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Article

Identification, Classification of the MIKC-Type MADS-Box Gene Family, and Expression Analysis of Female and Male Flower Buds in Walnut (Juglans regia, Juglandaceae)

by
Caihua Guo
1,2,
Olumide Phillip Fesobi
1,2,
Zhongrong Zhang
1,2,
Xing Yuan
1,2,
Haochang Zhao
1,2,
Shaowen Quan
1,2,* and
Jianxin Niu
1,2,*
1
Department of Horticulture, College of Agriculture, Shihezi University, Shihezi 832003, China
2
Xinjiang Production and Construction Corps Key Laboratory of Special Fruits and Vegetables Cultivation Physiology and Germplasm Resources Utilization, Shihezi 832003, China
*
Authors to whom correspondence should be addressed.
Horticulturae 2025, 11(7), 787; https://doi.org/10.3390/horticulturae11070787
Submission received: 12 May 2025 / Revised: 29 June 2025 / Accepted: 30 June 2025 / Published: 3 July 2025
(This article belongs to the Section Genetics, Genomics, Breeding, and Biotechnology (G2B2))
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Abstract

MIKC-type MADS-box transcription factors constitute one of the largest gene families in plants, playing pivotal roles in regulating plant growth and development, hormone signaling transduction, and responses to biotic and abiotic stresses. However, there have been no reports on the systematic identification and characterization of MIKC-type MADS-box proteins in walnuts. In this study, we identified 52 JrMADS genes in the walnut genome and transcriptome, and categorized them into 14 subfamilies through structural domain and phylogenetic tree analysis. It was found that these genes were unevenly distributed across 16 chromosomes. Within the MIKC-type MADS-box gene family, we identified three pairs of tandem-duplicated genes and 40 pairs of segmental duplicated genes, indicating that segmental duplication was the primary mechanism of gene amplification in walnut. Ka/Ks analysis showed that the family genes have undergone purifying selection during evolutionary processes. The promoter was predicted to contain cis-acting elements related to growth, development, plant hormones, and stress response. Expression profile analysis showed that JrMADS genes have different expression patterns in various tissues and developmental stages of male and female flower buds. Notably, an ancient clade of TM8 (JrMADS43) genes was found, which is absent in Arabidopsis but present in other flowering plants. Another gene, TM6 gene (JrMADS4), belongs to the AP3 subfamily and is a clade that has diverged from tomatoes. Through qPCR analysis, we verified the differential expression of JrMADS genes at different developmental stages (MB-1/2/3 and FB-1/2/3), with JrMADS5, JrMADS8, JrMADS14, JrMADS24, JrMADS40, JrMADS46, JrMADS47, JrGA3ox1, and JrGA3ox3 showing significantly higher expression in male than in female flower buds. In summary, our results provide valuable information for further biological functions research on MIKC-type MADS-box genes in walnut, such as flower organ development, and lays a solid foundation for future studies.

1. Introduction

The MADS-box genes represent the largest family of transcription factors found in plants, animals, and fungi [1]. The name of this family originates from the abbreviation of the four initially discovered genes: MAINTENANCE OF MINICHROMOSOME1 (MCM, yeast), AGAMOUS (AG, Arabidopsis), DEFICIENS (DEF, fruit fly), and SERUM RESPONSE FACTOR (SRF, human). MADS-box genes play a central role in many plant species, especially in seed germination, vegetative growth, and transition to flowering; control of flowering time; and regulating the characteristics of floral meristems and organs, ovule development, and fruit ripening [2,3,4,5,6,7,8]. These genes encode proteins that contain a conserved domain of 50–60 amino acids, known as the MADS (M) domain. Based on phylogenetic analysis and gene structure, the MADS-box gene family can be divided into two categories: type I (SRF-like) and type II (MEF2-like) [9]. In plants, It can be further classified into five subcategories, Mα, Mβ, Mγ, MIKCc, and MIKC*, in which Mα, Mβ, and Mγ belong to type I. The type II MADS-box gene family can be further subdivided into two subgroups based on the intervening regions, MIKCC and MIKC* [10,11], also known as MIKC-type genes. type I genes are generally shorter in length, have a simple structure, and only contain the MADS domain. MIKC-type genes contain a conserved domain architecture with an N-terminally located DNA-binding M domain, followed by the I- (intervening) and K-box domain, which are crucial for dimerization and higher-order complex formation. Finally, a highly variable C (C-terminal) domain is present, which may play roles in protein complex formation and transcriptional regulation [5,12,13,14,15].
The majority of MIKC-type MADS-box members are floral organ-determining genes, playing crucial roles in regulating flower primordia, floral morphogenesis, and flowering time. Many MIKC* members are usually involved in male gametophyte development [16,17], while MIKCC genes play a role in all stages of flower development, including 12 subclasses: APETALA1 (AP1/SQUA), APETALA3/PISTILLATA (AP3/PI or DEF/GLO), AGAMOUS/SEEDSTICK (AG/STK), AGAMOUS-LIKE6 (AGL6), AGL12, AGL15, AGL17, BSISTER (BS), SUPPRESSOR OF OVEREXPRESSION OF CONSTANS 1 (SOC1/TM3), SHORT VEGETATIVE PHASE (SVP), FLOWERING LOCUS C (FLC), and SEPALLATA (SEP) [18,19]. For instance, SOC1 is a downstream gene of the floral regulator CO (COSTANS), and its overexpression leads to early flowering [20]. SVP is regulated by the autonomous and gibberellin pathways, and its protein binds to the promoter region of SOC1 and FT genes, inhibiting their expression [21]. FLC, a floral regulator repressor, delays flowering [22]. In the development of Arabidopsis thaliana inflorescence, STM interacts with FT-FD and AGL24-SOC1 complexes, upregulating the flower meristem identity genes [23]. The FT-FD complex acts as a transient stimulus, and a regulatory mechanism exists to reduce the level of the FT-FD complex by regulating FD expression during the flowering transition [24]. Additionally, AGL79 promoted early flowering by interacting with the SOC1 and repressing the expression of Terminal Flower 1 (TFL1) [25]. SlMADS1 can directly interact with SlMC (SlMACROCALYX) to regulate the tomato sepal growth, root system development, and plant height [26]. The soybean NJCMS2A gene may lead to cytoplasmic male sterility in soybean [27]. Overexpression of PavFUL led to polypodium formation and early flowering in Arabidopsis and interacted with PavLFY, PavSOC1, PavAP1, and PavSEP to co-regulate flowering and polypodium formation [28]. Sorbitol signaling induces loquat bud formation through EjCAL, and EjERF12 binds to the EjCAL promoter and regulates its expression [29]. The expression level of EjAGL17 reaches its highest at the transition stage of bud development, and its ectopic expression in Arabidopsis shows early flowering characteristics [30]. VvMADS39 can be regulated by the upstream BPC transcription factors, and acts synergistically with other proteins to maintain floral meristem characteristics and promote fruit and ovule development [31]. However, little is known regarding the MADS-box gene family in walnut, particularly regarding its role in floral development [32].
Walnuts are widely grown over the world as an essential economic tree with significant nutritional value, as well as being high in protein, fat and carbohydrates, trace elements. However, in actual production, the distribution of flower buds is uneven, resulting in significantly more male flowers than female flowers. This leads to the consumption of a large quantity of tree nutrients, which limits the growth and development of female flowers and reduces the number of fruits produced [33]. Therefore, to study the mechanism of flower bud differentiation in walnuts, finding the key genes or transcription factors that regulate the differentiation of male and female flower buds is of great importance for the production of walnuts. In this study, we performed a whole-genome analysis to identify members of the MIKC-type MADS-box gene family. Bioinformatics approaches were used to investigate their physical features, phylogenetic relationships, gene structure, gene duplication, and protein interactions. In addition, second and third generation transcriptome data were used to explore the expression patterns of this gene family in different tissues and at different developmental stages, and were validated by qRT-PCR. This study lays the foundation for further exploration of the regulatory role of this family of genes.

