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Article

Genome-Wide Identification and Expression Analysis of the DA1 Gene Family in Sweet Potato and Its Two Diploid Relatives

by
Zelong Zhou
1,†,
Jianzhi Huang
1,†,
Yuehui Wang
1,
Shixiang He
1,
Jing Yang
1,
Ying Wang
1,
Wenxing Li
1,
Yi Liu
1,
Ran Xu
1,
Yunhai Li
2 and
Lian Wu
1,*
1
School of Breeding and Multiplication (Sanya Institute of Breeding and Multiplication), Hainan University, Sanya 572025, China
2
State Key Laboratory of Plant Cell and Chromosome Engineering, CAS Centre for Excellence in Molecular Plant Biology, Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing 100101, China
*
Author to whom correspondence should be addressed.
†
These authors contributed equally to this work.
Int. J. Mol. Sci. 2024, 25(5), 3000; https://doi.org/10.3390/ijms25053000
Submission received: 8 January 2024 / Revised: 28 February 2024 / Accepted: 4 March 2024 / Published: 5 March 2024
(This article belongs to the Collection Genetics and Molecular Breeding in Plants)
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Versions Notes

Abstract

:
The DA1-like gene family plays a crucial role in regulating seed and organ size in plants. The DA1 gene family has been identified in several species but has not yet been reported in sweet potatoes. In this study, nine, eleven, and seven DA1s were identified in cultivated sweet potato (Ipomoea batatas, 2n = 6x = 90) and its two diploid wild relatives, I. trifida (2n = 2x = 30) and I. triloba (2n = 2x = 30), respectively. The DA1 genes were classified into three subgroups based on their phylogenetic relationships with Arabidopsis thaliana and Oryza sativa (rice). Their protein physiological properties, chromosomal localization, phylogenetic relationships, gene structure, promoter cis-elements, and expression patterns were systematically analyzed. The qRT-PCR results showed that the expression levels of four genes, IbDA1-1, IbDA1-3, IbDA1-6, and IbDA1-7, were higher in the sweet potato leaves than in the roots, fiber roots, and stems. In our study, we provide a comprehensive comparison and further the knowledge of DA1-like genes in sweet potatoes, and provide a theoretical basis for functional studies.

1. Introduction

The sizes of plant seeds and organs are important yield traits. An important topic in developmental biology is size regulation. Multiple genes involved in seed size regulation have been identified in plants, including DA1 [1], SGD1 [2], GS3 [3], OsLG3 [4], LGY3 [5], GW5 [6], GW2 [7], GW8 [8], qGL3 [9], GS10 [10], GL4 [11], TGW2 [12], and GS9 [13]. DA1 is an important gene that regulates seed size. This gene encodes a ubiquitin receptor. A protein sequence analysis has indicated that DA1 contains two typical ubiquitin interaction motifs (UIMs) and a single zinc-binding (LIM) domain. Through ubiquitination, ubiquitin proteins regulate transcription factors’ and regulatory proteins’ stability and activity, which is a critical regulatory function in the growth and determination of seed size [14].
The DA1-like gene family has been identified in several crops: four genes in rapeseed (Brassica napus) [15], eleven genes in soybean (Glycine max) [16], six genes in maize (Zea mays L.) [17], three genes in oat (Avena sativa L.) [18], twenty-one genes in cotton (Gossypium spp.) [19], and four genes in rice (Oryza sativa L.) [20]. The DA1 gene was initially discovered to regulate the size of leaves, seeds, flowers, and siliques in Arabidopsis thaliana. Additionally, abscisic acid (ABA) upregulates AtDA1 expression, thereby positively regulating seed size [1]. The DA2 gene encodes a ring-type E3 ubiquitin ligase that interacts with DA1, thereby influencing seed size. Decreasing the expression level of BnDA1 in rapeseed significantly increased seed size, yield, and biomass [15]. In rice, HDR3, as a UIM-type active ubiquitin receptor, increases its ubiquitination level by interacting with GW6a. This slows down the degradation of the 26S proteasome. This effect promotes the grain filling rate, which increases the final grain size and weight [21]. The interaction of TaDA1 and TaGW2, homologous genes of Arabidopsis thaliana DA1, has been found in wheat. Both genes regulate grain size and have a genetic additive effect. The excellent haplotypes TaDA1-2A and TaGW2-6B are associated with a high 1000-grain weight, and they also exhibit additive effects [22]. The upregulation of the ZmDA1 or ZmDAR1 genes promotes grain development and enlargement, leading to increased yield in maize. In addition, ZmDR1 regulates starch synthesis, promotes starch accumulation, and enhances its quality [17]. Therefore, improving crop yield and quality can be achieved by varying the DA1-like gene family’s expression levels.
Ipomoea batatas (L.) Lam. is a type of sweet potato that belongs to the Convolvulaceae group. It is known for its high yield, strong adaptability, and wide cultivation areas, making it a significant staple crop and industrial raw material [23,24]. The completion of the genome sequencing of sweet potato (I. batatas (L.) Lam., 2n = B1B1B2B2B2B2 = 6x = 90) [25] and its two diploid species, I. trifida NCNSP0306 (2n = 2x = 30) and I. triloba NCNSP0323 (2n = 2x = 30) [26], has made it possible to analyze the sweet potato gene family using bioinformatics [27,28,29,30,31,32]. However, the DA1-like gene family in sweet potatoes has not been reported in these whole-genome studies.
In this study, we identified DA1-like genes in sweet potato and two diploid species at the whole-genome level. We examined the gene architectures, promoter cis-acting elements, expression patterns, and evolutionary relationships of these DA1-like genes in several tissues. Moreover, the expression profiles of particular DA1-like genes were confirmed using quantitative real-time polymerase chain reaction (qRT-PCR) tests in four different tissue sites. These results supply crucial information for examining the roles of DA1-like genes in sweet potatoes.

