Genome-wide investigation of the ZF-HD gene family in Tartary buckwheat (Fagopyrum tataricum)
ZF-HD is a family of genes that play an important role in plant growth, development, some studies have found that after overexpression AtZHD1 in Arabidopsis thaliana, florescence advance, the seeds get bigger and the life span of seeds is prolonged, moreover, ZF-HD genes are also participate in responding to adversity stress. The whole genome of the ZF-HD gene family has been studied in several model plants, such as Arabidopsis thaliana and rice. However, there has been little research on the ZF-HD genes in Tartary buckwheat (Fagopyrum tataricum), which is an important edible and medicinal crop. The recently published whole genome sequence of Tartary buckwheat allows us to study the tissue and expression profiles of the ZF-HD gene family in Tartary buckwheat on a genome-wide basis.
In this study, the whole genome and expression profile of the ZF-HD gene family were analyzed for the first time in Tartary buckwheat. We identified 20 FtZF-HD genes and divided them into MIF and ZHD subfamilies according to phylogeny. The ZHD genes were divided into 5 subfamilies. Twenty FtZF-HD genes were distributed on 7 chromosomes, and almost all the genes had no introns. We detected seven pairs of chromosomes with fragment repeats, but no tandem repeats were detected. In different tissues and at different fruit development stages, the FtZF-HD genes obtained by a real-time quantitative PCR analysis showed obvious expression patterns.
In this study, 20 FtZF-HD genes were identified in Tartary buckwheat, and the structures, evolution and expression patterns of the proteins were studied. Our findings provide a valuable basis for further analysis of the biological function of the ZF-HD gene family. Our study also laid a foundation for the improvement of Tartary buckwheat crops.
KeywordsTartary buckwheat ZF-HDs Genome-wide Fruit development Expression patterns
Days after pollination
Leucine zipper-associated HD
Mini zinc finger
- PHD finger
Finger-like domain associated with an homeodomain
Zinc finger motif-associated homeodomain
Specialized genetic networks regulate plant growth by encoding various proteins. Proteins containing transcription factors that bind to specific nucleotide sequences play an important role in different stages of plant growth, flowering, fruiting, and resistance to stress [1, 2]. A homeodomain (HD), as an NDA domain (BD), has 60 conserved amino acid sequences and encodes a homeobox (HB) gene in all eukaryotic transcription factors . The 60 amino acids of the homeodomain fold into a characteristic three-helix structure called recognition helix, which attaches to the main sulcus of DNA to form a special connection to DNA [4, 5]. The HD protein is involved in the development of plants and animals by regulating the expression pattern of target genes . Most HD proteins are related to the protein-protein interactions and other domains/motifs with regulatory functions . Proteins with homologous domains are divided into six different families according to different themes: leucine zipper-associated HD (HD-Zip), zinc finger motif-associated HD (ZF-HD), WUSCHEL-related homeobox (WOX), Bell-type HD, finger-like domain associated with an HD (PHD finger), and knotted-related homeobox (KNOX) proteins .
The zinc finger structure is an important structure and is composed of zinc ions and cysteine or histidine (in most cases) . The zinc finger, as an important motif, is widely found in a variety of regulatory proteins, can specifically bind to DNA/RNA sequences, and participates in protein interactions [9, 10]. Zinc finger units are divided into many categories according to Cys and differences in residues, such as C3hC2H2 and C2C2 . As a family, the zinc finger homeodomain (ZF-HD) proteins, containing HD proteins and a zinc finger related to the homeodomain, were first identified in the C4 plant Flaveria .
At present, the ZF-HD gene family has been identified in Arabidopsis thaliana, rice (Oryza sativa) and tomato (Solanum lycopersicum) [12, 13, 14]. Arabidopsis thaliana has 17 ZF-HD members, and they act as transcription factors, have unique physiological characteristics, and play a very important role in the development of flowers . Drought, salinity and abscisic acid (ABA) can induce AtZHD1 to bind to the ERD1 promoter region  Three genes were found in the ZF-HD gene family of Arabidopsis thaliana, and its mini zinc finger (MIF) gene and protein sequence encoded by congeners are highly similar to the ZF domain of the ZF-HD protein but have no HD domain [12, 16]. The phylogeny and sequence analyses of MIF and ZHD genes were conducted, suggesting that the ZHD gene has plant specificity and that almost all genes have no introns . Until now, the origin and evolution of the ZHD and MIF gene populations remain unclear . In soybean, GmZF-HD1 and GmZF-HD2 bind to the promoter region of the gene encoding calmodulin subtype 4 (GmCaM4), and the expression of GmZF-HD1 and GmZF-HD2 increased after inoculation with pathogenic bacteria . In tomato, the ZF-HD gene family was found to be related to fruit development as well as to stress . The ZF-HD gene family may play a similar role in Tartary buckwheat.
