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Article

Genome-Wide Identification, Classification and Expression Analyses of MADS-Box Genes Reveal Their Role in Stem Gall Formation and Expansion of Zizania latifolia

by
Zhiping Zhang
1,
Meng Xiao
1,
Sixiao Song
1,
Yifeng Jiang
1,
Xinrui Zhu
1,
Lingtong Shi
1,
Xiaomeng Zheng
1,
Jiezeng Jiang
1 and
Minmin Miao
1,2,3,*
1
College of Horticulture and Landscape Architecture, Yangzhou University, Yangzhou 225009, China
2
Joint International Research Laboratory of Agriculture and Agri-Product Safety of Ministry of Education of China, Yangzhou University, Yangzhou 225009, China
3
Key Laboratory of Plant Functional Genomics of the Ministry of Education, Yangzhou University, Yangzhou 225009, China
*
Author to whom correspondence should be addressed.
Agronomy 2023, 13(7), 1758; https://doi.org/10.3390/agronomy13071758
Submission received: 13 May 2023 / Revised: 14 June 2023 / Accepted: 25 June 2023 / Published: 29 June 2023
(This article belongs to the Section Horticultural and Floricultural Crops)
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Abstract

:
MADS-box genes are important transcription factors in eukaryotes that are involved in regulating the growth and development of many plants. Jiaobai is a special aquatic vegetable formed through the stem expansion of Zizania latifolia infected by Ustilago esculenta and is wildly cultivated in Southeast Asia. To date, the biological function of MADS-box genes in Z. latifolia remains largely unknown. In this study, a genome-wide search for MADS-box genes in Z. latifolia was performed, and 87 MADS-box genes were identified. According to the phylogenetic analysis, there were 27 type I genes and 60 type II genes. The type I genes were mainly distributed in the Mα, Mβ and Mγ subfamilies, and the type II genes were divided into 14 subfamilies and unevenly distributed across 17 chromosomes. The encoded protein sequences ranged from 91 to 433 amino acids, the number of exons was 1~14, and most type I genes did not contain introns. The promoter region contained a large number of functional domains related to light response, plant hormones, stress and the meristem. An analysis of the expression patterns showed that there were massive differences in the expression of the MADS-box genes in different tissues, with six genes highly expressed in leaves and eight genes highly expressed in the stem apical meristem. Photoperiod and temperature were found to regulate the formation of the stem gall (SG) of Z. latifolia, and short-day (SD) conditions had an obvious promoting effect. ZlMADS57 and ZlMADS85 were significantly increased in SG under SD. Further analysis of the expression levels of MADS-box genes during the developmental stages of Z. latifolia found that the ZlMADS45, ZlMADS57, ZlMADS81 and ZlMADS85 were highly expressed 10 days before the SG formation, and ZlMADS66 reached its highest value on the day when the SG began forming. ZlMADS14, ZlMADS15, ZlMADS32, ZlMADS36, ZlMADS59 and ZlMADS71 were highly expressed after SG expansion, indicating that the MADS-box gene may play an important role in regulating SG formation and the subsequent expansion of Jiaobai. This study provides a reference for the functional identification of the MADS-box gene family and establishes a foundation for regulating the maturity of Z. latifolia.

1. Introduction

The MADS-box gene transcription factor family widely exists in animals, plants, fungi and other eukaryotes [1]. The name “MADS” is derived from the initial letters of the four types of MADS-box genes: MINICHROMOSEME MAINTENANCE1 (MCM1) in Saccharomyces cerevisiae [2], AGAMOUS (AG) in Arabidopsis [3], DEFICIES (DEF) in Antirrhinum majus [4] and SERUM RESPONSE FACTOR (SRF) in Homo sapiens [5]. MADS-box genes contain a highly conserved region composed of 58~60 amino acids in the N-terminus, which functions in combination with DNA and is an important component involved in transcriptional activation [6,7]. Based on the conserved domains and phylogenetic relationships, MADS-box proteins can be categorized into two types: Type I (M-type) and Type II (MIKC type). Type I contains three subfamilies, Mα, Mβ and Mγ, and Type II contains not only the MADS conservative domain but also the intervening domain, the keratin-like domain, and the C-terminal domain. Type II can be further divided into two subfamilies: MIKC* and MIKCC [8,9]. The MIKCC-group genes in the most recent common ancestor (MRCA) of extant angiosperms form approximately 17 clades: AG, STK, AGL12, AP3/DEF, PI/GLO, OsMADS32, Bsister/GGM13, SEP1/AGL2, AGL9/SEP1, AGL6, SQUA/AP1, FLC, SVP/StMADS11, SOC1/TM3, TM8, AGL15 and AGL17 [10,11].
In plants, the function of MIKCC-type genes has been extensively studied among MADS-box genes, as they are conserved among various plants and play an important role in regulating plant growth, development and other processes. These processes include the development of plant flowers and vegetative organs, flowering time, fruit maturity and senescence, axillary bud dormancy and germination, as well as participation in responses to various abiotic stressors [12,13,14,15,16,17,18,19,20,21]. The genome-wide identification, classification and functional annotation of the MADS-box gene family have been extensively studied in Arabidopsis, rice, wheat, potato, sweet potato and other plants [14,17,19,20,22].
Jiaobai is a symbiont of Zizania latifolia and Ustilago esculenta. It is widely cultivated in southeast Asia as a common aquatic vegetable [23,24]. Its edible organ, the swollen stem gall, is rich in polysaccharides, amino acids, flavonoids and phenols, and is thought to be beneficial to human health [25]. Jiaobai is the second largest aquatic vegetable, and its production has become a key industry in several provinces in China [23]. Understanding the mechanism of stem gall (SG) formation is of significant importance for improving the yield and quality of Jiaobai. Traditionally, the formation of stem gall was thought to be closely related to the temperature and photoperiod [23,26]. However, it remains unclear which of these two factors plays a more important role. Among the cultivated Jiaobai varieties in China, there are two types based on the temperature required for stem gall formation: the low-temperature type and the high-temperature type. According to the response to the photoperiod, there are also two main types of cultivars: single-season crop plants, which are short-day (SD) plants, with stem gall (SG) formation occurring in the fall when days become shorter, and two-season plants that are harvested twice in the summer and fall in China [23,27]. Z. latifolia is relatively closely related to rice, and the two species can be distantly hybridized, suggesting that their homologous genes may have similar functions [24,28]. OsMADS14 and OsMADS15 are considered the main regulatory factors for early flowering under SD in rice, and they are homologous genes that encode AP1 in Arabidopsis [14]. AP1 is an important MADS-box gene that activates the expression of meristem-determining genes involved in initiating flower development and promoting flowering in Arabidopsis. In wheat, the AP1 homologous gene VRN1 also has a similar function [29]. StMADS1 and StMADS13 are homologous genes of AP1 and OsMADS14/15, involved in tuberization and subsequent tuber development under SD, while four other StMADS genes are highly expressed in stolons and/or young tubers in potatoes [17]. Furthermore, the SQUA/AP1-like genes IbMADS79 and IbMADS19 participate in the tuberous root formation and development in sweet potatoes [20]. Therefore, we speculate that the MADS-box genes likely play an important role in the initiation and expansion of SG in Z. latifolia. However, to date, the biological function of MADS-box genes in transcriptional regulation in Z. latifolia remains largely unknown.
The recent successful assembly of the Z. latifolia genome at the chromosome level using Nanopore sequencing and Hi-C scaffolding [24] has provided a basis for analyzing the MADS-box gene family in Z. latifolia. Based on the whole genome, this study identified the MADS-box gene of Z. latifolia, analyzed its physical and chemical properties, evolutionary relationship, gene structure and conserved motifs, and analyzed the expression patterns of the MADS-box gene in leaf and stem apical meristem (SAM) and the initial stage of SG formation under different photoperiods and temperatures. Furthermore, the expression changes of highly expressed genes in the stem during the developmental stage of Z. latifolia its were studied. It was of great significance to deeply study the MADS-box transcription factor family for clarifying the unique mechanism of SG expansion in Jiaobai and expanding the understanding of new functions of MADS-box genes in regulating plant growth and development.