2. Results

2.1. Identification of DA1-like Genes in Sweet Potato and Two Diploid Relatives

We used three strategies to comprehensively identify all of the members of the DA1-like gene family in sweet potato and its two diploid relatives. There are 27 genes in the DA1-like gene family that have been identified. Respectively, in the sweet potato and its two diploid relatives, there are 9, 7, and 11 DA1-like family members. By sequencing these DA1-like genes, the physicochemical properties were determined in I. batatas. The IbDA1 CDS lengths ranged from 471 bp (IbDA1-9) to 1992 bp (IbDA1-2). There were 157 amino acids in IbDA1 (IbDA1-9) and 664 amino acids in IbDA1-2; the MW values differed from 17.91 kDa to 74.59 kDa; and the isoelectric point values ranged from 5.09 (IbDA1-6) to 7.49 (IbDA1-2). Among all of the DA1-like genes, only IbDA1-2 had a PI value greater than seven, indicating that it was a basic protein. There were Thr, Ser, and Tyr phosphorylation sites on all of the IbDA1s. The aliphatic index varied from 64.49 (IbDA1-6) to 85.64 (IbDA1-9). IbDA1-2 and IbDA1-9 had aliphatic indices exceeding 85, suggesting thermophilia. The GRAVY scores of all of the IbDA1 proteins were less than 0, which indicates that they are hydrophilic proteins. The subcellular localization predictions indicated that IbDA1-1, IbDA1-3, IbDA1-6, IbDA1-7, and IbDA1-8 would be found in the nucleus; IbDA1-4, IbDA1-5, and IbDA1-9 would be detected in the cytoplasm; and IbDA1-2 would be detected in the endoplasmic reticulum (Table 1).
A DA1-like gene can be found on chromosomes 5, 5, and 8 of I. batatas, I. trifida, and I. triloba, respectively. LG11 contained three IbDA1s (IbDA1-1, -2, and 9), two were on LG1 (IbDA1-7 and -8) and LG4 (IbDA1-4 and -5), and LG5 (IbDA1-6) and LG15 (IbDA1-3) had one each in I. batatas. Two ItfDA1s were detected on LG1 (ItfDA1-1 and -2) and LG12 (ItfDA1-4 and -6), and one each on LG3 (ItfDA1-7), LG5 (ItfDA1-5), and LG6 (ItfDA1-3) in I. trifida. The DA1-like genes in I. triloba had the highest number of members, with one or two genes on each chromosome (Figure 1).

