Chrysosplenium, a perennial herb in the family Saxifragaceae, prefers to grow in low light and moist environments and is divided into two sections of Alternifolia and Oppositifolia based on phyllotaxy. Although there has been some progress in the phylogeny of Chrysosplenium over the years, the phylogenetic position of some species is still controversial. In this study, we assembled chloroplast genomes (cp genomes) of 34 Chrysosplenium species and performed comparative genomic and phylogenetic analyses in combination with other cp genomes of previously known Chrysosplenium species, for a total of 44 Chrysosplenium species. The comparative analyses revealed that cp genomes of Chrysosplenium species were more conserved in terms of genome structure, gene content and arrangement, SSRs, and codon preference, but differ in genome size and SC/IR boundaries. Phylogenetic analysis showed that cp genomes effectively improved the phylogenetic support and resolution of Chrysosplenium species and strongly supported Chrysosplenium species as a monophyletic taxon and divided into three branches. The results also showed that the sections of Alternifolia and Oppositifolia were not monophyletic with each other, and that C. microspermum was not clustered with other Chrysosplenium species with alternate leaves, but with C. sedakowii into separate branches. In addition, we identified 10 mutational hotspot regions that could serve as potential DNA barcodes for Chrysosplenium species identification. In contrast to Peltoboykinia, the clpP and ycf2 genes of Chrysosplenium were subjected to positive selection and had multiple significant positive selection sites. We further detected a significant positive selection site on the petG gene between the two sections of Chrysosplenium. These evolutionary characteristics may be related to the growth environment of Chrysosplenium species. This study enriches the cp genomes of Chrysosplenium species and provides a reference for future studies on its evolution and origin.

Carthamus tinctorius L. 1753 (Asteraceae), also called safflower, is a cash crop with both edible and medical properties. We analyzed and reported the safflower mitogenome based on combined short and long reads obtained from Illumina and Pacbio platforms, respectively. This safflower mitogenome mainly contained two circular chromosomes, with a total length of 321,872 bp, and encoded 55 unique genes, including 34 protein-coding genes (PCGs), 3 rRNA genes, and 18 tRNA genes. The total length of repeat sequences greater than 30 bp was 24,953 bp, accounting for 7.75% of the whole mitogenome. Furthermore, we characterized the RNA editing sites of protein-coding genes located in the safflower mitogenome, and the total number of RNA editing sites was 504. Then, we revealed partial sequence transfer events between plastid and mitochondria, in which one plastid-derived gene (psaB) remained intact in the mitogenome. Despite extensive arrangement events among the three mitogenomes of C. tinctorius, Arctium lappa, and Saussurea costus, the constructed phylogenetic tree based on mitogenome PCGs showed that C. tinctorius has a closer relationship with three Cardueae species, A. lappa, A. tomentosum, and S. costus, which is similar to the phylogeny constructed from the PCGs of plastid genomes. This mitogenome not only enriches the genetic information of safflower but also will be useful in the phylogeny and evolution study of the Asteraceae.

The genus Carthamus Linnaeus, which belongs to the tribe Cardueae in the Asteraceae family, originated in the Mediterranean region and consists of approximately 20 species worldwide. Understanding the phylogeny of the Carthamus is crucial for the cultivation of C. tinctorius. Although chloroplast genomes are widely used for species identification and evolutionary studies, there have been limited investigations on the chloroplast genomes of Carthamus species. In this study, we assembled the chloroplast genomes of C. persicus, C. tinctorius × C. persicus, and C. lanatus and combined them with the five chloroplast genomes of C. tinctorius for comparative genomic analysis. The sizes of the chloroplast genomes of C. lanatus, C. persicus, and C. tinctorius × C. persicus were 152,602 bp, 153,177 bp, and 153,177 bp, respectively. Comparative analysis showed that the chloroplast genome structures of the four Carthamus species were highly conserved. Additionally, the phylogenomic analysis demonstrated that the plastid genome and angiosperms353 dataset significantly improved the phylogenetic support of Carthamus species. This analysis supported Carthamus as a monophyletic taxon and its internal division into the sect. Carthamus and sect. Atractylis. The Carthamus was closely related to Carduncellus, Femeniasia, Phonus, and Centaurea. In conclusion, this study not only expands our understanding of the cp genomes of Carthamus species but also provides support for more comprehensive phylogenetic studies of Carthamus.