Common buckwheat (Fagopyrum esculentum) is produced in southwestern China and has spread to all continents. Tartary buckwheat (Fagopyrum tataricum) grows in the mountainous areas of southwestern China, northern India, Bhutan and Nepal . Tartary buckwheat is currently the only widely grown single-sex only food crop, has a balanced essential amino acid composition in its seed protein, and has a total protein content that is higher than that in many food crops [8, 19]. The ZF-HD gene family has been widely studied in many plants, some studies have found that after overexpression AtZHD1 in Arabidopsis thaliana, florescence advance, the seeds get bigger and the life span of seeds is prolonged [12, 20], moreover, ZF-HD genes are also participate in responding to adversity stress . However, the ZF-HD gene family in Tartary buckwheat has not been studied, at present, the ARF, AP2, NAC, MADS genes family have been studied deeply in Tartary Buckwheat [22, 23, 24, 25]. Because of the important role of the ZF-HD gene in various physiological processes, it is of great significance to systematically study the Tartary buckwheat ZF-HD family. The recently completed genome sequence of Tartary buckwheat provides an opportunity to reveal the tissue, expression and evolution characteristics of the ZF-HD gene family in Tartary buckwheat at the whole genome level. In this paper, the exon-intron structure, motif composition, genomic structure, chromosome location, sequence homology and expression pattern of 20 Tartary buckwheat ZF-HD genes are introduced in detail. In addition, the phylogenetic relationship between the ZF-HD gene family in Arabidopsis thaliana and Tartary buckwheat was compared. Through global expression analysis, the degree of participation of ZF-HD gene family members in different biological processes of Tartary buckwheat was determined. The role of the FtZF-HD gene in the development of buckwheat fruit was studied in detail, which provided a valuable clue for the functional characterization of the buckwheat ZF-HD gene family members in the growth and development of Tartary buckwheat.
Identification of the FtZF-HD genes in Tartary buckwheat
To identify the FtZF-HD genes in Tartary buckwheat, all possible FtZF-HD members in the Tartary buckwheat genome were mined using two BLAST methods, multiple FtZF-HD genes from the Tartary buckwheat genome were isolated by these two methods, and since the buckwheat genome was sequenced using a genome-wide shotgun strategy, some of these FtZF-HD genes may be redundant even though they were on different scaffolds. In this study, we identified a total of 20 ZF-HD genes, and we named them FtZHD1~FtZHD17 and FtMIF1~FtMIF3 based on their physical location on the chromosomes (Additional file 2: Table S1).
For the Tartary buckwheat FtZF-HD proteins, FtZHD14 was the smallest protein with 83aa, and the largest protein was FtZF-HD5 (330aa) (Additional file 2: Table S1). The molecular weight of the proteins ranged from 9.33 kda to 34.95 kda; PI from 4.94 (FtMIF1) to 10.15 (FtZHD10). The predicted subcellular localization results showed that all proteins are located in the nuclear region.
Phylogenetic analysis of the ZF-HD gene family in Tartary buckwheat
Structural analysis and motif composition of the Tartary buckwheat ZF-HD gene family
To further study the characteristic region of the FtZF-HD protein, the structures of 20 FtZF-HD proteins were analyzed by online MEME analysis. According to the results of the MEME analysis, a schematic map was constructed to characterize the structure of the FtZF-HD protein. We identified 10 conserved motifs, named motifs 1 to 10 (Fig. 2c). It is worth noting that all FtZF-HD genes had motif3 domains, and most (60%) had motif2 domains. Interestingly, motif4 was a domain that was specific to the ZHD family, and motif1 and motif5 were detected in almost all ZHD genes. Motif6 and motif10 were detected specifically in the FtPinG0001863300.01 and FtPinG0009244100.01genes. Motif8 and motif9 existed in four genes each. Motif7, motif8, and motif9 only existed in a few genes. Compared with the MIF gene family, the ZHD gene family showed obvious differences, and the functional differences in the ZHD gene in Tartary buckwheat were probably due to the subfamily-specific distribution of conserved motifs. The same motifs in three MIF genes and the same motifs in several subpopulations of ZHD indicated that there were conserved motifs in the subpopulations, but the function of these conserved motifs remains to be clarified.
