Tandem Duplicates Research Articles

Phylogenomic Analysis and Functional Characterization of the APETALA2/Ethylene-Responsive Factor Transcription Factor Across Solanaceae.

The AP2/ERF family constitutes one of the largest groups of transcription factors in the Solanaceae. AP2/ERF contributes to various plant biological processes, including growth, development, and responses to various stresses. The origins and functional diversification of AP2/ERF within the Solanaceae family remain poorly understood, primarily because of the complex interactions between whole-genome duplications (WGDs) and tandem duplications. In this study, a total of 1282 AP2/ERF proteins are identified from 7 Solanaceae genomes. The amplification of AP2/ERF genes was driven not only by WGDs but also by the presence of clusters of tandem duplicated genes. The conservation of synteny across different chromosomes provides compelling evidence for the impact of the WGD event on the distribution pattern of AP2/ERF genes. Distinct expression patterns suggest that the multiple copies of AP2/ERF genes evolved in different functional directions, catalyzing the diversification of roles among the duplicated genes, which was of great significance for the adaptability of Solanaceae. Gene silencing and overexpression assays suggest that ERF-1 members' role in regulating the timing of floral initiation in C. annuum. Our findings provide insights into the genomic origins, duplication events, and function divergence of the Solanaceae AP2/ERF.

Comparative characterization and phylogenetic analysis of complete mitogenome of three taxonomic confused groupers and the insight to the novel gene tandem duplication in Epinephelus

Epinephelus bilobatus, Epinephelus maculatus and Epinephelus longispinis are three groupers that share common morphological characteristics and coloration patterns and have been morphologically confused and misidentified with each other for a long time. Complete mitochondrial genomes of the three groupers were determined and analyzed in this study. Mitogenomes of E. bilobatus, E. maculatus and E. longispinis were 17, 354 bp, 17, 066 bp and 17, 221 bp in size respectively and consisted of 13 protein-coding genes, two ribosomal RNA genes and one control region. However, different from most teleosts, which contain canonical 22 tRNAs, more numbers of tRNAs were identified in the three groupers with 27 tRNAs in E. bilobatus and E. longispinis and 25 tRNAs in E. maculatus. The increased number of tRNAs was due to the presence of tandemly duplicated tRNA-Asp genes that were located between tRNA-Ser and COII genes (six duplications in E. bilobatus and E. longispinis, four duplications in E. maculatus). Intact gene tandem duplication was an uncommon feature that was found in the typical teleost mitogenomes. The phylogenetic trees of the 32 groupers (genus Epinephelus) that were constructed based on 12 protein-coding genes revealed that Epinephelus species with tandemly duplicated tRNA-Asp genes were clustered into one monophyletic group, distinct from other Epinephelus species without any duplication features, which indicated that tandemly duplicated tRNA-Asp genes may be the particular linage-specific characteristics that evolve from a common ancestor and have the ability to distinguish them from other Epinephelus species. The results of the mitogenomes comparative analyses of the three groupers revealed the genetic distance of mitogenomes between each two species to be 0.062 (E. bilobatus vs E. maculatus), 0.091 (E. bilobatus vs E. longispinis) and 0.087 (E. maculatus vs E. longispinis). All values were far greater than the minimum value of 0.020 for species identification, which shows that they were three independent species at molecular level. Regarding the relationships between the three groupers, E. bilobatus was found to be more closely related to E. maculatus in comparison to E. longispinis. The results provide valuable molecular data for the species identification and phylogenetic analyses on E. bilobatus, E. maculatus and E. longispinis, and also provided a new insight into the tandem gene duplication features of Epinephelus mitogenomes.

Genome-wide identification of CaWD40 proteins reveals the involvement of a novel complex (CaAN1-CaDYT1-CaWD40-91) in anthocyanin biosynthesis and genic male sterility in Capsicum annuum