2. Materials and Methods

2.1. Identification of MADS-Box Family Members in Z. latifolia

The MADS-box protein sequences of Arabidopsis and Oryza sativa were obtained from the TAIR database (https://www.arabidopsis.org/ (accessed on 15 June 2022)) and the Rice Genome Annotation Project database (RGAP, http://rice.uga.edu/ (accessed on 15 June 2022)). The Z. latifolia genomic information was downloaded from the National Genomics Data Center (NGDC, https://ngdc.cncb.ac.cn/ (accessed on 12 February 2022)) [24]. The MADS-box protein sequences of Arabidopsis and rice were used as query sequences to search for the MADS-box gene family members in the Z. latifolia genome by BLASTP, and the threshold was set to E < 10−5, the consistency of sequence alignment (% identity) and the length of the comparison area that matches the comparison (alignment length) were set to 70% and 50%, respectively. After removing the repeat sequence, the candidate genes were further confirmed by the online tool CD-search (Conserved Domain Database, CDD v3.20, https://www.ncbi.nlm.nih.gov/Structure/cdd/wrpsb.cgi/ (accessed on 10–13 October 2022)), Plant TFDB v5.0 (Plant Transcription Factor Database, http://planttfdb.gao-lab.org/prediction.php (accessed on 16 October 2022)), and HMMER Services on EMBL-EBI (Hidden Markov Models database v2.41.2, https://www.ebi.ac.uk/Tools/hmmer/search/hmmscan (accessed on 18–20 October 2022)). Furthermore, the localization of all candidate MADS box genes on chromosomes was also examined, redundant sequences with the same chromosome location were deleted, and finally, the MADS box family genes of Z. latifolia were obtained.

2.2. Analysis of Protein Properties, Subcellular Localization and Tertiary Structure of MADS-Box Genes in Z. latifolia

Using the ExPASY protparam tool (https://web.expasy.Org/protparam/ (accessed on 15 October 2022)) analysis tool to analyze the number of amino acids, molecular weight, isoelectric point, instability index, aliphatic index and grand average of hydropathicity, and the subcellular localization of MADS-box proteins was predicted by the Plant-mPLoc v2.0 online tools (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/ (accessed on 19 October 2022)). The tertiary structures of ZlMADS-box proteins were performed by SWISS-Model (https://swissmodel.expasy.org/ (accessed on 7 June 2023)) with the GMQE value > 0.5.

2.3. Phylogenetic Tree Construction of MADS-Box Proteins in Z. latifolia

The full-length MADS-box protein sequences of Z. latifolia, Arabidopsis and rice were multiple aligned by Cluster X1.83, and the complete alignment results were saved as the PHYLIP format. The maximum likelihood method (ML) phylogenetic tree was constructed by FastTree v2.1.11 [30] using LG model which selected by MEGA 7.0 software (Find Best-Fit Substitution Model). Then, the phylogenetic tree was displayed with FigTree v1.4.3 and finally edited and annotated using Adobe Illustrator CS6.

2.4. Chromosomal Localization, Collinearity Analysis and SSRs Predictions of MADS-Box Genes in Z. latifolia

Chromosome information and gene annotation document (GFF) of the Z. latifolia genome (GWHBFHI00000000.1) were downloaded from the National Genomics Data Center (https://ngdc.cncb.ac.cn/ (accessed on 12 February 2022)). The corresponding position on chromosomes, duplication events and the collinearity relationships of MADS-box gene in Z. latifolia, and the collinearity relationships of the orthologous MADS-box genes among the genomes of Z. latifolia, Arabidopsis and rice were performed and visualized using the MCScanX tool and the Multiple Synteny Plot tool in TBtools-II (Toolbox for Biologists) v1.120 software, respectively. The Simple Sequence Repeats (SSRs) of ZlMADS-box genes were predicted using MicroSatellite identification tool v2.1 on Misa-web-IPK Gatersleben website (https://webblast.ipk-gatersleben.de/misa/ (accessed on 10 June 2023)) with the parameters of default.

2.5. Conserved Motif and Gene Structure Analysis of the MADS-Box Family in Z. latifolia

Conserved motifs of ZlMADS-box family protein were obtained by the MEME Suite v5.5.2 (https://meme-suite.org/meme/tools/meme (accessed on 16 October 2022)), in which the number of motifs was set to 10, and the width of conserved loci was 6~50. The gene structures of ZlMADS-box genes were analyzed using the online tool GSDS v2.0 (Gene Structure Display Server, http://gsds.gao-lab.org/ (accessed on 16 October 2022)) combined with the genome and CD sequence information of Z. latifolia. The phylogenetic tree, conserved motif and gene domain of the ZlMADS-box family were visually analyzed using the Gene Structure View (Advanced) tool in TBtools-II v1.120 software.

2.6. Cis-Acting Element Analysis of MADS-Box Genes in Z. latifolia

The genome sequence (fasta sequence) and gene structure annotation information (gff file) of Z. latifolia were prepared. The gff3 sequence extraction tool in TBtools-II v1.120 was used and only extract parameters of 2000-bp upstream of CDs was adopted to obtained the promoter regions of ZlMDAS genes, then the cis-acting elements were analyzed by the Plantcare online tool (http://bioinformatics.psb.ugent.be/webtools/plantcare/html/ (accessed on 25 October 2022)). After the general transcriptional regulatory elements and unknown functional elements were removed, the results were visualized using the Gene Structure View (Advanced) tool in TBtools-II v1.120 software.