2. Materials and Methods

2.1. Plant Materials

The walnut cultivar ‘wen185’ was cultivated under natural conditions in the southern part of Xinjiang Uyghur Autonomous Region, China (40.50° N, 81.28° E), located in the upper reaches of the Tarim River. The area has long daylight hours and a large diurnal temperature range. The plant materials were collected from walnut trees that had been cultivated for 10 years. Samples were collected from 31 May to 12 June 2021. The developmental stages of floral buds at sampling were histologically verified by paraffin sectioning, and the location was in the same walnut orchard. However, female flower buds were collected at three time points: before (FB-1), during (FB-2), and after (FB-3) the female flower bud transition period. Male flower buds (MB-1, MB-2, and MB-3) were obtained at the same time intervals as female flower buds. Each time period sample was composed of a mixture of three flower buds, with three biological replicates for each treatment, totaling nine flower buds. The samples were wrapped in foil paper, quickly frozen in liquid nitrogen, and then stored at −80 °C for RNA extraction.

2.2. Identification of MIKC-Type MADS-Box Genes in Walnut

The whole walnut genome sequence data set was downloaded from the National Center for Biotechnology Information (NCBI) (https://www.ncbi.nlm.nih.gov/) for the identification and annotation of MADS-box transcription factors. The specific identification approach for MIKC-type MADS-box genes involved utilizing the BLASTP tool (BLAST+ version 2.9.0) [34] to search the walnut transcriptome database using functionally annotated MIKC-type MADS-box Arabidopsis orthologs as queries. Following this preliminary screening, we obtained the hidden Markov model (HMM) profile of the MADS and K domains (PF00319 and PF01486) with an E-value of 1 × 10−10 from the pfam database (http://pfam.xfam.org/). Furthermore, the protein sequences found by both algorithms were combined and manually edited to remove the redundant. Subsequently, the sequence was submitted to online tools such as SMART (http://smart.embl.de/) and NCBI-CDD (https://www.ncbi.nlm.nih.gov/cdd) for the prediction of candidate protein domains. Finally, the Expasy Protparam tool (https://web.expasy.org/protparam/) and WoLF PSORT II (https://www.genscript.com/wolf-psort.html?src=leftbar) were used to predict the physicochemical characteristics and subcellular localization of MIKC-type MADS-box genes, respectively.

2.3. Phylogenetic Relationship Analysis of MIKC-Type MADS-Box Genes in Walnut

To investigate the evolutionary relationships among MIKC-type MADS-box genes in walnut, multiple sequence alignments were constructed using the neighbor-joining method and validated using the maximum likelihood method. These alignments included the identified MIKC-type MADS-box proteins of walnut, together with Arabidopsis (https://www.arabidopsis.org/), grape, and poplar (https://phytozome-next.jgi.doe.gov/), and apple (https://www.rosaceae.org/). The parameters for the neighbor-joining analysis were set to the Poisson distribution model in MEGA 7.0 software, and the bootstrap value was set to 1000 [35]. The classification of the MIKC-type family in walnut was according to the topology of the phylogenetic tree and the established classification of the MIKC-type MADS-box gene family in the other four plants. The phylogenetic tree was displayed and manipulated using the EvolView (https://evolgenius.info//evolview-v2/).

2.4. Exon–Intron Structural and Conserved Motif Analysis

The conserved domains of each MIKC-type MADS-box protein sequence were predicted using the MEME tool (http://meme-suite.org/) [36]. The maximum number of motifs found in this process was 20, with all other parameters set to their default values. Additionally, the DNA and cDNA sequences of walnut MIKC-type MADS-box genes were compared by online Gene Structure Display Server 2.0 (http://gsds.gao-lab.org/) to predict the intron and exon structures. On this basis, combined with the walnut genome annotation information, the gene structure, and conserved motifs of walnut MIKC-type MADS-box genes, the genes were visualized with TBtools (Version 2_025) [37].

2.5. Chromosome Localization, Duplication Events, and Divergence Time Estimation with Other Plants

The physical position and chromosomal distribution information of MIKC-type MADS-box genes were obtained using the MapInspect software (http://mapinspect.software.informer.com/) [38]. The tandem and segmental duplication events of the family members in walnut were studied using the MCScanX and TBtools toolkits, and the segmental duplication events and collinearity relationships in different species were analyzed [39]. The non-synonymous substitution rate (Ka), synonymous substitution rate (Ks), and the Ka/Ks ratio were calculated to assess the molecular selection effect using the TBtools tool. Genes with Ks values above 0.3, as well as Ka and Ks values of 0, were discarded as they could be due to sequence saturation or mismatches [37]. The divergence time of the JrMADS genes were calculated with reference to Koch [40] and Huang [41], and the R package was used to draw the figure.

2.6. Functional Annotation Analysis

Gene Ontology (GO) enrichment analysis was carried out on protein sequences from walnut using the eggNOG-mapper (Version 2.1.12) tool. Subsequently, the generated files were imported into the R package (Version 4.4.2) for visualization.

2.7. Cis-Acting Elements and Protein Interaction Network Analysis

The cis-acting elements in the promoter region (2000 bp upstream of the starting codon) of the JrMADS were predicted by the online program of PlantCARE (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/) [42].

