledgments We thank all contributors for their work and would like to thank the editor and reviewers for their valuable comments and suggestions. Authors’ contribution DQ and JWH designed and managed the experiments. HXG and XLZ drafted and revised the manuscript. HXG and DNZ conducted the sequence data analysis. XLZ performed the experiments. The authors have carefully read and approved the final manuscript. Funding This research was supported by the following fundings: National Key R&D Program of China (2022YFD1600500), Academic Backbone Project of Northeast Agricultural University (20XG04), China Postdoctoral Science Foundation (2022MD713724) and Central Government Supports the Reform and Development Fund Talent Training Project of Local Colleges and Universities, China. Availability of data and materials All data generated or analyzed during this study are included in this published article and its supplementary information fles. The raw RNA-Seq data in this manuscript are available for downloading from the NCBI Sequence Read Archive (BioProject ID: PRJNA1021373). https://www.ncbi.nlm.nih.gov/sra/PRJNA1021373 Ethics approval and consent to participate The fruits of blackcurrant in this study was collected from the germplasm nursery of Northeast Agricultural University, Harbin, China. No specific permits are required for sample collection. The use of plant parts in present study compiles with international, national and/or institutional guidelines. Consent for publication Not applicable. Competing interests The authors declare that they have no competing interests. References Bordonaba JG, Terry LA. Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars. J Agric Food Chem. 2008;56(16):7422–30. Woznicki TL, Sonsteby A, Aaby K, Martinsen BK, Heide OM, Wold AB, Remberg SF. 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Int J Mol Sci. 2015;16(9):22938–56. de Jong M, Wolters-Arts M, Schimmel BCJ, Stultiens CLM, de Groot PFM, Powers SJ, Tikunov YM, Bovy AG, Mariani C, Vriezen WH, et al. Solanum lycopersicum AUXIN RESPONSE FACTOR 9 regulates cell division activity during early tomato fruit development. J Exp Bot. 2015;66(11):3405–16. Su LY, Bassa C, Audran C, Mila I, Cheniclet C, Chevalier C, Bouzayen M, Roustan JP, Chervin C. The Auxin Sl-IAA17 Transcriptional Repressor Controls Fruit Size Via the Regulation of Endoreduplication-Related Cell Expansion. Plant Cell Physiol. 2014;55(11):1969–76. Jia P, Xing LB, Zhang CG, Zhang D, Ma JJ, Zhao CP, Han MY, Ren XL, An N. MdKNOX19, a class II knotted-like transcription factor of apple, plays roles in ABA signalling/sensitivity by targeting ABI5 during organ development. Plant Sci. 2021;302:13. Huang W, Hu N, Xiao ZN, Qiu YP, Yang Y, Yang J, Mao X, Wang YC, Li ZG, Guo HW. A molecular framework of ethylene-mediated fruit growth and ripening processes in tomato. Plant Cell. 2022;34(9):3280–300. Waterhouse AL. Determination of total phenolics. Current protocols in food analytical chemistry 2002, 6(1):I1. 1.1-I1. 1.8. Zou YP, Chang SKC, Gu Y, Qian SY. Antioxidant Activity and Phenolic Compositions of Lentil (Lens culinaris var. Morton) Extract and Its Fractions. J Agric Food Chem. 2011;59(6):2268–76. Grabherr MG, Haas BJ, Yassour M, Levin JZ, Thompson DA, Amit I, Adiconis X, Fan L, Raychowdhury R, Zeng Q. Trinity: reconstructing a full-length transcriptome without a genome from RNA-Seq data. Nat Biotechnol. 2011;29(7):644. Juskyte AD, Mazeikiene I, Stanys V. Analysis of R Genes Related to Blackcurrant Reversion Virus Resistance in the Comparative Transcriptome of Ribes nigrum cv. Aldoniai Plants-Basel. 2022;11(22):11. Additional Declarations No competing interests reported. Supplementary Files SupplementalTables.doc Cite Share Download PDF Status: Posted Version 1 posted You are reading this latest preprint version Research Square lets you share your work early, gain feedback from the community, and start making changes to your manuscript prior to peer review in a journal. As a division of Research Square Company, we’re committed to making research communication faster, fairer, and more useful. We do this by developing innovative software and high quality services for the global research community. Our growing team is made up of researchers and industry professionals working together to solve the most critical problems facing scientific publishing. Also discoverable on Platform About Our Team In Review Editorial Policies Advisory Board Help Center Resources Author Services Accessibility API Access RSS feed Manage Cookie Preferences © Research Square 2026 | ISSN 2693-5015 (online) Privacy Policy Terms of Service Do Not Sell My Personal Information {"props":{"pageProps":{"initialData":{"identity":"rs-3330314","acceptedTermsAndConditions":true,"allowDirectSubmit":true,"archivedVersions":[],"articleType":"Research Article","associatedPublications":[],"authors":[{"id":237113263,"identity":"c72682cc-c8e5-4d0a-907a-98b2010b61f1","order_by":0,"name":"Huixin Gang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Huixin","middleName":"","lastName":"Gang","suffix":""},{"id":237113264,"identity":"38a7c11d-7b2e-4751-9166-8a1d0df3caee","order_by":1,"name":"Xuelin Zhang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Xuelin","middleName":"","lastName":"Zhang","suffix":""},{"id":237113265,"identity":"ac1b87e5-6877-411f-b8e6-c905a6513cc4","order_by":2,"name":"Danni Zhang","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Danni","middleName":"","lastName":"Zhang","suffix":""},{"id":237113266,"identity":"ee4f509b-1b93-4578-a773-33bd6e49d52c","order_by":3,"name":"Junwei Huo","email":"","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":false,"submittingAuthor":false,"prefix":"","firstName":"Junwei","middleName":"","lastName":"Huo","suffix":""},{"id":237113267,"identity":"5ccd95af-7ff8-4ae5-91d2-606768bf1d1c","order_by":4,"name":"Dong Qin","email":"data:image/png;base64,iVBORw0KGgoAAAANSUhEUgAAAZAAAAAyAQMAAABI0h/eAAAABlBMVEX///8AAABVwtN+AAAACXBIWXMAAA7EAAAOxAGVKw4bAAAA1UlEQVRIiWNgGAWjYDCCAyCiwqa+n72x8cEH4rWcSWOc2XO42XAG0VoY2w4zbpiR3ibNQYwOvuO9h1+8bUtjNpB82CDNwGAnp9tAQIvkmXNplnPO2bCZSyc2GBcwJBubHSCgxeBGjpkxT1kaj+XsxIbkGQwHErcRp4XtsITBzYMNh3mI1GL8mKftsIHBDcbGZqK0SJ45Y8Y450xagmRPYjPjDAMi/MJ3vMf4w5sKmwR+9uPPf3yosJMjqAUI2CR4EO4krBwEmD/wEFY0CkbBKBgFIxkAAII9SpUvbKL6AAAAAElFTkSuQmCC","orcid":"","institution":"Northeast Agricultural University","correspondingAuthor":true,"submittingAuthor":false,"prefix":"","firstName":"Dong","middleName":"","lastName":"Qin","suffix":""}],"badges":[],"createdAt":"2023-09-06 08:29:29","currentVersionCode":1,"declarations":"","doi":"10.21203/rs.3.rs-3330314/v1","doiUrl":"https://doi.org/10.21203/rs.3.rs-3330314/v1","draftVersion":[],"editorialEvents":[],"editorialNote":"","failedWorkflow":false,"files":[{"id":44213418,"identity":"aaacb0f7-0005-4d2e-b1c1-ee6c2f343cfe","added_by":"auto","created_at":"2023-10-06 21:06:48","extension":"png","order_by":1,"title":"Figure 1","display":"","copyAsset":false,"role":"figure","size":284549,"visible":true,"origin":"","legend":"\u003cp\u003eCharacteristics of fruit development and ripening in the two varieties of ‘Heifeng’ and ‘Adelinia’. (A) Fruit weight, (B) transverse, and (C) longitudinal diameters during fruit development and ripening. (D) Phenotypes at four stages in ‘Heifeng’ and ‘Adelinia’.\u003c/p\u003e","description":"","filename":"Fig.1CharacteristicsoffruitdevelopmentandripeninginthetwovarietiesofHeifengandAdelinia..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/5ca986a67ef01a76834ca0fb.png"},{"id":44213417,"identity":"14547456-24d1-4d36-8e89-c50568912ac4","added_by":"auto","created_at":"2023-10-06 21:06:48","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":238501,"visible":true,"origin":"","legend":"\u003cp\u003eThe alignment results against the gene ontology (GO) database.\u003c/p\u003e","description":"","filename":"Fig.2ThealignmentresultsagainstthegeneontologyGOdatabase..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/f1f98850aff741f6da673c3a.png"},{"id":44214820,"identity":"e78ffec6-57b8-4f2f-bfb9-48473b5d31d4","added_by":"auto","created_at":"2023-10-06 21:14:48","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":228778,"visible":true,"origin":"","legend":"\u003cp\u003eKOG function classification of consensus sequence\u003c/p\u003e","description":"","filename":"Fig.3KOGfunctionclassificationofconsensussequence.png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/bdbdeba34fe401f9a78ccc9a.png"},{"id":44214818,"identity":"4d8d8a00-e061-46c2-80be-2eb102a0d6a1","added_by":"auto","created_at":"2023-10-06 21:14:48","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":135128,"visible":true,"origin":"","legend":"\u003cp\u003eAnalysis of expression differences between 'Adelinia' and 'Heifeng' fruits at the same stage.\u003c/p\u003e\n\u003cp\u003e(A) Up-regulated and down-regulated DEGs in ‘Adelinia’ and ‘Heifeng’. (B) Statistical results of significant enrichment of KEGG.\u003c/p\u003e","description":"","filename":"Fig.4AnalysisofexpressiondifferencesbetweenAdeliniaandHeifengfruitsatthesamestage..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/8657987dc4970b146820ed64.png"},{"id":44214819,"identity":"9beacfe2-042e-409f-85ff-e4696bdfeb71","added_by":"auto","created_at":"2023-10-06 21:14:48","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":276637,"visible":true,"origin":"","legend":"\u003cp\u003eThe top 20 enriched KEGG pathways of DEGs.\u003c/p\u003e\n\u003cp\u003eNote: Y1–Y4 indicate the young fruit, expansion fruit, veraison fruit, and ripe fruit of ‘Adelinia’. H1-H4 indicates the young fruit, expansion fruit, veraison fruit, and ripe fruit of ‘Heifeng’.\u003c/p\u003e","description":"","filename":"Fig.5Thetop20enrichedKEGGpathwaysofDEGs..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/beeab8c1730543ba50ca355a.png"},{"id":44213425,"identity":"b6e623b3-4d5c-48f6-8f9a-f6894b8f783d","added_by":"auto","created_at":"2023-10-06 21:06:48","extension":"png","order_by":6,"title":"Figure 6","display":"","copyAsset":false,"role":"figure","size":398177,"visible":true,"origin":"","legend":"\u003cp\u003eGenes expression in AsA biosynthesis and recycling pathway during fruit ripening.\u003c/p\u003e\n\u003cp\u003e(A) AsA biosynthesis and recycling pathway in the blackcurrant fruits. (B) The correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e) of all the genes identified in AsA biosynthesis and recycling pathway with AsA content in the four fruit developmental stages of ‘Adelinia’ and ‘Heifeng’.\u003c/p\u003e","description":"","filename":"Fig.6GenesexpressioninAsAbiosynthesisandrecyclingpathwayduringfruitripening..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/ad9fc19b525942bc168edcc4.png"},{"id":44216648,"identity":"2198daf9-3922-48c0-af0a-4a4358bc261c","added_by":"auto","created_at":"2023-10-06 21:22:48","extension":"png","order_by":7,"title":"Figure 7","display":"","copyAsset":false,"role":"figure","size":255069,"visible":true,"origin":"","legend":"\u003cp\u003eGenes expression in flavonoids biosynthesis pathway during fruit ripening.\u003c/p\u003e\n\u003cp\u003e(A) Flavonoid biosynthesis pathway in the blackcurrant fruits. (B) The correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e) of all the genes identified in flavonoid biosynthesis pathway with flavonoid content in the four fruit developmental stages of ‘Adelinia’ and ‘Heifeng’.\u003c/p\u003e","description":"","filename":"Fig.7Genesexpressioninflavonoidsbiosynthesispathwayduringfruitripening..