2.2. Evolutionary Analysis of the DA1-like Genes

We constructed a phylogenetic tree for thirty-nine DA1 proteins (i.e., nine in I. batatas, seven in I. trifida, eleven in I. triloba, eight in Arabidopsis thaliana, and four in Oryza sativa) to study the evolutionary relationship of DA1 in I. batatas, I. trifida, I. triloba, A. thaliana, and O. sativa (Figure 2). Considering their evolutionary divergence, the DA1 proteins found in sweet potatoes and their diploid relatives can be classified into three subclasses (Groups I to III). As a result, (for I. batatas, I. trifida, I. triloba, A. thaliana, and O. sativa) the DA1s were distributed in three groups: Group I (12: 5, 1, 5, 1, 0), Group II (12: 0, 2, 2, 7, 1), and Group III (15: 4, 4, 4, 0, 3).

2.3. An Analysis of the Conserved Motif and Exon and Intron Structures of DA1 Genes from Sweet Potato and Its Two Closest Relatives

An analysis of the sequence motifs in nine IbDA1s, seven ItfDA1s, and eleven ItbDA1s was performed using the MEME website. Ten highly conserved motifs were also identified. The number of motifs varied among the members, ranging from zero to nine. Over half of the members (16 DA1s) contained all ten motifs. Most of the DA1s contained eight of these conserved motifs, and only a few DA1s differed in their number and types of motifs, such as IbDA1-9 (without a motif), IbDA1-3 (lacking motifs 3, 6, and 10), and IbDA1-1 (lacking motifs 4 and 10) (Figure 3a). We analyzed the exon and intron structures to comprehensively understand the structural characteristics of the DA1 genes. In the cultivated sweet potato, IbDA1-2 had the largest number of exons (thirteen), whereas IbDA1-4 and IbDA1-5 had the smallest number of exons (three). Comparing the cultivated sweet potato and its two diploid wild relatives, we observed a substantial variation in the number of exons per gene, ranging from a maximum of thirteen to a minimum of only two exons. The DA1-like genes had distinct functional differences during evolution, according to these results (Figure 3b).

2.4. Sweet Potato and Its Two Diploid Relatives: A Cis-Element Analysis

Cis-elements participate in the regulation of gene expression. We analyzed the cis-regulatory elements present in the 2000 bp promoter regions of the IbDA1s, ItbDA1s, and ItfDA1s in order to investigate the molecular regulatory mechanisms of the DA1s. According to Figure 4, the elements can be grouped into five groups based on their predicted functions: core elements, binding sites, developmentally regulated elements, hormone-responsive elements, stress-responsive elements, and light-responsive elements.
All of the IbDA1 members contained the core elements CAAT-box, TATA-box, MYC, and TCA; however, TCA was absent in IbDA1-4. The developmental transcription factor MYB was identified in all IbDA1s, whereas the majority of the IbDA1s contained P-box (IbDA1-1, -2, -4, -5, and -6) and O2-site (IbDA1-2, -3, -6, and -7) elements. Among these, IbDA1-6 exhibited the highest diversity of developmental elements, including MYB, P-box, O2-site, WUN-motif, ATCT-motif, GC-motif, and HD-zip. In most of the IbDA1s, hormone- and light-responsive elements were observed, including CGTCA motifs for the MeJA themes, TCA themes for the SA themes, and AE-box motifs for the light motifs; however, these elements were absent from IbDA1-3. Additionally, the IbDA1 promoters exhibited a high abundance of drought-responsive MYC elements among the abiotic elements.
We analyzed two wild sweet potato relatives to clarify the characteristics of cis-acting elements in the DA1s’ promoter regions. Similar to the IbDA1s, the ItbDA1s and ItfDA1s have CAAT-boxes and TATA-boxes in their promoter regions as well as the developmental element MYB. In addition, most of the ItbDA1s and ItfDA1s had the developmental elements O2-site and P-box, the hormone elements CGTCA motif and TCA, the abiotic stress elements MYB, MYC, and STRE, and the light-responsive elements GATA-motif and AE-box.

