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Article

Genome-Wide Identification and Characterization of the ANS Gene Family in Pomegranate (Punica granatum L.)

by
Huihui Ni
,
Heming Suo
,
Xuan Zhang
,
Lei Hu
,
Fangyu Yuan
,
Maowen Zhang
and
Shuiming Zhang
*
Department of Ornamental Horticulture, School of Horticulture, Anhui Agricultural University, Hefei 230036, China
*
Author to whom correspondence should be addressed.
Horticulturae 2023, 9(4), 468; https://doi.org/10.3390/horticulturae9040468
Submission received: 3 March 2023 / Revised: 21 March 2023 / Accepted: 6 April 2023 / Published: 7 April 2023
(This article belongs to the Special Issue Research on Pomegranate Germplasm, Breeding, Genetics and Multiomics)
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Abstract

:
Anthocyanidin Synthase (ANS) is a key enzyme in the later stages of the anthocyanin biosynthetic pathway, and its role is to convert colorless leucoanthocyanidins to colored anthocyanidins. In this study, a total of 75 members of the pomegranate ANS family were identified and divided into four groups (Group I, Group Ⅱ, Group Ⅲ and Group Ⅳ) based on evolutionary relationships. The 75 ANS gene family members were unevenly distributed on seven of the eight chromosomes of pomegranate. The results of the physical and chemical property analysis showed that 93.33% of the proteins were acidic proteins, 6.67% were alkaline proteins, 28% of the proteins were stable proteins and 72% were unstable proteins. Protein secondary structure analysis showed that α-Spiral and irregular curl are the main structural elements. Analysis of the conserved structural domains of the proteins showed that all 75 ANS family members contained one DIOX -N subfamily structural domain and one 2OG-FeII_Oxy subfamily structural domain. The results of subcellular localization showed that all 75 ANS family members of pomegranate were localized in the cytoplasm. Analysis of the transcriptome data showed that the expression of the pomegranate ANS genes were variety-specific and period-specific.

1. Introduction

Pomegranate (Punica granatum L.) belongs to the genus Punica in the family of Pomegranate and is an excellent fruit tree that combines ecological, economic and social benefits, ornamental value and health functions [1]. Pomegranates have a long history of cultivation and were one of the first known edible fruits [2]. The pomegranate originated in the Middle East [3] and gradually developed in China after Zhang Qian’s mission to the west in the Han Dynasty. It has developed rapidly in China in recent years. Currently, Shandong Zaozhuang, Xinjiang Yecheng, Anhui Huaiyuan and Sichuan Huili are famous pomegranate cultivation areas in China [4]. Pomegranates are increasingly popular in the consumer market for their sweetness, high economic value, nutritional value, medicinal value and health functions [5]. The red color of pomegranate seeds is the result of the accumulation of anthocyanins, which have many physiological functions, not only for the plant itself to differentiate cells and prevent the occurrence of diseases, but also for human health, such as antioxidation and the prevention of cardiovascular diseases [6]. The results of Wang, D et al. [7] showed that pomegranate juice and peels have strong antioxidant effects, with pomegranate polyphenols and anthocyanins being the active substances that exert antioxidant effects. Therefore, it is important to clarify the mechanism of its regulation of pomegranate seed color.
The colors that appear in plants are determined by the presence of different pigment substances in the plant, which mainly include flavonoids, carotenoids, betaines and chlorophyll, among which anthocyanins are the most abundant type of flavonoid pigments [8]. Anthocyanins are secondary metabolites of plants and are water-soluble natural pigments of the flavonoid group. In their natural form, anthocyanins are extremely unstable and easily combine with sugar molecules to form anthocyanidins [9]. Anthocyanins are an important class of secondary metabolites in the flavonoid family, mainly found in the vesicles of epidermal cells; are responsible for the color development of leaves, flowers, fruits, seeds and other organs; giving flowers, fruits, seed coats and other organs of plants red, blue, purple and other colors; and are the main pigment substances for plant coloring [10,11]. They also play an important role in insect pollination, growth hormone transport, the protection of leaves from UV damage, pest and disease suppression and root tumor induction [12].
Anthocyanidin Synthase (ANS) is involved in a series of metabolic reactions and is a 2-ketoglutarate-dependent enzyme belonging to a family of glutamate-dependent oxygenases [13]. The main chain of ANS contains thirteen strands, of which eight form a jellyroll or double-stranded helix topology. The jellyroll forms a hydrophobic cavity, one end of which forms the active site [14]. Anthocyanidin Synthase is the key enzyme at the end of the anthocyanidin synthase pathway, and catalyzes the conversion of colorless leucoanthocyanidins to colored anthocyanidins. ANS belongs to the dioxygenase gene family in the flavonoid pathway and catalyzes a number of two-electron oxidations, such as hydroxylation, desaturations and oxidative ring closures [15]. Current studies have shown that ANS expression regulates the accumulation of anthocyanins and the color of fruit and flowers [16]. The ANS gene was first isolated from a mutant of maize by transposon tagging [17]. In addition, it has now been cloned in a variety of plants including Arabidopsis thaliana [14], Litchi chinensis [18], Mangifera indica [19], Malus pumila [20], Camellia sinensis [21] and Vitis vinifera [22]. Overexpression of ANS can increase anthocyanin accumulation, while reducing the expression of ANS significantly decreases the anthocyanin level in plants, and this leads to the production of white flowers [23,24].
Pomegranates are receiving more attention for their antioxidant and cardiovascular-disease-prevention properties, of which the main substance is anthocyanin. The ANS gene plays a crucial role in the synthesis of anthocyanins, and the identification and expression analysis of the ANS gene family in pomegranate has not been reported. In this study, members of the pomegranate ANS gene family were identified and characterized by bioinformatics tools, and their expression patterns in three different varieties of ‘Hongyushizi’, ‘Baiyushizi’ and ‘Tunisia’ and at different periods were analyzed to lay the foundation for studying the functions of the ANS gene family in pomegranate.