2.7. MicroRNA (miRNA) Target of MADS-Box Genes in Z. latifolia

Targeting miRNAs of ZlMADS-box genes were predicted using Submit target candidates tool on psRNATarget website v2.0 (https://www.zhaolab.org/psRNATarget/ (accessed on 8-10 June 2023)). Specifically, the nucleotide sequences of the ZlMADS-box family were uploaded, and the expected value was set as 4 and all other parameters were set as default.

2.8. Plant Growth and Sample Collection

A single-season Jiaobai line ‘YD-3′ was used as the experimental material. It was a strictly short-day plant and commonly planted in spring and harvested once a year in autumn under natural conditions. All plant materials were cultivated in the aquatic vegetable experiment station of Yangzhou University (119°42′ E, 32°24′ N), Yangzhou, Jiangsu, China. Seedlings at the stage with 3–4 leaves were planted in plastic pots (53.5 cm diameter × 37.5 cm height) filled with garden soil under natural conditions.
The seedlings in the 8-leaf stage were moved to the artificial climate room for 30 days of four different photoperiod and temperature treatments until the SG began to form. The four temperature and photoperiod combinations were LD + HT (LD, long day, 14 h day/10 h night; HT, high temperature, 35 °C day/28 °C night), LD + AT (AT, appropriate temperature for SG formation of Jiaobai, 24 °C day/18 °C night), SD + HT (SD, short day, 8 h day/16 h night), and SD + AT. Then, the 3rd (counted from the stem top to bottom) fully expanded leaf, the 0.5 cm of the upper part of SAM and the nodes of initial swollen SG were collected according to Zhang et al. [31] and frozen in liquid nitrogen for transcriptome sequencing and qRT-PCR.
In order to intensively comprehend the role of the ZlMADS-box genes during the SG formation and expansion, expression patterns of these genes in the stems during the developmental stage of Z. latifolia were analyzed using qRT-PCR. The experiment was conducted from April 2021 to October 2021 under natural conditions, and then repeated in 2022. The seedlings were planted on April 5, and collected the SAM (as described above) every 10 days from 120 days after transplanting (August 6) to about 10 days before the initial expansion of Z. latifolia, labeled as 120 d, 130 d, 140 d, 150 d and 160 d. From the initial stage of SG formation (September 25) to the maturity period of the Z. latifolia (October 13), the SG at the second node (0.5 cm thick) were collected every three days, and labeled as 170 d, 173 d, 176 d, 179 d, 182 d, 185 d and 188 d, respectively. All samples were quickly placed in liquid nitrogen and then stored in an ultra-low-temperature freezer at −80 °C. Each sample was collected from six independent plants, and three biological replications were performed.

2.9. Transcriptome Sequencing Analysis

RNA was extracted from all samples using RNAiso Plus (total RNA-extraction reagent) (Takara, Japan), and double-terminal sequencing was performed based on the Illumina HiSeq 2500 platform. The original contributions presented in the study were publicly available. The raw sequencing data have been deposited to the NCBI Sequence Read Archive database with NCBI BioProject Accession PRJNA931771. The clean reads were mapped to the Z. latifolia genome (GWHBFHI00000000.1). Gene expression levels were calculated by the FPKM (fragments per kilobase of exon model per million mapped reads). To ensure the specificity of the coexpression network, the genes with an average FPKM of less than 1 in each tissue were filtered out, and the remaining genes were used for further analysis. The heatmap was established based on the log2-transformed (FPKM) through TBtools-II v1.120 software.

2.10. Gene Expression Analysis by qRT-PCR

According to the MADS-box gene expression profile, several genes highly expressed in the leaf or in the SG at the initial stage of Jiaobai formation under different cultivation conditions were selected for qRT-PCR expression verification. The qRT-PCR-specific primers of ZlMADS-box genes were designed by Primer Premier 5. Z. latifolia actin was used as the reference gene [32]. qRT-PCR amplifications were carried out with the Bio-Rad CFX ConnectTM Real-Time system (BIO-Rad, Hercules, California, USA), and three technical replicates were set for each cDNA. The conditions of qRT-PCR were as follows: 95 °C initial denaturation for 3 min, and followed by 40 cycles of 10 s at 95 °C, 30 s at 56 °C, melt curve 60.0 to 95 °C and increment 0.5 °C for 0.05 s. The results were analyzed by the 2−ΔΔCT method [33]. The primers were synthesized by Sangon Biotech (Shanghai, China) and listed in Table S1.

2.11. Statistical Analysis

The Excel 2019 and the SPSS software v15.0 were used to process and analyze the data, and the software GraphPad Prism v9.0 was applied to draw the column graphs.

3. Results

3.1. Information on the ZlMADS-Box Gene Family

Through bioinformatics analysis, a total of 87 ZlMADS-box genes were identified in Z. latifolia (Table S2). According to their chromosome location information, the 87 ZlMADS-box genes were named ZlMADS1–ZlMADS87 in turn. After analyzing the physical and chemical properties of the ZlMADS gene and ZlMADS protein, it was found that the CD sequence length of the ZlMADS-box gene varies greatly, ranging from 276 (ZlMADS46) to 1302 bp (ZlMADS33). The ZlMADS-box proteins contained 91 to 433 amino acids with molecular weights of 10,171.78 to 47,586.95 U, and the PIs (isoelectric points) was between 4.17 (ZlMADS31) and 10.18 (ZlMADS9) Further study found that the GRAVY of all predicted ZlMADS-box proteins was negative, indicating that ZlMADS-box proteins were hydrophilic proteins, but the degree of hydrophilicity was different. Among the 87 ZlMADS-box protein members, only ZlMADS3 and ZlMADS39 had an instability index (II) less than 40.

3.2. Phylogenetic Analysis of MADS-Box Genes in Z. latifolia

To further analyze the phylogenetic relationship and predict the function of the ZLMADS-box transcription factor family, we constructed a phylogenetic tree from 102, 73 and 87 MADS-box proteins in Arabidopsis, rice and Z. latifolia (Figure 1). MADS-box proteins were mainly divided into two categories: type I and type II MADS-box proteins, and type II accounted for 68.9%, 42.2% and 60.3% of the total MADS-box proteins in Z. latifolia, Arabidopsis, and rice, respectively. The proportion of type II MADS-box proteins in Z. latifolia was the highest in the three species. Among them, there were 27 ZlMADS-box proteins of type I, including the Mα (ten), Mβ (five) and Mγ (twelve) subcategories, and 60 type II MADS-box proteins, including MIKCC (fifty seven) and MIKC* (three) (Table S3). The MIKCC group can be further divided into 13 subfamilies: AG/STK (eight), AGL6 (three), AGL12 (five), AGL15 (three), AGL17 (seven), AP3 (DEF) (two), OsMADS32 (two), PI (GLO) (two), Bsister (GGM13) (three), SEP (seven), SOC1 (TM3) (five), SQUA/AP1 (five), and SVP (five). In general, the ZlMADS-box proteins was well classified, and all members can be classified correctly, which showed that the MADS-box genes were highly conserved in plants. After further comparative analysis, MADS-box genes of Arabidopsis were usually concentrated in one cluster, which was more obvious in type I proteins, while the proteins of Z. latifolia and rice were cross distributed in each subfamily, which indicated that the ZlMADS-box proteins were phylogenetically closer to the rice MADS-box transcription factors.