BackgroundThe WD40 domain, one of the most abundant in eukaryotic genomes, is widely involved in plant growth and development, secondary metabolic biosynthesis, and mediating responses to biotic and abiotic stresses. WD40 repeat (WD40) protein has been systematically studied in several model plants but has not been reported in the Capsicum annuum (pepper) genome.ResultsHerein, 269, 237, and 257 CaWD40 genes were identified in the Zunla, CM334, and Zhangshugang genomes, respectively. CaWD40 sequences from the Zunla genome were selected for subsequent analysis, including chromosomal localization, phylogenetic relationships, sequence characteristics, motif compositions, and expression profiling. CaWD40 proteins were unevenly distributed on 12 chromosomes, encompassing 19 tandem duplicate gene pairs. The 269 CaWD40s were divided into six main branches (A to F) with 17 different types of domain distribution. The CaWD40 gene family exhibited diverse expression patterns, and several genes were specifically expressed in flowers and seeds. Yeast two-hybrid (Y2H) and dual-luciferase assay indicated that CaWD40-91 could interact with CaAN1 and CaDYT1, suggesting its involvement in anthocyanin biosynthesis and male sterility in pepper.ConclusionsIn summary, we systematically characterized the phylogeny, classification, structure, and expression of the CaWD40 gene family in pepper. Our findings provide a valuable foundation for further functional investigations on WD40 genes in pepper.

Comparative genomics reveals the molecular mechanisms of two drought-tolerant forages adapted to extreme environments in the northwest of China

Carex breviculmis, is a perennial herb with effective resistance of salt and drought stress in alkaline habitats and is widely used for forage production and turf management. We present its 469 Mb chromosome-level genome with 45,262 annotated protein-coding genes. The genome had 50.15 % repetitive sequences, mainly driven by TEs insertion in intronic regions. Phylogenetic analysis reveals C. breviculmis divergence from C. littledalei at 6.61 Mya, shared a WGD event with Ananas comosus about 103.12 Mya. Tandem duplicate genes rapidly expanded in sugar metabolism, amino acid synthesis, and phenylpropanoid biosynthesis. We identified 125/128 positively selected genes and 277/309 rapidly evolving genes in C. breviculmis and Achnatherum splendens, respectively, and analysis found that they were co-enriched in pathways like amino acid biosynthesis and fructose and mannose metabolism. In addition, a total of 51 convergent evolutionary genes were identified by two method, including stress-responsive genes like ACBP4, CIPK1, HMA5, OTC, SMT3, SPP2, VDE, VFB1, and VLN2. Finally, we reconstructed the sucrose metabolic pathways in C. breviculmis and A. splendens, and found that the major structural genes responsible for the regulation of sucrose metabolic pathways were significantly amplified. This study lays the foundation for future research on their environmental adaptations, and potential genetic breeding.

AmDEF2.7, a tandem duplicated defensin gene from Ammopiptanthus mongolicus, activated by AmWRKY14, enhances the tolerance of Arabidopsis to low temperature and osmotic stress

Ammopiptanthus mongolicus is an evergreen broad-leaved shrub growing in temperate regions. Plant defensins, a type of cysteine-rich small peptides, contribute to plant defense as antimicrobial peptides. In this study, we analyzed the evolution and expression patterns of the defensin gene family in A. mongolicus, and explored the function and regulatory mechanisms of the AmDEF2.7 gene in response to abiotic stress. Seven out of ten defensin genes had undergone segmental duplication and tandem duplication, especially, AmDEF2.6, AmDEF2.7, and AmDEF2.8, which were clustered on chromosome 9. The expression of multiple defensin genes was responsive to abiotic stress, with three defensin genes, including AmDEF2.7, showing significant induction during winter. Yeast expressing AmDEF2.7 gene exhibited increased resistance to freeze-thaw cycles and osmotic stress, while transgenic Arabidopsis overexpressing the AmDEF2.7 showed improved tolerance to both freezing and drought conditions. Furthermore, AmWRKY14 bound to the AmDEF2.7 gene promoter, and activated the expression of AmDEF2.7. These results highlighted the role of defensin AmDEF2.7 in the adaptation of A. mongolicus to temperate winter climate. This study expands our knowledge of plant defensin and provides support for clarifying the molecular mechanism of the adaptation of A. mongolicus to winter climate.

The genome assembly of Carex breviculmis provides evidence for its phylogenetic localization and environmental adaptation.

