Genome-wide identification of UGT gene family and their expression profiles analysis during fruit development in walnut (Juglans regia L.)

doi:10.21203/rs.3.rs-4288373/v1

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Genome-wide identification of UGT gene family and their expression profiles analysis during fruit development in walnut (Juglans regia L.)

https://doi.org/10.21203/rs.3.rs-4288373/v1

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Walnut (Juglans regia L.) possess the ability to prevent coronary heart disease and promote cardiovascular health, which can be attributed to their rich content of polyphenols, particularly flavonoids. The biosynthesis of flavonoids is reliant on the catalytic activity of uridine diphosphate glycosyltransferase (UGT). However, the identification of UGTs in walnut has not been reported. In the current study, a total of 124 UGT genes containing the PSPG box were identified from the walnut genome. Based on phylogenetic analysis, the 124 UGT could be classified into 16 distinct groups, which exhibited an uneven distribution across the 16 chromosomes. Subcellular localization prediction analysis revealed that approximately 78.23% of walnut UGT proteins were predominantly localized in the cytoplasmic compartment. Furthermore, motif annotation confirmed that motifs 1, 2, and 3 represented conserved structural features within UGT proteins, while interestingly, around 56.5% of walnut UGT members lacked introns. In conjunction with transcriptome analysis and quantitative expression, approximately 39% of UGT genes in walnut exhibited high expression levels during early fruit development. Correlation analysis between UGT genes expression and phenolic content in walnut indicated that JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 might be involved in phenolic biosynthesis in walnut. This comprehensive study provides a comprehensive overview of the UGT genes in walnut, serving as a valuable reference and theoretical foundation for further investigations into the biological functions of JrUGTs in flavonoid biosynthesis.

Walnut

UDP-glycosyltransferase

Gene family

Gene expression

Walnut (Juglans regia L.) is a member of the Juglans family and extensively cultivated worldwide. Many evidence suggested that walnuts possess the ability to prevent coronary heart disease and promote cardiovascular health, which can be attributed to their rich content of polyphenols, particularly flavonoids (Feldman et al. 2002; Mc et al. 2012). Flavonoids, as secondary metabolites in plants, represent a class of polyphenolic compounds that exhibit potent biological activity and are widely distributed throughout the plant kingdom. The biosynthesis of flavonoids relies on the catalytic function of uridine diphosphate glycosyltransferase family (UGT), especially Uridinediphosphate Glycosyltransferase (Shirley et al. 2001). Glycosylation, an important modification reaction in plants, plays a crucial role in growth and response to biotic and abiotic stresses, and it also enhances solubility, stability, transferability, and diversity of various plant secondary metabolites such as flavonoids (Vogt et al. 2000; Bowles et al. 2006; Yang et al. 2018; Naeem et al. 2021).

Glycosylation modification is catalyzed by glycosyltransferases (GTs), which are highly diverse, multifunctional enzymes belonging to a polymorphic gene family (Mackenzie et al. 1997). According to the latest update on CAZy (http://www.cazy.org/Glycosyl Transferases.html), GTs can be classified into 115 distinct species based on amino acid sequence similarity, catalytic mechanism, and the presence of conserved sequence motifs (Chen et al. 2022). Among these, the GT1 family, commonly referred to as uridine diphosphate glycosyltransferases (UGTs), represents the predominant group in plants and exerts significant influence on plant growth and development (Yonekura et al. 2011). The UGTs not only glycosylate receptor molecules, such as anthocyanins, flavonols, flavonoids, saponins, sterol terpenes, phenylpropanes and plant hormones but also play a crucial role in the detoxification and inactivation of foreign organisms and plant-pathogen interactions (Vogt et al. 2000; Caputi et al. 2012). The UGT protein exhibits a highly conserved sequence of 44 amino acids (aa) near the C-terminal region and is referred to as the plant secondary product glycosyl transferase (PSPG) (Zhang et al. 2020). Moreover, UGT demonstrates broad substrate specificity in plants by recognizing multiple receptor molecules selectively (Taguchi et al. 2000; Lim et al. 2003).