2. Materials and Methods

2.1. Plant Materials

The varieties of pomegranate tested were ‘Hongyushizi’, ‘Baiyushizi’ and ‘Tunisia’. Samples were taken from pomegranate seeds at 40, 80, and 120 days after bloom, with three biological replicates for each variety and period. The seeds of pomegranates were quick-frozen in liquid nitrogen and stored at ultra-low temperatures at −80 °C. Samples were sent to Guangdong GENE DENOVO Company (Guangzhou, China) for transcriptome sequencing.

2.2. Identification of Members of the ANS Gene Family of Pomegranate

The whole genome sequence of pomegranate (ASM765513v2) was downloaded from NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 20 October 2021). Searching on NCBI (https://www.ncbi.nlm.nih.gov/, accessed on 20 October 2021) yielded sequences of Arabidopsis thaliana containing the structural domain of Anthocyanidin Synthase (PF03171, PF14226). The obtained sequences were used as probes and homology searches were performed by local BLASTP (E-value less than 1 × 10−10) against pomegranate’s proteins in order to prevent the erroneous loss of sequences with low similarity to the probes but containing the structural domain of ANS [25]. We also used HMMER to validate our results. Then, we finally obtained all the members of the pomegranate ANS gene family.

2.3. Analysis of Physicochemical Properties and Prediction of Secondary Structure of Pomegranate ANS Proteins

The physicochemical properties, such as molecular weight and theoretical isoelectric point, of the pomegranate ANS proteins were predicted using the online tool Expasy (http://web.expasy.org/, accessed on 21 October 2021) [26].
Subcellular localization analysis of the pomegranate ANS family was conducted using the online tool Plant-mPLoc in Cell-Ploc 2.0 (http://Plant-mPLoc server (sjtu.edu.cn), accessed on 21 October 2021).
The online software SPOMA (https://npsa-prabi.ibcp.fr/cgi-bin/npsa_automat.pl?page=npsa_sopma.html, accessed on 22 October 2021) [27] was used to predict the secondary structure of the pomegranate ANS gene family proteins.

2.4. Construction of Phylogenetic Trees and Mapping of Gene Structures

The phylogenetic tree was constructed using MEGA X software for the pomegranate ANS gene family proteins according to the neighbor-joining (NJ) method, with the check parameter step (bootstrap) set to 1000 and all other parameters set to default values [28]. The gene structure was mapped by Gene Structure View in TBtools software (version number: v1.098685) [29].

2.5. Structural and Conserved Motif Domain Analysis of the Pomegranate ANS Protein

CD search (NCBI Conserved Domain Search (nih.gov), accessed on 23 October 2021) [30] and SMART were used for protein conserved domain analysis. The conserved motifs of the pomegranate ANS family of proteins were analyzed using the online tool MEME (http://meme-suite.org, accessed on 23 October 2021) [31], where the parameters of MEME were set to a maximum number of motifs of 10 and occurrences of a single motif of zero, or one per sequence [32].

2.6. Chromosome Positioning and Co-Linearity Analysis

Chromosomal localization of pomegranate ANS family genes based on pomegranate whole genome annotation information was performed using Gene Location Visualization from GTF/GFF in TBtools software [30]. MCScanX in TBtools software was used to identify gene duplication patterns and collinearity analysis. The Simple Ka/Ks Calculator in TBtools software was used for calculating the Ka/Ks values [33].

2.7. RNA Extraction, Library Construction and Sequencing

Total RNA was extracted using Trizol reagent kit (Invitrogen, Carlsbad, CA, USA). RNA quality was assessed on an Agilent 2100 Bioanalyzer (Agilent Technologies, Palo Alto, CA, USA) and checked using RNase free agarose gel electrophoresis. After total RNA was extracted, mRNA was enriched by Oligo (dT) beads. Then, the enriched mRNA was fragmented into short fragments using fragmentation buffer and reverse transcripted into cDNA with random primers [34]. Second-strand cDNA were synthesized by DNA polymerase I, RNase H, dNTP and buffer. Then, the cDNA fragments were purified with a QiaQuick PCR extraction kit (Qiagen, Venlo, The Netherlands), end repaired, poly(A) added and ligated to Illumina sequencing adapters. The ligation products were size selected by agarose gel electrophoresis, PCR amplified and sequenced using Illumina HiSeq2500 by Gene Denovo Biotechnology Co. (Guangzhou, China).

2.8. Raw Data Filtering, GO Enrichment and Transcriptome Expression Analysis

To obtain high quality clean reads, reads were further filtered by fastp [35] (version 0.18.0). The parameters were as follows: (1) removing reads containing adapters; (2) removing reads containing more than 10% of unknown nucleotides (N); (3) removing low quality reads containing more than 50% of low quality (Q-value ≤ 20) bases.
To analyze the expression characteristics of pomegranate ANS genes, Log2 based on the Fragments Per kb per Million reads (FPKM) value was used to create a heat map with the HeatMap tool in TBtools software.