3.3. Analysis of MADS Protein Conserved Motifs and Gene Structure

To clarify the conserved structure of the MADS-box gene encoding protein in Z. latifolia, we predicted the conserved motif structure of 87 ZlMADS-box proteins (Figure 2A,B and Figure S1). In general, the number and type of motifs contained in the same subfamily were very similar, and there were differences among subgroups, which may be related to the functional differentiation of subgroups. Mα, Mβ and Mγ and MIKC* subgroup genes have two–four conserved motifs, while the majority of MIKCC-like genes contain more motifs, which can be up to eight. Annotated with SMART online tools, motifs 1 and motifs 3 were MADS-box domains, and the motif 1 domain encoded 29 proteins, which appeared in all ZlMADS-box proteins; except for 6 type I proteins, the other 81 ZlMADS-box gene family members contained motif 3, and motif 1 was adjacent to motif 3. Motif 2 was a K-box domain encoding 41 proteins, which only appeared in 83.3% of the type II MADS-box proteins but not in type I. Motifs 4–10 were structures with unknown functions; motifs 2, 5, 6 and 9 only appear in type II proteins; motifs 4, 7 and 8 were found in some MADS-box proteins; and further analysis found that motif 8 only appeared in the Mγ-subfamily, which may be the unique domain of the subgroup.
In the Type I group, except for four ZlMADS-box genes containing one intron, the other 23 ZlMADS-box genes had one long fragment of exons. On the other hand, the number of exons in Type II genes varies from 2 to 11. The number of exons of three genes in the MIKC* subfamily was the largest, with 10–11 exons. The distribution pattern of exons of most genes in the same MIKCC subgroup was consistent and regular, and the number of exons among genes in each subgroup was basically the same, such as AGL6, AGL12, AGL15, AP3(DEF), OsMADS32, SEP, SOC1 and SQUA/AP1 (Figure 2C).

3.4. Chromosome Distribution and Duplication Events of MADS-Box Genes

By extracting the chromosomal location information of the MADS-box gene of Z. latifolia, it was found that only one gene (ZlMADS87) was located on a fragment that had not been completely assembled, and the remaining 86 members were unevenly distributed on 17 chromosomes, as shown in Figure 3A. Chr02 and Chr04 contained the most MADS-box gene subfamilies, with more than 10 members, accounting for 19.5% and 13.7% of the total, respectively, followed by Chr 03 and Chr 09, which all contained 7 ZlMADS-box genes, and other chromosomes contained fewer members, between 1 and 5 genes. The Type I MADS proteins were distributed on the 9 chromosomes. The Mα subfamily was widely distributed on Chr02, Chr03, Chr04, Chr05, Chr08, Chr09, Chr15, Chr04 and Chr16. Eight ZlMADS-box genes on Chr02 were in the Mγ subfamily, and the remaining 4 members were sporadically distributed on Chr04, Chr05, Chr09 and Chr12. In contrast, the Mβ subfamily group was relatively concentrated and only distributed on Chr02 and Chr04. The Type II MADS-box genes were widely and unevenly distributed on all the chromosomes, in which AGL6, AGL12, AGL15, AP3 (DEF), OsMADS32, PI, BS, SOC1 and SVP subfamily gene members were the most dispersed, and each gene was independently distributed on one chromosome, while in the other five type II MADS protein subfamilies, at least two gene family members were co-located on one chromosome. Several gene clusters existed on Chr02, Chr03, Chr04, Chr09, Chr11, and Chr15; among them, two gene clusters were distributed on Chr02, especially in the interval 29455.1~19580 kb containing six Mβ subfamily group genes.
Collinearity analysis of Z. latifolia genome was carried out using McScanX, and 78.2% of the MADS box genes originated from gene duplication (blue, green and red lines) (Figure 3A). Six genes located on Chr02 formed four tandem duplications, in which 83.39% belonged to Type I group. It was also found that tandem duplications could occur between different subgroups, e.g., ZltMADS5 belonged to MIKCC, while ZlMADS6 belonged to Mβ. Sixty-two genes were found with their counterparts on inter-chromosome segmental duplications, of which 16 genes belonged to type I group and the other genes belonged to type II group. Further analysis showed that there were only one or two collinear relationships in type I group, and 26.7% of genes had two collinearity relationships (green lines). About 47.8% of type I MADS-box genes had more than two collinear relationships, of which 8 genes had three collinear relationships (red lines). The frequency of segmental duplication relationships in Type II MADS-box genes was 1.41 times higher than that in Type I MADS-box genes. The collinearity analysis statistics of MADS box family genes in three species of Z. latifolia, Arabidopsis, and rice were further analyzed. As shown in Figure 3B, six MADS-box genes in the Arabidopsis genome and nine MADS-box members in Z. latifolia constituted a total of 11 collinear gene pairs, and 52 MADS-box genes from the rice genome and 72 MADS-box genes from Z. latifolia constituted a total of 111 collinear gene pairs, indicating that the vast majority of ZlMADS-box genes could find orthologous genes in rice. SSRs analysis indicated that most ZlMADS-box genes were predicted to have not involved an SSR sequence, only 13 ZlMADS-box genes contained SSRs, and there were two SSRs involved in ZlMADS81 (Table S4).

3.5. Analysis of Cis-Acting Elements of MADS-Box Gene Promoter Regions in Z. latifolia

The ZlMADS-box genes had a wide variety of cis-acting elements and diverse functions (Figure 4, Table S5). A total of 2200 elements were detected in the 2000-bp promoter regions of 87 MADS-box genes. Among these regulatory elements, light response elements accounted for a large proportion, with a total of 927, and were distributed in all ZlMADS-box genes. Each gene had different numbers of light response elements; ZlMDAS58 had the most (up to 21), while ZlMDAS51 had the least (only 3). There were many types of hormone response elements, including abscisic-acid response element (278), MeJA response element (256), gibberellin-responsiveness (88), auxin response element (66), and salicylic-acid response element (49). In addition, MADS-box genes in Z. latifolia also contain a variety of cis-acting elements involved in anaerobic induction, low-temperature response and drought inducibility.

