Genomic Insights into Adaptation to Karst Limestone and Incipient Speciation in East Asian Platycarya spp. (Juglandaceae).

When challenged by similar environmental conditions, phylogenetically distant taxa often independently evolve similar traits (convergent evolution). Meanwhile, adaptation to extreme habitats might lead to divergence between taxa that are otherwise closely related. These processes have long existed in the conceptual sphere, yet molecular evidence, especially for woody perennials, is scarce. The karst endemic Platycarya longipes and its only congeneric species, Platycarya strobilacea, which is widely distributed in the mountains in East Asia, provide an ideal model for examining the molecular basis of both convergent evolution and speciation. Using chromosome-level genome assemblies of both species, and whole-genome resequencing data from 207 individuals spanning their entire distribution range, we demonstrate that P. longipes and P. strobilacea form two species-specific clades, which diverged around 2.09 million years ago. We find an excess of genomic regions exhibiting extreme interspecific differentiation, potentially due to long-term selection in P. longipes, likely contributing to the incipient speciation of the genus Platycarya. Interestingly, our results unveil underlying karst adaptation in both copies of the calcium influx channel gene TPC1 in P. longipes. TPC1 has previously been identified as a selective target in certain karst-endemic herbs, indicating a convergent adaptation to high calcium stress among karst-endemic species. Our study reveals the genic convergence of TPC1 among karst endemics and the driving forces underneath the incipient speciation of the two Platycarya lineages.

Regulation of exogenous sugars on the biosynthesis of key secondary metabolites in Cyclocarya paliurus.

The biosynthesis and accumulation of secondary metabolites play a vital role in determining the quality of medicinal plants, with carbohydrate metabolism often influencing secondary metabolism. To understand the potential regulatory mechanism, exogenous sugars (sucrose, glucose/fructose) were applied to the leaves of Cyclocarya paliurus, a highly valued and multiple function tree species. The results showed that exogenous sugars enhanced the accumulation of soluble sugar and starch while increasing the enzyme activity related to carbohydrate metabolism. In addition, the plant height was increased by a mixture of exogenous mixed sugars, the addition of sucrose promoted the net photosynthetic rate, while all types of exogenous sugars facilitated the accumulation of flavonoids and terpenoids. Based on weighted gene co-expression network analysis (WGCNA), two key gene modules and four candidate transcription factors (TFs) related to carbohydrate metabolism and secondary metabolite biosynthesis were identified. A correlation analysis between transcriptome and metabolome data showed that exogenous sugar up-regulated the expression of key structural genes in the flavonoid and terpenoid biosynthetic pathway. The expression levels of the four candidate TFs, TIFY 10A, WRKY 7, EIL 3 and RF2a, were induced by exogenous sugar and were strongly correlated with the key structural genes, which enhanced the synthesis of specific secondary metabolites and some plant hormone signal pathways. Our results provide a comprehensive understanding of key factors in the quality formation of medicinal plants and a potential approach to improve the quality.

Insights into the Hormone-Regulating Mechanism of Adventitious Root Formation in Softwood Cuttings of Cyclocarya paliurus and Optimization of the Hormone-Based Formula for Promoting Rooting.

Adventitious root (AR) formation is vital for successful cutting propagation in plants, while the dynamic regulation of phytohormones is viewed as one of the most important factors affecting AR formation. Cyclocarya paliurus, a hard-to-root plant, is faced with the bottleneck of cloning its superior varieties in practice. In this study, ten treatments were designed to figure out the best hormone-based formula for promoting AR formation in softwood cuttings and explore their hormone-regulating mechanisms. Both the rooting process and the rooting parameters of the softwood cuttings were significantly affected by different hormone-based formulas (p < 0.05), while the greatest rooting rate (93%) and root quality index were achieved in the H3 formula (SR3:IR3 = 1:1). Significant differences in the measured phytohormone concentrations, as well as in their ratios, were detected among the cuttings sampled at various AR formation stages (p < 0.05), whereas the dynamics for each phytohormone varied greatly during AR formation. The transcriptome analysis showed 12,028 differentially expressed genes (DEGs) identified during the rooting process of C. paliurus cuttings, while the KEGG enrichment analysis indicated that a total of 20 KEGG terms were significantly enriched in all the comparison samples, with 253 DEGs detected in signal transduction. Furthermore, 19 genes with vital functions in regulating the hormone signaling pathway were identified by means of a WGCNA analysis. Our results not only optimize a hormone-based formula for improving the rooting of C. paliurus cuttings but also provide an insight into the hormonal regulatory network during AR formation in softwood C. paliurus cuttings.

Molecular mechanism of thiamine in mitigating drought stress in Chinese wingnut (Pterocarya stenoptera): Insights from transcriptomics.