3. Results

3.1. Identification, Physicochemical Characterization and Subcellular Localization of Pomegranate ANS Gene Family Members

A total of 75 candidate ANS genes from pomegranate were screened in this study, and the physicochemical properties of ANS proteins were analyzed by the online tool ExPASy. The results (Table 1) showed that the number of amino acids in the protein ranged from 289 to 442. The molecular weight of the pomegranate ANS family proteins ranged from 32,101.26 Da (PgANS59) to 49,422.13 Da (PgANS62), with an average molecular weight of 40,507.81 Da. The theoretical isoelectric point ranged from 4.98 (PgANS58) to 9.11 (PgANS36), with an average theoretical isoelectric point of 5.83. Of these, 93.33% were acidic proteins (theoretical PI < 7) and 6.67% were basic proteins (theoretical PI > 7), indicating that most of the pomegranate ANS exhibited acidic proteins. Hydrophilicity analysis showed that the pomegranate ANS proteins were all hydrophilic. Based on the instability factor, 28% of these were stable proteins (instability coefficient < 40) and 72% of these were unstable proteins (instability coefficient > 40), indicating that the majority of the pomegranate ANS were unstable proteins. The subcellular localization of all pomegranate ANS gene family proteins were localized in the cytoplasm, indicating that pomegranate ANS proteins were non-secretory and carried out metabolic activities within the cell.

3.2. Predicted Secondary Structure of the Pomegranate ANS Gene Family Proteins

Structural predictions (Table 2) showed that all members of the pomegranate ANS proteins were composed of α-helices, irregular coils, extended chains and β-turns. The lowest proportion of these was β-turns, all below 10%, with most around 6%. The majority of proteins had around 35% alpha-helices, with extended chains accounting for around 20% and the highest proportion of irregular coils at around 40%. From the results, it appeared that irregular coiling and α-helix were the main constituent forms of the secondary structure of the pomegranate ANS proteins, with β-turns and extended chains being the secondary constituent forms.

3.3. Phylogenetic and Genetic Structure Analysis of the Pomegranate ANS Family

To investigate the phylogenetic relationships of the pomegranate ANS proteins, a phylogenetic tree was constructed. The results (Figure 1a) show that the 75 ANS family members of pomegranate can be divided into four groups according to their distance of kinship, named Group I, Group II, Group III and Group IV, each containing 43, 13, 16 and 3 members, respectively. According to the analysis, the 75 pomegranate ANS proteins formed 29 paralogous proteins pairs. For example, PgANS7 and PgANS11, PgANS8 and PgANS12, PgANS23 and PgANS38, PgANS19 and PgANS29 and PgANS13 and PgANS21 were paralogous proteins pairs. Of these, 21 pairs had 100% support from the 1000 bootstrapping test, except for 8 paralogous proteins pairs, including PgANS8 and PgANS12, PgANS23 and PgANS39, PgANS17 140 and PgANS45, PgANS1 and PgANS4, PgANS34 and PgANS38, PgANS71 and PgANS72, PgANS40 and PgANS41, PgANS53 and PgANS54. This illustrates the robustness of the constructed phylogenetic tree, the similarity in protein sequences of the 21 pairs of pomegranate ANS proteins and their close affinity.
Structural mapping of the pomegranate ANS gene was carried out using Gene Structure View in TBtools software. The results (Figure 1b) showed that each member of the pomegranate ANS gene family had two–five CDSs (8 members had two CDSs, 29 members had three CDSs, 36 members had four CDSs and 2 members had five CDSs), with little variation in the number of CDSs between members and a relatively simple gene structure.

3.4. Analysis of Protein Structures and Conserved Motifs of Members of the Pomegranate ANS Family

Information on the location of the conserved structural domains of the pomegranate ANS proteins were analyzed using CD search and SMART online software and mapped using the “My Domains” function of the online software ProSite, in conjunction with a phylogenetic tree. The results (Figure 2a) showed that 75 pomegranate ANS protein sequences contained one DIOX -N [36] (non-haem dioxygenase in morphine synthesis N-terminal) subfamily structural domain and one 2OG-FeII_Oxy [37] (2-oxoglutarate Fe(II) oxygenase, 2-ketoglutarate-Fe2+ oxidase) subfamily with a characteristic polypeptide sequence of the Anthocyanidin Synthase family. The ANS enzyme oxidized colorless leucoanthocyanidins to colored anthocyanidins via Fe2+ and (2-oxoglutarate) ions and belonged to the family of dioxygenases.
Analysis of the conserved motifs of the pomegranate ANS proteins was conducted using the online tool MEME, which yielded 10 potentially conserved motifs (Figure 2b), with different motifs indicated by different colored boxes. The 10 obtained motifs were named as Motif 1–Motif 10. All members of the pomegranate ANS family contained Motif 1, Motif 3, Motif 4 and Motif 6 conserved motifs, and all protein motifs were highly conserved in their order of arrangement. Distribution of motifs in Group I: (1) all had Motif 8, excepting PgANS44; (2) all had Motif 3, Motif 6, Motif 4, Motif 2 and Motif 5; (3) Motif 7, except for PgANS6, PgANS43, PgANS51, PgANS65 and PgANS66; (4) all had Motif 9, excepting PgANS24, PgANS32, PgANS50, PgANS43, PgANS65 and PgANS66; (5) all had Motif 10, excepting PgANS29, PgANS24, PgANS32, PgANS43, PgANS65 and PgANS66. The distribution of motifs in Group II: (1) all had Motif 8, Motif 3, Motif 7, Motif 9, Motif 6, Motif 1 and Motif 4; (2) all had Motif 2, excepting PgANS71 and PgANS72; (3) all had Motif 5, excepting PgANS49 and PgANS72; (4) all had Motif 10, excepting PgANS55, PgANS62 and PgANS72. Distribution of motifs in Group III: (1) all had Motif 8, excepting PgANS59; (2) all had Motif 3, Motif 9, Motif 6, Motif 1, Motif 4, Motif 2 and Motif 5; (3) all had Motif 7, excepting PgANS57; (4) all had Motif 10, excepting PgANS52, PgANS68, PgANS53 and PgANS54. Distribution of motifs in Group IV: (1) all had Motif 8, excepting PgANS75; (2) all members of Group IV contained Motif 3, Motif 7, Motif 9, Motif 6, Motif 1, Motif 4, Motif 2, Motif 5 and Motif 10.