3.6. Short Days Promote the Formation of SG in Z. latifolia

In Z. latifolia plants cultivated under SD + AT for 15 days, the fleshy stem began to expand, and the SG was barely visible (Figure 5A,B), and the SG formation rate was about 12.3%; under the LD + HT, LD + AT and SD + HT conditions the SG has not yet entered the initial formation period. Compared with the LD + HT growth conditions, after 30 days of cultivation under LD + AT, SD + HT, and SD + AT, the junction of the leaf sheath and leaf of Z. latifolia plants gradually tightened, and the height of the three leaves in the middle of the plant was significantly different, and the height of the central widened leaf was significantly lower than that of the adjacent leaves, and there were obvious nodes and internodes in the SG, with a diameter of approximately 1.4 ± 0.15 cm, while under LD + HT, the SAM was only a small shrinking growth point, and no nodes were observed (Figure 5C,D). Z. Latifolia grown under SD + HT and SD + AT conditions, the SG formation rates reached 96.5% and 98.7%, respectively, which were significantly higher than that under LD + AT conditions (Figure 5E).

3.7. Expression Analysis of ZlMADS-Box Genes under Different Photoperiods and Temperature Conditions

Through cluster analysis of transcriptome data of leaves, SAM and SG of Jiaobai plants under different photoperiod and temperature conditions by our research group, a heatmap map of the MADS gene expression pattern of Z. latifolia was constructed (Figure 6). Only two Mα genes in 27 type I groups were expressed in all tissues, but there was no significant difference among the samples of each group. One MIKC* gene (ZlMADS26) was only expressed in leaves, and there was no difference in the expression level when treated with different photoperiods and temperatures. Although more than half of MIKCC genes were expressed, there were significant differences in the expression pattern among the genes in each subgroup. For example, five SVP genes and five SQUA/AP1 genes were highly expressed in all samples, while none of the two AP3 (DEF) genes and five SEP genes were expressed, and only two of the eight AG/STK genes were expressed.
The AG/STK-like gene (ZlMADS34), AGL6-like gene (ZlMADS49) and AGL17-like gene (ZlMADS64) were only expressed in leaves under four cultivation conditions; the AGL15-like gene (ZlMADS2) was only expressed in leaves under LD + AT conditions. In addition, we also found that ZlMADS1, ZlMADS28, ZlMADS43, ZlMADS71 and ZlMADS86 were expressed in all samples, but the expression levels in leaves were significantly higher than that in SAMs.
However, one PI (GLO)-like gene (ZlMADS75), two OsMADS32-like genes (ZlMADS14, ZlMADS36), one SQUA/AP1-like gene (ZlMADS85), one AGL17-like gene (ZlMADS66), and three AGL12-like genes (ZlMADS32, ZlMADS45, and ZlMADS70) were specifically expressed in SAM or SG. Further study found that the expression of ZlMADS36 in SAM under LD + HT was obviously higher than that in the swollen SG treated with LD + AT, SD + HT and SD + AT, which may be a negative regulator for Jiaobai formation; ZlMADS66 and ZlMADS75 were specifically expressed in the SG at the initial expansion stage under AT cultivation; ZlMADS85 was only expressed during SG formation, especially under SD. ZlMADS45 was specifically expressed under HT, but the difference between SAM under LD + HT or SG under SD + HT was not obvious.
In addition, the expressions of ZlMADS3, ZlMADS15, ZlMADS54, ZlMADS55, ZlMADS59, and ZlMADS65 in SAM and SG were significantly higher than that in leaves. Expression of ZlMADS65 in SAM under LD + HT was significantly lower than that in the LD + AT, SD + HT and SD + AT with stem expansion, implying that it may be an important positive regulator of the initial formation of Jiaobai. The expression of ZlMADS15 and ZlMADS59 in SG under LD + AT and SD + AT was higher than that in LD + HT and SD + HT, indicating that these two genes may play an important role in the formation of Jiaobai stimulated by temperature. Furthermore, another SQUA/AP1-like gene (ZlMADS57) was expressed at significantly higher levels in SG under SD, especially under SD + HT conditions, than under LD.
Eight ZlMADS-box genes with FPKM values > 2 were analyzed by RT-qPCR to further verify the RNA-seq data during SG swelling under the conditions of LD + HT, LD + AT, SD + HT, and SD + AT cultivation (Figure 7). The change trend of the expression levels of the ZlMADS-box genes was consistent with the results of RNA-seq data.

3.8. Expression Analysis of MADS-Box Genes during the Developmental Stage of Z. latifolia

Before the SG formation of Z. latifolia, there was almost no change in the meristem tissue at the top of the stem, and at 170 d after transplanting under SD, the stem began swelling, followed by rapid expansion, and reached maturity at 188 d after transplanting (Figure 8A). In order to clarify the function of ZlMADS-box gene in the SG formation and expansion, we further analyzed the expression changes of 12 ZlMADS-box genes found from transcriptome data that were highly expressed in the stem and significantly increased when the stem began to swell during the different growth periods of Z. latifolia. Except for ZlMADS75, the expression levels of the other 11 ZlMADS-box genes exhibited obvious and regular changes during different developmental stages of Z. latifolia (Figure 8B). Overall, the expression levels of each gene did not change significantly from 120 to 150 days after transplantation, but gradually peaked from 160 to 188 d after cultivation. According to the peak time when the expression level of each gene reached maximum, 11 ZlMADS-box genes could be roughly divided into four categories. The first group included ZlMADS45, ZlMADS57, ZlMADS81 and ZlMADS85, which had consistently high expression levels at 150 days after transplantation, with a peak occurring 10 days before the SG formation (160 days). The gene expression level in the second group reached its highest value on the day when the SG began forming (170 d), including ZlMADS66. The peak time of the expression level in the third and fourth group occurred in the early stage (173 days) and the middle to late stage (179 and 182 days) after SG expansion, and the former included ZlMADS14 and ZlMADS15, while the latter included ZlMADS32, ZlMADS36, ZlMADS59 and ZlMADS71, respectively.