2.5. Different Tissues Containing DA1-like Genes Analyzed via RNA-Seq

To identify the expression of the DA1 gene in different tissues, we analyzed the published transcriptome data of “Xushu 18”. This was performed to clarify the potential function of the DA1 gene in sweet potatoes. A heat map was created using fragments per kilobase of the exon model per million mapped fragments (FPKM). Usually, a gene’s FPKM value is greater than 1, indicating its expressibility and functionality. In this study, the FPKM of IbDA1-6 in the sweet potatoes was greater than 1 in eight different tissues, indicating that IbDA1-6 is expressible and functional. IbDA1-1 was highly expressed in six tissues but not in the fibrous root (FR) or leaf. The FPKM values of IbDA1-2, -3, -4, -7, -8, and -9 were almost all less than 1 in the eight sweet potato tissues, indicating that they were non-functional (Figure 5).
DA1 expression was also investigated in I. trifida and I. triloba based on RNA-seq data collected from six tissues (flowers, flower buds, leaves, root 1, root 2, and stem). In I. triloba, a total of five ItbDA1s were highly expressed in the flowers: ItbDA1-2, -4, -9, -10, and -11. The flower buds expressed high levels of six ItbDA1s (i.e., ItbDA1-2, -4, -8, -9, -10, and -11). In the leaves, ItbDA1-2, -3, -4, -7, -8, -9, and -10 were highly expressed. There were five ItbDA1s (ItbDA1-2, -3, -8, -9, and -10) found at high levels of expression in the roots (root 1 and root 2). In the leaves, six ItbDA1s were highly expressed (ItbDA1-2, -3, -4, -7, -9, and 10). Moreover, three ItbDA1s (i.e., ItbDA1-1, 5, and 6) showed low expression levels in all of the tissues (Figure 6a).
In all tissues of I. trifida, ItfDA1-1 showed a low level of expression. The expression levels of six ItfDA1s (i.e., ItfDA1-2, -3, -4, -5, and -7) were high across all of the tissues. Furthermore, ItfDA1-6 showed a low level of expression in all of the tissues and ItfDA1-3 showed a low level of expression in the flowers and roots (Figure 6b).

2.6. QRT-PCR of the Sweet Potato DA1-like Gene in Different Tissues

We analyzed the expression patterns of four genes (IbDA1-1, IbDA1-3, IbDA1-6, and IbDA1-7) in four different tissues to better understand the role of IbDA1 in sweet potato growth and development (Figure 7). The gene expression was high in leaves, but there were no significant differences in the expression in the roots, fibrous roots, and stems. The expression of IbDA1-1 was higher in the roots and fibrous roots than that of the other three genes.

3. Discussion

AtDA1 is one of the smaller gene families in Arabidopsis thaliana, containing only eight genes. These genes not only play a regulatory role in grain size, but also play an important role in stress in Arabidopsis thaliana [33,34]. DA1 encodes a ubiquitination receptor, so DA1 may interact with the ubiquitination substrate in the ubiquitination protease pathway to promote its degradation. Recent studies have shown that the DA1 gene family is involved in the ubiquitin protease pathway to regulate grain size [20,35]. At present, studies on the control of plant organ size by DA1 have been reported in a variety of crops, such as maize [17], rice [21], rape [15], wheat [22], and so on. These results indicate that DA1 has a relatively conservative function in crops and plays an important role in plant growth and development.
The DA1 gene family members have been systematically analyzed in a variety of crops such as Arabidopsis thaliana [1], rice [20], rapeseed [15], maize [17], cotton [19], oat [18], and soybean [27], but the DA1-like genes of sweet potato have not been analyzed. With the rapid development of sweet potato sequencing technology and genetic engineering, it is convenient to mine candidate genes related to sweet potato yield and quality. Although the polyploid genome of sweet potato hindered the process of identifying grain size genes by mapping cloning, the use of the regulators of grain size identified in the model rice and Arabidopsis thaliana plants was effective as a candidate gene validation method. Therefore, this study systematically identified the DA1-like gene family in sweet potato, which provides a favorable theoretical and genetic reference for the subsequent study of IbDA1 gene function.
In this study, we discovered DA1-like genes in sweet potatoes and their diploid relatives. Based on the evolutionary distance, the DA1-like genes were divided into three subgroups. According to the evolutionary tree, some DA1 proteins in sweet potatoes are closely related to OsDA1 proteins in rice and may have similar functions. These results suggest that DA1 proteins have differentiated significantly during long-term evolution; however, some DA1 proteins may still regulate organ size. According to the chromosomal location results, the sweet potato and its two diploid relatives have significantly different chromosomal distributions of DA1-like genes, suggesting the evolution of distinct variations among the species. In addition, we found that IbDA1-7 and IbDA1-8 are clustered in LG1; IbDA1-4 and IbDA1-5 are clustered in LG4; and IbDA1-2 and IbDA1-9 are clustered in LG11. Clustering has been extensively characterized in organisms such as fungi and plays an important role in transcriptional regulation mechanisms [36]. This suggests that clustering is conserved in Eukaryotes [37]. However, clustering exists only in sweet potatoes, but not in I. batatas and I. trifida, which is worth considering. We analyzed that the possible reason for this is that sweet potato has been artificially domesticated in the evolution of clustering so that its traits meet the needs of production. In the future, the functional verification of these cluster genes can be carried out to analyze the causes of the occurrence of these cluster genes. The physiological characteristics, phylogenetic relationships, gene structures, and promoter cis-elements of the DA1 proteins were systematically studied. The expression patterns and qRT-PCR analyses suggested that IbDA1-1 and IbDA1-6 might regulate sweet potato traits.