A growing number of putative UGT-coding genes have been discovered in plants, with 107 members identified in Arabidopsis thaliana, which were categorized into 14 groups based on their amino acid sequences and designated as groups A-N (Li et al. 2001). Subsequently, Zea mays was found to possess 147 UGT genes, Vitis vinifera had 181, Malus x domestica contained 241, Citrus grandis 145 and Camellia sinensis harbored 178. (Velasco et al. 2010; Li et al. 2014; Cui et al. 2016; Wu et al. 2020). Polygenic UGT family members collaborate synergistically to orchestrate intricate biochemical processes in plant cells, thereby exerting profound influence on diverse biological activities and functions. Currently, the functionality of UGT genes has been validated in multiple species. Yang et al (2023) demonstrated the functions of 276 glycosyltransferases in Nicotiana tabacum and observed a significant increase in the levels of quercetin-3-O-glucoside, quercetin-3-O-rutin, and kaempferol-3-O-rutin in transgenic tobacco leaves by overexpressing NtUGT217. In a study by Lim et al (2004), 91 UGT genes were identified in Arabidopsis thaliana using quercetin as a substrate, with 29 of them exhibiting catalytic activity towards related glycosylation reactions. Yao et al (2022) conducted a comprehensive genome-wide analysis of epimedium, revealing that only the recombinant EpGT60 protein exhibited activity against 8-enyl kaempferol and icariin, leading to the biosynthesis of key bioactive compounds pyridoside II and pyridoside I. However, despite the large number of UGT genes in the plant genome, the functional validation of signature proteins remains limited (Caputi et al. 2012).

The present study employed whole genome sequencing to identify the UGT gene family in walnut (JrUGT). Subsequently, sequence alignment and phylogenetic tree analysis were conducted to elucidate the genetic relationships among these UGTs in walnut. Furthermore, predictions regarding the physical and chemical properties of proteins, subcellular localization, chromosome distribution, collinearity, and gene structure were made for JrUGT. Based on this premise, the expression patterns of UGT genes in various developmental stages of walnut kernel pellicle were analyzed and subsequently validated using qRT-PCR. The correlation analysis between JrUGT and phenolic content establishes a fundamental for future investigations into the biosynthesis mechanism and accumulation pattern of walnut phenolics.

Identification of UGT gene family in walnut

The genome sequence information of walnut was primarily obtained from the Juglans genome database (http://www.juglandaceae.net/). Arabidopsis UGT members were predominantly retained from the TAIR database (https://www.arabidopsis.org/). The Hidden Markov Model HMM file for the conserved domain of UGT gene family (PF00201) was accessible through Pfam (http://pfam.xfam.org/). By employing the Arabidopsis UGT amino acid sequence as a query, BLASTP was utilized to search for proteins in the walnut genome with an e-value threshold set at 1e^− 15, thereby constructing UGT candidate dataset 1. The hmmsearch function in TBtools was employed to retrieve the UGT candidate data 2 by utilizing the HMM file of the UGT family as a probe. Subsequently, two sets of candidate data were combined and subjected to verification using the NCBI CDD program, confirming the presence of conserved domain PSPG box in all walnut UGT members.

Analysis of physicochemical properties of UGT gene family

The Expasy online database (http://www.expasy.org/) was utilized for the prediction of physicochemical properties of the amino acid sequence, encompassing relative molecular weight, theoretical isoelectric point, and amino acid length. Additionally, subcellular localization analysis of walnut UGT protein was performed using the PSORT II Prediction online software (https://psort.hgc.jp/form2.html).

Phylogenetic and chromosomal localization analysis

The UGT protein sequences of walnut, Arabidopsis, Zea mays and Pilosella officinarum were aligned with MUSCLE software. Subsequently, phylogenetic trees based on 1000 bootstrap replicates using the neighbor-joining (NJ) method were constructed in MEGA 11 software (Kumar et al. 2016). The genomic localization information of JrUGT members was visually analyzed using TBtools software, as reported by the walnut genome database.