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/51cfcede4d336a387d41245f.png"},{"id":44213426,"identity":"8b7a13db-deda-402e-be96-8d17309b95f4","added_by":"auto","created_at":"2023-10-06 21:06:48","extension":"png","order_by":8,"title":"Figure 8","display":"","copyAsset":false,"role":"figure","size":312198,"visible":true,"origin":"","legend":"\u003cp\u003eGenes expression in plant hormone signal transduction pathway during fruit ripening\u003c/p\u003e","description":"","filename":"Fig.8Genesexpressioninplanthormonesignaltransductionpathwayduringfruitripening.png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/5f0327abe75a9356d5f74727.png"},{"id":44213422,"identity":"1e50b28a-5938-47f7-b43b-e143344dd927","added_by":"auto","created_at":"2023-10-06 21:06:48","extension":"png","order_by":9,"title":"Figure 9","display":"","copyAsset":false,"role":"figure","size":901544,"visible":true,"origin":"","legend":"\u003cp\u003eThe expression of nine genes in the fruit of 'Adelinia' and 'Heifeng'.\u003c/p\u003e\n\u003cp\u003eLeft y-axis: Quantitative real-time PCR validation of DEGs at four stages. Right y-axis: Expression profiling of genes at four stages from RNA-seq data. Error bars represent ± SE of biological replicates.\u003c/p\u003e","description":"","filename":"Fig.9TheexpressionofninegenesinthefruitofAdeliniaandHeifeng..png","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/61cab9a5fa2d52acbc829af0.png"},{"id":50846220,"identity":"be91da2d-b07e-46b0-b956-fb37ba3028d9","added_by":"auto","created_at":"2024-02-08 09:38:12","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":3221606,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/f41e51d0-6278-4cec-9f2f-ed45528f6a5d.pdf"},{"id":44213420,"identity":"9e681336-0e7f-4c43-87fe-3635aa5c5caa","added_by":"auto","created_at":"2023-10-06 21:06:48","extension":"doc","order_by":3,"title":"","display":"","copyAsset":false,"role":"supplement","size":80384,"visible":true,"origin":"","legend":"","description":"","filename":"SupplementalTables.doc","url":"https://assets-eu.researchsquare.com/files/rs-3330314/v1/21e7d89fd5204a5d8c2afb76.doc"}],"financialInterests":"No competing interests reported.","formattedTitle":"Transcriptome profiling reveals the regulatory mechanisms of ascorbic acid and flavonoid synthesis and metabolic processes in fruit development of Ribes nigrum","fulltext":[{"header":"1. Background","content":"\u003cp\u003eBlackcurrant (\u003cem\u003eRibes nigrum\u003c/em\u003e L.) is a perennial shrub that produces sweet and sour fruit packed with a wide range of nutrients. Biochemical analysis of blackcurrants has revealed the presence of numerous components, including flavonoids, polyunsaturated fatty acids, structural and non-structural carbohydrates, non-volatile organic acids, tannins, and astragals[\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e]. Ascorbic acid (AsA), which can enhance antioxidant capacity and health potential[\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e], is also found in blackcurrants at higher levels than in raspberries and blueberries[\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e]. The high nutritional, medical, health, and cosmetic value of blackcurrant fruits is due to their bioactive components, which are effective in reducing thrombosis, inhibiting tumor growth[\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e], lowering cholesterol[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], inhibiting salmonella enteritidis[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and preventing major diseases such as asthma, cardiovascular disease, heart disease, and hypertension[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. In addition, blackcurrant is an effective anti-microbial agent[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and has the potential to be used as a disinfectant and antiseptic[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe antioxidant properties of blackcurrants are largely due to their phenolic compounds, such as ANC, which can act as hydrogen donors or electron transfer agents depending on their chemical structure. The antioxidant activity of ANC is directly related to its chemical structure, and the differences in the antioxidant activity of flavonoids are due to differences in the type, position, and number of methyl and hydroxyl groups[\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e]. Blackcurrant fruits have significant value in nutrition, medicine, health, and beauty. Their bioactive components can effectively reduce blood clot formation, inhibit tumor growth, lower cholesterol levels[\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e], suppress Salmonella enteritidis infection[\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e], and prevent/treat major diseases such as asthma, cardiovascular disease, heart disease, and hypertension[\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e]. Additionally, blackcurrants are effective antimicrobial agents[\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e] and hold potential in the production of disinfectants and preservatives[\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e].\u003c/p\u003e \u003cp\u003eThe rapid development of high-throughput sequencing technology has greatly improved the efficiency of gene discovery[\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e, \u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e]. The advancement of sequencing technology has provided powerful tools for scientists to study plant transcriptomes. Compared to woody plants, fruit trees have relatively less genomic information and have therefore been less extensively studied. Transcriptomes play a vital role in overcoming the bottleneck that hinders the development of fruit tree genomes. Currently, RNA-Seq has been widely applied to most fruit trees, such as sweet orange[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e, \u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e], apple[\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e, \u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e], blackberry[\u003cspan citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e, \u003cspan citationid=\"CR18\" class=\"CitationRef\"\u003e18\u003c/span\u003e], kiwi[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], blueberry[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], lychee[\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e], Chinese cherry[\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e], dragon fruit[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e], pear[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], strawberry[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], raspberry[\u003cspan citationid=\"CR26\" class=\"CitationRef\"\u003e26\u003c/span\u003e], plum[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and others. It has been used to investigate the synthesis of carotenoids in yellow-fleshed kiwi[\u003cspan citationid=\"CR27\" class=\"CitationRef\"\u003e27\u003c/span\u003e], gene expression changes during the maturation and development of blueberry[\u003cspan citationid=\"CR20\" class=\"CitationRef\"\u003e20\u003c/span\u003e], plum[\u003cspan citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e], and kiwi fruits[\u003cspan citationid=\"CR19\" class=\"CitationRef\"\u003e19\u003c/span\u003e], genetic differences among different varieties of dragon fruit[\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e] and strawberry fruits[\u003cspan citationid=\"CR25\" class=\"CitationRef\"\u003e25\u003c/span\u003e], changes in the cuticle layer during different stages of navel orange fruit[\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e], and the production of differentially expressed genes (DEGs) under Huanglongbing bacteria and decline viruses in citrus[\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e]. However, the fruit tree genome still faces several challenges, including imperfect bioinformatics studies and the difficulty of identifying key genes responsible for physiological changes. The quality of blackcurrant fruit not only determines its appearance, storage potential, and transportability but also affects subsequent product development and quality. The Northeast region of China has abundant blackcurrant germplasm resources and multiple varieties but lacks comprehensive blackcurrant varieties with superior economic traits. To explore the genetic basis of blackcurrant fruit quality, this study utilized RNA-Seq combined with de novo transcriptome assembly to analyze the differences in fruit quality during ripening between two varieties, 'Adelinia' and 'Heifeng'. The key genes involved in AsA biosynthesis and recycling, flavonoid biosynthesis, and plant hormone signal transduction were identified by integrating physiological experimental results. This study aims to investigate the major candidate genes influencing fruit quality, laying the foundation for future research on gene function and high-quality blackcurrant fruit cultivation.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e \u003ch2\u003e2.1. Changes in fruit weight, transverse and longitudinal diameter during the development of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;\u003c/h2\u003e \u003cp\u003eDuring fruit growth and development, the variation in fruit weight and longitudinal and cross-sectional diameter between \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; was investigated (Fig.\u0026nbsp;\u003cspan refid=\"Fig1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). The single fruit weight of \u0026lsquo;Adelinia\u0026rsquo; was consistently greater than that of \u0026lsquo;Heifeng\u0026rsquo;, and the weight of the ripe fruit of \u0026lsquo;Adelinia\u0026rsquo; was three times of \u0026lsquo;Heifeng\u0026rsquo;. The most significant changes in fruit weight and transverse and longitudinal diameters occurred during the period from expansion to veraison, demonstrating an S-shaped trend of \"slow-fast-slow\". During fruit development, the longitudinal diameter developed faster, resulting in an oval fruit shape during the young fruit stage. After veraison, the transverse diameter gradually became larger than the longitudinal diameter, resulting in a spherical fruit shape.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec4\" class=\"Section2\"\u003e \u003ch2\u003e2.2. Changes in fruit quality during the development of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;\u003c/h2\u003e \u003cp\u003eThe contents of soluble solids and titratable acids in blackcurrant fruits increased with fruit development (Table\u0026nbsp;\u003cspan refid=\"Tab1\" class=\"InternalRef\"\u003e1\u003c/span\u003e). Soluble solids increased the slowest from the young fruit stage to the expansion stage and the fastest from the veraison stage to the ripe stage. The contents of soluble solids increased by 4.6% and 3.0% from the veraison stage to the ripe stage in \u0026lsquo;Heifeng\u0026rsquo; and \u0026lsquo;Adelinia\u0026rsquo; respectively. The contents of soluble solids in \u0026lsquo;Heifeng\u0026rsquo; were consistently higher than that in \u0026lsquo;Adelinia\u0026rsquo; throughout the fruit development, reaching 1.4 times higher than that of \u0026lsquo;Adelinia\u0026rsquo;at the ripe fruit stage.