2.8. Subcellular Localization

The complete coding sequences (CDS) of JrFUL genes (JrMADS5, JrMADS8, and JrMADS23) lacking stop codons were amplified and cloned into the 35S::eGFP vector to generate recombinant constructs (35S::JrMADS5-eGFP, 35S::JrMADS8-eGFP, and 35S::JrMADS23-eGFP). For transient expression, an Agrobacterium tumefaciens strain GV3101 carrying the recombinant plasmids was resuspended in 100 mL of infiltration buffer (0.042 g MES; 0.048 g MgCl·6H2O; 100 mM acetosyringone; pH 5.5) and adjusted to OD600 = 0.4–0.6, and injected into four-week-old leaves of Nicotiana benthamiana. After 24 h of darkness, subcellular localization was observed 48 h post-infiltration using a Nikon C2-ER (Nikon, Tokyo, Japan) confocal microscope equipped with a 488 nm argon laser (15% intensity), 515/30 nm emission filter, CFI Plan Apo λ 40× oil-immersion objective (NA = 1.3), and Z-stack scanning at 1 μm intervals [41].

2.9. Genes Expression and Quantitative Real-Time PCR

To study the expression profiles of JrMADS family members in female and male buds. Expression level data of female buds (FB-1, FB-2, FB-3) and male buds (MB-1, MB-2, MB-3) at different differentiation stages were obtained from transcriptomic data (Table S6). The FPKM values were calculated and normalized, followed by the generation of heatmaps using TBtools software. The qPCR system and protocol followed our previous description [43], with primers designed using the primer premier 6.0 software, and the Jr18SrRNA gene serving as the internal control. The primer sequences used in this study were shown in Table S1. The 2−ΔΔCt method was utilized to compute the relative expression levels of JrMADS genes [44].

3. Results

3.1. Characterization and Distribution of MIKC-Type MADS-Box Genes in Walnut

We identified 52 MIKC-type MADS-box genes from the walnut genome and named them JrMADS1-JrMADS52. The chromosomes location, coding sequence length, molecular weight (MW), subcellular localizations, and isoelectric point (PI) of these genes are shown in Table 1. These 52 JrMADS were found to be unequally distributed on 16 chromosomes. JrMADS7 had the shortest amino acid sequence, containing only 203 amino acids, while JrMADS33 was the longest, with 391 amino acid residues. The molecular weights of the MADS-box members ranged from 23.66 (JrMADS32) to 44.34 kDa (JrMADS33), with an average molecular weight of 28.48 kDa. The theoretical PI varied between 5.46 and 10.11. Prediction of the subcellular localization indicated that the majority of JrMADS genes were located in the nucleus. Additionally, one JrMADS protein (JrMADS30) was localized at the mitochondria, followed by two (JrMADS45 and JrMADS50) being predicted to be localized to the chloroplast. Interestingly, JrMADS52 is found in both the cytoplasm and nucleus (Table 1).

3.2. Phylogenetic Analysis of MIKC-Type MADS-Box Gene Family Members

To determine the evolutionary trend of MIKC-type MADS-box genes across different species, a bar chart was used to display the number of these genes in 20 species, including walnut (Figure 1). To clarify the classification and evolutionary status of walnut MIKC-type MADS-box genes, a phylogenetic tree was constructed using 261 full-length amino acid sequences from model plants Arabidopsis (46), grape (48), apple (53), poplar (62), and walnut (52) (Figure 2). Based on the clustering results, the MIKC-type MADS-box proteins were categorized into 14 subfamilies in walnut (Figure 2). Among them, only five members, namely, JrMADS27, JrMADS31, JrMADS33, JrMADS34 and JrMADS36, were classified as MIKC*. The other 47 genes were classified as MIKCC, which were categorized into 13 subfamilies: SVP (5), BS (3), AGL17 (5), AGL15 (3), AP3/PI (3), AG (3), AP1/SQUA (6), SEP (6), AGL6 (2), and FLC (2). The largest branch belongs to the SOC1 subfamily, which has seven MADS-box family members. An exception is that the TM8 subfamily does not exist in Arabidopsis, and the AGL12 (JrMADS32) and TM8 (JrMADS43) subfamilies each contain only one MADS-box member, making them the smallest branches.

3.3. Analysis of Conserved Motifs and Gene Structure in Walnut

Further, twenty conserved sequences of MIKC-type MADS-box proteins in walnut were identified using the online MEME software (Version 5.5.8). All JrMADS proteins contained a highly conserved MADS domain (motif 1), which consisted of 50 amino acids, and all subfamilies except MIKC* contained motif 1, motif 3, and motif 5 (Figure 3B and Figure S1). MIKCC-type proteins showed greater differences in K-box regions compared to MIKC*. In the MIKCC subfamily, motifs 2, 4, 6, 7, and 13 corresponded to the K-box domain, whereas the AGL 17 and AG subfamilies contained motifs 2, 4, and 7. Motifs 2, 4, and 6 belonged to the AP1, AGL6, and SEP subfamilies, while only the SOC1 subfamily included motifs 2, 4, and 13. However, in the MIKC* subfamily, the K-box domain only contained motif 2.
We investigated the gene structure by GSDS [45]. The results showed that JrMADS genes had varying numbers of exons (Figure 3C), ranging from 6 to 12. Gene structure analysis was an important part of studying the evolution of family members. It was found that the gene structures of different subfamilies differed greatly by comparing the number of exons, introns, and gene lengths between family members. However, apart from differences in gene length, the gene structure of different members of the same subfamily was very similar. JrMADS33 and JrMADS36 of the MIKC* subfamily could have up to 12, which differed from the structures of the other three subfamily members (exon number was 11). Therefore, the length of each intron was significantly shorter in MIKC*, and we speculated that this phenomenon might be related to the different regulatory functions of introns. The MIKCC members of the AGL15, AP1, AGL6, SEP, and TM8 subfamilies contained eight exons, and in the SVP, SOC1, and AGL12 subfamilies, except for JrMADS7and JrMADS20, other members contained seven exons. However, in the PI, FLC and AG subfamilies, JrMADS19 and JrMADS51 had eight exons. Additionally, all members of the BS subfamily contained six exons. Furthermore, the JrMADS12 gene was longer than other family members. We hypothesize that these differences may be due to different replication patterns of different genes during evolution, which eventually resulted in structural and functional changes.