Gene replication and collinearity analysis

The gene replication events were analyzed using the One Step MCScanX program and visualized with the TBtools program (Wang et al. 2012; Chen et al. 2020). To investigate the evolutionary relationship between walnut and Arabidopsis thaliana, the gene sequence of walnut was comprehensively analyzed (Chen et al. 2020).

Conserved sequence and gene structure analysis

The conserved motifs of walnut UGT members were identified using the MEME program (http://meme-suite.org/tools/meme). The conserved motifs recognized by MEME programs were further annotated using the NCBI CDD program, with a total of 10 motifs identified. Additionally, the walnut gene annotation files and coding sequences from its corresponding genome sequence comparison were inputted into the NCBI GSDS online tools (v2.0) at http://gsds.cbi.pku.edu.cn/ to display information on exons and introns of walnut UGT.

Expression pattern analysis of 13 JrUGTs

The kernel pellicle of the walnut varieties 'NongHe 1' (‘NH1’) and 'FenHe 4' (‘FH4’) were collected at different developmental stages, including the hard core stage, the fatty stage and the mature stage. These samples were obtained from the walnut planting resource nursery of the horticultural experimental station of Shanxi Agricultural University. Subsequently, all samples were rapidly frozen in liquid nitrogen and stored at -80℃ in an ultra-low temperature refrigerator for future use. Three biological replicates were performed on each sample. The kernel pellicle of two walnut varieties, ‘NH1’ and ‘FH4’, at various developmental stages were submitted to Baimaike Biotechnology Co., Ltd. for transcriptome sequencing analysis. Transcript abundance was quantified using fragments per kilobase transcript per million fragment mapping (FPKM) values derived from RNA-seq reads. Each gene exhibited distinct expression levels. The tissue-level data was averaged and presented as log² values, followed by the generation of a heat map using TBTOOLS software (Anders et al. 2010).

The total RNA was extracted using the improved CTAB method (Feng et al., 2013), and the first strand cDNA synthesis was performed using the HiScript II 1st Strand cDNA Synthesis Kit (+ gDNA wiper). Specific primers for 13 selected JrUGTs were designed using Primer Premier 5.0 software, and their sequences were provided in Table S1. The relative expression of JrUGTs was determined by qRT-PCR reaction system with the 2^-△△CT method (Livak et al. 2001).

Determination of phenolics

The kernel pellicle was freeze-dried and ground into a fine powder. 0.1 g of the i kernel pellicle powder was measured and added to 5 mL of a 50% methanol solution. Ultrasound-assisted extraction was conducted for 30 min at 50℃, followed by centrifugation at 12000 r /min for 10 min to obtain the supernatant. This process was repeated twice, and the resulting supernatants were combined and adjusted to a constant volume before being analyzed using HPLC. The content of phenolic compounds was determined following the methods described by Lu et al (2017) and Yang et al (2020). The regression equation for the standard curve is presented (Table S2).

Identification of UGT gene family members in walnut

In order to identify JrUGT, UGT proteins in walnut genome database were screened by using HMM file (PF00201) as a query. The screening criterion was set at an e-value < 1. We employed two screening methods, namely BLASTP and hmmsearch, resulting in the identification of 174 potential UGT genes in walnut. To verify the presence of conserved domains, TBtools was employed, and any UGT proteins lacking a PSPG box were manually excluded. Ultimately, 124 JrUGT genes were obtained, and designated as JrUGT1-124.

The 124 JrUGTs genes encoded proteins with diverse physicochemical properties (Table S3). Each member of the JrUGT family had a protein length ranging from 216 to 611 aa, with an average length of 474 aa. The theoretical molecular weight of the protein ranges from 29.29 to 61.50 KDa, with an average value of 52.90 KDa. The theoretical isoelectric point (pI) ranges from 5.17 to 8.1, with an average pI of 5.85. According to the PSORT II Prediction online tool for subcellular localization prediction, majority of the UGT proteins in walnut (78.23%) were localized in the cytoplasm, followed by mitochondria (12.10%). Additionally, eleven UGT proteins were found in the endoplasmic reticulum and one protein was located extracellularly within the cell wall.

