Background: MYB transcription factors are a kind of DNA binding protein that can specifically interact with the promoter region. Members of MYB TFs are widely involved in plant growth and development, secondary metabolism, stress response, and hormone signal transduction. However, there is no report of comprehensive bioinformatics analysis on the MYB family of Casuarina equisetifolia. Results: In this study, bioinformatics methods were used to screen out 182 MYB transcription factors from the Casuarina equisetifolia genome database, including 69 1R-MYB, 107 R2R3-MYB, 4 R1R2R3-MYB, and 2 4R-MYB. The C. equisetifolia R2R3-MYB genes were divided into 29 groups based on the phylogenetic topology and the classification of the MYB superfamily in Arabidopsis thaliana, while the remaining MYB genes (1R-MYB, R1R2R3-MYB, and 4R-MYB) was divided into 19 groups. Moreover, the conserved motif and gene structure analysis shown that the members of the CeqMYBs were divided into the same subgroups with mostly similar gene structures. In addition, many conserved amino acids in the R2 and R3 domains of CeqMYBs by WebLogo analysis, especially tryptophan residues (W), with 3 conserved W in R2 repeat and 2 conserved W in R3 repeat. Combining promoter and GO annotation analysis, speculated on the various biological functions of CeqMYBs, thus 32 MYB genes were selected to further explore its response to salt stress by using qPCR analysis technique. Most CeqMYB genes were differentially regulated following multiple salt treatments. Conclusions: Seven genes (CeqMYB164, CeqMYB4, CeqMYB53, CeqMYB32, CeqMYB114, CeqMYB71 and CeqMYB177) were assigned to the “response to salt stress” by GO annotation. Among them, the expression level of CeqMYB4 was up-regulated under various salt treatments, indicating CeqMYB4 might participated in the response to salt stress. Our results provide important information for the biological function of C. equisetifolia, as well as offer candidate genes for further study of salt stress mechanism.

The potassium transporter group of the HAK/KUP/KT (high-affinity K⁺)/KUP (K⁺ uptake)/KT (K⁺ transporter) family plays a crucial role in plant growth and development as well as in environmental adaptation such as tolerance to salt stress. HAK/KUP/KT genes and their functions have been characterized for a number of plant species, but they remain unknown for Casuarina equisetifolia, an important tree species for coastal protection in southern China and many other countries. In this study, 25 HAK genes were identified in the C. equisetifolia genome. Their gene structure, conserved motif, phylogeny, and expression were comprehensively and systematically analyzed to understand their functions. All HAK genes were relatively conserved and could be divided into four clusters. The expression level of two particular genes, CeqHAK11 and CeqHAK6, increased significantly with the duration of salt treatment. To further elucidated their function in response to salt stress, subcellular localization, and their functional analysis were developed. Results revealed that CeqHAK11 and CeqHAK6 were localized on the plasma membrane, which mainly mediated high-affinity K⁺ uptake. Overexpression of CeqHAK6 or CeqHAK11 in Arabidopsis showed higher germination and survival rates and longer root length than wild-type (WT) under salt stress, suggesting that both genes improve tolerance to salt stress. Moreover, CeqHAK6 and CeqHAK11 improved their ability to tolerate salt stress by increasing the K⁺/Na⁺ ratio and antioxidant enzyme activities (CAT, POD, and SOD), and decreasing reactive oxygen species (ROS) accumulation. Consequently, CeqHAK6 and CeqHAK11 were verified as potassium transport proteins and could be applied for further molecular breeding for salt tolerance in C. equisetifolia or other crops to increasing salt tolerance.