\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab1\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 1\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eChanges in quality parameters during fruit development of different cultivars of blackcurrant\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"8\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c5\" colnum=\"5\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c6\" colnum=\"6\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c7\" colnum=\"7\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c8\" colnum=\"8\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eVarieties\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eDevelopmental stage\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eThe soluble solids (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eTitratable acid (%)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c5\"\u003e \u003cp\u003eAscorbic acid (mg/g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colspan=\"2\" nameend=\"c7\" namest=\"c6\"\u003e \u003cp\u003eTotal phenol (mg/100g)\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c8\"\u003e \u003cp\u003eFlavonoid (mg/100g)\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eHeifeng\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyoung fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e7.6\u0026thinsp;\u0026plusmn;\u0026thinsp;0.52cd\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.34\u0026thinsp;\u0026plusmn;\u0026thinsp;0. 02d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e17.52\u0026thinsp;\u0026plusmn;\u0026thinsp;0.04b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e11.29\u0026thinsp;\u0026plusmn;\u0026thinsp;0.93b\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eexpansion fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.2\u0026thinsp;\u0026plusmn;\u0026thinsp;0.64c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.97\u0026thinsp;\u0026plusmn;\u0026thinsp;0.11b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.56\u0026thinsp;\u0026plusmn;\u0026thinsp;0.32b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e13.68\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e3.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.69c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003everaison fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.97b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e11.33\u0026thinsp;\u0026plusmn;\u0026thinsp;0.10c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.13\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05h\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.26\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eripe fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e15.9\u0026thinsp;\u0026plusmn;\u0026thinsp;0.72a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.45ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e7.62\u0026thinsp;\u0026plusmn;\u0026thinsp;0.08f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.55\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03f\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e2.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.21d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\" morerows=\"3\" rowspan=\"4\"\u003e \u003cp\u003eAdelinia\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eyoung fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e5.3\u0026thinsp;\u0026plusmn;\u0026thinsp;0.44d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1.63\u0026thinsp;\u0026plusmn;\u0026thinsp;0.05c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e16.61\u0026thinsp;\u0026plusmn;\u0026thinsp;0.91a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e18.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e12.23\u0026thinsp;\u0026plusmn;\u0026thinsp;0.27a\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eexpansion fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e6.4\u0026thinsp;\u0026plusmn;\u0026thinsp;0.37d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e2.45\u0026thinsp;\u0026plusmn;\u0026thinsp;0.12ab\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e15.87\u0026thinsp;\u0026plusmn;\u0026thinsp;0.99b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e15.39\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e3.58\u0026thinsp;\u0026plusmn;\u0026thinsp;0.34c\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003everaison fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e8.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.62c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.07\u0026thinsp;\u0026plusmn;\u0026thinsp;1.02a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e10.18\u0026thinsp;\u0026plusmn;\u0026thinsp;0.07d\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e0.16\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01g\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e0.32\u0026thinsp;\u0026plusmn;\u0026thinsp;0.06e\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003eripe fruit\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e11.5\u0026thinsp;\u0026plusmn;\u0026thinsp;0.43b\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e3.36\u0026thinsp;\u0026plusmn;\u0026thinsp;0.58a\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c5\"\u003e \u003cp\u003e8.85\u0026thinsp;\u0026plusmn;\u0026thinsp;0.03c\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c6\"\u003e \u003cp\u003e1.05\u0026thinsp;\u0026plusmn;\u0026thinsp;0.01e\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colspan=\"2\" nameend=\"c8\" namest=\"c7\"\u003e \u003cp\u003e2.24\u0026thinsp;\u0026plusmn;\u0026thinsp;0.28d\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003ctfoot\u003e \u003ctr\u003e\u003ctd colspan=\"8\"\u003eNote: Data are expressed as mean\u0026thinsp;\u0026plusmn;\u0026thinsp;standard deviation. Different letters within the same column indicate significant differences (p\u0026thinsp;\u0026lt;\u0026thinsp;0.05) based on Duncan's multiple range test.\u003c/td\u003e\u003c/tr\u003e \u003c/tfoot\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eThe contents of titratable acid in \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; increased most rapidly from the young fruit stage to the expansion stage, with the content increasing by 0.63% and 0.82%, respectively. The rate of increase in titratable acid gradually slowed down from the expansion stage to the ripe stage. The content of titratable acid in \u0026lsquo;Adelinia\u0026rsquo; was consistently higher than that of \u0026lsquo;Heifeng\u0026rsquo; throughout fruit development, reaching 1.3 times higher than that of \u0026lsquo;Heifeng\u0026rsquo;.\u003c/p\u003e \u003cp\u003eThe AsA content in the fruit showed a continuous decreasing trend during growth and development, with the highest content in the young fruit stage and the lowest in the ripe fruit. The AsA content decreased most from the expansion stage to the veraison stage, with \u0026lsquo;Heifeng\u0026rsquo; decreasing by 4.23 mg/g and \u0026lsquo;Adelinia\u0026rsquo; decreasing by 5.69 mg/g. The AsA content in the young, expansion, and ripe fruit of \u0026lsquo;Adelinia\u0026rsquo; was higher than that of 'Heifeng.\u003c/p\u003e \u003cp\u003eThe changing trend of total phenols and total flavonoids was similar during fruit growth and development, with the maximum value observed in the young fruit stage and the minimum value in veraison fruit, which slightly increased in ripe fruit.\u003c/p\u003e \u003cp\u003eThe contents of total phenols and flavonoids from the expansion stage to the veraison stage decreased the most in 'Heifeng' and \u0026lsquo;Adelinia\u0026rsquo;. The content of total phenols decreased by 13.6 mg/100g and 15.23 mg/100g in 'Heifeng' and \u0026lsquo;Adelinia\u0026rsquo;, respectively. The content of total flavonoids decreased by 5.11 mg/100g and 7.95 mg/100g in 'Heifeng' and \u0026lsquo;Adelinia\u0026rsquo;, respectively. The contents of total phenols and flavonoids in \u0026lsquo;Adelinia\u0026rsquo; fruit were consistently higher than those in 'Heifeng' fruit throughout the fruit development. The highest total phenolic content was 1.9 times that of 'Heifeng', and the highest total flavonoid content was 1.5 times that of 'Heifeng'.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec5\" class=\"Section2\"\u003e \u003ch2\u003e2.3. Transcriptome sequencing quality of blackcurrant fruit and denovo-assemble of unigenes\u003c/h2\u003e \u003cp\u003eIn order to explore the difference in gene expression in the two varieties during fruit ripening, transcriptome sequencing was performed on young, expansion, veraison, and ripe fruit stages of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. After removing low-quality reads, a total of 186.71 Gb reads were obtained from 24 samples, with an average of 6.74 Gb per sample (Table S2). The Q30 base percentage of all samples was greater than 94.08%, and GC content ranged from 44%-47%, indicating good sequencing quality and credible results.\u003c/p\u003e \u003cp\u003eThen the clean reads of all the samples were used for de novo assembly, and a total of 220,757 transcripts and 116,793 unigene were obtained. The average length was 1,129 bp and 729 bp respectively. The N50 was 1,998 and 1,342 respectively (Table S3), indicating that the assembly results were credible. Unigene length ranged from 150 to 16,047 bp, with most unigenes being 300 to 2,000 bp in length.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec6\" class=\"Section2\"\u003e \u003ch2\u003e2.4. Gene function annotation, GO, and KOG functional classification\u003c/h2\u003e \u003cp\u003eAll assembled unigenes were annotated with databases. A total of 23,788, 41,786, 22,044, 35,830, 41,872, 29,652, 64,522, and 66,902 unigenes were matched with COG, GO, KEGG, KOG, Pfam, Swissprot, eggNOG and NR databases, respectively. A total of 70,224 unigene were identified. There were 8,257, 13, 7,701, 12,554, 16,740, 13,041, 19,607, and 19,977 unigene genes with length\u0026thinsp;\u0026ge;\u0026thinsp;1,000 bp (Table S4).\u003c/p\u003e \u003cp\u003eGO annotation was performed on 41,786 unigenes, which are included in 54 branches. Twenty branches of biological processes involve 110,234 annotated information, accounting for 45.82% of all annotated information. The branches with the most unigenes are the \"metabolic process\" with 30,074 annotated information and the \"cellular process\" with 24,290 annotated information. In the cellular component category, 17 branches involve 78,115 annotated information, accounting for 32.47% of all annotated information. The branches with the most annotated information are \"cell\" and \"cell part,\" both with 17,483 annotated information. In the molecular function category, 17 branches involve 52,216 unigenes, accounting for 21.71% of all annotated information. The branches with the most molecular function are \"catalytic activity\" with 23,645 unigenes and \"binding\" with 20,421 unigenes (Fig.\u0026nbsp;\u003cspan refid=\"Fig2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe KOG database has a wide range of functions related to life activities, with a total of 35,830 genes annotated and divided into 26 categories represented by letters A to Z. The \"General function prediction only\" accounts for 20.56% of all gene sequences, with 7,368 genes in this category. The next most abundant category is \"Posttranslational modification, protein turnover, chaperones\", which accounts for 10.48% with 3,755 genes. Additionally, there are 3,202 genes involved in \"Translation, ribosome structure, and biogenesis\", accounting for 8.94% of the total sequence. Furthermore, there are 2,762 genes involved in \"Signal transduction mechanisms\", 2,488 genes involved in \"Amino acid transport and metabolism\", and 2,102 genes involved in \"Carbohydrate transport and metabolism\". The category with the fewest number of genes is \"cell motility\", with only 21 genes, followed by \"extracellular structures\" and \"Nuclear structure\" with 123 and 181 genes, respectively. The number of genes in each category is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig3\" class=\"InternalRef\"\u003e3\u003c/span\u003e.