Phylogenetic and chromosomal localization analysis

The classification and phylogenetic relationship of UGT proteins in walnut were investigated by constructing a phylogenetic tree based on UGT protein sequences from walnut, Arabidopsis thaliana, Zea mays, and Pilosella officinarum. The main clusters of walnut UGT family members consisted of 18 subgroups, with a total of 124 JrUGTs being classified into these previously identified subgroups (Fig. 1). Notably, JrUGTs were absent from groups F and Q, while the majority of them clustered within groups E (25), G (16), D (19), L (15), and A (15). It has been reported that AtUGT71B1, AtUGT72B1, and AtUGT88A1 of Arabidopsis were involved in flavonoid biosynthesis. Sequence analysis found JrUGT4, JrUGT13, JrUGT31, JrUGT32, JrUGT43, JrUGT44, JrUGT45, JrUGT57, JrUGT58, JrUGT59 JrUGT60, JrUGT61, JrUGT62, JrUGT67, JrUGT68, JrUGT69, JrUGT80, JrUGT81, JrUGT93, JrUGT108, JrUGT111, JrUGT114, JrUGT117, JrUGT118, JrUGT121 clustered with AtUGT71B1, AtUGT72B1, and AtUGT88A1, suggesting that these JrUGT may be involved in flavonoid biosynthesis in walnut.

Among the 124 JrUGTs identified, 124 JrUGTs were located on 16 chromosomes of walnut (Fig. 2). Among the 16 walnut chromosomes, chromosome 9 contained only one UGT family member, while chromosome 1 contains 17 UGT genes. Chromosome 7 contained 15 UGT genes, chromosome 3 contained 13 UGT genes, and chromosomes 2 and 15 each contained only 2 UGT genes. This unbalanced distribution of UGT gene in walnut on chromosome indicates that there was genetic variation in walnut during evolution.

Gene replication and collinearity analysis

Gene replication events play a pivotal role in the formation of gene families. In order to elucidate the expansion and evolution mechanism of UGT gene family in walnut, the potential gene replication events in walnut genome were further investigated. The detection of UGT gene replication events in walnut was performed using Tbtools software. A total of five genes located on chromosomes 1, 2, 4, 9, 10, and 14 were identified to undergo four gene replication events (Fig. 3). Notably, chromosomes 1 and 10 exhibited the highest frequency of tandem repeat events (three occurrences each), implying that UGT genes might have originated from such replication events which could be considered as key drivers for UGT evolution. Furthermore, by comparing the DNA sequence similarity between the UGT gene of walnut and the homologous genes of Arabidopsis thaliana (Fig. 4), a collinear relationship between 39 walnut genes and 36 Arabidopsis genes was found. These conserved genes are likely to possess crucial functions across different species.

Conserved sequence and gene structure analysis

To further elucidate the conserved domain characteristics of the walnut UGT family, 10 motifs were generated using the online tool MEME and then numbered from 1 to 10. Notably, motif 1 and motif 3 corresponded to the highly conserved PSPG box within the UGT family. The distribution pattern of these motifs among different types of walnut UGT members was depicted (Fig. 5). Remarkably, our findings revealed that members belonging to the same group exhibited either identical or similar conserved motifs. The characteristic sequence motif 1–3, present in all walnut UGT proteins, was considered to be the glycosyltransferase recognition site for glycosyl-donor. With a few exceptions, most walnut UGT proteins exhibited the following characteristics: motif 6 was located proximal to motif 2, and motif 5 was also positioned near motif 8. In the majority of sequences, motif 4 appeared at the beginning while motif 7 occured at the end. The protein's sequence typically follows this pattern: motif 4-5-8-6-2-10-1-3-9-7; however, variations existed among certain proteins.