RWP-RK is a plant-specific family of transcription factors, involved in nitrate response, gametogenesis, and nodulation. However, genome-wide characterization, phylogeny, and the regulation of RWP-RK genes in the nodulating and non-nodulating plant species of nitrogen-fixing clade (NFC) are widely unknown. Therefore, we identified a total of 292 RWP-RKs, including 278 RWP-RKs from 25 NFC species and 14 RWP-RKs from the outgroup, Arabidopsis thaliana. We classified the 292 RWP-RKs in two subfamilies: the NIN-like proteins (NLPs) and the RWP-RK domain proteins (RKDs). The transcriptome and phylogenetic analysis of RWP-RKs suggested that, compared to RKD genes, the NLP genes were just upregulated in nitrate response and nodulation. Moreover, nodule-specific NLP genes of some nodulating NFC species may have a common ancestor (OG0002084) with AtNLP genes in A. thaliana. Further, co-expression networks of A.thaliana under N-starvation and N-supplementation conditions revealed that there is a higher correlation between expression of AtNLP genes and symbiotic genes during N-starvation. In P. vulgaris, we confirmed that N-starvation stimulated nodulation by regulating expression of PvNLP2, closely related to AtNLP6 and AtNLP7 with another common origin (OG0004041). Taken together, we concluded that different origins of the NLP genes involved in both N-starvation response and specific expression of nodulation would contribute to the evolution of nodulation in NFC plant species. Our results shed light on the phylogenetic relationships of NLP genes and their differential regulation in nitrate response of A. thaliana and nodulation of NFC.

Chrysosplenium macrophyllum Oliv., belonging to the family Saxifragaceae, is a traditional and unique Chinese herbal medicine. However, the lack of adequate molecular markers has hampered the progress regarding population genetics and evolution within this species. In this research, we used the DNBSEQ-T7 Sequencer (MGI) sequencing assay to analyze the transcriptome profiles of C. macrophyllum. SSR markers were developed on the basis of transcriptomic sequences and further validated on C. macrophyllum and other Chrysosplenium species. The genetic diversity and structure of the 12 populations were analyzed by using polymorphic expressed sequence tag simple sequence repeat (EST-SSR) markers. A potential pool of 3127 non-redundant EST-SSR markers were identified for C. macrophyllum in this study. The developed EST-SSR markers had high amplification rates and cross-species transferability in Chrysosplenium. Our results also showed that the natural populations of C. macrophyllum had a high level of genetic diversity. Genetic distance, principal component analysis, and popular structure analysis revealed that all 60 samples clustered into two major groups that were consistent with their geographical origins. This study provided a batch of highly polymorphic EST-SSR molecular markers that were developed via transcriptome sequencing. These markers will be of great significance for the study of the genetic diversity and evolutionary history of C. macrophyllum and other Chrysosplenium species.

Understanding the molecular mechanisms of seed germination and seedling growth is vital for mining functional genes for the improvement of plant drought in a desert. Tamarix hispida is extremely resistant to drought and soil salinity perennial shrubs or trees. This study was the first to investigate the protein abundance profile of the transition process during the processes of T. hispida seed germination and seedling growth using label-free proteomics approaches. Our data suggested that asynchronous regulation of transcriptomics and proteomics occurs upon short-term seed germination and seedling growth of T. hispida. Enrichment analysis revealed that the main differentially abundant proteins had significant enrichment in stimulus response, biosynthesis, and metabolism. Two delta-1-pyrroline-5-carboxylate synthetases (P5CS), one Ycf3-interacting protein (Y3IP), one low-temperature-induced 65 kDa protein-like molecule, and four peroxidases (PRX) were involved in both water deprivation and hyperosmotic salinity responses. Through a comparative analysis of transcriptomics and proteomics, we found that proteomics may be better at studying short-term developmental processes. Our results support the existence of several mechanisms that enhance tolerance to salinity and drought stress during seedling growth in T. hispida.