3.5. Chromosome Positioning

Analysis of the chromosomal positioning of the 75 pomegranate ANS family genes based on the gene location file showed (Figure 3) that the 75 ANS genes were unevenly distributed on seven of the eight pomegranate chromosomes, Chr1, Chr2, Chr3, Chr4, Chr5, Chr6 and Chr8, with no genes being positioned on Chr7. The largest number of pomegranate ANS genes were distributed on Chr4, with 19 gene members; Chr2 had 15 gene members; Chr1 had 14 gene members; Chr5 had 10 gene members; Chr3 had 9 gene members; Chr8 had 5 gene members; and Chr6 had the lowest number of ANS genes distributed on it, with only 3 gene members.
Further analysis revealed tandem duplication between PgANS45 and PgANS28, as well as fragment duplication. Ten pairs of fragment duplication genes were identified, which were PgANS16 and PgANS14, PgANS15 and PgANS18, PgANS1 and PgANS4, PgANS53 and PgANS54, PgANS15 and PgANS22, PgANS71 and PgANS47, PgANS25 and PgANS31, PgANS57 and PgANS68, PgANS32 and PgANS24 and PgANS36 and PgANS56, suggesting possible functional similarities between some gene members of the pomegranate ANS family.
In addition, we calculated Ka/Ks ratios for 11 pomegranate ANS gene pairs to assess the selective pressure between duplicated pomegranate ANS genes. The results showed that the Ka values of the 11 pomegranate ANS gene pairs ranged from 0.10 to 0.51, and the Ks values ranged from 1.20 to 4.38. Additionally, all the values of Ka/Ks were less than one (Table 3).

3.6. Summary of RNA Sequencing Data

The output of RNA-seq date is shown in Table 4. After filtering low-quality, adapter-polluted and high content of unknown base (N) reads, we acquired a total of 1,200,021,858 clean reads (from 35,721,370 to 53,391,468 for each sample) and 178,302,270,781 clean bases (from 5,322,204,950 to 7,882,028,946 for each sample) in 27 libraries. An average quality value of Q20 (sequencing base quality score > 20) was 96.89% for each library.

3.7. Expression Analysis of the Pomegranate ANS Gene Family

The expression characteristics of pomegranate ANS genes were analyzed in three different varieties (Honhyushi, Baiyushizishi and Tunisia) 40, 80 and 120 days after the blooming period (Figure 4). Out of 75 aforementioned pomegranate ANS genes, we only detected the expression of 64 genes in the transcriptome datasets, while PgANS5, PgANS7, PgANS8, PgANS13, PgANS18, PgANS38, PgANS44, PgANS55, PgANS59, PgANS69 and PgANS72 were not detected in any samples, possibly due to special expression patterns that cannot be examined in our libraries. The 21 genes (PgANS27, PgANS73, PgANS36, PgANS2, PgANS50, PgANS31, PgANS64, PgANS21, PgANS25, PgANS56, PgANS23, PgANS10, PgANS43, PgANS60, PgANS46, PgANS54, PgANS33, PgANS34, PgANS35, PgANS42 and PgANS62) had a similar expression pattern in the three different varieties; that is, the expression level of the genes tended to increase and then decrease as the growth period progressed, with the highest expression level at 80 days after the blooming period (T2). This suggested that most PgANS genes had a period-specific expression pattern.
Analysis of the expression of ANS genes in the three varieties at T1 (40 days after the blooming period) showed that PgANS17, PgANS74, PgANS41, PgANS37, PgANS57, PgANS40 and PgANS11 were expressed at the highest levels in the red variety, ‘Hongyushizi’. PgANS51, PgANS22, PgANS45, PgANS61 and PgANS26 were expressed at the highest levels in ‘Baiyushizi’. Additionally, PgANS32, PgANS1, PgANS14 and PgANS65 had the highest expression levels in Tunisia.
Analysis of the expression of ANS genes in the three varieties at T2 (80 days after the blooming period) showed that PgANS23, PgANS67, PgANS9, PgANS54, PgANS57 and PgANS40 were expressed at the highest levels in ‘Hongyushizi’. PgANS51, PgANS36, PgANS2, PgANS31, PgANS64, PgANS21, PgANS20 and PgANS29 were expressed at the highest levels in ‘Baiyushizi’. Additionally, PgANS42 and PgANS71 were expressed at the highest levels in Tunisia.
Analysis of the expression of ANS genes in the three varieties at T3 (120 days after the blooming period) showed that PgANS12, PgANS75, PgANS16, PgANS65, PgANS41, PgANS30, PgANS24, PgANS6, PgANS28, PgANS26, PgANS63, PgANS4, PgANS70, PgANS49, PgANS48, PgANS47, PgANS66, PgANS33 and PgANS62 were expressed at the highest levels in the red variety, ‘Hongyushizi’. Additionally, PgANS42 was expressed at the highest levels in Tunisia. However, PgANS3 and PgAMS39 had the highest expression levels in the white variety, ‘Baiyushizi’.