3.1. Evolution of the DA1-like Gene Family in Sweet Potato and Its Two Diploid Relatives

The studied sweet potato and its two diploid relatives contained 27 DA1s, i.e., 9 IbDA1s, 11 ItbDA1s, and 7 ItfDA1s (Figure 1). These genes were relatively stable in terms of their structure and physical and chemical properties and had high functional conservation. Some genes had closer evolutionary relationships with rice OsDA1, such as IbDA1-1, IbDA1-7, and IbDA1-6. OsDA1 is homologous to AtDA1, has a high affinity for ubiquitin, and interacts with OsUBP15 to regulate rice grain size [20]. These results suggest that IbDA1-1, IbDA1-7, and IbDA1-6 may regulate organ size in sweet potatoes. The IbDA1 gene family in sweet potatoes comprises nine members, surpassing the numbers found in rapeseed, corn, oat, rice, and Arabidopsis thaliana; however, this is less than in cotton and soybean. In addition, more DA1-like genes were identified in I. triloba than in I. trifida, out of the two diploid wild relatives of sweet potatoes. The genome size may influence the diversity of DA1-like gene numbers and positions among species.
Over half of the DA1s contained ten conserved motifs. This indicates that these motifs have been relatively conserved (Figure 3a). The role of exons in gene diversity evolution is crucial [38]. Sweet potato and its two diploid relatives differ in the number of exons and introns in some DA1s. (Figure 3b). The Group I IbDA1-1 genes contained 10 exons. This group’s homologous genes, ItfDA1-1 and ItbDA1-1, contained 3 exons, and IbDA1-2 possessed 13 exons, whereas ItbDA1-5 contained the smallest number (only 2). These results suggest that DA1s evolved into genes with different functions in sweet potatoes and their diploid relatives. This is due to the differences in the structure of these exons.

3.2. Differences in the Functions of the DA1-like Gene Family in Growth and Development between Sweet Potato and Its Two Diploid Relatives

The regulation of plant organ size by DA1-like genes has been reported in several species [21,39,40,41] However, the function of DA1-like genes in regulating organ size in sweet potatoes has not been reported. We analyzed the cis-elements of these genes to explore the potential function of DA1-like genes in the growth and development of sweet potato and its two diploid relatives. As an extensively expressed transcription factor family in plants, the MYB transcription factor family actively participates in plant development and stress responses by binding specifically to MYB cis-elements in target gene promoters [42]. In this study, we found that the DA1-like promoter region contained MYB cis-acting elements in both the cultivated sweet potatoes and its two diploid relatives, indicating that the DA1-like gene family may interact with MYB transcription factors and thus participate in the growth, development, and stress resistance responses of sweet potatoes. In addition, we found that most of the DA1-like gene promoter regions contained many elements related to the response to hormonal and abiotic stresses. This provides a basis for studying the DA1-like gene family in sweet potatoes under stress conditions.
We focused on analyzing the expression pattern of DA1 in different tissues to further explore the potential function of DA1-like genes in the growth and development of sweet potato and its two diploid relatives. The results showed that the expressions of IbDA1-2, IbDA1-4, IbDA1-5, and IbDA1-9 were downregulated in the eight tissues (stem, leaf, FR, ISR, RS, RB, DE, and PE). The stem showed high levels of IbDA1-6 expression, and it was expressed in all the tissues. The expression levels of the ItfDA1s and ItbDA1s exhibited significant variation across six tissues in I. trifida and I. triloba. For instance, although ItbDA1-1 was not detected in any tissue, ItbDA1-2 was highly expressed in all of the tissues. These results suggest that DA1-like genes may play different roles in the growth and development of sweet potatoes. Furthermore, qRT-PCR was used to validate the expression profiles of DA1s across various sweet potato tissues. Interestingly, IbDA1-1, IbDA1-3, IbDA1-6, and IbDA1-7 were highly expressed in the sweet potato leaves, indicating that they might be important functional genes involved in regulating sweet potato leaf development.

