- The ABC transporter and HMA gene families are important in the control of cadmium (Cd) accumulation in crops. - Results: Here we describe the ABC transporter and HMA gene families in flax to provide a comprehensive overview of its evolution and some support towards the functional annotation of its members. - The 198 ABC transporter and 12 HMA genes identified in the flax genome were classified into eight ABC transporter and four HMA subfamilies based on their phylogenetic analysis and domains ’ composition. - Ten motifs were identified from all ABC transporter and HMA genes. - Both the ABC transporter and HMA gene families were highly conserved among subfamilies of flax and with those of Arabidopsis . - Gene ontology analyses suggested that most flax ABC transporter and HMA genes had a role in ATP binding, transport, catalytic activity, ATPase activity, and metal ion binding. - The RNA-Seq analysis of eight different organs demonstrated diversified expression profiling patterns of the genes and revealed their functional or sub- functional conservation and neo-functionalization.. - Full list of author information is available at the end of the article. - Conclusion: Characterization of the ABC transporter and HMA gene families will help in the functional analysis of candidate genes in flax and other crop species.. - The structure of the TMDs is highly diverse and varies in the number of transmembrane helices, whereas the NBDs have highly conserved helices [4]. - The two members of the ABCB gene family in Arabidopsis ( AtABCB1 and AtABCB2 ) are auxin trans- porters and the overexpression of AtABCB1 causes the elongation of hypocotyl cells [11, 12]. - Several members of the ABCC subfamily are responsible for phytate trans- port as exemplified in Arabidopsis ( AtABCC5 ) [13], maize ( ZmABCC4 ) [14] and rice ( OsABCC13) [15]. - These cited studies pro- vide an overview of the importance of both ABC transporter and HMA genes in various plant species, but no systemic studies have been reported in flax.. - The initial draft of the flax genome sequence was pro- duced using whole-genome shotgun (WGS) sequencing with short reads obtained on the Illumina sequencing platform [30]. - Thus, the availability of this recent update of the flax genome (version 2.0) constitutes a genomic resource that allows the identification of gene families, evolutionary relation- ships, and structural analyses. - To date, ABC transporter and HMA gene families have been studied in several plant species including Oryza sativa and Arabidopsis thaliana [32], Zea mays [33, 34], Brassica rapa [7], Brassica napus [35], Triticum aestivum [36], and Vitis vinifera [37]. - In this research work, we hypothesized that either whole genome duplications (WGDs) or tan- dem events contributed to the expansion of the ABC transporter and HMA gene families in flax. - Therefore, we studied the phylogenetic relationships, gene annota- tion, physicochemical properties, chromosomal distribu- tion, gene synteny, protein-protein interactions (PPIs), and gene duplications of all predicted ABC transporter and HMA genes of the flax genome to understand their evolution and hypothesize their putative functions. - ABC transporter and HMA genes in flax and their physicochemical properties. - A total of 198 ABC transporter and 12 HMA genes were identified in the flax genome reference sequence of CDC Bethune [31]. - The ABC transporter and the HMA genes were classified into eight and four subfamilies, respectively. - Further, several studies have also confirmed almost an identical localization of ABC transporter and HMA genes [7, 44].. - Annotation and phylogenetic analysis of ABC transporter and HMA genes in plant species. - In brief, based on the predicted function(s), most of the LuABC and LuHMA genes confirmed the presence of ABC transporter, ATP binding, and heavy metal ATPase domain functions. - the LuABCE subfamily en- codes to RNAse l inhibitor protein, whereas members of the LuABCG subfamily were involved in regulating pleiotropic drug resistance. - Unrooted phylogenetic trees were constructed using the protein sequences for each of the eight ABC trans- porter and four HMA gene subfamilies of Linum usita- tissimum , Arabidopsis thaliana , Populus trichocarpa , Vitis vinifera , and Brachypodium distachyon (Fig. - Based on the phylogenetic relationships of flax and other species, the ABC transporter genes were divided into eight subfamilies: ABCA-ABCG and ABCI (Fig. - The distribution patterns of both ABC transporter and HMA genes and their subfamilies among five species are given in Table 1.. - Chromosomal localization, Syntenic relationships, and duplication of ABC transporter and HMA genes. - Only one ABC transporter gene ( LuABCI16 ) was a singleton. - Eight of the nine flax Cd candidate genes were of the segmental type and one ( LuHMA4 ) was a tandem duplication.. - Thus, segmental duplications played a dominant role in the expansion of the ABC transporter and HMA gene families in flax and confirmed our hypothesis.. - Of the ten motifs, motif 6 was prevalent in both ABC transporter and HMA proteins except in ABCB, ABCD, and ABCF subfamilies. - Of the nine flax Cd candidate genes, three ( LuABCG71, LuABCG72, and LuABCG73 ) consistently exhibited 9–10 of these motifs and similar gene lengths. - However, distinct motif compo- sitions existed among most of the subfamilies.. - Specifically, 19 ABC transporter genes, including LuABCC7 , LuABCC9–. - The number of exons was highly Table 1 The distribution patterns of ABC transporter and HMA. - ABC transporter ABCD 5 2 3 1 4. - 1 Phylogenetic relationships of eight subfamilies of the ABC transporter proteins (a-h) and four subfamilies of HMA proteins (i) in five species. - Arabidopsis thaliana has 129 ABC transporter and 8 HMA proteins (AtABC and AtHMA), Vitis vinifera has 181 and 8 (VvABC and VvHMA), Linum usitatissimum has 198 and 12 (LuABC and LuHMA), Populus trichocarpa has 192 and 12 (PtABC and PtHMA), and Brachypodium distachyon has 133 and 9 (BdABC and LuHMA). - conserved within ABC transporter gene subfamilies. - To predict the regulatory functions of the LuABC and LuHMA genes in flax, we performed gene ontology (GO) analyses. - The expression patterns of the LuABC and LuHMA genes in the root, seed, ovary, and five different stages of embryo development (heart, globular, torpedo, ma- ture, and cotyledon) from RNA-Seq data are pre- sented in a heatmap (Fig. - 2 Chromosomal locations of the orthologous ABC transporter and HMA genes of flax and Arabidopsis . - Eight of the nine flax Cd candidate genes were highly expressed in different tissues, including LuABCG71, LuABCG72, and LuABCG73 in root and seed (Fig. - correlation coefficient ( r ) of the syntenic pairs across the eight different tissues used in our research. - How- ever, a few of the ABC proteins did not interact:. - Here, we identified 198 ABC transporter and 12 HMA genes in flax, accounting for 0.484% of the total 43,384 an- notated genes of its reference sequence [30]. - 4 Expression profiling of the 160 differentially expressed genes in eight different tissues based on log fold-changes, including 143 LuABC genes (a), nine LuHMA genes (b), and eight potential Cd candidate genes (c). - The remaining 48 LuABC and one each HMA ( LuHMA5 ) and Cd ( LuABCG58 ) genes were discarded because they were represented by less than 5 RPM (reads per million) after normalization of the data. - The results of the phylogenetic relationships between the genes were consistent with previous findings in Arabidop- sis thaliana, Brassica rapa, and Brassica napus [7, 35].. - Flax had more ABC transporter and HMA genes than any of the other five species investigated which included the dicots Arabidopsis thaliana , Vitis vinifera , Populus tricho- carpa , and the monocot Brachypodium distachyon , des- pite having a smaller genome than all of them except Arabidopsis . - In general, the LuABC and LuHMA genes were scattered on the phylogenetic tree, suggesting that the expansion of these gene families occurred before evo- lutionary divergence of the common ancestor.. - Our findings suggest an average estimated duplication diver- gence time of 8.59 MYA for LuABC and LuHMA genes, consistent with the most recent (3.7–9 MYA) WGD of the flax genome. - In flax, four types of gene duplications were observed in the 210 ABC transporter and HMA genes. - Segmental (WGD) duplications (77.14%) contributed by far the most to the expansion of the two gene families in flax, a common mode of expansion of gene families