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec7\" class=\"Section2\"\u003e \u003ch2\u003e2.5. DEGs analysis of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; fruits at the same stage\u003c/h2\u003e \u003cp\u003eThe expression levels of unigenes in the fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were compared at four developmental stages, namely, young fruit, expansion fruit, veraison fruit, and ripe fruit stages. A total of 483 DEGs were detected in the young fruit stage, including 241 up-regulated genes and 242 down-regulated genes. In the expansion stage, 1,909 DEGs were detected, including 1,020 up-regulated genes and 889 down-regulated genes. In the veraison fruit stage, 1,548 genes were differentially expressed, including 752 up-regulated genes and 796 down-regulated genes. In the ripe fruit stage, 2,822 genes were differentially expressed, including 1,602 up-regulated genes and 1,220 down-regulated genes (Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, A).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eThe KEGG enrichment analysis of the DEGs in the fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; at the same stage is shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig4\" class=\"InternalRef\"\u003e4\u003c/span\u003e, B. Specifically, DEGs in the young fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were significantly enriched in monoterpenoid biosynthesis (ko00902). DEGs in the expansion fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were significantly enriched in plant hormone signal transduction (ko04075), DNA replication (ko03030), plant-pathogen interaction (ko04626), and mismatch repair (ko03430) pathway. DEGs in the veraison fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were significantly enriched in photosynthesis-antenna proteins (ko00196), flavonoid biosynthesis (ko00941), and plant-pathogen interaction (ko04626) pathway. DEGs in the ripe fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were significantly enriched in glyoxylate and dicarboxylate metabolism (ko00630), carbon metabolism (ko01200), citrate cycle (TCA cycle, ko00020), flavonoid biosynthesis (ko00941), photosynthesis-antenna proteins (ko00196), alanine, aspartate and glutamate metabolism alanine (ko00250), cutin, suberine and wax biosynthesis (ko00073), pyruvate metabolism (ko00620) pathway.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec8\" class=\"Section2\"\u003e \u003ch2\u003e2.6. DEGs analysis of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; fruits during ripening\u003c/h2\u003e \u003cp\u003eTo investigate the DEGs during fruit ripening, we compared young fruit with expansion fruit, expansion fruit with veraison fruit, and veraison fruit with ripe fruit in both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. The number of DEGs was 1499 (810 up-regulated DEGs and 689 down-regulated DEGs) and 2311 (1163 up-regulated DEGs and 1148 down-regulated DEGs) in the comparison group of young fruit with expansion fruit in \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;, respectively. The number of DEGs was 2250 (850 up-regulated DEGs and 1400 down-regulated DEGs) and 3746 (1767 up-regulated DEGs and 1979 down-regulated DEGs) in the comparison group of expansion fruit with veraison fruit in \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;, respectively. The number of DEGs was 1356 (1258 up-regulated DEGs and 98 down-regulated DEGs) and 960 (501 up-regulated DEGs and 456 down-regulated DEGs) in the comparison group of veraison fruit with ripe fruit in \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;, respectively (Table\u0026nbsp;\u003cspan refid=\"Tab2\" class=\"InternalRef\"\u003e2\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003cdiv class=\"gridtable\"\u003e\u003ctable float=\"Yes\" id=\"Tab2\" border=\"1\"\u003e \u003ccaption language=\"En\"\u003e \u003cdiv class=\"CaptionNumber\"\u003eTable 2\u003c/div\u003e \u003cdiv class=\"CaptionContent\"\u003e \u003cp\u003eUp-regulated and down-regulated DEGs in the young fruit, expansion fruit, veraison fruit, and ripe fruit stage\u003c/p\u003e \u003c/div\u003e \u003c/caption\u003e \u003ccolgroup cols=\"4\"\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c1\" colnum=\"1\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c2\" colnum=\"2\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c3\" colnum=\"3\"\u003e\u003c/div\u003e \u003cdiv align=\"left\" class=\"colspec\" colname=\"c4\" colnum=\"4\"\u003e\u003c/div\u003e \u003cthead\u003e \u003ctr\u003e \u003cth align=\"left\" colname=\"c1\"\u003e \u003cp\u003eCompared group\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c2\"\u003e \u003cp\u003eTotal DEGs\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c3\"\u003e \u003cp\u003eUp-regulated\u003c/p\u003e \u003c/th\u003e \u003cth align=\"left\" colname=\"c4\"\u003e \u003cp\u003eDown-regulated\u003c/p\u003e \u003c/th\u003e \u003c/tr\u003e \u003c/thead\u003e \u003ctbody\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY1 vs. Y2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1499\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e810\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e689\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY2 vs. Y3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2250\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e850\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1400\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eY3 vs. Y4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e1356\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1258\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e98\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH1 vs. H2\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e2311\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1163\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1148\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH2 vs. H3\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e3746\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e1767\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e1979\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003ctr\u003e \u003ctd align=\"left\" colname=\"c1\"\u003e \u003cp\u003eH3 vs. H4\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c2\"\u003e \u003cp\u003e960\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c3\"\u003e \u003cp\u003e501\u003c/p\u003e \u003c/td\u003e \u003ctd align=\"left\" colname=\"c4\"\u003e \u003cp\u003e456\u003c/p\u003e \u003c/td\u003e \u003c/tr\u003e \u003c/tbody\u003e \u003c/colgroup\u003e \u003c/table\u003e\u003c/div\u003e \u003c/p\u003e \u003cp\u003eAll DEGs in the comparison groups were mapped to KEGG pathway enrichment to analyze the enriched pathways. The DEGs in the comparison groups of Y1 vs Y2 and H1 vs H2 were both significantly enriched in plant hormone signal transduction (ko04075), Phenylpropanoid biosynthesis (ko00940), flavonoid biosynthesis (ko00941), Stilbenoid, diarylheptanoid and gingerol biosynthesis (ko00945), Starch and sucrose metabolism (ko00500), Phenylalanine metabolism (ko00360), Cutin, suberine, and wax biosynthesis (ko00073) pathway. The DEGs in the comparison groups of Y2 vs Y3 and H2 vs H3 were both significantly enriched in plant hormone signal transduction (ko04075), starch and sucrose metabolism (ko00500), phenylpropanoid biosynthesis (ko00360), homologous recombination (ko03440), phenylalanine metabolism (ko00360), flavonoid biosynthesis (ko00941), DNA replication (ko03030), Cutin, suberine, and wax biosynthesis (ko00073) pathway. While the DEGs in the comparison groups of Y3 vs Y4 and H3 vs H4 did not share the same significantly enriched pathway (Fig.\u0026nbsp;\u003cspan refid=\"Fig5\" class=\"InternalRef\"\u003e5\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec9\" class=\"Section2\"\u003e \u003ch2\u003e2.7. Identification of genes involved in AsA biosynthesis and recycling pathway during fruit ripening\u003c/h2\u003e \u003cp\u003eAccording to the gene expression level of transcriptome sequencing, crucial genes involved in AsA biosynthesis and recycling pathway were identified. Including 3 genes of glucose-6-phosphate isomerase (\u003cem\u003ePGI\u003c/em\u003e), 1 gene of mannose-6-phosphate isomerase (\u003cem\u003ePMM\u003c/em\u003e), 1 gene of GDP-mannose-3\u0026rsquo;,5\u0026rsquo;-epimerase (\u003cem\u003eGME\u003c/em\u003e), 2 genes of GDP-L-galactose phosphorylase (\u003cem\u003eGGP\u003c/em\u003e), 1 gene of Myo-inositol oxygenase (\u003cem\u003eMIOX\u003c/em\u003e) and 3 genes of L-galactono-1,4-lactone dehydrogenase (\u003cem\u003eGalLDH\u003c/em\u003e) associated with AsA biosynthesis. And 6 genes of ascorbate peroxidase (\u003cem\u003eAPX\u003c/em\u003e), 6 genes of ascorbate oxidase (\u003cem\u003eAO\u003c/em\u003e), 3 genes of monodehydroascorbate reductase (\u003cem\u003eMDHAR\u003c/em\u003e) and 1 gene of glutathione reductase (\u003cem\u003eGR\u003c/em\u003e) associated with AsA recycling. As shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig6\" class=\"InternalRef\"\u003e6\u003c/span\u003e, most of these genes were highly expressed in the young fruits of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. Such as the expression of the \u003cem\u003ePGI\u003c/em\u003e gene (c179793.graph_c1 and c181113.graph_c0) and \u003cem\u003eMDHAR\u003c/em\u003e (c160843.graph_c0) were highest in the young fruit and lowest in the veraison fruit in both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. The expression of the \u003cem\u003ePMM\u003c/em\u003e gene (c184228.graph_c0), \u003cem\u003eGME\u003c/em\u003e gene (c181107.graph_c0), \u003cem\u003eGalLDH\u003c/em\u003e gene (c178565.graph_c2), and \u003cem\u003eAO\u003c/em\u003e gene (c185688.graph_c1) was the highest in the young fruits and lowest in the ripe fruit.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eIn addition, we calculate the correlation coefficient (r\u003csup\u003e2\u003c/sup\u003e) of all the genes identified in AsA biosynthesis and recycling pathway with AsA content in the four fruit developmental stages of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. The expression of a \u003cem\u003ePMM\u003c/em\u003e gene (c184228.graph_c0) has a strong correlation (r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.84) with AsA content. Similarly, the expression of an \u003cem\u003eAPX\u003c/em\u003e (c176242.graph_c0), a \u003cem\u003eGME\u003c/em\u003e (c181107.graph_c0), and a \u003cem\u003ePGI\u003c/em\u003e (c181113.graph_c0) gene were strongly correlated with AsA content, with r\u003csup\u003e2\u003c/sup\u003e of 0.74, 0.71 and 0.71, respectively. These results revealed that the above genes may be crucial for AsA biosynthesis in blackcurrant. The expression of 2 \u003cem\u003eAPX\u003c/em\u003e (c176953.graph_c0, c172886.graph_c0) gene showed a strong negative correlation with AsA content, with \u0026minus;\u0026thinsp;0.93 and \u0026minus;\u0026thinsp;0.77, respectively. The expression of 2 \u003cem\u003eAO\u003c/em\u003e (c184540.graph_c1, c185556.graph_c0) genes has a strong negative correlation with AsA content, with r\u003csup\u003e2\u003c/sup\u003e of -0.83 and \u0026minus;\u0026thinsp;0.74, respectively. An \u003cem\u003eMDHAR\u003c/em\u003e (c172058.graph_c0) gene also showed a strong negative correlation (r\u003csup\u003e2\u003c/sup\u003e= -0.72) with AsA content. These genes may be crucial for the AsA recycling pathway in blackcurrant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec10\" class=\"Section2\"\u003e \u003ch2\u003e2.8. Identification of genes involved in flavonoid biosynthesis pathway during fruit ripening\u003c/h2\u003e \u003cp\u003eAs the flavonoid contents were variational during fruit ripening, we then identified the key genes involved in the flavonoid biosynthesis pathway. A total of 6 genes of 4-coumarate-CoA (\u003cem\u003e4CL\u003c/em\u003e), 3 genes of chalcone synthase (\u003cem\u003eCHS\u003c/em\u003e), 2 genes of chalcone isomerase (\u003cem\u003eCHI\u003c/em\u003e), 1 gene of Flavanone-3-hydroxylase (\u003cem\u003eF3H\u003c/em\u003e), 2 genes of dihydro flavonol reductase (\u003cem\u003eDFR\u003c/em\u003e), and 1 gene of leucoanthocyanidin dioxygenase (\u003cem\u003eLDOX\u003c/em\u003e), anthocyanin synthase (\u003cem\u003eANS\u003c/em\u003e), anthocyanidin reductase (\u003cem\u003eANR\u003c/em\u003e), flavonoid 3-O-glucosyl transferase (\u003cem\u003eUFGT\u003c/em\u003e) and leucoanthocyanidin reductase (\u003cem\u003eLAR\u003c/em\u003e) were identified that associated with flavonoid biosynthesis. Most of these genes were expressed at their highest levels in the young fruits, by the flavonoid contents during fruit ripening. The expressions of the \u003cem\u003e4CL\u003c/em\u003e gene (c183421.graph_c1), \u003cem\u003eCHS\u003c/em\u003e genes (c115942.graph_c0, c183294.graph_c1), \u003cem\u003eCHI\u003c/em\u003e genes (c157264.graph_c0, c163054.graph_c0), \u003cem\u003eDFR\u003c/em\u003e gene (c183820.graph_c4), \u003cem\u003eANS\u003c/em\u003e gene (c187123.graph_c2), ANR gene (c183720.graph_c1) and \u003cem\u003eLAR\u003c/em\u003e (c162573.graph_c1) were highest in the young fruit of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;, and were significantly down-regulated in the expansion fruits.