The diversity in intron-exon structure often plays a pivotal role in the evolutionary dynamics of gene families and provides supplementary evidence to support phylogenetic classifications. In order to further understand the gene structure, the intron-exon structure of UGT gene in walnut was analyzed. Out of the 124 UGT genes identified in this study, 47 contained a single intron, 70 were devoid of any introns, while 7 harbored two introns.

Expression patterns of UGT genes

The transcriptomic sequencing results of the kernel pellicle of two walnut varieties, ‘NH1’ and ‘FH4’, at different developmental stages were analyzed in this study to gain further insights into the expression pattern of UGT genes in walnut. The findings revealed that out of 118 UGT genes analyzed in walnut, JrUGT2, JrUGT11, JrUGT68, JrUGT113, JrUGT119 and JrUGT121 were not detected in the transcriptome data. A total of 19 JrUGTs (FPKM > 10) exhibited high transcription levels in ‘NH1’ and ‘FH4’, respectively (Fig. S1). The expression profiles of JrUGT39, JrUGT58, JrUGT75, and JrUGT118 were consistently elevated across all three developmental stages in both varieties, suggesting their potential involvement in diverse biological processes throughout growth and development. Group E, which represents the largest subset of the UGT gene family in walnut, exhibited predominant expression of UGT during the hard core stage in ‘NH1’ and ‘FH4’. Additionally, some genes showed higher expression levels in the mature stage compared to those observed in he hard core stage and the fatty stage. Similarly, Group G also displayed peak expression during the hard core stage with greater abundance detected in ‘FH4’ than ‘NH1’. Most UGT genes demonstrated elevated transcript levels during early fruit development stages but decreased as fruit matured.

In this study, we performed a comprehensive analysis of the walnut UGT gene family through phylogenetic tree analysis and transcriptome differential gene screening. Subsequently, 13 differentially expressed walnut UGT genes were randomly selected for validation using real-time fluorescence quantitative PCR. The qRT-PCR results were presented (Fig. 6). The gene expression levels of JrUGT39, JrUGT58, JrUGT88, and JrUGT95 were found to be higher in August, while JrUGT118 exhibited the lowest gene expression levels during this period. These findings were consistent with the transcriptome data analysis.

Analysis of phenolic substances in walnut kernel pellicle at different periods

HPLC was used to determine the content of ‘NH1’ and ‘FH4’ phenolics in walnut kernel pellicle at different periods. With the growth and development of walnut fruit, the content of phenolics also changed, and the results were shown (Table 1). The content of Gallic acid was the highest among the 11 substances and the content of GCG was the lowest. The content of C was the highest among catechins, and the content of C in ‘FH4’ was significantly higher than that of ‘NH1’. The contents of EC and GC showed a trend of continuous increase on the whole, and the contents of EGC and EGCG in the two varieties showed a trend of first increasing and then decreasing, and the content of EGC in ‘NH1’ fatty stage was significantly higher than that in other periods. The content of ‘FH4’ in syringate was higher than that of ‘NH1’ in different periods.

Correlation analysis of UGT gene expression and phenolic content in walnut

The pearson coefficient was employed to investigate the correlation between UGT gene expression and phenolic substance content in walnut, and the results were presented (Fig. 7). No significant correlation was observed between JrUGT36 and phenolic substances. Only JrUGT118 showed a positive correlation with EGCG and Chlorogenic acid (P < 0.05). JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 exhibited positive correlations with Vanillic acid (P < 0.01). Additionally, JrUGT38, JrUGT39, JrUGT67, JrUGT69, JrUGT75, and JrUGT82 were positively correlated with C, GC, and EC. These findings suggest that these identified UGTs may play a role in the biosynthesis of phenolic substances in walnut; thus providing valuable insights for further study on this topic.