Carex breviculmis is a perennial herb with good resistance and is widely used for forage production and turf management. It is important in ecology, environmental protection and biodiversity conservation, but faces several challenges due to human activities. However, the absence of genome sequences has limited basic research and the improvement of wild plants. We annotated the genome of C. breviculmis and conducted a systematic analysis to explore its resistance to harsh environments. We also conducted a comparative analysis of Achnatherum splendens, which is similarly tolerant to harsh environments. The assembled the genome comprises 469.01 Mb, revealing 37 372 genes with a BUSCO completeness score of 99.0 %. The genome has 52.03 % repetitive sequences, primarily influenced by recent LTR insertions that have contributed to its expansion. Phylogenetic analysis suggested that C. breviculmis diverged from C. littledalei ~6.61 million years ago. Investigation of repetitive sequences and expanded gene families highlighted a rapid expansion of tandem duplicate genes, particularly in areas related to sugar metabolism, synthesis of various amino acids, and phenylpropanoid biosynthesis. Additionally, our analysis identified crucial genes involved in secondary metabolic pathways, such as glycolysis, phenylpropanoid biosynthesis and amino acid metabolism, which have undergone positive selection. We reconstructed the sucrose metabolic pathway and identified significant gene expansions, including 16 invertase, 9 sucrose phosphate synthase and 12 sucrose synthase genes associated with sucrose metabolism, which showed varying levels of expansion. The expansion of these genes, coupled with subsequent positive selection, contributed to the ability of C. breviculmis to adapt to environmental stressors. This study lays the foundation for future research on the evolution of Carex plants, their environmental adaptations, and potential genetic breeding.

Characterization and stress-responsive regulation of CmPHT1 genes involved in phosphate uptake and transport in Melon (Cucumis melo L.)

BackgroundPhosphorus (P) deficiency, a major nutrient stress, greatly hinders plant growth. Phosphate (Pi) uptake in plant roots relies on PHT1 family transporters. However, melon (Cucumis melo L.) lacks comprehensive identification and characterization of PHT1 genes, particularly their response patterns under diverse stresses.ResultsThis study identified and analyzed seven putative CmPHT1 genes on chromosomes 3, 4, 5, 6, and 7 using the melon genome. Phylogenetic analysis revealed shared motifs, domain compositions, and evolutionary relationships among genes with close histories. Exon number varied from 1 to 3. Collinearity analysis suggested segmental and tandem duplications as the primary mechanisms for CmPHT1 gene family expansion. CmPHT1;4 and CmPHT1;5 emerged as a tandemly duplicated pair. Analysis of cis-elements in CmPHT1 promoters identified 14 functional categories, including putative PHR1-binding sites (P1BS) in CmPHT1;4, CmPHT1;6, and CmPHT1;7. We identified that three WRKY transcription factors regulated CmPHT1;5 expression by binding to its W-box element. Notably, CmPHT1 promoters harbored cis-elements responsive to hormones and abiotic factors. Different stresses regulated CmPHT1 expression differently, suggesting that the adjusted expression patterns might contribute to plant adaptation.ConclusionsThis study unveils the characteristics, evolutionary diversity, and stress responsiveness of CmPHT1 genes in melon. These findings lay the foundation for in-depth investigations into their functional mechanisms in Cucurbitaceae crops.

Protective efficacy and immune responses of largemouth bass (Micropterus salmoides) immunized with an inactivated vaccine against the viral hemorrhagic septicemia virus genotype IVa

Viral hemorrhagic septicemia virus (VHSV) poses a significant threat to the aquaculture industry, prompting the need for effective preventive measures. Here, we developed an inactivated VHSV and revealed the molecular mechanisms underlying the host's protective response against VHSV. The vaccine was created by treating VHSV with 0.05 % formalin at 16 °C for 48 h, which was determined to be the most effective inactivation method. Compared with nonvaccinated fish, vaccinated fish exhibited a remarkable increase in survival rate (99 %) and elevated levels of serum neutralizing antibodies, indicating strong immunization. To investigate the gene changes induced by vaccination, RNA sequencing was performed on spleen samples from control and vaccinated fish 14 days after vaccination. The analysis revealed 893 differentially expressed genes (DEGs), with notable up-regulation of immune-related genes such as annexin A1a, coxsackievirus and adenovirus receptor homolog, V-set domain-containing T-cell activation inhibitor 1-like, and heat shock protein 90 alpha class A member 1 tandem duplicate 2, indicating a vigorous innate immune response. Furthermore, KEGG enrichment analysis highlighted significant enrichment of DEGs in processes related to antigen processing and presentation, necroptosis, and viral carcinogenesis. GO enrichment analysis further revealed enrichment of DEGs related to the regulation of type I interferon (IFN) production, type I IFN production, and negative regulation of viral processes. Moreover, protein-protein interaction network analysis identified central hub genes, including IRF3 and HSP90AA1.2, suggesting their crucial roles in coordinating the immune response elicited by the vaccine. These findings not only confirm the effectiveness of our vaccine formulation but also offer valuable insights into the underlying immunological mechanisms, which can be valuable for future vaccine development and disease management in the aquaculture industry.