4. Material and Methods

4.1. Identification of DA1s

From the following websites: http://sweetpotato.plantbiology.msu.edu/ (accessed on 9 October 2023) and http://sweetpotato.plantbiology.msu.edu/ (accessed on 9 October 2023), we downloaded the genomic data of the sweet potato and two wild species. The protein sequences of the DA1 gene family in Arabidopsis thaliana were obtained from the TAIR11 database (https://www.arabidopsis.org/, accessed on 9 October 2023), and the protein sequences of the DA1 gene family in sweet potato and Arabidopsis thaliana (AT1G19270, AT4G36860, AT2G39830, AT5G66640, AT5G17890, AT5G66630, AT5G66620, and AT5G66610) were compared via BLAST. Meanwhile, Hidden Markov Model data of typical DA1 family protein knots were downloaded from the PFAM database (http://pfam.sanger.ac.uk, accessed on 10 October 2023), and the protein sequences containing characteristic domains were searched for using HMMER 3.0 software. The candidate proteins screened through the above two methods were merged, and multiple comparisons were performed using SnapGene 4.3.6 software. Incomplete or redundant sequences were removed manually, and screening results were checked using Pfam and SMART online analysis (http://smart.emblheidelberg.de/, accessed on 11 October 2023). Gene sequences without the DA1 gene family domains or containing incomplete DA1 domains were removed, and the sweet potato DA1 genes were finally obtained.

4.2. Distribution of DA1s on Chromosomes

Gene family members IbDA1, ItfDA1, and ItbDA1 were located using annotation files for I. batatas genomes (https://ipomoea-genome.org/, accessed on 9 November 2023), I. trifida, and I. triloba (http://sweetpotato.plantbiology.msu.edu/, accessed on 9 November 2023).

4.3. Prediction of DA1 Protein Characteristics

We calculated DA1’s molecular weight, theoretical PI, instability index, and hydrophilicity with ExPASy (https://www.expasy.org/, accessed on 15 October 2023). Plant-mPLoc (http://www.csbio.sjtu.edu.cn/bioinf/plant-multi/, accessed on 15 October 2023) was used to predict DA1 subcellular localization.

4.4. Analysis of the Phylogenetic Relationships among DA1s

CLUSTALX 1.8.3 was used to perform multiple sequence alignments of the inferred amino acid sequences of the DA1s from I. batatas, I. trifida, I. triloba, A. thaliana, and O. sativa. After importing the alignment findings into MEGA11, 1000 bootstrap repeats and the greatest likelihood approach were used to build a phylogenetic tree (www.megasoftware.net, accessed on 19 November 2023). Then, using iTOL (http://itol.embl.de/, accessed on 19 November 2023), the phylogenetic tree was created.

4.5. Detection of Domains and Analysis of Conserved Motifs in DA1s

Using MEME 5.3.0 software (https://meme-suite.org/meme/, accessed on 21 November 2023), a conserved domain analysis of the DA1s was carried out, with a maximum motif count of 10. The conserved domain and exon–intron structures of the DA1s were visualized by applying Tbtools v2.056. PlantCARE was used to anticipate the DA1 cis-elements.

4.6. Transcriptome Analysis

We used the published RNA-seq data of “Xushu 18” (PRJNA511028) to explore the expression pattern of DA1s in different tissues and organs of sweet potatoes [43]. From the Sweet Potato Genomics Resource (http://sweetpotato.plantbiology.msu.edu/, accessed on 25 November 2023), RNA-seq data for ItfDA1s and ItbDA1s were downloaded [26]. We plotted gene expression levels using TBtools.

4.7. Plant Material and qPCR

The sweet potato variety “HD7798”, independently bred by Hainan University, was utilized as the experimental material in this study, and four DA1-like genes were selected to analyze their expression patterns in leaves, stems, roots, and fibrous roots. IbDA1 primers were produced using Primer 6.0 (Table S1). Reverse transcription was performed using an M-MLV RTase cDNA Synthesis Kit (TaKaRa, Kyoto, Japan). qRT–PCR was performed using the Roche LightCycler ® 480II (Applied Biosystems, Foster City, CA, USA) System under the following conditions: 95 °C for 15 s, followed by 40 cycles of 95 °C for 15 s each, then 55 °C for 15 s, and 72 °C for 15 s. Relative quantification was performed using the 2–∆∆Ct method [44].