3.4. Chromosomal Locations, Gene Duplications, and Synteny Relationship of MIKC-Type MADS-Box Gene Family in Walnut

Fifty-two JrMADS genes were mapped to sixteen chromosomes in the walnut genome database (Figure 4A). The number of genes varied greatly on different chromosomes, with the highest number of genes (seven) distributed on chromosomes Chr1, Chr11, Chr13, and Chr16, followed by six, four, three, and three genes on chromosomes Chr10, Chr2, Chr3, and Chr8, respectively. Two genes were located on Chr6 and Chr7, and only one gene was found on other chromosomes.
Segmental duplications and tandem duplications of these JrMADS genes were investigated using TBtools and McscanX (Multiple Collinear Scan toolkit) method. Interestingly, three pairs of tandem duplication events were identified, as five or fewer genes within 100 kb of the same chromosome are generally considered to be tandem duplication genes. However, 40 pairs of genes exhibited segmental duplication events (Figure 4B). For the analysis of the selective constraints on the duplication of the JrMADS gene pair, we carried out combined analysis of Ka/Ks ratios and Ks values using the full-length gene sequences for estimation of divergence time (Figure 4C,D and Table S2). The calculated distribution of Ks values for homologous gene pairs (Jr-Jr) showed a mean value of approximately 1.4, indicating that the JrMADS genes experienced a major duplication event around 46 MYA (million years ago). A previous study estimated the genome-wide duplication of walnut to be 31 MYA [46], suggesting that the mass duplication of the JrMADS gene occurred much earlier. Additionally, the divergence times of the Jr-Jr, Jr-At, Jr-Vt, Jr-Pt, and Jr-Jm genomes were in the ranges of 7–160 MYA, 51–173 MYA, 11–72 MYA, 20–139 MYA, and 0.1–118 MYA, respectively. In principle, Ka/Ks ratios greater than, equal to, and less than 1 indicate accelerated evolution due to positive, neutral, and negative or stable selection, respectively [47,48]. The genomes of Jr-Jr, Jr-At, Jr-Vt, Jr-Pt and Jr-Jm have Ka/Ks ratios less than 1, suggesting that these genes are mainly subjected to purifying selection in the evolutionary process.

3.5. Cis-Regulatory Motif Analysis of MIKC-Type MADS-Box Genes

The potential cis-acting elements in the JrMADS promoter region were analyzed using the PlantCARE (Plant Cis-Acting Regulatory Elements) Database, and various cis-acting elements related to growth and development, plant hormone response, and stress induction were identified (Figure 5). Among them, cis-acting elements related to growth and development include CAT-box, RY-element, and GCN4_motif. The CAT-box, which was a cis-acting regulatory element related to meristem expression, was found to exist in 21 JrMADS genes. In addition, various cis-acting elements involved in plant hormone response were found in the promoter region, such as auxin response elements (AuxRR, AuxRR-core, and TGA-element), MeJA response elements (CGTCA-motif and TGACG-motif), salicylic acid response elements (TCA-element), gibberellin response elements (TATC-box, GARE-motif, and P-box), and ABA response elements (ABRE). Furthermore, there were also many types of stress-induced cis-acting elements in the JrMADS genes, such as ARE, LTR, drought inducibility (MBS), TC-rich repeats, and GC-motif. ARE, which is essential for anaerobic induction and was found in most promoters (52 JrMADS genes). The LTR cis-acting elements, which are involved in low-temperature responsiveness, were found in many promoters (28 JrMADS genes). TC-rich repeats, which are involved in defense and stress responsiveness, were detected in 23 JrMADS genes, while the GC-motif involved in anoxic-specific inducibility existed in 9 JrMADS genes. Among them, the abscisic acid-responsive element (ABRE) was the most abundant of all hormone-responsive elements, GCN4_motif was the most of growth and development related element, and the anaerobic-inducible element (ARE) was the most common in non-biological stress response, indicating it may play a key role in plants adapting to hypoxic environments (Figure 5 and Figure S2).

3.6. Transient Expression Analysis

We constructed a subcellular localization vector containing 35S::eGFP fusion protein and used it to infiltrate tobacco leaves and the results were shown in Figure 6. Green fluorescence was observed in both the cytoplasm and nucleus of tobacco leaf cells, serving as a positive control for empty loading. In contrast, for JrMADS5, JrMADS8 and JrMADS23 fluorescence was only observed in the nucleus of the cells, indicating that these proteins are subcellularly localized in the nucleus of the plant, which is consistent with the prediction results of the online software.

3.7. Analysis of the Expression Patterns of MIKC-Type MADS-Box Genes

We investigated the roles of JrMADS genes in walnut flower bud differentiation by examining the expression patterns of JrMADS family members in male and female flower buds at different developmental stages using the walnut cultivar ‘Wen 185’. A heat map was generated based on RNA-seq data (Figure 7). Seven A-class genes (JrMADS5, JrMADS8, JrMADS14, JrMADS23, JrMADS35, JrMADS40 and JrMADS45) were found to be significantly more expressed in male flower buds than in female ones. Similarly, members of the AP3/PI subfamily within the B-class genes (JrMADS4, JrMADS19, and JrMADS49) were also highly expressed in male flower buds. In the C and D-class genes AG/STK subfamily, JrMADS24 and JrMADS48 were highest in male flower buds. It is worth noting that the expression level of JrMADS51 in female flower buds was higher than that in male flower buds. The expression levels of E-class genes (JrMADS2, JrMADS9, JrMADS15, JrMADS28, JrMADS39, and JrMADS47) were similar to A- and C-classes, and were highly expressed in male flower buds. Simultaneously, the expression of the BS subfamily (JrMADS11, JrMADS30, and JrMADS42) in walnut male and female flower buds was examined. JrMADS11 was highly expressed in FB-1, MB-1, and MB-3 stages, JrMADS30 in FB-3 stage, while JrMADS42 had lower expression in both male and female flower buds. The expression of the SOC1 subfamily (JrMADS16, JrMADS17, JrMADS18, JrMADS22, JrMADS26, JrMADS38, JrMADS51), which can promote the formation of floral organs, was significantly less expressed in male than in female flower buds. In addition, AGL6 and AGL15 subfamilies were highly expressed in male flower buds, while most members of the SVP subfamily, except JrMADS12 which was highly expressed in FB-3, had higher expression in female flower buds. Finally, in the MIKC* family, JrMADS31 and JrMADS36 were expressed at low levels in flower buds, while JrMADS33 and JrMADS34 had higher expression in the MB-3 stage.

3.8. Functional Annotation Analysis

In this study, further Gene Ontology (GO) functional annotation analyses were performed to explore the biological and molecular functions of JrMADS proteins. The GO enrichment analyses consisted of three sections: biological process (BP), cellular component (CC) and molecular function (MF). A total of 45 JrMADS proteins were classified into 3 categories and 35 subcategories among the protein sequences annotated in the GO database (Figure 8A and Table S5). In terms of biological processes, these proteins were categorized into 25 subclasses, with the major subclasses being “regulation of transcription” (GO:0006355, 35 sequences, 67.3%), “transcription” (GO:0006351, 9 sequences and 17.3%), “oxidation-reduction process” (GO:0055114, 4 sequences, 7.7%), “maintenance of inflorescence meristem identity” (GO:0010077, 3 sequences, 5.8%) and “fruit development” (GO:0010154, 2 sequences, 3.8%). Among the cellular components, “nucleus” (GO:0005634), “cytoplasm” (GO:0005737) and “integral components of membranes” (GO:0016021) with 2, 1 and 1 sequences, respectively (3.8%, 1.9% and 1.9%). In the molecular function category, “DNA binding” (GO:0003677, 36 sequences, 69.2%) and “protein dimerization activity” (GO:0046983, 35 sequences, 67.3%) and “transcription factor activity, sequence-specific DNA binding” (GO:0003700, 34 sequences, 65.4%) had the highest representation. Furthermore, floral organ development includes carpel development (GO:0048440), floral whorl development (GO:0048438), flower development (GO:0009908), petal development (GO:0048441), plant ovule development (GO:0048481), sepal development (GO:0048442), and stamen development (GO:0048443) (Figure 8B).