Chromosome distribution and synchronous analysis of the FtZF-HD genes
There was an uneven distribution of the FtZF-HD genes on 7 buckwheat chromosomes (except Ft2). Chromosome 3 and chromosome 7, which were the two chromosomes with the most ZF-HD genes, each contained four ZF-HD genes, and three chromosomes had the lowest number of ZF-HD genes (2 ZF-HD genes)(Fig. 3). Gene replication plays an important role in the occurrence of new functions and gene amplification. To determine the fragment replication events between the genes, we adopted the standard . When the query coverage and consistency of the candidate genes are ≥80, they are thought to be repetitive genes. Chromosomal regions within the 200 kb range of two or more genes are defined as tandem replication events. Therefore, the analysis of Tartary buckwheat gene duplication showed that no tandem repeat gene was found on the chromosomes of Tartary buckwheat; however, 12 genes were involved in fragment repeat events, and the ZF-HD gene on chromosome 4 was involved in the most fragment repeat events (Fig. 3). These results suggest that some FtZF-HD genes may be produced by repeated fragments of the gene, and these replication events are the main driving force in the evolution of FtZF-HD.
Evolution analysis of the FtZF-HD gene family within several different species
We also use MEME web servers to search for conserved motifs shared by the ZF-HD proteins. The results showed (Fig. 5) that 10 different conserved motifs were identified. The genes of the same group had similar motifs, such as those in group d, which suggests potential functional similarities among the ZF-HD proteins. Motif 1 and motif 4 were conserved motifs shared by almost all ZF-HD proteins, indicating that ZF-HD had some highly conserved domains. Motif2 and motif3 were conserved domains shared by almost all ZF-HD proteins except for group d; therefore, group d may have evolved from other genes after the loss of motif2 and motif3 or other genes evolved after acquiring motif2 and motif3.
Expression patterns of the FtZF-HD genes in different plant tissues
Differential expression of the FtZF-HD genes during fruit development of Tartary buckwheat
A phylogenetic analysis and sequence analysis of the ZHD and MIF genes indicate that both genes are endemic to terrestrial plants and belong to two different groups of the same protein family (ZF-HD). Extensive research has shown that ZHD has been found in many terrestrial plants but not in algae . We identified 20 ZF-HD genes in Tartary buckwheat, including 17 ZHD genes and 3 MIF genes, which was slightly higher than the number found in Arabidopsis (17). The replication of genes can amplify the number of genes. For example, four large replication events occurred in the Arabidopsis genome, and more than half of ZF-HD genes may be produced by genomic replication [12, 28, 29]. Gene replication mechanisms include fragment replication, tandem replication and translocation (reverse transcription and replication translocation), which is an important factor in biological evolution . Among the mechanisms, fragment replication is one of the main contributors to the amplification of many gene families . The analysis of the chromosome distribution of the ZF-HD genes in Tartary buckwheat revealed there was fragment replication, but no tandem replication, which suggests that the gene fragment replication events greatly facilitates the expansion of the ZF-HD gene family in plants with smaller genomes. Moreover, a phylogenetic tree with Arabidopsis also suggests an evolutionary relationship between these two species. There are three main mechanisms for the variation of the exon and intron structures of a gene (the gain or loss, exonization or pseudoexonization and insertion or deletion of exon or intron), and each mechanism leads to a gene structural difference [15, 32, 33, 34]. The presence of a few introns in the Tartary buckwheat ZF-HD gene may be due to the variations in the participation of these three mechanisms. To study the phylogenetic relationship between Tartary buckwheat and dicotyledon, we constructed collinear relationship maps between Tartary buckwheat and seven plant species (Fig. 4). Finally, 23 colinear ZF-HD gene pairs of Tartary buckwheat and soybean were identified. The number of orthologous events was far greater than that between Tartary buckwheat and rice, which was consistent with the closer evolutionary distance between Tartary buckwheat and soybean .