Comparative genomics and evidence for an unusual polyamine oxidation pathway in aquatic duckweed (Spirodela polyrhiza L.)

Polyamines (PA) cellular levels are maintained through a balance between synthesis and catabolism, achieved by two classes of enzymes polyamine oxidases (PAOs) and copper amine oxidases (CuAO). Here we investigated the occurrence, molecular evolution and role(s) of PAOs and CuAO gene families in aquatic duckweed and their comparison with other aquatic plants -sea eelgrass, bladderwort, and Lotus. We identified eight bona fide PAO genes (SpPAO1–SpPAO8) and one SpCuAO1 in the greater duckweed genome from three genome assemblies. Interestingly, duckweed PAO genes increased their number through a tandem duplication event, while contrary to this CuAO genes were significantly lost to a single gene SpCuAO1. Phylogenetic analysis revealed that tandemly duplicated SpPAO2–7 share close similarity to well-known terminal catabolism (TC) pathway PAO genes while SpPAO1 and SpPAO8 seem to segregate along with back conversion (BC) participating known PAO genes, suggesting that all tandem duplicated PAOs are involved in TC pathway which is contrary to known trend in land plants where CuAOs are mainly involved in TC pathway. Comparative transcript abundance studies indicated that all eight PAOs and one CuAO gene respond to multiple stresses and principal component analysis identifies SpPAO4 as a highly active gene in response to multiple stresses. Results showed that oxidation of higher polyamines (SPD/SPM) through the TC pathway is diversified in duckweeds. Taken together this study reveals unique insights into the genomic losses and gains of polyamine metabolism possibly involved in achieving the structural and physiological adaptations required for aquatic lifestyle of duckweeds.

The biosynthesis of asiaticoside and madecassoside reveals tandem duplication–directed evolution of glycoside glycosyltransferases in the Apiales

Certain plant species within the Apiales order accumulate triterpenoid saponins that feature a distinctive glucose-glucose-rhamnose (G-G-R) sugar chain attached at the C-28 position of the pentacyclic triterpene skeleton. Until recently, the genomic basis underlying the biosynthesis and evolution of this sugar chain has remained elusive. In this study, we identified two novel glycoside glycosyltransferases (GGTs) that can sequentially install the sugar chain’s second D-glucose and third L-rhamnose during the biosynthesis of asiaticoside and madecassoside, two representative G-G-R sugar chain–containing triterpenoid saponins produced by Centella asiatica. Enzymatic assays revealed the remarkable substrate promiscuity of the two GGTs and the key residues crucial for sugar-donor selectivity of the glucosyltransferase and rhamnosyltransferase. We further identified syntenic tandem gene duplicates of the two GGTs in the Apiaceae and Araliaceae families, suggesting a well-conserved genomic basis underlying sugar chain assembly that likely has evolved in the early ancestors of the Apiales order. Moreover, expression patterns of the two GGTs in pierced leaves of C. asiatica were found to be correlated with the production of asiaticoside and madecassoside, implying their involvement in host defense against herbivores and pathogens. Our work sheds light on the biosynthesis and evolution of complex saponin sugars, paving the way for future engineering of diverse bioactive triterpenoids with unique glycoforms.

Genome-Wide Identification Analysis of GST Gene Family in Wild Blueberry Vaccinium duclouxii and Their Impact on Anthocyanin Accumulation.

Vaccinium duclouxii, a wild blueberry species native to the mountainous regions of southwestern China, is notable for its exceptionally high anthocyanin content, surpassing that of many cultivated varieties and offering significant research potential. Glutathione S-transferases (GSTs) are versatile enzymes crucial for anthocyanin transport in plants. Yet, the GST gene family had not been previously identified in V. duclouxii. This study utilized a genome-wide approach to identify and characterize the GST gene family in V. duclouxii, revealing 88 GST genes grouped into seven distinct subfamilies. This number is significantly higher than that found in closely related species, with these genes distributed across 12 chromosomes and exhibiting gene clustering. A total of 46 members are classified as tandem duplicates. The gene structure of VdGST is relatively conserved among related species, showing closer phylogenetic relations to V. bracteatum and evidence of purifying selection. Transcriptomic analysis and qRT-PCR indicated that VdGSTU22 and VdGSTU38 were highly expressed in flowers, VdGSTU29 in leaves, and VdGSTF11 showed significant expression in ripe and fully mature fruits, paralleling trends seen with anthocyanin accumulation. Subcellular localization identified VdGSTF11 primarily in the plasma membrane, suggesting a potential role in anthocyanin accumulation in V. duclouxii fruits. This study provides a foundational basis for further molecular-level functional analysis of the transport and accumulation of anthocyanins in V. duclouxii, enhancing our understanding of the molecular mechanisms underlying anthocyanin metabolism in this valuable species.