3.9. Regulatory Relationships of Genes Involved in Flower Development

To investigate the roles of genes associated with male and female flower bud differentiation, floral organ development, and gibberellin biosynthesis enzymes in walnut, we conducted a correlation analysis between MIKC-type MADS-box genes and these genes. Furthermore, we utilized correlation coefficients to construct a network diagram illustrating the relationships between JrMADS genes and the seven floral organ genes. Among them, JrLFY showed positive correlations with 19 JrMADS genes and negative correlations with 4. JrTFL1 exhibited positive and negative correlations with 11 and 13 genes, respectively. JrSTM displayed positive correlations with 10 JrMADS genes. The remaining four flowering genes (JrFT, JrCO, JrFD and JrBLH8) each demonstrated positive correlations with six genes (Figure 9A and Table S4). We also constructed a correlation network diagram between JrMADS genes and three gibberellin-related genes, which were positively correlated with seven JrMADS genes.
Additionally, following the Arabidopsis association model, we constructed a protein interaction network for 52 JrMADS genes in walnut using STRING 12.0 software (Figure 9B, Table S3). The JrMADS proteins were primarily categorized into groups associated with the developmental regulation of plant floral organs. Class A, represented by AtAP1, AtFUL and AtCAL (JrMADS5/8/14/23/35/40). Class B comprised AtAP3 (JrMADS4, which, in other species, has been further identified as the TM6 gene and JrMADS19 in developmental analyses) and AtPI (JrMADS47). Class C genes, represented by AtAG (JrMADS24/46), and Class E genes, represented by AtSEP3 (JrMADS2/28), occupied important places in the overall MADS-box protein interaction network in walnut, interacting with many JrMADS proteins. In addition to genes controlling flowering time, numerous MADS-box genes contribute to regulating the transition from vegetative to reproductive growth in plants. For instance, FLC (JrMADS7) and SVP (JrMADS1/21/37/41) act as inhibitors of flowering, while AGL24 (JrMADS12) and SOC1 (JrMADS17/38/43) function as promoters of flowering. There are also genes involved in the regulation of non-long-day dependent pathways, related to gibberellin in plants, and complete the regulation of flowering by activating genes like LFY.

3.10. qRT-PCR to Verify the Expression Pattern of JrMADS Genes and Gibberellin Biosynthetic Enzymes

To characterize the DEGs of JrMADS under male and female flower buds, we compared the expression levels of JrMADS genes in male and female buds at different developmental stages (Table S5). Ten differentially expressed JrMADS genes (FPKM > 0.5) were identified in walnut male and female flower buds using RNA-seq, and the gibberellin biosynthetic enzymes JrGA3ox1 and JrGA3ox3 genes were also selected to analyze their relative transcript abundance using qRT-PCR (Figure 10). The results showed that JrMADS5, JrMADS8, JrMADS14, JrMADS24, JrMADS40, JrMADS46, JrMADS47, JrGA3ox1, and JrGA3ox3 genes were significantly highly expressed in male flower buds (MB) (p < 0.05), while JrMADS37 was significantly upregulated in female buds (FB) (p < 0.05). No significant differences in expression were observed for JrMADS1 and JrMADS17 between male and female buds. Transcriptomic data also revealed that JrMADS40 was more highly expressed in FB-2, but less expressed in MB-2, consistent with the qRT-PCR results, which further confirmed the accuracy of the RNA-seq data.