\u003c/p\u003e \u003cp\u003eSimilarly, the expression of a \u003cem\u003eDFR\u003c/em\u003e (c183820.graph_c4), an \u003cem\u003eANR\u003c/em\u003e gene showed a strong correlation (both r\u003csup\u003e2\u003c/sup\u003e\u0026thinsp;=\u0026thinsp;0.88) with flavonoid content. The expression of 2 \u003cem\u003eCHI\u003c/em\u003e (c157264.graph_c0, c163054.graph_c0) with r\u003csup\u003e2\u003c/sup\u003e of 0.87and 0.83, and 2 \u003cem\u003eCHS\u003c/em\u003e (c115942.graph_c0, c183294.graph_c1) genes with r\u003csup\u003e2\u003c/sup\u003e of 0.82 and 0.80 showed strong correlation with flavonoid content. A \u003cem\u003e4CL\u003c/em\u003e and a \u003cem\u003eLAR\u003c/em\u003e gene were also strongly correlated with flavonoid content, with r\u003csup\u003e2\u003c/sup\u003e of 0.73 and 0.71, respectively. These results indicated that the above genes may be crucial for flavonoid biosynthesis in blackcurrant (Fig.\u0026nbsp;\u003cspan refid=\"Fig7\" class=\"InternalRef\"\u003e7\u003c/span\u003e).\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec11\" class=\"Section2\"\u003e \u003ch2\u003e2.9. Identification of DEGs involved in plant hormone signal transduction pathway\u003c/h2\u003e \u003cp\u003eTo explore changes in gene expression related to plant hormone signal transduction, we analyzed the DEGs involved in auxin, cytokinine, gibberellin, ethylene, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid signal transduction pathways. A total of 31 DEGs were detected involved in the auxin signal transduction pathway, and 18 genes were expressed highest in the young fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; (Fig.\u0026nbsp;\u003cspan refid=\"Fig8\" class=\"InternalRef\"\u003e8\u003c/span\u003e). These genes included 2 \u003cem\u003eARG7\u003c/em\u003e genes(c171898.graph_c0, c167577.graph_c0), 4 \u003cem\u003eAUX10A\u003c/em\u003e genes (c177750.graph_c0, c179267.graph_c0, c173102.graph_c0, c178278.graph_c0), \u003cem\u003eAUX28\u003c/em\u003e (c182867.graph_c3), \u003cem\u003eAUX6B\u003c/em\u003e (c163920.graph_c0), 2 \u003cem\u003eGH3.1\u003c/em\u003e genes (c179273.graph_c0, c187153.graph_c1), \u003cem\u003eGH3.5\u003c/em\u003e (c175332.graph_c0), \u003cem\u003eGH3.9\u003c/em\u003e (c175298.graph_c0), \u003cem\u003eIAA7\u003c/em\u003e (c159328.graph_c0), \u003cem\u003eIAA9\u003c/em\u003e (c177889.graph_c1), \u003cem\u003eIAA14\u003c/em\u003e (c184998.graph_c1), 2 \u003cem\u003eIAA26\u003c/em\u003e genes (, c176546.graph_c1, c176147.graph_c3), \u003cem\u003eIAA27\u003c/em\u003e gene (c179588.graph_c0). This indicates that the auxin signal transduction pathway plays an important role in the young fruit stage. We also found that the expression of \u003cem\u003eARG7\u003c/em\u003e (c174082.graph_c0), \u003cem\u003eIAA7\u003c/em\u003e (c159328.graph_c0), and \u003cem\u003eIAA11\u003c/em\u003e (c164719.graph_c0) was significantly higher in \u0026lsquo;Adelinia\u0026rsquo; than \u0026lsquo;Heifeng\u0026rsquo; in the expansion fruit stage. The expression of \u003cem\u003eAGR7\u003c/em\u003e (c167451.graph_c0) was significantly higher in \u0026lsquo;Adelinia\u0026rsquo; than \u0026lsquo;Heifeng\u0026rsquo; in the expansion and veraison fruit stages. As the fruit grows rapidly in the expansion stage, these genes may be responsible for the difference in volume and weight between \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; fruits. A total of 10 DEGs were detected as involved in the cytokinine signal transduction pathway. Three genes including \u003cem\u003eARR3\u003c/em\u003e (c178666.graph_c0), \u003cem\u003eARR9\u003c/em\u003e (c173285.graph_c1), and \u003cem\u003eAHP\u003c/em\u003e gene (c181299.graph_c0) were expressed at their highest level in the young fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. Two genes including \u003cem\u003eARR1\u003c/em\u003e (c179842.graph_c0) and \u003cem\u003eARR12\u003c/em\u003e (c183630.graph_c1) were expressed highest in the expansion fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. The \u003cem\u003eHK2\u003c/em\u003e (c185911.graph_c0) gene was expressed at its highest level in the ripe fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. A total of 4 DEGs were detected involved in the gibberellin signal transduction pathway. The expression of the \u003cem\u003ePIF3\u003c/em\u003e (c182315.graph_c1) and \u003cem\u003eGAI\u003c/em\u003e (c151333.graph_c0) gene was higher in the young and expansion fruit than veraison and ripe fruit of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. While the expression of the \u003cem\u003eGID1B\u003c/em\u003e (c184501.graph_c3) gene was expressed higher in the veraison and ripe fruit than in young and expansion fruit of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. A total of 5 DEGs were detected involved in the jasmonic acid signal transduction pathway. Four genes including \u003cem\u003eTIFY3B\u003c/em\u003e (c179099.graph_c0), \u003cem\u003eTIFY11B\u003c/em\u003e (c183995.graph_c4), \u003cem\u003eMYC2\u003c/em\u003e (c182574.graph_c0), and \u003cem\u003ebHLH14\u003c/em\u003e (c167287.graph_c0) were expressed highest in the young fruit of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003cp\u003eA total of 4 genes were detected involved in the ethylene signal transduction pathway. Two genes of \u003cem\u003eEBF1\u003c/em\u003e (c168698.graph_c0, c168698.graph_c1) were expressed higher in the young and ripe fruit stage than expansion and veraison fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. In addition, the expression of \u003cem\u003eEBF1\u003c/em\u003e (c168698.graph_c0, c168698.graph_c1) and \u003cem\u003eEIN3\u003c/em\u003e (c180971.graph_c4) genes was higher in the expansion fruit of \u0026lsquo;Heifeng\u0026rsquo; than \u0026lsquo;Adelinia\u0026rsquo;. A total of 11 DEGs were detected involved in abscisic acid signal transduction. The \u003cem\u003ePYL8\u003c/em\u003e (c169965.graph_c0) gene was expressed highest in the young fruit stage. One of the \u003cem\u003eABI5\u003c/em\u003e (c186178.graph_c0) genes was expressed highest in the expansion fruit stage, while another \u003cem\u003eABI5\u003c/em\u003e (c172267.graph_c0) gene was expressed highest in the veraison fruit stage. The \u003cem\u003ePP2C\u003c/em\u003e (c177710.graph_c0) gene was expressed highest in the ripe fruit stage. In addition, the expression of \u003cem\u003ePP2C\u003c/em\u003e (c176253.graph_c0) was higher in the expansion fruit of \u0026lsquo;Adelinia\u0026rsquo; than \u0026lsquo;Heifeng\u0026rsquo;. The expression of another \u003cem\u003ePP2C\u003c/em\u003e (c177479.graph_c0) and \u003cem\u003ePYR1\u003c/em\u003e (c141701.graph_c0) gene was higher in the ripe fruit of \u0026lsquo;Heifeng\u0026rsquo; than in \u0026lsquo;Adelinia\u0026rsquo;. A total of 8 DEGs were detected involved in the brassinosteroid signal transduction pathway. Three genes including \u003cem\u003eSIK\u003c/em\u003e (c169178.graph_c0, c186784.graph_c1) and \u003cem\u003eTHX\u003c/em\u003e (c156073.graph_c0) genes were expressed higher in the veraison and ripe fruit stages than in young and expansion fruit stages of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. In addition, \u003cem\u003eCYCD3\u003c/em\u003e (c173925.graph_c0, c178067.graph_c0) gene was expressed highest in the young fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;. Three DEGs were detected as involved in the salicylic acid signal transduction pathway. \u003cem\u003ePR1\u003c/em\u003e (c181784.graph_c1) gene was expressed higher in the ripe fruit, while \u003cem\u003eTGA3\u003c/em\u003e (c174510.graph_c1) gene was expressed higher in the expansion fruit stage of both \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng. The result indicated that these DEGs involved in the plant hormone signal transduction pathway may play an important role in the fruit growth and development of blackcurrant.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec12\" class=\"Section2\"\u003e \u003ch2\u003e2.10. Validation of DEGs by qRT-PCR\u003c/h2\u003e \u003cp\u003eTo confirm the accuracy and reliability of the transcriptome analysis results, three ascorbic acid biosynthesis and recycling-related genes, five flavonoid biosynthesis genes, and one hormone signal transduction-related gene were selected. The gene expression levels at different developmental stages were further examined by qRT-PCR, as shown in Fig.\u0026nbsp;\u003cspan refid=\"Fig9\" class=\"InternalRef\"\u003e9\u003c/span\u003e. For all nine genes, the qRT-PCR analysis results had the same expression trend as the FPKM data from RNA-seq.