Glycosylation is modified by GTs, which can be divided into at least 111 families, of which the UGT gene family is the largest family (Paquette et al, 2003). Currently, the UGT gene family has been found and analyzed in some plants such as Arabidopsis, Triticum aestivum, Zea mays and Morella rubra (Li et al, 2001; He et al, 2018; Li et al, 2014; Ren et al., 2022). However, there was a lack of information regarding the UGT gene family in walnut. Through BLASTP and hmmsearch screenings along with conserved domain verification, 124 JrUGTs were identified. Compared to other species, the number of UGT genes in walnut showed lower numbers, that 152 UGT genes in Morella rubra and 168 UGT genes in Prunus persica (Wu et al. 2017). This observation suggests that the amplification of the walnut UGT gene family was not significant, possibly due to the absence of genome-wide duplication events in walnut. The protein encoded by the UGT gene of walnut exhibits a wide range of amino acid lengths and molecular weights. The intron content in walnut UGT family members was 43.5%, which was lower compared to Arabidopsis (58%) (Li et al. 2001), Linum usitatissimum (55%) (Barvkar et al. 2012), and Zea mays (60%) (Li et al. 2014). To functionally identify UGT genes in walnut, the phylogenetic tree was constructed to classify the 124 identified UGT genes into 16 groups. In the evolutionary process of higher plants, groups A, L, D, G, and E of the UGT gene family were considered as rapidly evolving groups (Caputi et al. 2012), and these five groups also contain the highest number of UGT gene family members in walnut, consistent with the aforementioned results. The phylogenetic tree showed that group E contained the most UGT genes (25), accounting for 20.2% of the total UGT genes in walnut. There were 17, 22 and 35 UGT genes in group E in Arabidopsis, Linum usitatissimum and Zea mays, respectively, indicating the expansion of group E in different plant species (Barvkar et al., 2012; Caputi et al., 2012). JrUGTs were not distributed in group F and group Q. Group F was first identified in Arabidopsis Thaliana (Li et al., 2001), and group Q was first identified in Zea mays (Li et al., 2014), and this group of genes was thought to be related to cytokinin glycosylation.

124 JrUGTs were located on 16 chromosomes. In Dendrobium nobile and Gossypium, the distribution of genes on chromosomes usually existed in clusters with high homology, and the distribution of UGT gene family on chromosomes of walnut also showed this feature (Ren et al, 2020; Huang et al., 2015). Qiao et al. (2019) argued that gene replication events include five modes, namely WGD, TD, DSD, PD and TRD. Tandem repeat events play an important role in the expansion of gene families. In Broussonetia papyrifera, TD was the main driver of expansion of the BpUGT gene family (Wang et al, 2021). In the UGT family of walnut, a total of 5 tandem replicators were obtained, and four tandem replication events occurred. Therefore, tandem repetition events continue to occur in UGT gene families, which is a continuous process throughout the evolutionary history of UGT gene families. By analyzing the conserved motifs of UGT proteins in walnut, 10 different motifs were found, and all UGT proteins contained motif 1–3. The UGT protein of walnut in group E did not contain motif 9. These specific motifs may lead to differentiation of UGTs function in walnut.

Understanding the spatiotemporal expression patterns of genes is crucial for predicting their functional roles. In ‘NH1’, a total of 62 UGT genes exhibited significant expression (FPKM > 1) during kernel pellicle development, while ‘FH4’ showed the expression of 52 UGT genes with FPKM > 1. Notably, both varieties displayed higher expression levels of JrUGT58 and JrUGT118 in group E throughout fruit development. Additionally, JrUGT75 demonstrated the highest level of expression within group G. Interestingly, ‘FH4’ exhibited a tenfold increase in FPKM value for JrUGT67 compared to ‘NH1’; conversely, JrUGT69 displayed an opposite pattern of expression between these two varieties. These specific gene expressions may potentially impact the metabolite composition in walnut fruits. Correlation analysis between UGT gene expression and phenolics content in walnut showed that JrUGT36 had no significant correlation with phenolics. JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75 and JrUGT82 were significant correlation with C, GC, EC and Vanillic acid. This discovery improved the basis for subsequent research on phenolic biosynthesis of walnut.