Genome-wide identification and expression analysis of the WRKY gene family in Rhododendron henanense subsp. lingbaoense.

This work explored the characteristics of the WRKY transcription factor family in Rhododendron henanense subsp. lingbaoense (Rhl) and the expression patterns of these genes under abiotic stress by conducting bioinformatics and expression analyses. RhlWRKY genes were identified from a gene library of Rhl. Various aspects of these genes were analyzed, including genetic structures, conserved sequences, physicochemical properties, cis-acting elements, and chromosomal location. RNA-seq was employed to analyze gene expression in five different tissues of Rhl: roots, stems, leaves, flowers, and hypocotyls. Additionally, qRT-PCR was used to detect changes in the expression of five RhlWRKY genes under abiotic stress. A total of 65 RhlWRKY genes were identified and categorized into three subfamilies based on their structural characteristics: Groups I, II, and III. Group II was further divided into five subtribes, with shared similar genetic structures and conserved motifs among members of the same subtribe. The physicochemical properties of these proteins varied, but the proteins are generally predicted to be hydrophilic. Most proteins are predicted to be in the cell nucleus, and distributed across 12 chromosomes. A total of 84 cis-acting elements were discovered, with many related to responses to biotic stress. Among the identified RhlWRKY genes, there were eight tandem duplicates and 97 segmental duplicates. The majority of duplicate gene pairs exhibited Ka/Ks values <1, indicating purification under environmental pressure. GO annotation analysis indicated that WRKY genes regulate biological processes and participate in a variety of molecular functions. Transcriptome data revealed varying expression levels of 66.15% of WRKY family genes in all five tissue types (roots, stems, leaves, flowers, and hypocotyls). Five RhlWRKY genes were selected for further characterization and there were changes in expression levels for these genes in response to various stresses. The analysis identified 65 RhlWRKY genes, among which the expression of WRKY_42 and WRKY_17 were mainly modulated by the drought and MeJA, and WRKY_19 was regulated by the low-temperature and high-salinity conditions. This insight into the potential functions of certain genes contributes to understanding the growth regulatory capabilities of Rhl.

Genome-wide identification and expression analysis of protein arginine methyltransferase and JmjC domain-containing family in apple.

Histone methylation is an important type of histone modification that regulates gene expression in plants. In this study, we identified 14 arginine methylation-related genes (Protein Arginine Methyltransferase, MdPRMT) and 32 demethylation-related genes (JmjC Domain-Containing Family, MdJMJ) in apple. Furthermore, we investigated the phylogenetic relationship, chromosome distribution, gene structure, motif analysis, promoter sequence analysis, and expression patterns of MdPRMT and MdJMJ genes. Homology analysis showed a high degree of conservation and homology between PRMT and JMJ genes in Arabidopsis and apple. We identified the types of duplicated genes in the MdJMJ and MdPRMT gene families, found a large number of whole-genome duplicates (WGD) gene pairs and a small number of tandem duplicates (TD) pairs, transposed duplication (TRD) gene pairs as well as proximal duplicates (PD) pairs, and discussed the possible evolutionary pathways of the gene families from the perspective of duplicated genes. Homology analysis showed a high degree of conservation and homology between PRMT and JMJ genes in Arabidopsis and apple. In addition, the promoter regions of MdPRMT and MdJMJ contain numerous cis-acting elements involved in plant growth and development, hormone response, and stress responses. Based on the transcriptional profiles of MdPRMT and MdJMJ in different tissues and developmental stages, it was found that MdPRMT and MdJMJ may play multiple roles in apple growth and development, for example, MdJMJ21 may be involved in the regulation of apple endosperm formation. MdPRMT and MdJMJ exhibit different expression patterns in response to hormone signaling in apple, MdJMJ3, MdJMJ18, MdJMJ30, MdPRMT2, MdPRMT13, and MdPRMT14 may play roles in apple response to drought stress, while the expression of MdJMJ13, MdPRMT3, MdPRMT4, and MdPRMT6 is affected by cold stress. Our study provides a foundation for determining the functional roles of MdPRMT and MdJMJ genes in apple.