3.9. miRNA Targets and Tertiary Structure of MADS-Box Gene Family in Z. latifolia

The miRNA Targets and tertiary structures were conducted on the 12 ZlMADS-box gene family mentioned above. A total of 43 miRNAs were predicted to target 11 ZlMADS-box genes, while ZlMADS75 was predicted to have no miRNA targeting. On the other hand, different ZlMADS-box genes were predicted to be the target genes of different number of miRNAs; for example, ZlMADS30 and 57 were predicted to be target gene of 10 different miRNAs, while ZlMADS32, 71 and 81 were targeted by just one miRNA. Three miRNAs, osa-miR426, cme-miR854 and aly-miR3441-3p.2, were predicted to inhibit ZlMADS 66, 71 and 85 through translation, while other miRNAs regulated ZlMADS gene expression through the cleavage effect (Table S6).
The results of tertiary-structure analysis indicate that the proportions of α-helix, β-sheet, random coil and extended strand involved in the ZlMADS box proteins were different, resulting in certain differences in their spatial structure. The tertiary structures in the same subfamily were relatively similar, such as ZlMADS14 and 36 in OsMADS32 subfamily, ZlMADS57 and 85 in SQUA/AP1 subfamily. However, there were significant differences in spatial structure among subfamilies, such as AGL17-like ZlMADS66 exhibiting an α-helix, β-sheet and random coil, and AG-like ZlMADS71 which mainly exhibits an α-helix and β-sheet, while SOC1-like ZlMADS81 has no β-sheet (Figure S2).

4. Discussion

In this study, 87 MADS-box genes were identified in Z. latifolia. The number of MADS-box families varies greatly among different species, and 107, 75, 57, 153 and 95 MADS-box genes were identified in Arabidopsis, rice, Brachypodium distachyon, potato and sweet potato, respectively [14,17,20,22,34]. It was believed that this phenomenon may be due to the diversity and selectivity of biological evolution. Even in the same family in the classification system, the number of MADS-box genes varies greatly due to gene duplication events such as whole genome replication, tandem replication and fragment replication; for example, the MADS-box genes in Arabidopsis, Chinese cabbage and Brassica napus in the mustard family were 107, 167 and 307, respectively [22,35,36]. Comparative genomics studies have shown that Z. latifolia and rice evolved from the same ancestral species [24]. During the evolutionary process, some gene families of Z. latifolia and rice may have been lost or duplicated, which may be the reason for the difference in the number of MADS box genes between Z. latifolia and rice [24].
According to the MADS-box subgroup classifications of A. thaliana and rice [14,18,19], ZlMADS-box proteins were distinguished into type-I (M-type) proteins and type-II (MIKC-type) proteins, and then 27 M-types could be further divided into three groups, Mα, Mβ and Mγ. The MIKC-type contained 60 genes, which could be divided into two groups: MIKC* and MIKCC. The presence or absence of the 17 distinct clades in MIKCC group genes in the most recent common ancestor (MRCA) of extant angiosperms were different, and TM8-like genes and FLC-like genes were frequently completely lost in most gramineous plants, such as Z. mays, S. bicolor, S. italica, rice and B. distachyon [11]. However, it was reported that several FLC-like genes exist in the wheat genome [19]. Different classifications of MADS-box genes among species indicate that gene evolution and function were extremely complex, the retention of duplicate genes in different species was different, and MADS-box genes of the same classification in different species were subject to different constraints in the evolutionary process [37]. In this study, TM8- and FLC-like genes were lost in the genome sequence of Z. latifolia. Previous studies have shown that the clades of AG- and STK-like genes and the clades of AGL2- and AGL9-like genes cannot be divided in the complete phylogeny of MIKCC-group MADS-box genes, and the AG- and STK-like genes may be the sister clades [11,14,19]. The STK- and AGL9-like genes were not further subdivided in many species, such as rice, potato, sweet potato and orchids [14,17,18,20]. In this study, several MADS-box genes were also classified into mixed category, such as the clades of AG- and STK-like genes and the clades of AGL2- and AGL9-like genes.
Genome-wide duplication analysis could better analyze the evolutionary relationships of ZlMAD-box genes. It was found that a large number of ZlMAD-box genes resulted from the gene duplication events. Previous studies have shown that whole genome duplication events play an important role in the expansion of the MADS-box transcription factor family [35,37]. There were differences in evolutionary expansion between M-type and MIKC type, tandem duplications mainly contributed to the M-type genes expansion whereas inter-chromosome segmental duplication existed mainly in the MIKC type genes [17,38]. In the Z. latifolia genome, Type I MAD-box genes have more tandem repeat events within chromosomes than Type II type MAD-box genes, and mostly Type II type MAD-box genes have corresponding segmental duplication gene pairs. The collinearity analysis of potato MAD-box gene found that tandem duplication can occur between different subgroups, indicating that gene replication not only led to the expansion of MADS-box gene family, but also led to functional diversification [17]. A similar result was also found in this study. The results of collinearity analysis in the genomes of Z. latifolia and rice indicated that the vast majority of ZlMAD-box genes can be found as orthologous genes in rice, indicating that these genes may have similar functions.
Different from the quantitative relationship of MADS-box genes in different species, MADS-box genes have similar gene structures in different species and have similar numbers of exons in many species, such as Arabidopsis, rice, wheat, and apple [14,19,39]. MIKC-type gene sequences were generally long and contain more exons, the gene structure was more complex than that of M-type genes, and MADS-box proteins in the same subgroup on the phylogenetic tree contain almost identical motif types [14,17,18]. Similar results were found in ZlMADS-box genes. Furthermore, according to the expression results, most genes in the same subgroup had consistent expression patterns. For example, all AP3 (DEF)-like and SEP-like genes were not expressed in leaves and SAMs, while OsMADS32-like genes were specifically expressed in SAMs. However, some gene-expression patterns showed differences, such as the SQUA/AP1 and AG/STK subgroups. The genes derived from the same original group could be divided into different functional modules with different spatiotemporal characteristics of expression in the process of evolution to avoid functional redundancy and generate subfunctions and new functions [40].