4. Discussion

Members of the MIKC-type MADS-box gene family are widely distributed in eukaryotes [49]. With the development of genome sequencing technology, genome-wide analyses of the MIKC-type MADS-box gene family have been identified and classified in a large number of many dicots and monocots, including Arabidopsis [50], wheat [51], soybean [52], tomato [53], pineapple [54], banana [55], grape [56], Chinese jujube [57], apple [58], and poplar [59], among others. However, little is known about the MADS-box gene family in walnut.
As a diploid, the genome size of walnut is 606 Mb [60], while the genome sizes of Arabidopsis, rice, grape, apple, and poplar are 125 Mb, 430 Mb, 475 Mb, 742 Mb, and 485 Mb, respectively [61,62,63,64]. Only 52 walnut MIKC-type MADS-box genes were identified and characterized in this study, as well as the number of this family, which is similar to that of Arabidopsis (46), grape (48), apple (53), or poplar (63) (Figure 2), despite the much larger genome size of walnut compared to Arabidopsis and rice. Thus, it appears that there is no correlation between gene number and genome size in these plants, which may be due to complex historical events such as massive loss of genomic duplicates or limited gene duplication, but the specific reasons require further exploration.
The molecular weights and isoelectric points of MIKC-type MADS-box proteins from different members of the family also differed, suggesting that their functions may vary. In addition, through tobacco transient transformation experiments, it was confirmed that the JrMADS5, JrMADS8, and JrMADS23 (homologous to FUL-like genes) were located in the nucleus, consistent with online software predictions (Table 1). Phylogenetic analyses showed that these 52 family members could be classified into 14 groups, including 47 MIKCC-type MADS-boxes and five MIKC*-type MADS-boxes, based on protein sequence similarity and clustering, with group nomenclature derived from Arabidopsis, grape, and poplar. However, the TM8 gene (JrMADS43) is evolutionarily significant, yet its function remains poorly understood across angiosperms. Phylogenetic studies indicate that the TM8 lineage has been lost independently at least five times, making it the second most frequently lost MADS-box lineage after FLC [65]. The widespread loss of TM8 in model species such as Arabidopsis, maize and rice, in contrast to its retention in walnut, suggests that it may have a lineage-specific functional importance. Although TM8 has not been definitively characterized as having a functional role in any species, emerging evidence suggests a conserved involvement in floral development. For example, expression studies in grape link TM8 to flower formation [66], and ectopic expression in tomato alters reproductive structures [67]. These combined evolutionary and functional implications highlight the critical need for further investigation of JrMADS43, particularly in walnuts.
Exon–intron structural diversity is an essential component of gene family evolution. In exon–intron analyses, the number of exons is highly variable (ranging from 6 to 12). Furthermore, a previous study showed that the ancestral copy of a MADS-box gene had six introns and seven exons [68]. This diversity results from exon/intron gain/loss, exonization/pseudoexonization, and insertion/deletion during evolution. These three mechanisms lead to differing gene structure and function [69]. Furthermore, the corresponding JrMADS proteins contained MADS-related domains (Figure S1), suggesting that these genes were conserved during evolution. These motifs may have specific functions given the different functions of MIKC-type MADS-box transcription factors, a phenomenon that deserves further investigation.
We found that members of the MIKC-type MADS-box gene family were symbolized according to their location on the walnut chromosome. Among identified genes, six were located on six different chromosomes (Figure 4A). This uneven distribution may be due to specific retention and spread of genes during polyploidisation. Polyploidisation and duplication of gene regions (tandem duplication and segmental duplication) have been reported to be an important mechanism for the generation and expansion of gene families in plants [70]. Tandem gene duplication events occur frequently during plant evolution, leading to the expansion of gene families. Interestingly, a total of three tandem duplication pairs and 40 segmental duplication pairs of genes were found in the walnut MIKC-type MADS-box gene family (Figure 4B); the results suggest that the amplification of JrMADS genes may be the result of gene duplication during genome polyploidisation and evolution.
Through GO enrichment analysis, we sought to gain a better understanding of the functional roles of MIKC-type MADS-box genes in various biological processes. Our study’s results indicated that these genes are involved in transcriptional regulation, flower and fruit development, oxidation–reduction processes, as well as meristem determination, among other crucial biological processes. The performance of these functions is closely associated with transcriptional regulation, and these genes play an indispensable role in the growth and development of plants.
Cis-acting elements are important components of plant regulatory networks, contributing to a deeper understanding of transcriptional regulation and revealing the functions of related genes [71]. In this study, we predicted the promoter sequences of walnut MIKC-type MADS-box genes and identified 18 cis-acting elements primarily involved in growth, development, and responses to stress and hormones, consistent with findings in other woody plants [72,73]. Notably, anaerobic induction elements (ARE) were found in the promoters of most JrMADS genes, suggesting potential roles in low-oxygen adaptation similar to their function in apple flower development [74,75]. Other stress-related elements, such as MBS (drought response), further support the involvement of JrMADS genes in abiotic stress responses, as demonstrated in poplar studies [76]. Of particular interest were hormone-responsive elements, including ABRE (ABA signaling), TGACG/CGTCA motifs (MeJA response), and GARE-motifs/P-box (gibberellin signaling), implying that JrMADS genes may integrate hormonal and environmental cues to regulate floral development, a mechanism established in Arabidopsis [77]. Specifically, enrichment of gibberellin-responsive elements aligned with male-biased expression of gibberellin biosynthesis genes JrGA3ox1 and JrGA3ox3, which showed significant upregulation in male flower buds (p < 0.05). This pattern mirrors that observed in cucumber sex determination [78]. This suggests gibberellin biosynthesis enzymes may play an important role in male flower buds and remains to be investigated. Consistent with these findings, JrMADS37 (an SVP homolog regulating flowering time in kiwifruit) exhibited significantly higher expression in female flower buds in walnut, indicating potential dual functions in flowering time control and female flower bud differentiation [79]. This parallels AGAMOUS (AG) in Arabidopsis, where AG specifies carpel identity via conserved C-function, and SVP represses flowering by integrating environmental cues [80]. Conversely, JrAP1 (JrMADS14/JrMADS40) exhibited significantly higher expression levels in male flower buds than in female flower buds, contrasting with canonical AP1 roles in sepal/petal specification but aligning with Populus [81]. JrAP1 may interact with SEP-like partners to regulate stamen development, as observed in Arabidopsis [82]. While this study reveals the potential role of JrMADS genes in hormone signaling and floral development, their causal mechanisms still require further validation through functional experiments.

5. Conclusions

In this study, we identified 52 MIKC-type MADS-box genes in walnut, classifying them into 14 subfamilies, including MIKCC-type (comprising 13 subfamilies) and MIKC*-type, with uneven distribution across all 16 chromosomes. Structural analysis confirmed that all JrMADS proteins contain conserved MADS domains, with genes within the same subfamily exhibiting similar exon–intron patterns. Evolutionary investigations indicated segmental duplication as the predominant mechanism driving gene family expansion. Complementary co-expression networks, subcellular localization predictions, and GO annotations further supported putative roles in organogenesis and stress response pathways. While these integrated analyses provide compelling functional hypotheses, validation through transgenic or genome-editing approaches remains essential. Collectively, our findings establish a molecular framework for understanding floral organ development and stress adaptation mechanisms in walnut, while multi-omics characterization of promoters, gene structures, and expression dynamics provides valuable candidate resources for future functional genomics and precision breeding initiatives.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/horticulturae11070787/s1, Figure S1. Protein domain of MIKC-type MADS-box gene family; Figure S2. Number of cis-acting elements of MIKC-type MADS-box family members in walnuts; Figure S3. Logo image of 20 motifs of JrMADS family members; Table S1. Primers involved in this article; Table S2. Ka/Ks values and divergence times of MIKC-type MADS-box gene pairs in walnut; Table S3. Walnut–Arabidopsis blastp result; Table S4. Correlation network of MIKC-type MADS-box genes with genes related to floral organ development; Table S5. GO term data for JrMADS family members in walnut; Table S6. FPKM expression values of JrMADS genes in walnut male/female flower buds across differentiation stages.

Author Contributions

C.G., formal analysis, and writing—original draft preparation. O.P.F., investigation. Z.Z., investigation. X.Y., investigation. H.Z., investigation. S.Q., supervision. J.N., conceptualization, project administration, funding acquisition, and manuscript revision. All authors have read and agreed to the published version of the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China (No. 32060668) and the important National Science and Technology Specific projects of Xinjiang (No. 201130102–1-4). The funding institution was not involved in the design of the study, collection, analysis, and interpretation of data, or in writing the manuscript.

Data Availability Statement

The original contributions presented in this study are included in the article. Further inquiries can be directed to the corresponding author.

Acknowledgments

We would like to thank the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.

Conflicts of Interest

The authors declare no conflicts of interest.