The ZF-HD transcription factors are involved in various biological processes such as the response of plants to abiotic stress and the development of plants by phytohormones [34, 36]. Most of the previous reports on the ZF-HD gene were about the regulation of the ZF-HD gene in response to abiotic stress, but there were few reports on plant development. Members of the ZF-HD gene family are expressed in floral tissues in plants (e.g. Arabidopsis) . It was revealed that the ZF-HD gene family regulates flower development. Our research shows that the 20 ZF-HD genes identified from Tartary buckwheat were indeed expressed during the growth and development of plants, so there is no pseudogene. However, in the study of fruit development, we found that five genes were not involved in the regulation of fruit development. It is worth noting that FtMIF3, which belongs to the same subfamily as FtMIF1 and FtMIF2, is not expressed in the fruit, and when the motifs of the three were compared (Fig. 2), we found that they had the same motifs. This result leads us have great interest in exploring the reasons for the differences among the genes because after comparing their protein sequences (Additional file 1: Figure S1), we found that although the three motifs are the same, the amino-acid residues at the 126th to 139th positions of the amino acid sequence encoded by the FtMIF3 gene differ significantly from those of FtMIF1 and FtMIF2. We speculate that it is highly likely that a mutation in the FtMIF3 gene caused the protein it encodes to lose its ability to express itself in the fruit. For FtMIF1 and FtMIF2, their differences in amino acid sequences have little effect, and the expression of FtMIF1 and FtMIF2 in fruit development has a 0.998 correlation. Although not all genes are involved in fruit development, it is equally exciting to find that 15 genes are expressed in the fruit. Before this study, the expression of the ZF-HD protein family in fruit was only reported in grape (Vitis vinifera) and tomato, and there was only a potential role in grape fruit development [14, 15].
We found four grape genes that were expressed in fruits in the phylogenetic trees containing multiple plants (Fig. 5) (GSVIVT01003614001, GSVIVT01009128001, GSVIVT01012772001 and GSVIVT 01011413001, in groups a and b, respectively) . These genes have similar to the motifs of those of Tartary buckwheat in the respective groups. For example, the genes in group a have motif1 and motif3, and the genes, except for FtPinG006040000.01, in group b have motif1, motif2, motif3 and motif4. It can be seen that the relationship between the grape genes and Tartary buckwheat genes in group b is closer than that in group a.
The size of monocotyledonous fruit is related to endosperm development [37, 38], in dicotyledonous plants, the final size of the fruit is related to the number and size of cotyledon cells . Studies have pointed out that usually the initial growth of the endosperm rather than the late growth of the embryo is mainly related to the size of the fruit [40, 41, 42]. Reports on the development of Tartary buckwheat fruit indicated that the size of Tartary buckwheat fruit is mainly related to cell division during embryonic development, and most Tartary buckwheat fruits reach the maximum state on the 13th to 25th day after pollination. . Therefore, in this study, the early stage of fruit development is the key period in determining the size of the fruit. In our research on fruit development, we found that there are 8 genes that are more highly expressed in the earlier stages than in the other two stages. Fruit development is controlled by various transcriptional regulatory networks. These networks involve transcription factors, for example, members of the ARF family, which can regulate hormones such as AUX and ABA . FtARF 2 may have the ability to integrate signals, thus prolonging the cycle of embryonic development, increasing the cycle of cell division and increasing the accumulation of storage materials in the fruit tissue . The relationship between FtMIF1, FtMIF2 and FtZHD17 and the FtARF gene family needs further study; however, FtMIF1, FtMIF2 and FtZHD17 may be related to the fruit size of Tartary buckwheat. In addition, FtZHD12, which has a 0.999 correlation with FtZHD17, may also be related to fruit size. In conclusion, the analysis of FtZF-HD gene expression in the tissues and fruit laid a foundation for breeding new Tartary buckwheat varieties.
20 FtZF-HD genes were identified in Tartary buckwheat, and the structures, evolution and expression patterns of the proteins were studied. Our findings provide a valuable basis for further analysis of the biological function of the ZF-HD gene family. Our study also laid a foundation for the improvement of Tartary buckwheat crops.