The analysis of RNA-seq data during the formation of Jiaobai showed that most members of the type I MADS-box gene were not expressed or the expression level was extremely low, while the expression level of the type II MADS-box gene was relatively high, which laid a foundation for the important regulatory role of the type II MADS box gene in the organ development and morphogenesis of Z. latifolia. Type II MADS-box genes played important roles in numerous biological process in plants [41,42,43,44]. The MDAS-box gene function in regulating leaf structure was not conserved, and multiple subgroups were involved. AGL8/FUL displayed a function in the regulation of leaf characteristics by affecting vascular bundle development in Arabidopsis [45]. In tomato, AP1/FUL MADS-box gene SlMBP20 was involved in leaf development [46], and overexpression of SEP-like gene SIMBP21 altered leaf morphology [44]. AtAGL11 homologous gene BnaAGL11 regulated leaf morphogenesis and senescence in Brassica napus [47]. Z. latifolia ZlMADS1, ZlMADS28, ZlMADS34, ZlMADS43, ZlMADS49, ZlMADS64 and ZlMADS71 were highly expressed in leaves, indicating that these genes were probably involved in leaf growth and development.
Previous studies have suggested that photoperiod and temperature jointly determined the season and period of SG enlargement in Z. latifolia [23]. This study found that both SD and AT have the effect of inducing the swelling of SG, but SD has a more significant effect on promoting the SG formation, especially in the HT season when the formation and development of Jiaobai were inhibited. In sweet potato, the MADS-box genes IbMADS3, IbMADS4, IbMADS79, IbMADS17 and IbMADS20 may participate in the initiation of tuberous root differentiation and development [20,48,49]. Gao et al. [17] found that StMADS1, StMADS3, StMADS11, StMADS13, StMADS16 and StMADS29 were homologous genes of SQUA/AP1, AGL12, SVP and SOC1 (TM3) in Arabidopsis, and highly expressed in storage organs, which may play central role in tuberization and following tuber development. These studies indicated that multiple MADS-box genes were jointly involved in the formation and enlargement of vegetative organs. Several ZlMADS genes showed tissue-specific expression profiles. ZlMADS14, ZlMADS32, ZlMADS36, ZlMADS45, ZlMADS66, ZlMADS70, ZlMADS75 and ZlMADS85 were specifically expressed in SAM or SG. Besides that, the expression of ZlMADS3, ZlMADS15, ZlMADS54, ZlMADS55, ZlMADS59, and ZlMADS65 in stem were significantly higher than that in leaves. Among these genes, ZlMADS15, ZlMADS59, ZlMADS57, ZlMADS66, ZlMADS75, and ZlMADS85 were highly expressed in the SG under AT and/or SD, indicating that these genes displayed the function in the regulation of stem gall expansion of Jiaobai. Furthermore, expression pattern of MADS-box genes during the developmental stage of Z. latifolia cultivated under normal conditions, indicating that the ZlMADS-box gene synergistically regulated the formation and development of SG in Z. latifolia. ZlMADS45, ZlMADS57, ZlMADS66, ZlMADS81 and ZlMADS85 were probably involved in the initial formation of SG, and the ZlMADS14, ZlMADS15, ZlMADS32 and ZlMADS36 may play roles in SG expansion in the later stage.
A large number of studies on the photoperiod regulation of flowering in Arabidopsis and rice have shown that AP1-like clades of MADS-box genes were downstream targets of Flowering Locus T (FT) [50,51]. Additionally, the SQUA/AP1 homologous genes StMADS1 and StMADS13 were direct downstream genes of the FT-like gene StSP6A during potato tuberization, and they were involved in the photoperiod regulation of storage-organ formation [17]. Although AP1-like genes have different functions during evolution, they play a role in the transition from vegetative to reproductive growth and storage organ formation induced by photoperiod. The expression of ZlMADS57 and ZlMADS85 in the SQUA/AP1 subgroup was significantly upregulated under SD conditions. According to the common pathway components of flowering and tuber setting, we speculate that these two genes may be the key genes for photoperiod regulation of the initial formation of the fleshy stem of Jiaobai. In addition, the transcriptional activity of some key MADS-box genes are regulated by temperature in many plants to play roles in regulating flowering and breaking dormancy [52,53]. In this study, we also found that the expression of several MADS-box genes changed significantly in initial swollen SG under different temperature conditions, but their specific functions need to be further studied.
In conclusion, 87 MADS-box genes were identified in the whole genome of Z. latifolia in this study, and the classification and evolutionary relationship of ZlMADS-box proteins were constructed by sequence alignment and phylogenetic methods. The analysis of chromosome mapping showed that ZlMADS-box genes were unevenly distributed on 17 chromosomes, and a large number of ZlMAD-box genes were resulted from the gene duplication events. The gene structure and motif pattern of the ZlMADS-box family in the same subgroups were similar. The cis-acting elements of the ZlMADS-box gene promoters were mainly light response, ABA response, MeJA response and anaerobic induction. When combined with the expression pattern, the important role of the MADS-box gene family in the formation and following expansion of Jiaobai by photoperiod and temperature regulation was revealed. Furthermore, SQUA/AP1 genes ZlMADS57 and ZlMADS85 displayed a function in the regulation of SG formation under SD. The conclusion of this study laid a foundation for further revealing the function of MADS-box genes and provided an important reference for regulating the maturity time of Jiaobai.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/agronomy13071758/s1, Table S1: Primer sequences for qRT-PCR; Table S2: Identification and protein physic-chemical analysis of the ZlMADS-box gene family; Table S3: Phylogenetic relationship information of MADS-box genes in the Z. latifolia; Table S4: Predicted SSRs in the genomic sequences of MADS-box gene family in Z. latifolia; Table S5: Cis-acting elements in the promoter region of MADS-box gene family in the Z. latifolia; Table S6: Predicted miRNAs targeting MADS-box gene family in the Z. latifolia; Figure S1: Analysis of MADS-protein motifs in Z. latifolia; Figure S2: Tertiary structure of MADS-box gene family in Z. latifolia.