4. Discussion

The metabolism and regulation of plant anthocyanins have become a hot spot in scientific research in recent years. Anthocyanidin Synthase (ANS), a key enzyme in the later stages of the anthocyanidin synthesis pathway, has received widespread attention. The ANS gene has been cloned from most plants and has been shown to be an important structural gene for anthocyanin biosynthesis [38]. Anthocyanins readily combine with glycosides to form anthocyanidins, which, for the plant itself, not only give the plant a bright color and thus attract pollinators and foragers, facilitating pollination and seed dispersal [39], but also play a protective role in plant growth and development. In humans, anthocyanins have antioxidant, anti-aging and anti-vascular sclerosis effects [40,41]. ANS catalyzes the conversion of colorless anthocyanins to colored anthocyanins, which affects the accumulation of anthocyanins and determines the formation of flower and fruit coloration [42]. ANS is a family of iron- and 2-O-ketoglutarate-dependent dioxygenases, and the binding site for iron ions and 2-O-ketoglutarate in the conserved structural domain is the active central structure present in the cytochrome P450 family of genes [43]. In this study, a total of 75 members of the pomegranate ANS genes were screened by sequence alignment to predict and analyze the composition, structural characteristics and physicochemical properties of the proteins they encode. The results showed that members of the pomegranate ANS proteins contain conserved structural domains of the 2-ketoglutarate-Fe2+-dioxygenase family, typical of the plant dioxygenase family of genes.
We performed covariance analysis of the members of the pomegranate ANS gene family and identified 11 co-linear gene pairs. Further analysis of these 11 pairs of genes showed that all gene pairs had Ka/Ks values less than one, indicating that they underwent strong purifying selection and played a key role in the evolution of the ANS genes [44].
The results of GO enrichment analysis showed that 64 genes were involved in metabolic processes, single organism processes and catalytic activities, and 3 genes were involved in the cell and cell part, respectively. It is conjectured that these genes may be involved in the growth and development of pomegranate fruits.
In this study, we examined the transcriptome data of ‘Hongyushizi’, ‘Baiyushizi’ and ‘Tunisia’ varieties at different fruit ripening periods using transcriptome sequencing technology. Studies have shown that the expression of the ANS gene was species-specific, with expression levels of dark > light > white/no color varieties; for example, in Saussurea medusa, ANS expression was higher in the red line than in the white and green lines of the healing tissue [45]. The expression level of purple kale (Brassica oleracea var. Capitata Linnaeus) ANS was significantly higher than that of green kale [46]. The expression of ANS was significantly higher in the pink petal line of peach (Prunus persica) than in the white petal line [47]. In this study, PgANS12, PgANS75, PgANS16, PgANS65, PgANS41, PgANS30, PgANS24, PgANS6, PgANS28, PgANS26, PgANS63, PgANS4, PgANS70, PgANS49, PgANS48, PgANS47, PgANS66, PgANS33 and PgANS62 were expressed at the highest levels in the red variety, ‘Hongyushizi’, confirming that the ANS gene was expressed at a higher level in the darker varieties than in the white/stainless varieties. We speculated that these genes played an important role in the formation of pomegranate seed color. However, PgANS39 and PgANS3 were expressed at the highest levels in ‘Baiyushizi’ during the T3 period. We speculated that the expression of these genes may have inhibited the accumulation of anthocyanins, thereby giving pomegranate seeds a white color.
A comparison of the expression pattern of the ANS genes in the same variety at different periods revealed that there was period of specificity in the expression pattern of the ANS genes in pomegranate. The 21 genes (PgANS27, PgANS73, PgANS21, et al.) showed a trend of increasing and then decreasing expression as the growth period progressed. The highest expression levels were found at 80 days after the bloom period, which is consistent with the study by Wang Chunhui et al. [48] on structural genes related to ANS in the fruit of the ‘Hongyang’ kiwi fruit mutant. It suggested that the regulatory mechanisms of the relevant structural genes differ in the synthesis of anthocyanosises and play different roles in the anthocyanidin synthase pathway. Further experiments are needed to verify the effect of PgANS expression on the color of pomegranate seeds.

5. Conclusions

In this study, a genome-wide analysis of the phylogenetic relationships, intron/exon structures, motif composition and expression characteristics of PgANS genes was performed. A total of 75 ANS members from pomegranate were identified and divided into four groups. The results of gene expression analysis indicated that the PgANS genes have both a variety-specific and a period-specific expression pattern. The results of this study further elucidated the expression pattern of pomegranate ANS genes in pomegranate seeds, and ultimately lay a certain theoretical foundation for the breeding of new anthocyanin-rich pomegranate varieties.

Author Contributions

Methodology, X.Z.; software, L.H. and M.Z.; validation, H.N. and H.S.; formal analysis, H.N.; data curation, H.N. and F.Y.; writing—original draft preparation, H.N. and H.S.; writing—review and editing, S.Z.; visualization, X.Z. and H.N.; supervision, S.Z.; funding acquisition, S.Z.; H.S was the co-first author of this article. All authors have read and agreed to the published version of the manuscript.

Funding

This research and the APC were funded by the Anhui Provincial Natural Science Foundation, grant number 2008085MC100, and the Anhui Provincial Natural Science Research Project Fund, grant number KJ2019ZD19.

Data Availability Statement

The original transcriptome data used in this study have been submitted to the NCBI, and the data are stored in the SRA database. the accession number is “PRJNA914887”.

Conflicts of Interest

The authors declare no conflict of interest.