Identification of ZF-HD genes in Tartary buckwheat
We downloaded the Tartary buckwheat genome from the Tartary buckwheat genome project (TBGP; http://www.mbkbase.org/Pinku1/) . The ZF-HD gene family of Tartary buckwheat was searched by two BLASTP methods. The hidden Markov model (HMM) file corresponding to the ZF-HD_dimer domain (PF04770) was downloaded from the Pfam protein family database (http://pfam.xfam.org/). The existence of ZF-HD_dimer core sequences was verified by PFAM and SMART programs. Finally, 20 genes containing ZF-HD domain were screened from tartary buckwheat genome. By using the tools from the ExPASy website (https://web.expasy.org/compute_pi/), we obtained the sequence length, molecular weight, isoelectric point and subcellular localization of the identified 20 ZF-HD proteins.
Using the ZF-HD_dimer domain sequence of the FtZF-HD proteins, we used the default parameter ClustalW to compare several protein sequences. To determine the exon-intron structure of the FtZF-HD genes, the predicted coding sequence was compared with the corresponding full-length sequence by the Gene Structure Display Server (GSDS: http://gsds.cbi.pku.edu.cn) online program. Using the MEME online program (http:/meme.nbcr.net/meme/intro.html) to analyze the protein sequences under the following parameters: the optimum motif width was 6 ~ 200; the maximum number of motifs was 10.
Chromosomal distribution and gene duplication of FtZF-HD genes
The method of FtZF-HD genes mapping to the chromosomes of tartary buckwheat was refer to Liu et al. . Analysis of FtZF-HD genes replication events using Multiple collinear scanning toolkits (MCScanX). The syntenic relationship between FtZF-HD genes and ZF-HD genes from selected plants were determined by using the Dual Systeny Plotter software (https://github.com/CJ-Chen/TBtools).
Phylogenetic analysis and classification of the FtZF-HD gene family
Using ZF-HD protein sequences (Arabidopsis thaliana, maize, rice and soybean) downloaded from the UniProt database (https://www.uniprot.org/) constructed phylogenetic trees. We using the NJ method of Geneious R11 to derived the phylogenetic tree. The parameters were the Jukes-Cantor model, and global alignment with free end gaps.
This study used the Tartary buckwheat (XiQIAO) germplasm provided by Professor Wang Anhu of Xichang University. We planted materials in the experimental field of the College of Life Sciences of Sichuan Agricultural University (Lat. 29°97′ N, 102°97′ E, Alt. 580 m), Ya’an, Sichuan, China . The samples including flower, the fruit from three (13, 19, and 25, DAP) different developmental fruit stages, and the stem, root, and leaf of mature tartary buckwheat were collected separately and quickly put into liquid nitrogen and stored at − 80 °C for further use.
Expression analysis of the FtZF-HD genes using real-time PCR
Using the Primer3 software (http://bioinfo.ut.ee/primer3/) designed the RT-qPCR primers (Additional file 5: Table S4). Quantitative real-time PCR analysis was used to analyze the identified genes. Using the FtH3 gene as an internal control, the standard RT-qPCR with SYBR Premix Ex Taq II (TaKaRa) was repeated at least three times on a CFX96 Real Time System (BioRad). The data were analyzed by the 2−(∆∆Ct) method, and the relative mRNA expression data were obtained .
We used Origin Pro 2018b (OriginLab Corporation., Northampton, Massachusetts, USA) statistics program to analyze all the data, and the means were compared by the least significant difference test (LSD) at significance levels of 0.05 and 0.01 .
We thank all the colleagues in our laboratory for providing useful discussions and technical assistance. We are very grateful to the editor and reviewers for critically evaluating the manuscript and providing constructive comments for its improvement.
M.-Y.L. planned and designed the research, and analyzed data. X.-X.W. wrote the original manuscript. X.-X.W. and Z.-T.M. determined the expression of genes by qRT-PCR. T.-R.Z. and L.H. identified FtZF-HD family genes and visualized their structures. Q.W. and Z.-Z.T. performed the evolutionary analysis of FtZF-HD genes and several different species. T.-L.B. and C.-L.L. performed FtZF-HD genes chromosome distribution, gene replication and synchronous analysis. W.-J.S. planted and collected plant materials. H.C., M.-Y.L. and W.-J.S. reviewed and edited the manuscript. H.C. supervised the research. M.-Y.L. and X.-X.W. contributed equally. All authors read and approved the final manuscript.