Author Contributions

Conceptualization, methodology, writing—review and editing Z.Z. and M.M.; validation, formal analysis, investigation and data curation, M.X., S.S., Y.J., X.Z. (Xinrui Zhu), L.S. and X.Z. (Xiaomeng Zheng); software and visualization, M.X. and Y.J.; supervision, M.M. and J.J.; writing—original draft preparation, Z.Z. and M.X. All authors have read and agreed to the published version of the manuscript.

Funding

This research was supported by Jiangsu Agriculture Science and Technology Innovation Fund (CX (20)3104), Jiangsu Modern Agricultural (Vegetable) Industrial Technology System (JATS [2022] 495), Open Funds of the Jiangsu Key Laboratory of Crop Genomics and Molecular Breeding (ML202006), Project of Jiangsu Province Science and Technology (BE2017380), and the 58th batch of the China Postdoctoral Science Foundation (2015M580478).

Data Availability Statement

The original contributions presented in the study were publicly available. The raw sequencing data have been deposited to the NCBI Sequence Read Archive database with NCBI BioProject Accession PRJNA931771.

Conflicts of Interest

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

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Figure 1. Phylogenetic analysis of Arabidopsis, rice and Z. latifolia MADS-box proteins. Blue circles, green circles and red circles indicate Arabidopsis, rice and Z. latifolia proteins, respectively.
Figure 1. Phylogenetic analysis of Arabidopsis, rice and Z. latifolia MADS-box proteins. Blue circles, green circles and red circles indicate Arabidopsis, rice and Z. latifolia proteins, respectively.
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Figure 2. Phylogenetic relationships, conserved motifs and gene structure of MADS-box proteins in Z. latifolia. (A) ML tree of ZlMADS-box proteins. (B) Conserved motif distribution of ZlMADS-box proteins. (C) Gene structure of MADS-box genes in Z. latifolia.
Figure 2. Phylogenetic relationships, conserved motifs and gene structure of MADS-box proteins in Z. latifolia. (A) ML tree of ZlMADS-box proteins. (B) Conserved motif distribution of ZlMADS-box proteins. (C) Gene structure of MADS-box genes in Z. latifolia.
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Figure 3. Chromosome distribution and collinearity analysis of MADS-box genes in Z. latifolia. (A) chromosomal distribution and interchromosomal relationships of MADS-box genes in Z. latifolia. Gene IDs with the same color represent genes of the same MADS-box subfamily, and the blue, green, yellow, red, and black names represent the Mα, Mβ, Mγ, MIKC*, and MIKCC subfamilies, respectively. Dots with the same color represent a collinear relationship between genes. Blue lines, green lines and red lines represent one, two or three duplicated gene pairs, respectively. (B) Synteny analysis of MADS-box genes between Z. latifolia, Arabidopsis and rice. The blue lines indicate collinear MADS-box gene pairs. The gray lines indicate all collinear blocks.
Figure 3. Chromosome distribution and collinearity analysis of MADS-box genes in Z. latifolia. (A) chromosomal distribution and interchromosomal relationships of MADS-box genes in Z. latifolia. Gene IDs with the same color represent genes of the same MADS-box subfamily, and the blue, green, yellow, red, and black names represent the Mα, Mβ, Mγ, MIKC*, and MIKCC subfamilies, respectively. Dots with the same color represent a collinear relationship between genes. Blue lines, green lines and red lines represent one, two or three duplicated gene pairs, respectively. (B) Synteny analysis of MADS-box genes between Z. latifolia, Arabidopsis and rice. The blue lines indicate collinear MADS-box gene pairs. The gray lines indicate all collinear blocks.
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Figure 4. Cis-acting element distribution in promoter region of the Z. latifolia MADS-box genes.
Figure 4. Cis-acting element distribution in promoter region of the Z. latifolia MADS-box genes.
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Figure 5. Effects of different temperatures and photoperiods on SG formation in Z. latifolia. The morphological characteristics of Z. latifolia (A,C) and the external morphology and longitudinal section of the apical meristem of Jiaobai (B,D) at 15 and 30 days after different photoperiod and temperature treatments. Red lines: the height of the central (1st leaf counted from the stem top) leaf position; red circle: the connecting of the leaf blade and the leaf sheath. (E) Stem gall formation rate of Z. latifolia. HT: high temperature, 35 °C day/28 °C night; LD: long day, 14 h day/10 h night; AT: appropriate temperature for SG formation of Jiaobai, 24 °C day/18 °C night, SD: short day, 8 h day/16 h night. Four different photoperiods and temperature treatments were used: LD + AT, LD + HT, SD + HT, and SD + AT. The same as below. Vertical bars represent standard errors of means (n = 15). Different lowercase letters above the column indicate significant differences (p < 0.05) among treatments.
Figure 5. Effects of different temperatures and photoperiods on SG formation in Z. latifolia. The morphological characteristics of Z. latifolia (A,C) and the external morphology and longitudinal section of the apical meristem of Jiaobai (B,D) at 15 and 30 days after different photoperiod and temperature treatments. Red lines: the height of the central (1st leaf counted from the stem top) leaf position; red circle: the connecting of the leaf blade and the leaf sheath. (E) Stem gall formation rate of Z. latifolia. HT: high temperature, 35 °C day/28 °C night; LD: long day, 14 h day/10 h night; AT: appropriate temperature for SG formation of Jiaobai, 24 °C day/18 °C night, SD: short day, 8 h day/16 h night. Four different photoperiods and temperature treatments were used: LD + AT, LD + HT, SD + HT, and SD + AT. The same as below. Vertical bars represent standard errors of means (n = 15). Different lowercase letters above the column indicate significant differences (p < 0.05) among treatments.
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Figure 6. Expression patterns of all ZlMADS-box genes in different tissues during SG formation in response to photoperiod and temperature conditions. FPKM values of ZlMADS-box genes were normalized, and the blue and yellow colors indicate the expression levels of ZlMADS-box genes from low to high in the row scale.
Figure 6. Expression patterns of all ZlMADS-box genes in different tissues during SG formation in response to photoperiod and temperature conditions. FPKM values of ZlMADS-box genes were normalized, and the blue and yellow colors indicate the expression levels of ZlMADS-box genes from low to high in the row scale.
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Figure 7. Relative expression levels of 8 MADS-box genes during SG formation in response to photoperiod and temperature conditions using qRT-PCR. Different lowercase letters above the column indicate significant differences (p < 0.05) between samples of each treatment.
Figure 7. Relative expression levels of 8 MADS-box genes during SG formation in response to photoperiod and temperature conditions using qRT-PCR. Different lowercase letters above the column indicate significant differences (p < 0.05) between samples of each treatment.
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Figure 8. Morphology and expression profile of MADS-box genes of Z. latifolia at different developmental stages. (A) The morphological characteristics of Z. latifolia. (B) Expression profile of MADS-box genes during the different developmental stages of Z. latifolia. 120–188 d: days after transplantation. 170 d: the initial stage of SG formation. White box: morphology of the SG.
Figure 8. Morphology and expression profile of MADS-box genes of Z. latifolia at different developmental stages. (A) The morphological characteristics of Z. latifolia. (B) Expression profile of MADS-box genes during the different developmental stages of Z. latifolia. 120–188 d: days after transplantation. 170 d: the initial stage of SG formation. White box: morphology of the SG.
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MDPI and ACS Style

Zhang, Z.; Xiao, M.; Song, S.; Jiang, Y.; Zhu, X.; Shi, L.; Zheng, X.; Jiang, J.; Miao, M. Genome-Wide Identification, Classification and Expression Analyses of MADS-Box Genes Reveal Their Role in Stem Gall Formation and Expansion of Zizania latifolia. Agronomy 2023, 13, 1758. https://doi.org/10.3390/agronomy13071758

AMA Style

Zhang Z, Xiao M, Song S, Jiang Y, Zhu X, Shi L, Zheng X, Jiang J, Miao M. Genome-Wide Identification, Classification and Expression Analyses of MADS-Box Genes Reveal Their Role in Stem Gall Formation and Expansion of Zizania latifolia. Agronomy. 2023; 13(7):1758. https://doi.org/10.3390/agronomy13071758

Chicago/Turabian Style

Zhang, Zhiping, Meng Xiao, Sixiao Song, Yifeng Jiang, Xinrui Zhu, Lingtong Shi, Xiaomeng Zheng, Jiezeng Jiang, and Minmin Miao. 2023. "Genome-Wide Identification, Classification and Expression Analyses of MADS-Box Genes Reveal Their Role in Stem Gall Formation and Expansion of Zizania latifolia" Agronomy 13, no. 7: 1758. https://doi.org/10.3390/agronomy13071758

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