across vari- ous plant species [58–60]. - Most of the LuABC and LuHMA gene pairs underwent purifying selection. - However, nine pairs ex- hibited neo-functionalization, indicating new function(s) for the two genes of the pairs. - The structural diversity mainly contributed to the evolution of the gene families as indicated by evolutionary studies [64]. - Taken together, these analyses suggest that the ABC transporter and HMA gene families in flax expanded over evolutionary time through gene duplication events.. - A comprehensive sequence analysis of the ABC trans- porter and HMA gene families in flax was performed.. - We identified 198 LuABC and 12 LuHMA genes that clustered into eight ABC transporter (ABCA-ABCG and ABCI) and four HMA subfamilies. - These analyses will be foundational to future investigations into the biological functions of ABC transporter and HMA genes in flax, and will be especially helpful in conjunc- tion with a marker association study for Cd accumula- tion in flax.. - Identification of ABC transporter and HMA genes, and their duplications. - Two methods were used to identify ABC transporter genes in flax. - The ABC transporter do- mains included ABC transporter (PF00005), ABC-2 transporter (PF01061), ABC transporter transmembrane region (PF00664), cytochrome c polymerization (CYT) (PF01458) or mammalian cell entry (mce) related pro- tein (PF02470). - After merging the results, ABC transporter and HMA genes were further screened on the basis of their domain composition. - A total of 745 ABC transporter and HMA proteins were identi- fied among all species other than those of Arabidopsis thaliana. - Sequences of the 15 chromosomes of flax (version 2.0) were obtained from NCBI under GenomeProject ID no.. - The obtained protein sequences of ABC transporter and HMA genes were fur- ther verified for ABC/HMA domain compositions using the NCBI-Conserved Domain database (http://www.. - For the identification of flax Cd-associated genes, sev- eral ABC transporter and HMA responsive genes were used as a reference. - Phylogenetic characterization of ABC transporter and HMA genes, and synonymous ( Ks ) and non-synonymous ( Ka ) substitution rates for duplicated genes. - The results of the interaction network were visualized using Cytoscape (version . - Thus, expression results were presented for the eight tissues by comparing them to those of the anther.. - The GO analysis for ABC transporter and HMA genes in flax was conducted using the Phytozome database (http://phytozome.jgi.doe.gov/pz/portal.html) with key- word search options against the flax genome.. - Interaction network of ABC transporter genes (a) and HMA genes (b).. - Schematic representations of the two most stable genes among nine Cd candidate genes based on their conserved gene. - Basic information on the ABC transporter and HMA genes identified in flax.. - Gene duplications of the syntenic gene pairs in flax.. - Gene ontology (GO) of ABC transporter and HMA genes in flax.. - The authors wish to thank Nick Manseau for careful editing of the manuscript.. - The funders had no role in the design of the study. - in the collection, analysis, or interpretation of the data. - in the writing of the manuscript. - The ABC transporter structure and mechanism:. - functional and structural aspects of the ATP-hydrolyzing subunits/domains.. - In silico genome-wide analysis of the ATP-binding cassette transporter gene family in soybean ( Glycine max L.) and their expression profiling. - Genome-wide identification, evolution, and expression analysis of the ATP-binding cassette transporter gene family in Brassica rapa . - Plant cuticular lipid export requires an ABC transporter. - A member of the pleiotropic drug resistance family of ATP binding cassette transporters is required for the formation of a functional cuticle in Arabidopsis . - The ABC transporter ABCG36 is required for cadmium tolerance in rice.. - The ABC transporter ATPDR8 is a cadmium extrusion pump conferring heavy metal resistance.. - Inventory and general analysis of the ATP-binding cassette ( ABC ) gene superfamily in maize ( Zea mays L. - Genome-wide analysis and expression profiling of the HMA gene family in Brassica napus under cd stress. - 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