Background: Sucrose nonfermenting-1 (SNF1)-related protein kinases (SnRKs) play crucial roles in plant signaling pathways and stress adaptive responses by activating protein phosphorylation pathways. However, there have been no comprehensive studies of the SnRK gene family in the widely planted salt-tolerant tree species Casuarina equisetifolia. Here, we comprehensively analyze this gene family in C. equisetifolia using genome-wide identification, characterization, and profiling of expression changes in response to salt stress. Results: A total of 26 CeqSnRK genes were identified, which were divided into three subfamilies (SnRK1, SnRK2, and SnRK3). The intron–exon structures and protein‑motif compositions were similar within each subgroup but differed among groups. Ka/Ks ratio analysis indicated that the CeqSnRK family has undergone purifying selection, and cis-regulatory element analysis suggested that these genes may be involved in plant development and responses to various environmental stresses. A heat map was generated using quantitative real‑time PCR (RT-qPCR) data from 26 CeqSnRK genes, suggesting that they were expressed in different tissues. We also examined the expression of all CeqSnRK genes under exposure to different salt concentrations using RT-qPCR, finding that most CeqSnRK genes were regulated by different salt treatments. Moreover, co-expression network analysis revealed synergistic effects among CeqSnRK genes. Conclusions: Several CeqSnRK genes (CeqSnRK3.7, CeqSnRK3.16, CeqSnRK3.17) were up-regulated following salt treatment. Among them, CeqSnRK3.16 expression was significantly up-regulated under various salt treatments, identifying this as a candidate gene salt stress tolerance gene. In addition, CeqSnRK3.16 showed significant expression change correlations with multiple genes under salt stress, indicating that it might exhibit synergistic effects with other genes in response to salt stress. This comprehensive analysis will provide a theoretical reference for CeqSnRK gene functional verification and the role of these genes in salt tolerance.

Research Highlights: High variation of genetic diversity and differentiation among 27 seed sources within 14 natural provenances and 13 land race samples of Casuarina equisetifolia were found. High proportions of monoecious individuals may be present in some populations, as indicated by severe heterozytote deficiency and inbreeding found in many provenances and land races. The most probable origins of the land races were inferred according to the values of pairwise provenance differentiation and Nei’s genetic distances. Targeted introductions and testing of unrelated new accessions of C. equisetifolia from the Pacific and Philippines was proposed to identify Ralstonia-resistant genotypes. Background and Objectives: Casuarina equisetifolia was introduced to China a hundred years ago and has become a critically important tree species in coastal protection since the 1950s. Despite its importance, patterns of genetic variation, genetic relationships among natural provenances and probable origins of the land races remain unresolved. This has become a concern in China where Ralstonia solanacearum bacterial wilt has devastated plantations that are known to be from a narrow genetic base that urgently needs to be broadened. Materials and Methods: Fourteen natural provenances from Australia, Pacific islands and Southeast Asia, and 13 land race samples from parts of Asia and Africa outside the natural range were genotyped using 13 SSR (Simple Sequence Repeats) markers to characterize their allelic variation and genetic relationship. Results: Significant genetic diversity and differentiation among 27 seed sources within 14 provenances and 13 land race samples of C. equisetifolia was indicated. Significant heterozygote deficiency and inbreeding was indicated for a number of provenances, perhaps indicating a high proportion of monoecious parents in these populations. The most probable origins of the land races of the introduced countries were suggested according to the values of pairwise provenance differentiation (F_ST) and Nei’s genetic distances. Conclusions: We found significant genetic diversity and genetic differentiation among seed sources of C. equisetifolia. While individual land races do not appear to lack diversity, we were able to infer the origins of some, allowing targeted introductions of unrelated material to be made. In the case of the Chinese land race, targeting and testing new accessions from the Pacific and the Philippines may be a good strategy to identify Ralstonia-resistant genotypes.

The Tabebuia alliance is widely planted in tropical and subtropical countries. However, the lack of accurate taxonomic information for many commercial cultivars leads to commercial disputes. We sought to clarify the background of commercial cultivars in China and to provide a robust basis for patent protection. We collected 82 Tabebuia cultivars and 12 reference individuals, then developed large-scale single nucleotide polymorphism (SNP) markers using genotyping-by-sequencing. We obtained 73,219 SNPs from the test samples, which represented a substantial increase compared with previous studies of Tabebuia species. Based on these SNPs and a clustering analysis, we detected six species among the samples, as well as an uncertain population which may be a hybrid between Handroanthus impetiginosus (Mart. ex DC) Mattos and H. heptaphyllus (Vell.) Mattos. An analysis of genetic diversity to assess germplasm resources indicated that excessive heterozygosity of these cultivars in China, which may explain the genetic basis of “novel-preferred” selection in ornamental plants or a narrow genetic background. After rigorous selection, we identified numerous high-quality molecular markers for cultivar identification, then constructed a concise fingerprint with 54 SNPs. Our work clarifies the background information of commercial cultivars of Tabebuia in China and provides information for cultivar identification. It will serve as an important technical resource for genetic studies, and it will help to protect patents that involve Tabebuia species.