4.8. Statistical Analysis

The student’s two-tailed t test was used for significant difference analysis between two samples. One-way ANOVA analyses followed by Tukey’s test (p < 0.05) were used for pairwise multiple comparisons. All of the analyses were performed using SPSS 24.0 software.

5. Conclusions

In this study, DA1-like genes are presented for the first time in sweet potato. Its two diploid relatives I. trifida and I. triloba were shown to have abundant genetic variation and diverse expression patterns, suggesting that the DA1 gene family members may play different roles in the growth and development in these plants. This study provides a theoretical basis for further exploration of the function of the DA1-like genes in the growth and organ size regulation of sweet potatoes. However, further functional analyses are needed to clarify their role.

Supplementary Materials

The following supporting information can be downloaded at: https://www.mdpi.com/article/10.3390/ijms25053000/s1.

Author Contributions

L.W., R.X. and Y.L. (Yunhai Li) designed the experiments, L.W. wrote the manuscript. Z.Z. and J.H. performed most of the experiments and analyzed the data. S.H., J.Y., and Y.W. (Ying Wang) collected and downloaded the data. Y.L. (Yi Liu), W.L. and Y.W. (Yuehui Wang) designed the quantitative primers and performed the RT-PCR experiments. All authors have read and agreed to the published version of the manuscript.

Funding

The Project of Sanya Yazhou Bay Science and Technology City (SCKJ-JYRC-2022-64), the Natural Science Foundation of Hainan Province (322RC592), and the National Natural Science Foundation of China (31960159 and 32360083) supported this study.

Institutional Review Board Statement

Not applicable.

Informed Consent Statement

Not applicable.

Data Availability Statement

All data and conclusions are included in this paper.

Conflicts of Interest

The authors declare no conflicts of interest.