Abbreviations

The following abbreviations are used in this manuscript:
HMMHidden Markov model
NCBINational Center for Biotechnology Information
CDDConserved Domain Database
PITheoretical isoelectric point
MWMolecular weight
NJNeighbor joining
CDSCoding sequence
DEGsDifferential expression genes
GOGene Ontology

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Figure 1. The number of MIKC-type MADS-box gene in plants.
Figure 1. The number of MIKC-type MADS-box gene in plants.
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Figure 2. Phylogenetic tree of MIKC-type MADS-box gene family of walnut (purple ball) and that of Arabidopsis (green ball), grape (blue ball), poplar (yellow ball) and apple (red ball). The multiple sequence alignment and construction of phylogenetic tree were performed with MEGA7.0 using neighbor-joining method with 1000 bootstrap replicates. These numbers represented bootstrap values.
Figure 2. Phylogenetic tree of MIKC-type MADS-box gene family of walnut (purple ball) and that of Arabidopsis (green ball), grape (blue ball), poplar (yellow ball) and apple (red ball). The multiple sequence alignment and construction of phylogenetic tree were performed with MEGA7.0 using neighbor-joining method with 1000 bootstrap replicates. These numbers represented bootstrap values.
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Figure 3. (A) Phylogenetic relationship of MIKC-type MADS-box genes in walnut. (B) Motif composition and distribution of MIKC-type MADS-box proteins in walnut. Colored boxes represent conserved motifs. (C) Gene structure analysis of selected MIKC-type MADS-box genes in walnut. yellow rectangles indicate exons, black lines represent introns, and green rectangles denote untranslated regions (UTRs).
Figure 3. (A) Phylogenetic relationship of MIKC-type MADS-box genes in walnut. (B) Motif composition and distribution of MIKC-type MADS-box proteins in walnut. Colored boxes represent conserved motifs. (C) Gene structure analysis of selected MIKC-type MADS-box genes in walnut. yellow rectangles indicate exons, black lines represent introns, and green rectangles denote untranslated regions (UTRs).
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Figure 4. (A) Chromosomal localization of MIKC-type MADS-box genes in walnut. The chromosome number was indicated to the left of each chromosome. The MADS-box genes were marked at the approximate position on the chromosomes. (B) Genomic locations of JrMADS and segmental duplicated gene pairs in the walnut genome. Gray lines in the background indicated the collinear blocks within the whole walnut genome, and red lines represent the segmental duplication pairs between the JrMADS. (C) Synteny analysis of MIKC-type MADS-box genes between Arabidopsis thaliana (At), Juglans mandshurica (Jm), Vitis vinifera (Vv), and Populus trichocarpa (Pt). The gray lines in the background represent the collinear blocks within Juglans regia (Jr) and other plant genomes, while the red lines highlight represented orthologous gene pairs with the MADS-box gene family. Chromosome numbers are shown at the top or bottom of each horizontal bar. (D) Divergence time between the five genomes. The vertical coordinate indicates the divergence time, which is given in MYA (million years ago).
Figure 4. (A) Chromosomal localization of MIKC-type MADS-box genes in walnut. The chromosome number was indicated to the left of each chromosome. The MADS-box genes were marked at the approximate position on the chromosomes. (B) Genomic locations of JrMADS and segmental duplicated gene pairs in the walnut genome. Gray lines in the background indicated the collinear blocks within the whole walnut genome, and red lines represent the segmental duplication pairs between the JrMADS. (C) Synteny analysis of MIKC-type MADS-box genes between Arabidopsis thaliana (At), Juglans mandshurica (Jm), Vitis vinifera (Vv), and Populus trichocarpa (Pt). The gray lines in the background represent the collinear blocks within Juglans regia (Jr) and other plant genomes, while the red lines highlight represented orthologous gene pairs with the MADS-box gene family. Chromosome numbers are shown at the top or bottom of each horizontal bar. (D) Divergence time between the five genomes. The vertical coordinate indicates the divergence time, which is given in MYA (million years ago).
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Figure 5. Promoter cis-elements analysis of MIKC-type MADS-box genes in walnut. The different colored boxes represent different cis-acting elements, some of which may overlap.
Figure 5. Promoter cis-elements analysis of MIKC-type MADS-box genes in walnut. The different colored boxes represent different cis-acting elements, some of which may overlap.
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Figure 6. Transient subcellular localization of JrMADS5, JrMADS8 and JrMADS23 proteins in Nicotiana benthamiana mesophyll cells. From left to right: green fluorescence (GFP), mCherry nuclear localization signal, bright field, and merged field. Scale bar = 75 μm.
Figure 6. Transient subcellular localization of JrMADS5, JrMADS8 and JrMADS23 proteins in Nicotiana benthamiana mesophyll cells. From left to right: green fluorescence (GFP), mCherry nuclear localization signal, bright field, and merged field. Scale bar = 75 μm.
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Figure 7. Expression profiles of candidate JrMADS genes in tissues or different developmental stages. (A) Heatmap of JrMADS genes expressed differently in three development periods of female and male flower buds (FB/MB-1, FB/MB-2, and FB/MB-3) (Second generation transcriptome). (B) Heatmap of the JrMADS genes between female flower buds and male flower buds (Full-length transcriptome). The color scale represents log2(FPKM+1) values.
Figure 7. Expression profiles of candidate JrMADS genes in tissues or different developmental stages. (A) Heatmap of JrMADS genes expressed differently in three development periods of female and male flower buds (FB/MB-1, FB/MB-2, and FB/MB-3) (Second generation transcriptome). (B) Heatmap of the JrMADS genes between female flower buds and male flower buds (Full-length transcriptome). The color scale represents log2(FPKM+1) values.
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Figure 8. (A) Gene Ontology enrichment analysis of JrMADS family members in walnut. Colors represent GO categories: green for Biological Process (BP), orange for Cellular Component (CC), blue for Molecular Function (MF). (B) GOChord plot of top 10 ranked overrepresented GO terms belonging to the Biological Process subontology for MIKC-type MADS-box gene in walnut. Left arc: genes; right arc: GO terms; connecting ribbons show significant gene-term associations (colored by target GO term).
Figure 8. (A) Gene Ontology enrichment analysis of JrMADS family members in walnut. Colors represent GO categories: green for Biological Process (BP), orange for Cellular Component (CC), blue for Molecular Function (MF). (B) GOChord plot of top 10 ranked overrepresented GO terms belonging to the Biological Process subontology for MIKC-type MADS-box gene in walnut. Left arc: genes; right arc: GO terms; connecting ribbons show significant gene-term associations (colored by target GO term).
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Figure 9. (A) Correlation network of MIKC-type MADS-box genes with genes related to floral organ development. The line’s shape and color represents positive and negative correlation, respectively, while its thickness represents the strength of correlation. The size and color shade of the dots represent the number of correlated objects. (B) Protein interaction network of specific JrMADS proteins. Nodes represent proteins, with schematic 3D structural motifs shown within them. Line colors denote interaction evidence types, and thick lines indicate high-confidence interactions. The network was constructed using twenty-nine input proteins and ten predicted functional partners.
Figure 9. (A) Correlation network of MIKC-type MADS-box genes with genes related to floral organ development. The line’s shape and color represents positive and negative correlation, respectively, while its thickness represents the strength of correlation. The size and color shade of the dots represent the number of correlated objects. (B) Protein interaction network of specific JrMADS proteins. Nodes represent proteins, with schematic 3D structural motifs shown within them. Line colors denote interaction evidence types, and thick lines indicate high-confidence interactions. The network was constructed using twenty-nine input proteins and ten predicted functional partners.
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Figure 10. Relative expression levels of differentially expressed JrMADS genes in three periods of male (MB-1/2/3) and female (FB-1/2/3) flower buds in walnut. X-axis represents different developmental periods. y-axis represents the relative expression levels of genes. Data are presented as mean ± SEM (n = 3 independent biological replicates). Statistical significance was determined by one-way ANOVA.
Figure 10. Relative expression levels of differentially expressed JrMADS genes in three periods of male (MB-1/2/3) and female (FB-1/2/3) flower buds in walnut. X-axis represents different developmental periods. y-axis represents the relative expression levels of genes. Data are presented as mean ± SEM (n = 3 independent biological replicates). Statistical significance was determined by one-way ANOVA.
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Table 1. Characteristics of the MIKC-type MADS-box genes in walnut.
Table 1. Characteristics of the MIKC-type MADS-box genes in walnut.
Gene NameGene IDProteinChromosomeProtein Length (aa)MW (kDa)PISubcellular Location
JrMADS1gene25279XP_018806751.1Chr1122825.676.13nucleus
JrMADS2gene25477XP_018807034.1Chr1625228.647.67nucleus
JrMADS3gene27721XP_018810079.1Chr1327031.369.30nucleus
JrMADS4gene28276XP_018810897.1Chr0822525.869.33nucleus
JrMADS5gene29720XP_018813004.1Chr0224828.728.73nucleus
JrMADS6gene29996XP_018813442.1Chr0821925.298.97nucleus
JrMADS7gene31372XP_018815444.1Chr1020323.785.96nucleus
JrMADS8gene32213XP_018816775.1Chr0125528.989.14nucleus
JrMADS9gene32212XP_018816776.1Chr0124528.138.68nucleus
JrMADS10gene3078XP_018817259.1Chr0525529.036.21nucleus
JrMADS11gene32624XP_018817273.1Chr1323127.056.01nucleus
JrMADS12gene33316XP_018818329.1Chr1322325.516.65nucleus
JrMADS13gene33390XP_018818423.1Chr0324828.437.03nucleus
JrMADS14gene33459XP_018818517.1Chr1624628.348.50nucleus
JrMADS15gene33549XP_018818571.1Chr1024528.099.05nucleus
JrMADS16gene34631XP_018820266.1Chr0220523.749.79nucleus
JrMADS17gene34707XP_018820385.1Chr0723226.749.00nucleus
JrMADS18gene34785XP_018820468.2Chr0922625.939.36nucleus
JrMADS19gene35264XP_018821032.1Chr1127130.979.29nucleus
JrMADS20gene39166XP_018826847.1Chr1623126.718.91nucleus
JrMADS21gene39189XP_018826905.1Chr1622125.256.61nucleus
JrMADS22gene40183XP_018828400.1Chr1022125.609.20nucleus
JrMADS23gene3840XP_018828587.1Chr1524828.739.50nucleus
JrMADS24gene41314XP_018830028.1Chr0324227.849.45nucleus
JrMADS25gene41827XP_018830769.1Chr0124728.739.30nucleus
JrMADS26gene41823XP_018830813.1Chr0120723.858.70nucleus
JrMADS27gene4912XP_018833820.2Chr0634638.655.87nucleus
JrMADS28gene6737XP_018836792.1Chr1324528.248.33nucleus
JrMADS29gene6905XP_018837041.1Chr1122626.018.69nucleus
JrMADS30gene7096XP_018837323.1Chr0327532.266.84mitochondrion
JrMADS31gene8020XP_018838782.1Chr1036641.125.46nucleus
JrMADS32gene9597XP_018840982.1Chr0120823.668.27nucleus
JrMADS33gene12230XP_018845246.2Chr1639144.346.92nucleus
JrMADS34gene12804XP_018846114.1Chr1335540.606.36nucleus
JrMADS35gene13376XP_018846950.1Chr1024928.539.78nucleus
JrMADS36gene15119XP_018849786.1Chr1633738.145.47nucleus
JrMADS37gene16258XP_018851438.1Chr1128132.369.63nucleus
JrMADS38gene16410XP_018851691.1Chr1221825.219.42nucleus
JrMADS39gene16863XP_018852400.1Chr1324528.358.67nucleus
JrMADS40gene16864XP_018852402.2Chr1325930.019.22nucleus
JrMADS41gene22497XP_018852859.2Chr0822925.795.92nucleus
JrMADS42gene22569XP_018858716.2Chr1624428.597.07nucleus
JrMADS43gene371XP_035541113.1Chr0220423.7410.11nucleus
JrMADS44gene11730XP_035541415.1Chr1425529.076.04nucleus
JrMADS45gene29718XP_035543716.1Chr0224627.676.35chloroplast
JrMADS46gene1703XP_035544964.1Chr0424628.109.42nucleus
JrMADS47gene28585XP_035546734.1Chr0621324.757.76nucleus
JrMADS48gene34708XP_035547562.1Chr0726029.678.84nucleus
JrMADS49gene9586XP_035547898.1Chr0125629.249.39nucleus
JrMADS50gene8967XP_035550066.1Chr0121224.836.13chloroplast
JrMADS51gene40184XP_035550324.1Chr1020824.019.10nucleus
JrMADS52gene8205XP_035551623.1Chr1124628.459.30cytoplasm and nucleus
Note: MW: molecular weight; PI: isoelectric point.
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Guo, C.; Fesobi, O.P.; Zhang, Z.; Yuan, X.; Zhao, H.; Quan, S.; Niu, J. Identification, Classification of the MIKC-Type MADS-Box Gene Family, and Expression Analysis of Female and Male Flower Buds in Walnut (Juglans regia, Juglandaceae). Horticulturae 2025, 11, 787. https://doi.org/10.3390/horticulturae11070787