Funding this research was supported by the National Natural Science Foundation of China (31500289), and the National Key R&D Program of China (2018YFD1000706). Funds were used for the design of the study and collection, analysis, and interpretation of data and in writing the manuscript, as well as in the open access payment.
Ethics approval and consent to participate
The tartary buckwheat accessions (XIQIAO) materials used in the experiment were supplied by Professor Wang Anhu of Xichang University. These plant materials are widely used all over the world. and no permits are required for the collection of plant samples. The plant materials are maintained in accordance with the institutional guidelines of the College of Life Sciences, Sichuan Agricultural University, China. This article did not contain any studies with human participants or animals, and did not involve any endangered or protected species.
Consent for publication
The authors declare that they have no competing interests.
- 7.Kawagashira N, Ohtomo Y, Murakami K, Matsubara K, Kawai J, Carninci P, Hayashizaki Y, Kikuchi S, Higo K: Multiple Zinc Finger Motifs with Comparison of Plant and Insect 2001(12):368–369.Google Scholar
- 14.Khatun K, Nath UK, Robin AHK, Park JI, Lee DJ, Kim MB, Chang KK, Lim KB, Nou IS, Chung MY. Genome-wide analysis and expression profiling of zinc finger homeodomain ( ZHD ) family genes reveal likely roles in organ development and stress responses in tomato. BMC Genomics. 2017;18(1):695.CrossRefGoogle Scholar
- 16.Wei H, Hong M. Characterization of a novel putative zinc finger gene MIF1: involvement in multiple hormonal regulation of Arabidopsis development. Plant J. 2010;45(3):399–422.Google Scholar
- 17.HC P, ML K, SM L, JD B, DJ Y, CO L, JC H, SY L, MJ C, WS C. Pathogen-induced binding of the soybean zinc finger homeodomain proteins GmZF-HD1 and GmZF-HD2 to two repeats of ATTA homeodomain binding site in the calmodulin isoform 4 (GmCaM4) promoter. Nucleic Acids Res. 2007;35(11):3612–23.CrossRefGoogle Scholar
- 19.QF C: Buckwheat plant science: Beijing, China: Science Press; 2012.Google Scholar
- 23.Liu M, Fu Q, Ma Z, Sun W, Huang L, Wu Q, Tang Z, Bu T, Li C, Chen H: Genome-wide investigation of the MADS gene family and dehulling genes in tartary buckwheat (Fagopyrum tataricum). 2019.Google Scholar
- 24.Liu M, Ma Z, Sun W, Huang L, Wu Q, Tang Z, Bu T, Li C, Chen H: Genome-wide analysis of the NAC transcription factor family in Tartary buckwheat (Fagopyrum tataricum). 2019, 20.Google Scholar
- 25.Liu M, Sun W, Ma Z, Zheng T, Huang L, Wu Q, Zhao G, Tang Z, Bu T, Li C, et al. Genome-wide investigation of the AP2/ERF gene family in tartary buckwheat (Fagopyum Tataricum); 2019. p. 19.Google Scholar
- 27.Liu M, Ma Z, Zheng T, Sun W, Zhang Y, Jin W, Zhan J, Cai Y, Tang Y, Wu Q: Insights into the correlation between physiological changes in and seed development of tartary buckwheat (Fagopyrum tataricum Gaertn ) Bmc Genomics 2018, 19(1):648.Google Scholar
- 30.Hongzhi K, Landherr LL, Frohlich MW, Jim LM, Hong M, Depamphilis CW. Patterns of gene duplication in the plant SKP1 gene family in angiosperms: evidence for multiple mechanisms of rapid gene birth. Plant J. 2010;50(5):873–85.Google Scholar
- 35.Zhang L, Li X, Ma B, Gao Q, Du H, Han Y, Li Y, Cao Y, Qi M, Zhu Y, et al. The Tartary Buckwheat Genome Provides Insights into Rutin Biosynthesis and Abiotic Stress Tolerance, vol. 10; 2017. p. 1224–37.Google Scholar
- 42.H W, L B, U W. Molecular physiology of legume seed development. Annu Rev Plant Biol. 2004;56(56):253–79.Google Scholar
Open AccessThis article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creativecommons.org/licenses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article, unless otherwise stated.