\u003c/p\u003e \u003cp\u003e \u003c/p\u003e \u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cdiv id=\"Sec14\" class=\"Section2\"\u003e \u003ch2\u003e3.1. AsA biosynthesis and recycling in \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; during fruit ripening\u003c/h2\u003e \u003cp\u003eSeveral factors can influence the content of ascorbic acid (AsA) in plants, including variety, climate, growing conditions, cultivation practices, maturity at harvest, and storage conditions[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. For example, blackcurrant grown in high-latitude regions of Finland tend to have higher AsA content compared to those grown in lower latitudes[\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e]. The AsA content in blackcurrant fruits also varies under different light intensities, with shading leading to a decrease in AsA content[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. Additionally, the AsA content changes during the ripening process of blackcurrant fruits. Under natural growth conditions, the accumulation of AsA in blackcurrant begins in the early stages of berry growth and reaches a stable period at fruit maturity[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. This study also found that the AsA levels were highest during the early fruiting stage in the two varieties, 'Adelinia' and 'Heifeng', and gradually decreased as the fruits matured, with the lowest content observed at the ripe stage. In addition, the synthesis of AsA in blackcurrant occurs through the L-galactose pathway[\u003cspan citationid=\"CR30\" class=\"CitationRef\"\u003e30\u003c/span\u003e], which is consistent with apple (\u003cem\u003eMalus domestica\u003c/em\u003e), kiwifruit (\u003cem\u003eActinidia\u003c/em\u003e), tomato (\u003cem\u003eSolanum lycopersicum\u003c/em\u003e), and citrus fruits[\u003cspan citationid=\"CR31\" class=\"CitationRef\"\u003e31\u003c/span\u003e]. In the AsA recycling pathway of blackcurrant, ascorbate peroxidase (APX), dehydroascorbate reductase (DHAR), monodehydroascorbate reductase (MDHAR), and L-galactono-1,4-lactone dehydrogenase (GalLDH) collectively regulate the total AsA content in blackcurrant fruits[\u003cspan citationid=\"CR29\" class=\"CitationRef\"\u003e29\u003c/span\u003e]. In this study, a total of 26 genes were identified from the AsA biosynthesis and recycling pathway based on transcriptome sequencing results. Among them, the expression of a \u003cem\u003ePMM\u003c/em\u003e (c184228.graph_c0), an \u003cem\u003eAPX\u003c/em\u003e (c176242.graph_c0), a \u003cem\u003eGME\u003c/em\u003e (c181107.graph_c0), and a \u003cem\u003ePGI\u003c/em\u003e (c181113.graph_c0) gene have a strong correlation with AsA content. These genes may be crucial for the AsA biosynthesis pathway in blackcurrant. Similarly, the suppressed expression of the \u003cem\u003ePMM\u003c/em\u003e gene caused a substantial decrease in AsA content in tobacco[\u003cspan citationid=\"CR32\" class=\"CitationRef\"\u003e32\u003c/span\u003e]. In tomato, the overexpression of the \u003cem\u003eSlGMEs\u003c/em\u003e gene enhanced AsA accumulation[\u003cspan citationid=\"CR33\" class=\"CitationRef\"\u003e33\u003c/span\u003e]. However, the expression of 2 \u003cem\u003eAPX\u003c/em\u003e (c176953.graph_c0, c172886.graph_c0) gene, 2 \u003cem\u003eAO\u003c/em\u003e (c184540.graph_c1, c185556.graph_c0) gene, and an \u003cem\u003eMDHAR\u003c/em\u003e (c172058.graph_c0) gene showed strong negative correlation with AsA content. These genes may be crucial for the AsA recycling pathway in blackcurrant. Consist with our result, Shu et al. identified an \u003cem\u003eAcAO1\u003c/em\u003e and an \u003cem\u003eAcAPX2\u003c/em\u003e gene from comprehensive metabolic analysis of kiwifruit during ripening, and the overexpression of the two genes led to reduced L-AsA and total AsA content in tobacco[\u003cspan citationid=\"CR34\" class=\"CitationRef\"\u003e34\u003c/span\u003e]. The study on AsA indicated that the overexpression of chloroplastic monodehydroascorbate reductase (\u003cem\u003eMDHAR\u003c/em\u003e) led to an increase in AsA content, a decrease in DHA content, and an approximately twofold increase in the AsA/DHA ratio in tomato[\u003cspan citationid=\"CR35\" class=\"CitationRef\"\u003e35\u003c/span\u003e]. The DEGs identified in this work will be a valuable gene resource for exploring the molecular mechanism of AsA biosynthesis and breeding of blackcurrant with high AsA levels.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec15\" class=\"Section2\"\u003e \u003ch2\u003e3.2. Flavonoids biosynthesis in \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; during fruit ripening\u003c/h2\u003e \u003cp\u003eFlavonoid compounds, as a type of phenolic substance, possess powerful antioxidant and free radical scavenging activities. There is a wide variety of flavonoids, including anthocyanins, flavanols, flavones, flavanones, and flavonols[\u003cspan citationid=\"CR36\" class=\"CitationRef\"\u003e36\u003c/span\u003e]. Flavonoids play important roles in plant defense against microbial stress[\u003cspan citationid=\"CR37\" class=\"CitationRef\"\u003e37\u003c/span\u003e] as well as in promoting human health[\u003cspan citationid=\"CR38\" class=\"CitationRef\"\u003e38\u003c/span\u003e]. Among all the components of flavonoids, myricetin has the highest content in blackcurrants, followed by quercetin[\u003cspan citationid=\"CR39\" class=\"CitationRef\"\u003e39\u003c/span\u003e]. The content of flavonoids shows significant variations at different stages of fruit development. The flavonoid content in \u003cem\u003eRubus\u003c/em\u003e fruits[\u003cspan citationid=\"CR40\" class=\"CitationRef\"\u003e40\u003c/span\u003e] and navel oranges [\u003cspan citationid=\"CR41\" class=\"CitationRef\"\u003e41\u003c/span\u003e] has been observed to decline with fruit maturation. In this study, the peak flavonoid content was observed in young fruit, while the minimum was observed in fruits during the coloration stage. Similarly, most of the genes involved in flavonoid biosynthesis showed their maximum expression levels in young fruit, consistent with the changes in flavonoid contents during fruit ripening. The DEGs in the comparison groups Y1 vs Y2, H1 vs H2, Y2 vs Y3, and H2 vs H3 were significantly enriched in both phenylpropanoid biosynthesis and flavonoid biosynthesis pathway. These findings may account for the pronounced variations in flavonoid synthesis and accumulation at different stages of fruit development. The molecular mechanisms regulating flavonoid synthesis have been elucidated in many plants. In recent years, significant progress has also been made in research on flavonoid synthesis in fruit trees, including grapes, blueberries, apricots[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], blackberries[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e], and apples. Flavonoid synthesis begins with phenylalanine. The health benefits of blackcurrants are partly attributed to their high flavonoid content, so it is important to elucidate the molecular mechanism of flavonoid accumulation in blackcurrants. In melon, flavonoid accumulation is regulated by the up-regulation of \u003cem\u003e4CL\u003c/em\u003e, \u003cem\u003eF3H\u003c/em\u003e, \u003cem\u003eF3'H\u003c/em\u003e, \u003cem\u003eIFS\u003c/em\u003e, \u003cem\u003eFNS\u003c/em\u003e, and \u003cem\u003eFLS\u003c/em\u003e genes and the down-regulation of \u003cem\u003eANS\u003c/em\u003e, and \u003cem\u003eUFGT\u003c/em\u003e genes[\u003cspan citationid=\"CR43\" class=\"CitationRef\"\u003e43\u003c/span\u003e]. A study on apricot fruits has demonstrated the \u003cem\u003e4CL\u003c/em\u003e gene transcript level has a strong positive correlation with flavonoid contents[\u003cspan citationid=\"CR42\" class=\"CitationRef\"\u003e42\u003c/span\u003e]. \u003cem\u003eCHS\u003c/em\u003e was identified as the backbone gene for flavonoid biosynthesis in sweet orange[\u003cspan citationid=\"CR44\" class=\"CitationRef\"\u003e44\u003c/span\u003e]. Here, we identified 19 candidate genes associated with flavonoid biosynthesis. Among them, the expressions of \u003cem\u003e4CL\u003c/em\u003e gene (c183421.graph_c1), \u003cem\u003eCHS\u003c/em\u003e genes (c115942.graph_c0, c183294.graph_c1), \u003cem\u003eCHI\u003c/em\u003e genes (c157264.graph_c0, c163054.graph_c0), \u003cem\u003eDFR\u003c/em\u003e gene (c183820.graph_c4), \u003cem\u003eANS\u003c/em\u003e gene (c187123.graph_c2), \u003cem\u003eANR\u003c/em\u003e gene (c183720.graph_c1) and \u003cem\u003eLAR\u003c/em\u003e gene (c162573.graph_c1) followed the same trend as that of flavonoid content in fruits. These genes may be crucial for flavonoid biosynthesis in blackcurrants. Consistent with our results, Zhou et al. reported that the expression of \u003cem\u003eCHS\u003c/em\u003e, \u003cem\u003eDFR\u003c/em\u003e, and \u003cem\u003eANS\u003c/em\u003e genes peaked at the early stages, decreased when fruits turned white from green, and then increased during anthocyanin biosynthesis in strawberry[\u003cspan citationid=\"CR45\" class=\"CitationRef\"\u003e45\u003c/span\u003e]. Our results provide valuable information on the dynamic changes in flavonoid content and the molecular mechanism of flavonoid accumulation during the maturation of blackcurrants in the future.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec16\" class=\"Section2\"\u003e \u003ch2\u003e3.3. The difference between \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; in fruit weight\u003c/h2\u003e \u003cp\u003eFruit size is one of the most important indicators of fruit appearance and quality, and it is one of the breeding objectives that breeders cannot ignore. Fruit size is affected by a variety of factors such as varietal characteristics, genetic factors, plant hormones, cultivation, and management levels, and is a quantitative trait with multi-gene control. The fruit weight of different varieties of blackcurrant varies greatly[\u003cspan citationid=\"CR46\" class=\"CitationRef\"\u003e46\u003c/span\u003e], and our data also show that \u0026lsquo;Adelinia\u0026rsquo; fruit weight at maturity can be up to three times that of \u0026lsquo;Heifeng\u0026rsquo;. The influence of genetic factors on fruit size is mainly determined by cell division and expansion, and are the most fundamental factors in determining fruit size. Plant hormones play an important role in fruit development and fruit size. For example, BRASSINOSTEROID INSENSITIVE1 (\u003cem\u003eBRI1\u003c/em\u003e) Enlarges Cell Size by Regulating the Cooperation of BR-GA Signaling in Tomato[\u003cspan citationid=\"CR47\" class=\"CitationRef\"\u003e47\u003c/span\u003e]. Hence, the genes involved in the regulation of cell development and plant hormone signaling are important regulators of fruit size. When studying the influencing factors of fruit size in apricot, Huang et al. identified a series of DEGs involved in plant hormone signal transduction, biosynthesis, and metabolism based on the KEGG enrichment results[\u003cspan citationid=\"CR48\" class=\"CitationRef\"\u003e48\u003c/span\u003e]. Genes associated with fruit size in the early stage of fruit development in \u003cem\u003ePyrus pyrifolia\u003c/em\u003e significant enrichment in the plant hormone signal transduction passway of zeatin and gibberellin[\u003cspan citationid=\"CR49\" class=\"CitationRef\"\u003e49\u003c/span\u003e]. The main purpose of plant photosynthesis is to produce carbohydrates, an important energy material for plants to carry out various life activities. An adequate supply of carbohydrates plays an important role in plant growth[\u003cspan citationid=\"CR50\" class=\"CitationRef\"\u003e50\u003c/span\u003e]. Autotetraploid and diploid mulberry trees, with large fruit size differences, DEGs significant enrichment in the plant hormone signal transduction pathways, and many DEGs were involved in photosynthesis[\u003cspan citationid=\"CR51\" class=\"CitationRef\"\u003e51\u003c/span\u003e]. In our study, the difference in fruit size and weight between the two varieties increased significantly after the expansion stage, at the same time, KEGG pathway enrichment analysis revealed that DEGs were significantly enriched in the plant hormone signal transduction (ko04075) pathway in the expansion fruit stage of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo;, in photosynthesis-antenna proteins (ko00196) pathway in the veraison fruit stage and ripe fruit stage. Combined with the above results, we believe that the fruit size of blackcurrant is also affected by the regulation of hormone signals and photosynthesis. To clarify the specific regulation mechanism of different kinds of hormones on fruit size, we analyzed the DEGs involved in auxin, cytokinine, gibberellin, ethylene, abscisic acid, brassinosteroid, jasmonic acid, and salicylic acid signal transduction pathway. Among them, a total of 9 genes had different gene expression levels between the \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; varieties after the expansion stage. Such as \u003cem\u003eARG7\u003c/em\u003e (c174082.graph_c0), \u003cem\u003eIAA7\u003c/em\u003e (c159328.graph_c0), and \u003cem\u003eIAA11\u003c/em\u003e (c164719.graph_c0) in the expansion fruit stage. \u003cem\u003eAGR7\u003c/em\u003e (c167451.graph_c0) in the expansion and veraison fruit