Comprehensive Comparative Analysis of the JAZ Gene Family in Common Wheat (Triticum aestivum) and Its D-Subgenome Donor Aegilops tauschii.

JASMONATE-ZIM DOMAIN (JAZ) repressor proteins work as co-receptors in the jasmonic acid (JA) signalling pathway and are essential for plant development and environmental adaptation. Despite wheat being one of the main staple food crops, until recently, comprehensive analysis of its JAZ gene family has been limited due to the lack of complete and high-quality reference genomes. Here, using the latest reference genome, we identified 17 JAZ genes in the wheat D-genome donor Aegilops tauschii. Then, 54 TaJAZs were identified in common wheat. A systematic examination of the gene structures, conserved protein domains, and phylogenetic relationships of this gene family was performed. Five new JAZ genes were identified as being derived from tandem duplication after wheat divergence from other species. We integrated RNA-seq data and yield QTL information and found that tandemly duplicated TaJAZ genes were prone to association with spike-related traits. Moreover, 12 TaJAZ genes were located within breeding selection sweeps, including 9 tandemly duplicated ones. Haplotype variation analysis of selected JAZ genes showed significant association of TaJAZ7A and TaJAZ13A with thousand-grain weight. Our work provides a clearer picture of wheat JAZ gene evolution and puts forward the possibility of using these genes for wheat yield improvement.

AcMYB266, a key regulator of the red coloration in pineapple peel: a case of subfunctionalization in tandem duplicated genes.

Red fruit peel is an attractive target for pineapple breeding. Various pineapple accessions with distinct red coloration patterns exist; however, the precise molecular mechanism accounting for these differences remains unknown, which hinders the pineapple breeding process from combining high fruit quality with red peel. In this study, we characterized a transcription factor, AcMYB266, which is preferentially expressed in pineapple peel and positively regulates anthocyanin accumulation. Transgenic pineapple, Arabidopsis, and tobacco plants overexpressing AcMYB266 exhibited significant anthocyanin accumulation. Conversely, transient silencing of this gene led to decreased anthocyanin accumulation in pineapple red bracts. In-depth analysis indicated that variations of AcMYB266 sequences in the promoter instead of the protein-coding region seem to contribute to different red coloration patterns in peels of three representative pineapple varieties. In addition, we found that AcMYB266 was located in a cluster of four MYB genes exclusive to and conserved in Ananas species. Of this cluster, each was proved to regulate anthocyanin synthesis in different pineapple tissues, illustrating an interesting case of gene subfunctionalization after tandem duplication. In summary, we have characterized AcMYB266 as a key regulator of pineapple red fruit peel and identified an MYB cluster whose members were subfunctionalized to specifically regulate the red coloration of different pineapple tissues. The present study will assist in establishing a theoretical mechanism for pineapple breeding for red fruit peel and provide an interesting case for the investigation of gene subfunctionalization in plants.

Genome-Wide Identification and Expression Pattern Analysis of BAHD Acyltransferase Family in Taxus mairei.

BAHD acyltransferases are involved in catalyzing and regulating the secondary metabolism in plants. Despite this, the members of BAHD family and their functions have not been reported in the Taxus species. In this study, a total of 123 TwBAHD acyltransferases from Taxus wallichiana var. mairei genome were identified and divided into six clades based on phylogenetic analysis, of which Clade VI contained a Taxus-specific branch of 52 members potentially involved in taxol biosynthesis. Most TwBAHDs from the same clade shared similar conserved motifs and gene structures. Besides the typical conserved motifs within the BAHD family, the YPLAGR motif was also conserved in multiple clades of T. mairei. Moreover, only one pair of tandem duplicate genes was found on chromosome 1, with a Ka/Ks ratio < 1, indicating that the function of duplicate genes did not differentiate significantly. RNA-seq analysis revealed different expression patterns of TwBAHDs in MeJA induction and tissue-specific expression experiments. Several TwBAHD genes in the Taxus-specific branch were highly expressed in different tissues of T. mairei, suggesting an important role in the taxol pathway. This study provides comprehensive information for the TwBAHD gene family and sets up a basis for its potential functions.