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Figure 1. (a) Phylogenetic tree of the pomegranate ANS proteins and (b) genetic structure of the pomegranate ANS genes. UTR represents untranslated region, while CDS represents coding sequence.
Figure 1. (a) Phylogenetic tree of the pomegranate ANS proteins and (b) genetic structure of the pomegranate ANS genes. UTR represents untranslated region, while CDS represents coding sequence.
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Figure 2. Conserved structural domains of the pomegranate ANS proteins (a) and conserved motifs of the pomegranate ANS proteins (b).
Figure 2. Conserved structural domains of the pomegranate ANS proteins (a) and conserved motifs of the pomegranate ANS proteins (b).
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Figure 3. Location of the pomegranate ANS genes on the chromosome and gene duplication events. The red-colored names were the presence of co-linear genes.
Figure 3. Location of the pomegranate ANS genes on the chromosome and gene duplication events. The red-colored names were the presence of co-linear genes.
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Figure 4. Heat map of the expression of pomegranate ANS genes in 3 species at different growth periods. T1—40 days after blooming period, T2—80 days after blooming period, T3—120 days after blooming period, ‘Hongyu’—Hongyushizishizi, ‘Baiyu’—Baiyushizi.
Figure 4. Heat map of the expression of pomegranate ANS genes in 3 species at different growth periods. T1—40 days after blooming period, T2—80 days after blooming period, T3—120 days after blooming period, ‘Hongyu’—Hongyushizishizi, ‘Baiyu’—Baiyushizi.
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Table
Table 1. Information on the members of the pomegranate ANS gene family proteins.
Table 1. Information on the members of the pomegranate ANS gene family proteins.
Protein IDGene IDGene NameNumber of Amino AcidsMolecular Weight/(D)Isoelectric PointTotal Average HydrophilicityInstability FactorSubcellular Localization
XP_031403964.1XM_031548104PgANS135640,324.315.84−0.39253.57cytoplasm
XP_031393652.1XM_031537792PgANS233638,173.355.50−0.56346.13cytoplasm
XP_031384984.1XM_031529124PgANS333638,357.035.79−0.40544.57cytoplasm
XP_031390545.1XM_031534685PgANS433437,853.405.75−0.34450.46cytoplasm
XP_031376432.1XM_031520572PgANS535740,044.515.66−0.38250.46cytoplasm
XP_031377848.1XM_031521988PgANS635638,836.675.62−0.30552.91cytoplasm
XP_031393854.1XM_031537994PgANS735640,019.215.37−0.19244.10cytoplasm
XP_031393852.1XM_031537992PgANS835640,047.295.46−0.17244.14cytoplasm
XP_031376880.1XM_031521020PgANS936241,430.455.17−0.41340.53cytoplasm
XP_031393856.1XM_031537996PgANS1035640,055.225.14−0.19743.63cytoplasm
XP_031393853.1XM_031537993PgANS1138443,440.106.03−0.29546.39cytoplasm
XP_031393851.1XM_031537991PgANS1239043,645.105.77−0.25444.39cytoplasm
XP_031395560.1XM_031539700PgANS1335640,391.356.12−0.36640.64cytoplasm
XP_031380350.1XM_031524490PgANS1435840,565.395.29−0.37740.89cytoplasm
XP_031382717.1XM_031526857PgANS1534739,631.905.26−0.39040.16cytoplasm
XP_031386650.1XM_031530790PgANS1636641,504.685.75−0.35235.46cytoplasm
XP_031378923.1XM_031523063PgANS1735940,562.305.07−0.33836.23cytoplasm
XP_031382330.1XM_031526470PgANS1833938,929.065.13−0.48148.51cytoplasm
XP_031394087.1XM_031538227PgANS1936241,139.645.30−0.15248.30cytoplasm
XP_031378476.1XM_031522616PgANS2035939,580.336.09−0.18738.09cytoplasm
XP_031395559.1XM_031539699PgANS2137642,663.066.77−0.36638.50cytoplasm
XP_031391993.1XM_031536133PgANS2233838,634.905.78−0.45344.89cytoplasm
XP_031393855.1XM_031537995PgANS2336140,534.635.07−0.26545.11cytoplasm
XP_031401502.1XM_031545642PgANS2436641,173.195.26−0.37340.11cytoplasm
XP_031391176.1XM_031535316PgANS2635038,763.985.39−0.27754.46cytoplasm
XP_031378903.1XM_031523043PgANS2735339,091.585.80−0.24439.59cytoplasm
XP_031388526.1XM_031532666PgANS2836741,013.405.28−0.48744.63cytoplasm
XP_031390878.1XM_031535018PgANS2936941,918.315.38−0.22649.33cytoplasm
XP_031388423.1XM_031532563PgANS3035840,381.985.62−0.29542.66cytoplasm
XP_031372937.1XM_031517077PgANS3138843,418.396.28−0.31455.54cytoplasm
XP_031388151.1XM_031532291PgANS3237241,873.015.42−0.40243.37cytoplasm
XP_031383472.1XM_031527612PgANS3338043,218.425.38−0.39636.43cytoplasm
XP_031389012.1XM_03153315PgANS3435038,489.705.22−0.16936.97cytoplasm
XP_031391174.1XM_031535314PgANS3535339,037.425.34−0.22043.80cytoplasm
XP_031385121.1XM_031529261PgANS3636740,709.859.11−0.19541.41cytoplasm