In this study, a total of 124 JrUGTs protein sequences containing PSPG boxes were identified in the walnut genome, and they were categorized into 16 functional groups. The UGT protein motifs and gene structures in walnut exhibited similarities within the same group, while notable differences were observed between different groups. Significantly distinct expression patterns of UGT gene family members during various developmental stages were detected in the kernel pellicle of ‘NH1’ and ‘FH4’. Correlation analysis between UGT gene expression and phenolic content in walnut indicated that JrUGT6, JrUGT38, JrUGT39, JrUGT58, JrUGT69, JrUGT75, and JrUGT82 might be involved in phenolic biosynthesis in walnut. The results provided a basis for further study on the biosynthesis of phenolic substances in walnut.

Author Contributions

All authors contributed to the conception and design of the study. XZ and QL conceived and designed the experiment; DS, PY analysis data; DS and JY write papers; DS, GL and YS conducted experiments. XZ, QL, and JT reviewed the papers, and all authors read and approved the final manuscript.

Funding

This work was supported by the Basic Research Program of Shanxi Province (No. 20210302123396); the science and technology major project of Shanxi Province (No. 202201140601027) and the Key Research and Development Plan (Agriculture) of Jinzhong City (No. Y212013).

Conflict of Interest

All authors declare no conflict of interest.

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Table 1 Content analysis of phenolics in different stages (mg/100g). Different lowercase letters in the same line indicate significant difference in kernel pellicle at 0.05 level in different periods.

Developmental stage Substance name	NH1-6	NH1-7	NH1-8	FH4-6	FH4-7	FH4-8
C	866.71±2.71b	822.45±16.30c	746.36±12.40e	733.57±5.98e	785.20±11.82d	1797.77±13.64a
GC	168.22±2.31e	203.57±2.49c	355.09±2.61b	144.95±4.70f	185.72±4.39d	591.43±4.24a
EC	56.97±4.37e	230.58±2.91c	213.56±2.17d	57.44±2.91e	243.31±6.38b	315.93±2.13a
EGCG	209.25±6.12bc	254.38±28.44a	152.46±10.50d	231.61±1.67ab	232.77±4.42ab	191.65±2.40c
EGC	318.98±7.78d	646.48±13.98a	186.41±12.01f	282.21±15.76e	369.06±7.51b	344.71±4.98c
GCG	39.93±4.05b	8.18±0.40e	15.21±0.30cd	13.07±1.69d	17.52±0.58c	45.02±2.73a
ECG	359.08±25.51a	296.73±24.67b	177.76±4.80c	151.43±0.80cd	154.68±7.26cd	145.33±1.43d
Gallic acid	1390.92±46.78b	1535.90±11.41a	1223.70±4.08c	1086.18±11.21d	1243.39±19.68c	1218.43±15.23c
Chlorogenic acid	366.99±9.65c	263.89±7.50d	142.40±4.66e	547.09±32.01a	256.41±6.06d	449.65±11.78b
vanillic acid	185.66±5.70f	333.59±6.35c	321.29±2.72d	230.55±3.22e	451.43±11.37b	645.98±2.52a
Syringate	55.07±3.76e	97.62±7.02c	63.65±3.50e	76.84±6.60d	186.88±9.21a	139.95±1.75b

No competing interests reported.

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Genome-wide identification of UGT gene family and their expression profiles analysis during fruit development in walnut (Juglans regia L.)

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Introduction

Materials and methods

Phylogenetic and chromosomal localization analysis

Gene replication and collinearity analysis

Conserved sequence and gene structure analysis

Determination of phenolics

Results

Phylogenetic and chromosomal localization analysis

Gene replication and collinearity analysis

Conserved sequence and gene structure analysis

Analysis of phenolic substances in walnut kernel pellicle at different periods

Discussion

Conclusion

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