Abstract 4628: Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies

Abstract Optimal diagnosis, prognosis, and treatment selection for hematologic malignancies requires assessment of somatic mutations across a subset of clinically relevant genes. Here we present the analytical validation results of an expanded targeted next generation sequencing (NGS) panel of 141 clinically relevant genes for myeloid and lymphoid malignancies. Based on review of clinical guidelines, 141 genes including 79 myeloid and 84 lymphoid associated genes were selected. The expanded hematological panel interrogates all coding exons of the 141 genes to detect single nucleotide variants (SNVs) and insertions/deletions (indels). The panel also identifies copy number aberrations (CNAs) in 16 genes, FLT3 internal tandem duplicates (ITDs), select non-coding pathogenic variants, and patient sex. The custom hybrid capture-based assay utilizes genomic libraries created from 250 ng gDNA extracted from peripheral blood, bone marrow, or cell suspensions, followed by sequencing on Illumina® instruments. Concordance studies were performed on clinical samples previously assessed using an orthogonal NGS-based laboratory developed test for SNVs/indels/FLT3-ITDs or digital multiplexed ligation-dependent probe amplification for CNAs. In total, 308 patient samples were evaluated and included 72 (23.4%) acute myeloid leukemia and 58 (18.8%) myelodysplastic syndromes samples among other indications, of which 240 (77.9%) were bone marrow specimens. Analysis of concordance demonstrated a positive percent agreement (PPA) of 99.7% for SNVs (750/752), 99.5% for indels &amp;lt;25bp (206/207), and 95.7% for indels ≥ 25bp (22/23). Variants were from 45 genes including TP53, NPM1, and IDH1/2. PPA was 100% for FLT3-ITDs (38/38; size range 18-300 bp) and sex (294/294). For CNAs, PPA was 96.4% (132/137) and negative percent agreement (NPA) was 98.3% (931/947). Assay precision was determined using three replicates of 15 clinical samples for both intra and inter-assay precision using multiple operators, instruments, and reagent lots. 100% concordance was observed for SNVs (36/36), indels (11/11), a 24 bp FLT3-ITD (1/1), CNAs (24/24), and sex (15/15). Analytical specificity was assessed using 5 replicates of NA12878 showing specificity &amp;gt;99.99% for SNVs/indels with variant allele frequency ≥ 3%, and &amp;gt;99.99% for CNAs with copy number ≤0.85 or ≥1.15. Dilution series to determine analytical sensitivity are in progress. This study highlights the analytical validation of the expanded NGS panel for the detection of clinically informative genomic alterations in hematologic malignancies. Results from this validation study, when complete, will include at least 576 samples plus orthogonal testing. These data describe the performance of the assay to enable a comprehensive evaluation of genomic alterations using a single sample, further facilitating the use of broad NGS assays in patients with hematologic malignancies. Citation Format: Grant Hogg, Tong Liu, Helen Cao, Adib Shafi, Ashraf Shabaneh, Jon Williams, John Howitt, Amanda Williamson, Rachel Dango, Xiaojun Guan, Heidi Hoffmann, Michael Mooney, John Pruitt, Scott Parker, Henry Dong, Stan Letovsky, Li Cai, Eric A. Severson, Shakti H. Ramkissoon, Anjen Chenn, Marcia Eisenberg, Brian Caveney, Eyad Almasri, Taylor J. Jensen. Validation of an automated, scalable comprehensive genomic profiling assay for hematologic malignancies [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2024; Part 1 (Regular Abstracts); 2024 Apr 5-10; San Diego, CA. Philadelphia (PA): AACR; Cancer Res 2024;84(6_Suppl):Abstract nr 4628.

Genome-wide identification, structural characterization and gene expression analysis of the WRKY transcription factor family in pea (Pisum sativum L.)

BackgroundThe WRKY gene family is one of the largest families of transcription factors in higher plants, and WRKY transcription factors play important roles in plant growth and development as well as in response to abiotic stresses; however, the WRKY gene family in pea has not been systematically reported.ResultsIn this study, 89 pea WRKY genes were identified and named according to the random distribution of PsWRKY genes on seven chromosomes. The gene family was found to have nine pairs of tandem duplicates and 19 pairs of segment duplicates. Phylogenetic analyses of the PsWRKY and 60 Arabidopsis WRKY proteins were performed to determine their homology, and the PsWRKYs were classified into seven subfamilies. Analysis of the physicochemical properties, motif composition, and gene structure of pea WRKYs revealed significant differences in the physicochemical properties within the PsWRKY family; however, their gene structure and protein-conserved motifs were highly conserved among the subfamilies. To further investigate the evolutionary relationships of the PsWRKY family, we constructed comparative syntenic maps of pea with representative monocotyledonous and dicotyledonous plants and found that it was most recently homologous to the dicotyledonous WRKY gene families. Cis-acting element analysis of PsWRKY genes revealed that this gene family can respond to hormones, such as abscisic acid (ABA), indole-3-acetic acid (IAA), gibberellin (GA), methyl jasmonate (MeJA), and salicylic acid (SA). Further analysis of the expression of 14 PsWRKY genes from different subfamilies in different tissues and fruit developmental stages, as well as under five different hormone treatments, revealed differences in their expression patterns in the different tissues and fruit developmental stages, as well as under hormone treatments, suggesting that PsWRKY genes may have different physiological functions and respond to hormones.ConclusionsIn this study, we systematically identified WRKY genes in pea for the first time and further investigated their physicochemical properties, evolution, and expression patterns, providing a theoretical basis for future studies on the functional characterization of pea WRKY genes during plant growth and development.