AMA Style

Guo C, Fesobi OP, Zhang Z, Yuan X, Zhao H, Quan S, Niu J. Identification, Classification of the MIKC-Type MADS-Box Gene Family, and Expression Analysis of Female and Male Flower Buds in Walnut (Juglans regia, Juglandaceae). Horticulturae. 2025; 11(7):787. https://doi.org/10.3390/horticulturae11070787

Chicago/Turabian Style

Guo, Caihua, Olumide Phillip Fesobi, Zhongrong Zhang, Xing Yuan, Haochang Zhao, Shaowen Quan, and Jianxin Niu. 2025. "Identification, Classification of the MIKC-Type MADS-Box Gene Family, and Expression Analysis of Female and Male Flower Buds in Walnut (Juglans regia, Juglandaceae)" Horticulturae 11, no. 7: 787. https://doi.org/10.3390/horticulturae11070787

APA Style

Guo, C., Fesobi, O. P., Zhang, Z., Yuan, X., Zhao, H., Quan, S., & Niu, J. (2025). Identification, Classification of the MIKC-Type MADS-Box Gene Family, and Expression Analysis of Female and Male Flower Buds in Walnut (Juglans regia, Juglandaceae). Horticulturae, 11(7), 787. https://doi.org/10.3390/horticulturae11070787

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