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Figure 1. Chromosomal localization and distribution of DA1-like genes in I. batatas (a), I. trifida (b), and I. triloba (c).
Figure 1. Chromosomal localization and distribution of DA1-like genes in I. batatas (a), I. trifida (b), and I. triloba (c).
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Figure 2. Phylogenetic analysis of the DA1 proteins from five plant species (i.e., I. batatas, I. trifida, I. triloba, Arabidopsis thaliana, and Oryza sativa). In total, 39 DA1s were divided into three subgroups (Groups I to III) according to their evolutionary distance. The red stars, purple circles, green triangles, blue rectangles, and yellow check mark represent the 9 IbDA1s in I. batatas, the 7 ItfDA1s in I. trifida, the 11 ItbDA1s in I. triloba, the 8 AtDA1s in Arabidopsis thaliana, and the 4 OsDA1s in Oryza sativa, respectively.
Figure 2. Phylogenetic analysis of the DA1 proteins from five plant species (i.e., I. batatas, I. trifida, I. triloba, Arabidopsis thaliana, and Oryza sativa). In total, 39 DA1s were divided into three subgroups (Groups I to III) according to their evolutionary distance. The red stars, purple circles, green triangles, blue rectangles, and yellow check mark represent the 9 IbDA1s in I. batatas, the 7 ItfDA1s in I. trifida, the 11 ItbDA1s in I. triloba, the 8 AtDA1s in Arabidopsis thaliana, and the 4 OsDA1s in Oryza sativa, respectively.
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Figure 3. Conserved motifs and analysis of the exon–intron structure of the DA1 family in I. batatas, I. trifida, and I. triloba. (a) The phylogenetic tree shows that the DA1s are distributed in three subgroups (left), and the ten conserved motifs are shown in different colors. (b) Exon–intron structures of DA1s. The green boxes, yellow boxes, and black lines represent the UTRs, exons, and introns, respectively.
Figure 3. Conserved motifs and analysis of the exon–intron structure of the DA1 family in I. batatas, I. trifida, and I. triloba. (a) The phylogenetic tree shows that the DA1s are distributed in three subgroups (left), and the ten conserved motifs are shown in different colors. (b) Exon–intron structures of DA1s. The green boxes, yellow boxes, and black lines represent the UTRs, exons, and introns, respectively.
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Figure 4. Cis-element analysis of DA1s in I. batatas, I. trifida, and I. triloba. The cis-elements were divided into five broad categories. The intensity of the different colors represents the number of cis-elements in the DA1s’ promoters.
Figure 4. Cis-element analysis of DA1s in I. batatas, I. trifida, and I. triloba. The cis-elements were divided into five broad categories. The intensity of the different colors represents the number of cis-elements in the DA1s’ promoters.
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Figure 5. Expression pattern of the DA1-like genes in “Xushu 18” in different tissues. The eight tissues comprise stem, leaf, fibrous root (FR), initiated storage root (ISR), root stock (RS), root body (RB), distal end (DE), and proximal end (PE) tissues.
Figure 5. Expression pattern of the DA1-like genes in “Xushu 18” in different tissues. The eight tissues comprise stem, leaf, fibrous root (FR), initiated storage root (ISR), root stock (RS), root body (RB), distal end (DE), and proximal end (PE) tissues.
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Figure 6. Gene expression patterns of ItbDA1s (a) and ItfDA1s (b) in different organs (root 1, root 2, stem, leaf, flower, and flower bud).
Figure 6. Gene expression patterns of ItbDA1s (a) and ItfDA1s (b) in different organs (root 1, root 2, stem, leaf, flower, and flower bud).
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Figure 7. Analysis of IbDA1 gene expression in different tissues of “HD7798” using qRT-PCR. Note: data are shown as mean ± SEM (n = 3). (A–D) Expression levels of IbDA1-1 (A), IbDA1-3 (B), IbDA1-6 (C), and IbDA1-7 (D) in roots, fibrous roots, leaves, and stems. Different letters means significant at 5% probability levels according to Duncan’s new multiple range method.
Figure 7. Analysis of IbDA1 gene expression in different tissues of “HD7798” using qRT-PCR. Note: data are shown as mean ± SEM (n = 3). (A–D) Expression levels of IbDA1-1 (A), IbDA1-3 (B), IbDA1-6 (C), and IbDA1-7 (D) in roots, fibrous roots, leaves, and stems. Different letters means significant at 5% probability levels according to Duncan’s new multiple range method.
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Table 1. Characterization of IbDA1 gene family members in sweet potato.
Table 1. Characterization of IbDA1 gene family members in sweet potato.
Gene IDGene NameCDS (bp)Protein (aa)MW/kDapIPhosphorylation SiteAliphatic IndexGRAVYSubcellular Locations
SerThrTyr
g41815IbDA1-1140746953.156.2341221865.9−0.607nucleus
g46658IbDA1-2199266474.597.4952302685.41−0.111endoplasmic reticulum
g60449IbDA1-3138646252.516.2929211875.97−0.413nucleus
g15267IbDA1-465721925.406.22811777.34−0.2cytoplasm
g15269IbDA1-572924328.006.7912975.74−0.147cytoplasm
g17266IbDA1-6161453861.555.0952232164.49−0.75nucleus
g1178IbDA1-7159053059.805.245262174.1−0.45nucleus
g1183IbDA1-879526529.966.53014975.76−0.422nucleus
g46660IbDA1-947115717.915.2277785.64−0.049cytoplasm
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Zhou, Z.; Huang, J.; Wang, Y.; He, S.; Yang, J.; Wang, Y.; Li, W.; Liu, Y.; Xu, R.; Li, Y.; et al. Genome-Wide Identification and Expression Analysis of the DA1 Gene Family in Sweet Potato and Its Two Diploid Relatives. Int. J. Mol. Sci. 2024, 25, 3000. https://doi.org/10.3390/ijms25053000

AMA Style

Zhou Z, Huang J, Wang Y, He S, Yang J, Wang Y, Li W, Liu Y, Xu R, Li Y, et al. Genome-Wide Identification and Expression Analysis of the DA1 Gene Family in Sweet Potato and Its Two Diploid Relatives. International Journal of Molecular Sciences. 2024; 25(5):3000. https://doi.org/10.3390/ijms25053000

Chicago/Turabian Style

Zhou, Zelong, Jianzhi Huang, Yuehui Wang, Shixiang He, Jing Yang, Ying Wang, Wenxing Li, Yi Liu, Ran Xu, Yunhai Li, and et al. 2024. "Genome-Wide Identification and Expression Analysis of the DA1 Gene Family in Sweet Potato and Its Two Diploid Relatives" International Journal of Molecular Sciences 25, no. 5: 3000. https://doi.org/10.3390/ijms25053000

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