A chromosome-scale genome of Rhus chinensis Mill. provides new insights into plant-insect interaction and gallotannins biosynthesis.

Rhus chinensis Mill., an economically valuable Anacardiaceae species, is parasitized by the galling aphid Schlechtendalia chinensis, resulting in the formation of the Chinese gallnut (CG). Here, we report a chromosomal-level genome assembly of R. chinensis, with a total size of 389.40 Mb and scaffold N50 of 23.02 Mb. Comparative genomic and transcriptome analysis revealed that the enhanced structure of CG and nutritional metabolism contribute to improving the adaptability of R. chinensis to S. chinensis by supporting CG and galling aphid growth. CG was observed to be abundant in hydrolysable tannins (HT), particularly gallotannin and its isomers. Tandem repeat clusters of dehydroquinate dehydratase/shikimate dehydrogenase (DQD/SDH) and serine carboxypeptidase-like (SCPL) and their homologs involved in HT production were determined as specific to HT-rich species. The functional differentiation of DQD/SDH tandem duplicate genes and the significant contraction in the phenylalanine ammonia-lyase (PAL) gene family contributed to the accumulation of gallic acid and HT while minimizing the production of shikimic acid, flavonoids, and condensed tannins in CG. Furthermore, we identified one UDP glucosyltransferase (UGT84A), three carboxylesterase (CXE), and six SCPL genes from conserved tandem repeat clusters that are involved in gallotannin biosynthesis and hydrolysis in CG. We then constructed a regulatory network of these genes based on co-expression and transcription factor motif analysis. Our findings provide a genomic resource for the exploration of the underlying mechanisms of plant-galling insect interaction and highlight the importance of the functional divergence of tandem duplicate genes in the accumulation of secondary metabolites.

Tandemly duplicated MYB genes are functionally diverged in the regulation of anthocyanin biosynthesis in soybean.

Gene duplications have long been recognized as a driving force in the evolution of genes, giving rise to novel functions. The soybean (Glycine max) genome is characterized by a large number of duplicated genes. However, the extent and mechanisms of functional divergence among these duplicated genes in soybean remain poorly understood. In this study, we revealed that 4 MYB genes (GmMYBA5, GmMYBA2, GmMYBA1, and Glyma.09g235000)-presumably generated by tandem duplication specifically in the Phaseoleae lineage-exhibited a stronger purifying selection in soybean compared to common bean (Phaseolus vulgaris). To gain insights into the diverse functions of these tandemly duplicated MYB genes in anthocyanin biosynthesis, we examined the expression, transcriptional activity, induced metabolites, and evolutionary history of these 4 MYB genes. Our data revealed that Glyma.09g235000 is a pseudogene, while the remaining 3 MYB genes exhibit strong transcriptional activation activity, promoting anthocyanin biosynthesis in different soybean tissues. GmMYBA5, GmMYBA2, and GmMYBA1 induced anthocyanin accumulation by upregulating the expression of anthocyanin pathway-related genes. Notably, GmMYBA5 showed a lower capacity for gene induction compared to GmMYBA2 and GmMYBA1. Metabolomics analysis further demonstrated that GmMYBA5 induced distinct anthocyanin accumulation in Nicotiana benthamiana leaves and soybean hairy roots compared to GmMYBA2 and GmMYBA1, suggesting their functional divergence leading to the accumulation of different metabolites accumulation following gene duplication. Together, our data provide evidence of functional divergence within the MYB gene cluster following tandem duplication, which sheds light on the potential evolutionary directions of gene duplications during legume evolution.