stage. \u003cem\u003eEBF1\u003c/em\u003e (c168698.graph_c0, c168698.graph_c1) and \u003cem\u003eEIN3\u003c/em\u003e (c180971.graph_c4) gene in the expansion fruit. \u003cem\u003ePP2C\u003c/em\u003e (c176253.graph_c0) in the expansion fruit. \u003cem\u003ePP2C\u003c/em\u003e (c177479.graph_c0) and \u003cem\u003ePYR1\u003c/em\u003e (c141701.graph_c0) genes in the ripe fruit. \u003cem\u003eSlARF9\u003c/em\u003e affects cell division in early tomato fruit development, \u003cem\u003eSlARF9-OE\u003c/em\u003e fruit cells were on average bigger than in wild-type fruits, inversely the \u003cem\u003eSlARF9\u003c/em\u003e-RNAi lines, cells were smaller[\u003cspan citationid=\"CR52\" class=\"CitationRef\"\u003e52\u003c/span\u003e]. Silencing of \u003cem\u003eSl-IAA17\u003c/em\u003e resulted in the production of larger fruit than the wild type[\u003cspan citationid=\"CR53\" class=\"CitationRef\"\u003e53\u003c/span\u003e]. \u003cem\u003eMdKNOX19\u003c/em\u003e binds directly to the \u003cem\u003eMdABI5\u003c/em\u003e promoter to activate expression-enhanced ABA sensitivity-affected organ development and fruit size[\u003cspan citationid=\"CR54\" class=\"CitationRef\"\u003e54\u003c/span\u003e]. genome-edited mutants of tomato ETHYLENE INSENSITIVE 2(\u003cem\u003eSlEIN2\u003c/em\u003e) decreased auxin biosynthesis to reduce fruit size[\u003cspan citationid=\"CR55\" class=\"CitationRef\"\u003e55\u003c/span\u003e]. In brief, the above genes may be important influencing factors in cell enlargement and fruit enlargement, and they regulate plant growth and development by regulating the synthesis and metabolism of various hormones. The results provide new insights into the molecular mechanisms underlying fruit development and may be useful for improving fruit yield and quality.\u003c/p\u003e \u003c/div\u003e"},{"header":"4. Conclusions","content":"\u003cp\u003eIn this study, we generated a high-quality, functionally annotated reference transcriptome for blackcurrant. Based on the transcriptome, a comparison of different developmental stages and different varieties was performed and DEGs were detected. Detection of differentially expressed genes led to the identification of regulatory genes associated with the AsA biosynthesis and recycling, flavonoids biosynthesis pathways. A large number of differentially expressed genes were also found to be involved in plant hormone signal transduction. In addition, the accuracy of the RNA-Seq results was further verified by qRT-PCR. Our study provides insights into AsA biosynthesis and recycling, flavonoid biosynthesis, and fruit size regulation in blackcurrant.\u003c/p\u003e"},{"header":"5. Materials and methods","content":"\u003cdiv id=\"Sec19\" class=\"Section2\"\u003e \u003ch2\u003e5.1. Plant materials and growth condition\u003c/h2\u003e \u003cp\u003eTwo varieties of blackcurrants, 'Adelinia' and 'Heifeng', were used as samples in this study. These plants were grown in the germplasm nursery of Northeast Agricultural University, Harbin, China, at coordinates 44\u0026deg;04\u0026rsquo; N, 125\u0026deg;42\u0026rsquo; E, under natural conditions. Adelinia is an early-maturing variety, while Heifeng is a late-maturing variety. Four stages of fruit development were collected, including young fruit, expansion fruit, veraison fruit, and ripe fruit. Samples were immediately frozen in liquid nitrogen and stored at -80\u0026deg;C for RNA extraction.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec20\" class=\"Section2\"\u003e \u003ch2\u003e5.2. Determination of Soluble solids, titratable acid, Total phenol, flavonoids, and ascorbic acid\u003c/h2\u003e \u003cp\u003eThe soluble solids were measured using a refractometer, according to the GB/T 12295\u0026thinsp;\u0026minus;\u0026thinsp;1990 method. Titratable acid content was measured using acid-base titration, according to the GB/T 12295\u0026thinsp;\u0026minus;\u0026thinsp;1990 method. Total phenol was measured using the method reported by John[\u003cspan citationid=\"CR56\" class=\"CitationRef\"\u003e56\u003c/span\u003e] with some modifications. Flavonoid content was determined using the method reported by Zou[\u003cspan citationid=\"CR57\" class=\"CitationRef\"\u003e57\u003c/span\u003e]. According to the manufacturer's instructions, AsA content was determined using a Solarbio detection kit. Three fruits from different trees were used for each measurement.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec21\" class=\"Section2\"\u003e \u003ch2\u003e5.3. Library construction, and sequencing\u003c/h2\u003e \u003cp\u003eTotal RNAs were extracted from the collected fruit samples using the CTAB method. The RNA quality was assessed using agarose gel electrophoresis and RNA quantity was determined by an Agilent 2100 Bioanalyzer (Palo Alto, USA). Strand-specific RNA-Seq libraries were constructed and sequenced on the Illumina HiSeq 2000 platform by Biomarker Technologies (Beijing, China). Three biological replicates of each fruit sample were used.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec22\" class=\"Section2\"\u003e \u003ch2\u003e5.4. RNA-Seq data processing, and transcriptome assembly\u003c/h2\u003e \u003cp\u003eRaw RNA-Seq reads were processed using in-house Perl scripts to trim adapters and low-quality sequences. The transcriptome was assembled based on the left.fq and right.fq using Trinity[\u003cspan citationid=\"CR58\" class=\"CitationRef\"\u003e58\u003c/span\u003e] with the minimum Kmer coverage set to 2. Gene function was annotated based on NR (NCBI non-redundant protein sequences), Pfam (Protein family), KOG/COG/eggNOG (Clusters of Orthologous Groups of proteins), Swiss-Prot (A manually annotated and reviewed protein sequence database), KEGG (Kyoto Encyclopedia of Genes and Genomes) and GO (Gene Ontology) databases. GO functional enrichment analysis of genes in major KEGG enriched pathways was performed using BMKCloud (\u003cspan class=\"ExternalRef\"\u003e\u003cspan class=\"RefSource\"\u003e\u003ca href=\"http://www.biocloud.net\" target=\"_blank\"\u003ewww.biocloud.net\u003c/a\u003e\u003c/span\u003e\u003cspan address=\"http://www.biocloud.net\" targettype=\"URL\" class=\"RefTarget\"\u003e\u003c/span\u003e\u003c/span\u003e). For convenience, the young fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were labeled as Y1 and H1. The expansion fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were labeled as Y2 and H2, the veraison fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were labeled as Y3 and H3, and the ripe fruits of \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; were labeled as Y4 and H4, respectively.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec23\" class=\"Section2\"\u003e \u003ch2\u003e5.5. Identification of differentially expressed genes (DEGs)\u003c/h2\u003e \u003cp\u003eDifferential expression analysis was performed using the DESeq R package to identify DEGs between the two groups of samples collected at different stages of fruit development, including young fruit, expansion fruit, veraison fruit, and ripe fruit, for Adelinia and Heifeng varieties. For identifying differentially expressed genes (DEGs), a fold change (FC)\u0026thinsp;\u0026ge;\u0026thinsp;2 and false discovery rate (FDR)\u0026thinsp;\u0026lt;\u0026thinsp;0.01 were used as the screening criteria. FC represents the ratio of gene expression values in two groups. Genes with an adjusted P-value\u0026thinsp;\u0026lt;\u0026thinsp;0.05 determined by DESeq were assigned as differentially expressed.\u003c/p\u003e \u003c/div\u003e \u003cdiv id=\"Sec24\" class=\"Section2\"\u003e \u003ch2\u003e5.6. Quantitative Reverse Transcription PCR (qRT-PCR)\u003c/h2\u003e \u003cp\u003eGene expression analysis was performed for each sample using RT-qPCR. Total RNA from \u0026lsquo;Adelinia\u0026rsquo; and \u0026lsquo;Heifeng\u0026rsquo; fruit was extracted using the Biomarker Plant Total RNA Isolation Kit (Polysaccharides \u0026amp; Polyphenolics\u0026ndash;rich) according to the manufacturer\u0026rsquo;s instructions. The RNA concentration and quality were measured using an ultraviolet spectrophotometer (NanoPhotometer NP80, IMPLEN) and 1% agarose gel electrophoresis. cDNA was synthesized from 500 ng of the total RNA using ReverTra Ace qPCR RT Master Mix (TOYOBO) according to the manufacturer\u0026rsquo;s instructions. Specific primers for qRT-PCR are listed in Table \u003cspan refid=\"MOESM1\" class=\"InternalRef\"\u003eS1\u003c/span\u003e, which were designed using Premier 5.0.\u003c/p\u003e \u003cp\u003eThe qRT-PCR reaction was performed in a 20 \u0026micro;L reaction volume containing 10 \u0026micro;L of SYBR\u0026reg;Green Realtime PCR Master Mix (TOYOBO), 2 \u0026micro;L of cDNA, 0.8\u0026micro;L of each forward and reverse primer, and 6.4 \u0026micro;L ddH\u003csub\u003e2\u003c/sub\u003eO. The analysis was carried out on three biological replicates on a qTOWER3 fluorescence quantitative PCR instrument (Analytik Jena). qRT-PCR was carried out under the following conditions: denaturation at 95 ◦C for 3 min prior to 45 amplification cycles (95\u0026deg;C for 5 s, 60\u0026deg;C for 30 s), followed by melt-curve analysis (60\u0026ndash;95\u0026deg;C, 1\u0026deg;Cincrement for 15 s per step) to test the amplicon specificity. Relative expression was assessed by the 2\u003csup\u003e\u0026minus;∆∆CT\u003c/sup\u003e method using \u003cem\u003eActin\u003c/em\u003e (c178043.graph_c0) as the internal control gene[\u003cspan citationid=\"CR59\" class=\"CitationRef\"\u003e59\u003c/span\u003e].\u003c/p\u003e \u003c/div\u003e"},{"header":"Abbreviations","content":"\u003cdiv class=\"DefinitionList\"\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDEGs\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDifferentially expressed genes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eqRT-PCR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eQuantitative real-time PCR\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGene ontology\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKOG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eClusters of orthologous groups\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eKEGG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eKyoto encyclopedia of genes and genomes\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAO\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbate oxidas\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAPX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAscorbate peroxidase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGalDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eL-galactose dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGGP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGDP-L-galactose-phosphorylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGalLDH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eL-galactono-1,4-lactone dehydrogenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGME\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGDP-D-mannose3\u0026prime;, 5\u0026prime;-epimerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGMP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGDP-mannose pyrophosphorylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGPP\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eL-galactose-1-phosphate phosphatase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGSSH\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eOxidized glutathione\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMDHAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMonodehydroascorbate reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eMIOX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eMyoinositol oxygenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePGI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphoglucose isomerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphomannose isomerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003ePMM\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003ePhosphomannomutase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eGR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eGlutathione reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003e4CL\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003e4-coumarate-CoA\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChalcone synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eCHI\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eChalcone isomerase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eF3H\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFlavanone-3-hydroxylase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eDFR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eDihydro flavonol reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLDOX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLeucoanthocyanidin dioxygenase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANS\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnthocyanin synthase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eANR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAnthocyanidin reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eUFGT\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eFlavonoid 3-O-glucosyl transferase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eLAR\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eLeucoanthocyanidin reductase\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eARG\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eArgentina\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eAUX\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eAuxin\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003cdiv class=\"DefinitionListEntry\"\u003e \u003cdiv class=\"Term\"\u003eIAA\u003c/div\u003e \u003cdiv class=\"Description\"\u003e \u003cp\u003eIndole acetic acid.\u003c/p\u003e \u003c/div\u003e \u003c/div\u003e \u003c/div\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgments\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eWe thank all contributors for their work and would like to thank the editor and reviewers for their valuable comments and suggestions.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAuthors\u0026rsquo; contribution\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eDQ and JWH designed and managed the experiments. HXG and XLZ drafted and revised the manuscript. HXG and DNZ conducted the sequence data analysis. XLZ performed the experiments. The authors have carefully read and approved the final manuscript.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFunding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis research was supported by the following fundings: National Key R\u0026amp;D Program of China (2022YFD1600500), Academic Backbone Project of Northeast Agricultural University (20XG04), China Postdoctoral Science Foundation (2022MD713724) and Central Government Supports the Reform and Development Fund Talent Training Project of Local Colleges and Universities, China.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eAvailability of data and materials\u003c/strong\u003e\u003c/p\u003e\n\u003cp skip=\"true\"\u003eAll data generated or analyzed during this study are included in this published article and its supplementary information fles. The raw RNA-Seq data in this manuscript are available for downloading from the NCBI Sequence Read Archive (BioProject ID: PRJNA1021373). https://www.ncbi.nlm.nih.gov/sra/PRJNA1021373\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthics approval and consent to participate\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe fruits of blackcurrant in this study was collected from the germplasm nursery of Northeast Agricultural University, Harbin, China. No specific permits are required for sample collection. The use of plant parts in present study compiles with international, national and/or institutional guidelines.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eConsent for publication\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eNot applicable.\u0026nbsp;\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eCompeting interests\u0026nbsp;\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe authors declare that they have no competing interests.\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\u003cli\u003e\u003cspan\u003eBordonaba JG, Terry LA. Biochemical profiling and chemometric analysis of seventeen UK-grown black currant cultivars. J Agric Food Chem. 2008;56(16):7422\u0026ndash;30.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eWoznicki TL, Sonsteby A, Aaby K, Martinsen BK, Heide OM, Wold AB, Remberg SF. Ascorbate pool, sugars and organic acids in black currant (Ribes nigrum L.) berries are strongly influenced by genotype and post-flowering temperature. J Sci Food Agric. 2017;97(4):1302\u0026ndash;9.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eBender C, Killermann KV, Rehmann D, Weidlich HH. Effect of mash enzyme and heat treatments on the cellular antioxidant activity of black currant (Ribes nigrum), raspberry (Rubus idaeus), and blueberry (Vaccinium myrtillus) juices. CyTA-J Food. 2017;15(2):277\u0026ndash;83.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eLee Y, Pham TX, Bae M, Hu S, O'Neill E, Chun OK, Han MJ, Koo SI, Park YK, Lee JY. Blackcurrant (Ribes nigrum) Prevents Obesity-Induced Nonalcoholic Steatohepatitis in Mice. Obesity. 2019;27(1):112\u0026ndash;20.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eKim B, Bae M, Park YK, Ma H, Yuan T, Seeram NP, Lee JY. 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Comprehensive Transcriptome Profiling Reveals Long Noncoding RNA Expression and Alternative Splicing Regulation during Fruit Development and Ripening in Kiwifruit (Actinidia chinensis). Front Plant Sci. 2016;7:15.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eGupta V, Estrada AD, Blakley I, Reid R, Patel K, Meyer MD, Andersen SU, Brown AF, Lila MA, Loraine AE. RNA-Seq analysis and annotation of a draft blueberry genome assembly identifies candidate genes involved in fruit ripening, biosynthesis of bioactive compounds, and stage-specific alternative splicing. GigaScience. 2015;4:22.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eShu B, Li WC, Liu LQ, Wei YZ, Shi SY. Transcriptomes of Arbuscular Mycorrhizal Fungi and Litchi Host Interaction after Tree Girdling. Front Microbiol. 2016;7:14.\u003c/span\u003e\u003c/li\u003e \u003cli\u003e\u003cspan\u003eZhu YY, Li YQ, Xin DD, Chen WR, Shao X, Wang Y, Guo WD. 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Aldoniai Plants-Basel. 2022;11(22):11.\u003c/span\u003e\u003c/li\u003e\u003c/ol\u003e"}],"fulltextSource":"","fullText":"","funders":[],"hasAdminPriorityOnWorkflow":false,"hasManuscriptDocX":true,"hasOptedInToPreprint":true,"hasPassedJournalQc":"","hasAnyPriority":false,"hideJournal":true,"highlight":"","institution":"","isAcceptedByJournal":false,"isAuthorSuppliedPdf":false,"isDeskRejected":"","isHiddenFromSearch":false,"isInQc":false,"isInWorkflow":false,"isPdf":false,"isPdfUpToDate":true,"isWithdrawnOrRetracted":false,"journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true},"keywords":"Ribes nigrum L., Transcriptional analysis, AsA, Flavonoids, Plant hormone, qRT-PCR, Expression analysis","lastPublishedDoi":"10.21203/rs.3.rs-3330314/v1","lastPublishedDoiUrl":"https://doi.org/10.21203/rs.3.rs-3330314/v1","license":{"name":"CC BY 4.0","url":"https://creativecommons.org/licenses/by/4.0/"},"manuscriptAbstract":"\u003ch2\u003eBackground\u003c/h2\u003e \u003cp\u003eThe blackcurrant (\u003cem\u003eRibes nigrum\u003c/em\u003e L.) is rich in nutritional value. It is rich in ascorbic acid and flavonoids with excellent antioxidant properties. Fruit quality is an important factor affecting subsequent processing and production, yet the molecular mechanisms underlying the transcriptional regulation of blackcurrant fruit quality are largely unknown.\u003c/p\u003e\u003ch2\u003eResults\u003c/h2\u003e \u003cp\u003eIn the current study, comparative transcriptome analysis investigated the similarities and differences between two blackcurrant varieties: 'Adelinia' and 'Heifeng' at four fruit developmental stages (young, expansion, veraison, and ripe fruit). Additionally, we observed variations in a number of physiological indicators during the growth of blackcurrant fruits. The results indicate that, during the development of blackcurrant fruits, ascorbic acid content continued to decrease, whereas flavonoid content was lowest in the veraison period. The fruit size of 'Adelinia' variety was larger than that of 'Heifeng' variety throughout the developmental. Based on the results of the transcriptome sequencing data, a total of 4295 up-regulated genes and 3529 down-regulated genes were obtained between 'Adelinia' and 'Heifeng' varieties; A total of 6,349 up-regulated and 5,770 down-regulated genes were identified between the four developmental periods, respectively. The expression of 4 genes, \u003cem\u003ePMM\u003c/em\u003e, \u003cem\u003eAPX\u003c/em\u003e, \u003cem\u003eGME\u003c/em\u003e, and \u003cem\u003ePGI\u003c/em\u003e has a strong correlation with AsA content. while two \u003cem\u003eAPX\u003c/em\u003e, two \u003cem\u003eAO\u003c/em\u003e, and one \u003cem\u003eMDHAR\u003c/em\u003e genes showed a strong negative correlation with AsA content. A total of 19 genes related to flavonoid biosynthesis were screened. The KEGG pathway enrichment analysis showed that many DEGs were enriched in the plant hormone signal transduction (ko04075) pathway and the photosynthesis-antenna proteins (ko00196) pathway.\u003c/p\u003e\u003ch2\u003eConclusions\u003c/h2\u003e \u003cp\u003eWe predicted that the \u003cem\u003ePMM, APX, GME\u003c/em\u003e, and \u003cem\u003ePGI\u003c/em\u003e genes, which are positively correlated with ascorbic acid, may play an important role in the biosynthesis of AsA; while the negatively correlated \u003cem\u003eAPX, AO\u003c/em\u003e, and \u003cem\u003eMDHAR\u003c/em\u003e genes may be critical for AsA in the recycling pathway. \u003cem\u003eDFR, ANR, CHI, CHS, 4CL\u003c/em\u003e, and \u003cem\u003eLAR\u003c/em\u003e genes are important regulators of flavonoid synthesis. Also predicted the regulatory influences of various plant hormones on the fruit size of blackcurrants. We provide genetic resources for improving fruit quality and size as well as for rationalizing the use of plant growth regulators to improve fruit quality.\u003c/p\u003e","manuscriptTitle":"Transcriptome profiling reveals the regulatory mechanisms of ascorbic acid and flavonoid synthesis and metabolic processes in fruit development of Ribes nigrum","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2023-10-06 21:06:43","doi":"10.21203/rs.3.rs-3330314/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"10f20c8c-0ece-4b97-bb7d-b0c29b640378","owner":[],"postedDate":"October 6th, 2023","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"posted","subjectAreas":[],"tags":[],"updatedAt":"2024-02-08T09:29:59+00:00","versionOfRecord":[],"versionCreatedAt":"2023-10-06 21:06:43","video":"","vorDoi":"","vorDoiUrl":"","workflowStages":[]},"version":"v1","identity":"rs-3330314","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-3330314","identity":"rs-3330314","version":["v1"]},"buildId":"-HB7Z8yhvgn0wM9Nzuekk","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}