XP_031383168.1XM_031527308PgANS3735841,381.086.75−0.52435.12cytoplasm
XP_031383192.1XM_031527332PgANS3835439,158.465.62−0.27235.71cytoplasm
XP_031376436.1XM_031520576PgANS3937041,879.975.48−0.25241.88cytoplasm
XP_031379827.1XM_031523967PgANS4035841,371.035.91−0.48640.20cytoplasm
XP_031383167.1XM_031527307PgANS4135841,503.055.81−0.54243.97cytoplasm
XP_031395634.1XM_031539774PgANS4237341,126.146.35−0.13643.77cytoplasm
XP_031394103.1XM_031538243PgANS4332036,348.785.18−0.37934.24cytoplasm
XP_031391175.1XM_031535315PgANS4429632,917.575.87−0.23342.64cytoplasm
XP_031385605.1XM_031529745PgANS4535840,276.115.26−0.26449.52cytoplasm
XP_031398105.1XM_031542245PgANS4638042,899.825.08−0.31252.48cytoplasm
XP_031399235.1XM_031543375PgANS4736541,639.615.27−0.39033.34cytoplasm
XP_031380799.1XM_031524939PgANS4834639,478.705.42−0.47138.38cytoplasm
XP_031397740.1XM_031541880PgANS4933640,882.185.33−0.39144.01cytoplasm
XP_031390650.1XM_031534790PgANS5031936,283.507.06−0.54638.19cytoplasm
XP_031384128.1XM_031528268PgANS5131935,943.265.34−0.30030.71cytoplasm
XP_031390887.1XM_031535027PgANS5239344,373.416.11−0.32847.95cytoplasm
XP_031383134.1XM_031527274PgANS5337843,069.066.82−0.44332.77cytoplasm
XP_031390764.1XM_031534904PgANS5439544,855.055.43−0.43440.97cytoplasm
XP_031399302.1XM_031543442PgANS5543448,533.056.38−0.02636.22cytoplasm
XP_031407498.1XM_031551638PgANS5635639,585.566.71−0.18555.07cytoplasm
XP_031389388.1XM_031533528PgANS5739444,472.406.50−0.38349.59cytoplasm
XP_031380299.1XM_031524439PgANS5836942,082.854.98−0.29146.91cytoplasm
XP_031407500.1XM_031551640PgANS5928932,101.269.08−0.10449.69cytoplasm
XP_031374700.1XM_031518840PgANS6035439,399.375.76−0.01851.91cytoplasm
XP_031378238.1XM_031522378PgANS6139344,385.286.15−0.33436.41cytoplasm
XP_031399301.1XM_031543441PgANS6244249,422.136.56−0.03035.95cytoplasm
XP_031373129.1XM_031517269PgANS6337942,826.435.05−0.37446.89cytoplasm
XP_031374648.1XM_031518788PgANS6435439,488.266.05−0.15951.08cytoplasm
XP_031388742.1XM_031532882PgANS6530434,270.086.11−0.50942.64cytoplasm
XP_031383847.1XM_031527987PgANS6637141,943.068.93−0.48139.13cytoplasm
XP_031373149.1XM_031517289PgANS6736942,122.845.01−0.28246.38cytoplasm
XP_031390843.1XM_031534983PgANS6842948,221.938.02−0.37344.95cytoplasm
XP_031377515.1XM_031521655PgANS6937242,320.195.47−0.32643.29cytoplasm
XP_031397862.1XM_031542002PgANS7037642,150.935.41−0.34838.47cytoplasm
XP_031377516.1XM_031521656PgANS7137242,394.425.58−0.32345.43cytoplasm
XP_031377517.1XM_031521657PgANS7230935,244.095.51−0.37644.64cytoplasm
XP_031400740.1XM_031544880PgANS7330333,706.665.28−0.16645.19cytoplasm
XP_031400741.1XM_031544881PgANS7430133,484.225.06−0.18241.88cytoplasm
XP_031395563.1XM_031539703PgANS7531736,302.445.44−0.40935.63cytoplasm
Table
Table 2. Predicted secondary structure of the pomegranate ANS gene family proteins.
Table 2. Predicted secondary structure of the pomegranate ANS gene family proteins.
Gene Nameα-Helix (%)Extended Strand (%)β-Turn (%)Random Coil (%)
PgANS133.1517.425.0644.38
PgANS234.2319.056.8539.88
PgANS334.8219.355.3640.48
PgANS433.2317.965.3943.41
PgANS532.2118.217.8441.74
PgANS637.3617.985.0639.61
PgANS738.2016.295.9039.61
PgANS837.9216.576.4639.04
PgANS937.8516.025.5240.61
PgANS1039.8915.735.9038.48
PgANS1136.2015.896.7741.15
PgANS1233.8516.414.1045.64
PgANS1335.6715.736.4642.13
PgANS1434.0816.205.0344.69
PgANS1535.1617.295.4842.07
PgANS1633.6118.037.3840.98
PgANS1742.9015.606.9634.54
PgANS1838.0516.525.3140.12
PgANS1938.4015.196.3540.06
PgANS2030.9219.786.4142.90
PgANS2136.4416.496.1240.96
PgANS2234.9117.756.2141.12
PgANS2336.5716.075.2642.11
PgANS2437.9818.585.7437.70
PgANS2534.3915.616.3543.65
PgANS2635.4316.576.2941.71
PgANS2734.2817.565.3842.78
PgANS2835.9714.995.4543.60
PgANS2938.2115.187.0539.57
PgANS3038.5517.045.8738.55
PgANS3134.5416.495.9343.04
PgANS3236.8317.475.6540.05
PgANS3340.5316.327.1136.05
PgANS3435.1418.006.0040.86
PgANS3534.5619.555.1040.79
PgANS3632.4316.356.2744.96
PgANS3733.5217.605.5943.30
PgANS3835.8818.085.0840.96
PgANS3937.8416.495.9539.73
PgANS4030.7319.274.4745.53
PgANS4131.0117.605.5945.81
PgANS4230.2916.624.8348.26
PgANS4340.6217.196.8835.31
PgANS4437.8417.576.7637.84
PgANS4536.3119.276.9837.43
PgANS4640.5316.326.0537.11
PgANS4737.8116.995.4839.73
PgANS4835.2616.764.6243.35
PgANS4939.3416.396.2837.98
PgANS5040.4417.877.2134.48
PgANS5143.2617.557.5231.66
PgANS5233.5914.765.3446.31
PgANS5334.3918.256.6140.74
PgANS5432.4117.474.3045.82
PgANS5538.9420.975.9934.10
PgANS5634.5514.615.0645.79
PgANS5731.9818.535.5843.91
PgANS5836.0417.625.6940.65
PgANS5938.7518.695.8836.68
PgANS6036.1614.415.6543.79
PgANS6136.6416.286.3640.71
PgANS6238.6920.814.7535.75
PgANS6337.7317.685.5439.05
PgANS6437.2915.255.9341.53
PgANS6537.8317.767.5736.84
PgANS6638.2717.256.2038.27
PgANS6737.4018.975.4238.21
PgANS6835.4316.325.1343.12
PgANS6938.9816.406.1838.44
PgANS7038.5616.225.5939.63
PgANS7139.5216.945.9137.63
PgANS7236.2518.124.8540.78
PgANS7336.3015.516.6041.58
PgANS7437.2117.616.3138.87
PgANS7536.5917.035.6840.69
Table
Table 3. Estimated divergence period of the PgANS gene pairs. Ks, synonymous substitution rate; Ka, non-synonymous substitution rate.
Table 3. Estimated divergence period of the PgANS gene pairs. Ks, synonymous substitution rate; Ka, non-synonymous substitution rate.
Gene PairsKaKsKa/Ks
PgANS1–PgANS40.481.330.36
PgANS45–PgANS280.511.730.29
PgANS16–PgANS140.474.380.11
PgANS15–PgANS180.363.870.09
PgANS53–PgANS540.202.900.07
PgANS15–PgANS220.331.700.19
PgANS71–PgANS470.382.180.17
PgANS25–PgANS310.283.120.09
PgANS57–PgANS680.382.400.16
PgANS32–PgANS240.101.200.08
PgANS36–PgANS560.381.660.23
Table
Table 4. Summary of RNA Sequencing Data, Clean Reads Q20—Sequencing base quality score > 20 and GC Content–GC ratio of sequence bases before filtering.
Table 4. Summary of RNA Sequencing Data, Clean Reads Q20—Sequencing base quality score > 20 and GC Content–GC ratio of sequence bases before filtering.
SampleRaw ReadsRaw Bases (bp)Clean ReadsClean Bases (bp)Clean Reads Q20 (%)Clean Reads Ratio (%)GC Content (%)
Baiyushizi-T1(1)48,667,6387,300,145,70048,379,9527,219,608,23897.14%99.41%50.05%
Baiyushizi-T1(2)47,486,5547,122,983,10047,186,9447,050,368,79996.74%99.37%49.77%
Baiyushizi-T1(3)47,593,8127,139,071,80047,305,9007,039,879,17596.95%99.40%49.60%
Baiyushizi-T2(1)36,016,7665,402,514,90035,721,3705,322,204,95096.42%99.18%50.36%
Baiyushizi-T2(2)41,189,4046,178,410,60040,865,8306,095,053,04096.56%99.21%50.53%
Baiyushizi-T2(3)39,571,1505,935,672,50039,300,1025,862,500,28597.00%99.32%50.59%
Baiyushizi-T3(1)40,037,0726,005,560,80039,938,1465,907,058,37696.64%99.75%50.42%
Baiyushizi-T3(2)42,861,8926,429,283,80042,742,3806,337,181,50596.83%99.72%50.31%
Baiyushizi-T3(3)45,807,7606,871,164,00045,679,2746,745,209,77096.87%99.72%50.40%
Hongyushizi-T1(1)48,234,5747,235,186,10047,911,3507,148,996,85596.80%99.33%49.68%
Hongyushizi-T1(2)39,476,8445,921,526,60039,184,5885,847,426,00996.58%99.26%49.58%
Hongyushizi-T1(3)41,389,8846,208,482,60041,102,1386,137,884,64496.64%99.30%49.66%
Hongyushizi-T2(1)48,913,0267,336,953,90048,584,6587,251,639,21096.76%99.33%50.48%
Hongyushizi-T2(2)38,788,6425,818,296,30038,486,5325,744,610,57496.52%99.22%50.08%
Hongyushizi-T2(3)48,650,2767,297,541,40048,296,1127,208,671,02496.70%99.27%50.36%
Hongyushizi-T3(1)43,736,8046,560,520,60043,660,6626,324,509,06797.56%99.83%49.25%
Hongyushizi-T3(2)42,770,7086,415,606,20042,642,4526,357,074,52197.33%99.70%50.57%
Hongyushizi-T3(3)43,698,6106,554,791,50043,576,1386,466,042,71097.44%99.72%50.76%
Tunisia-T1(1)38,816,4945,822,474,10038,516,2725,739,905,92096.28%99.23%49.61%
Tunisia-T1(2)47,502,7107,125,406,50047,163,9907,039,556,37596.39%99.29%49.70%
Tunisia-T1(3)51,473,3167,720,997,40051,172,9367,638,003,16996.98%99.42%49.64%
Tunisia-T2(1)48,979,2427,346,886,30048,609,7087,242,609,08196.62%99.25%50.15%
Tunisia-T2(2)49,197,6287,379,644,20048,851,4047,265,839,88596.79%99.30%50.18%
Tunisia-T2(3)49,308,2427,396,236,30048,974,8567,303,980,12196.86%99.32%50.22%
Tunisia-T3(1)40,240,6506,036,097,50040,170,0005,888,045,00297.52%99.82%50.17%
Tunisia-T3(2)53,530,5128,029,576,80053,391,4687,882,028,94697.56%99.74%49.20%
Tunisia-T3(3)42,669,4486,400,417,20042,606,6966,236,383,53097.56%99.85%49.59%
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Ni, H.; Suo, H.; Zhang, X.; Hu, L.; Yuan, F.; Zhang, M.; Zhang, S. Genome-Wide Identification and Characterization of the ANS Gene Family in Pomegranate (Punica granatum L.). Horticulturae 2023, 9, 468. https://doi.org/10.3390/horticulturae9040468

AMA Style

Ni H, Suo H, Zhang X, Hu L, Yuan F, Zhang M, Zhang S. Genome-Wide Identification and Characterization of the ANS Gene Family in Pomegranate (Punica granatum L.). Horticulturae. 2023; 9(4):468. https://doi.org/10.3390/horticulturae9040468

Chicago/Turabian Style

Ni, Huihui, Heming Suo, Xuan Zhang, Lei Hu, Fangyu Yuan, Maowen Zhang, and Shuiming Zhang. 2023. "Genome-Wide Identification and Characterization of the ANS Gene Family in Pomegranate (Punica granatum L.)" Horticulturae 9, no. 4: 468. https://doi.org/10.3390/horticulturae9040468

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