Urban garden plants are frequently affected by drought, which can hinder their growth, development, and greening effect. Previous studies have indicated that Chinese wingnut (Pterocarya stenoptera) responds to drought stress by increasing the expression of thiamine synthesis genes. In this study, it was found that exogenous thiamine can effectively alleviate the negative effects of drought stress on plants. Forward transcriptome sequencing and physiological tests were further conducted to reveal the molecular mechanism of thiamine in alleviating drought stress. Results showed that exogenous thiamine activated the expression of eight chlorophyll synthesis genes in Chinese wingnut under drought stress. Moreover, physiological indicators proved that chlorophyll content increased in leaves of Chinese wingnut with thiamine treatment under drought stress. Photosynthesis genes were also activated in Chinese wingnut treated with exogenous thiamine under drought stress, as supported by photosynthetic indicators PIabs and PItotal. Additionally, exogenous thiamine stimulated the expression of genes in the auxin-activated signaling pathway, thus attenuating the effects of drought stress. This study demonstrates the molecular mechanism of thiamine in mitigating the effects of drought stress on non-model woody plants lacking transgenic systems. This study also provides an effective method to mitigate the negative impacts of drought stress on plants.

Metabolome and Transcriptome Analyses Unravel the Molecular Regulatory Mechanisms Involved in Photosynthesis of Cyclocarya paliurus under Salt Stress.

Photosynthesis is the primary life process in nature, and how to improve photosynthetic capacity under abiotic stresses is crucial to carbon fixation and plant productivity. As a multi-functional tree species, the leaves of Cyclocarya paliurus possess antihypertensive and hypoglycemic activities. However, the regulatory mechanism involved in the photosynthetic process of C. paliurus exposed to salinity has not yet been elucidated. In this study, the photosynthetic characteristics of C. paliurus seedlings, such as photosynthetic rate (Pn), stomatal conductance (Gs), and electron transfer rate (ETR), were investigated under different salt concentrations, while the metabolome and transcriptome analyses were conducted to unravel its molecular regulatory mechanisms. Salt stress not only significantly affected photosynthetic characteristics of C. paliurus seedlings, but also severely modified the abundance of metabolites (such as fumaric acid, sedoheptulose-7-phosphate, d-fructose-1,6-bisphosphate, and 3-phospho-d-glyceroyl phosphate) involved in central carbon metabolism, and the expression of photosynthetic genes. Through the co-expression network analysis, a total of 27 transcription factors (including ERFs, IDD, DOF, MYB, RAP) were identified to regulate photosynthetic genes under salt stress. Our findings preliminarily clarify the molecular regulatory network involved in the photosynthetic process of C. paliurus under salt stress and would drive progress in improving the photosynthetic capacity and productivity of C. paliurus by molecular technology.

Transcriptome Analysis Reveals the Role of GA3 in Regulating the Asynchronism of Floral Bud Differentiation and Development in Heterodichogamous Cyclocarya paliurus (Batal.) Iljinskaja.

Cyclocarya paliurus is an important medical plant owing to the diverse bioactive compounds in its leaves. However, the heterodichogamy with female and male functions segregation within protandry (PA) or protogyny (PG) may greatly affect seed quality and its plantations for medicinal use. To speculate on the factor playing the dominant role in regulating heterodichogamy in C. paliurus, based on phenotypic observations, our study performed a multi comparison transcriptome analysis on female and male buds (PG and PA types) using RNA-seq. For the female and male bud comparisons, a total of 6753 differentially expressed genes (DEGs) were detected. In addition, functional analysis revealed that these DEGs were significantly enriched in floral development, hormone, and GA-related pathways. As the dominant hormones responsible for floral differentiation and development, gibberellins (GAs) in floral buds from PG and PA types were quantified using HPLC-MS. Among the tested GAs, GA3 positively regulated the physiological differentiation (S0) and germination (S2) of floral buds. The dynamic changes of GA3 content and floral morphological features were consistent with the expression levels of GA-related genes. Divergences of GA3 contents at S0 triggered the asynchronism of physiological differentiation between male and female buds of intramorphs (PA-M vs. PA-F and PG-F vs. PG-M). A significant difference in GA3 content enlarged this asynchronism at S2. Thus, we speculate that GA3 plays the dominant role in the formation of heterodichogamy in C. paliurus. Meanwhile, the expression patterns of GA-related DEGs, including CPS, KO, GA20ox, GA2OX, GID1, and DELLA genes, which play central roles in regulating flower development, coincided with heterodichogamous characteristics. These results support our speculations well, which should be further confirmed.

Comprehensive Identification and Analyses of the GRF Gene Family in the Whole-Genome of Four Juglandaceae Species.

The GRF gene family plays an important role in plant growth and development as regulators involved in plant hormone signaling and metabolism. However, the Juglandaceae GRF gene family remains to be studied. Here, we identified 15, 15, 19, and 20 GRF genes in J. regia, C. illinoinensis, J. sigillata, and J. mandshurica, respectively. The phylogeny shows that the Juglandaceae family GRF is divided into two subfamilies, the ε-group and the non-ε-group, and that selection pressure analysis did not detect amino acid loci subject to positive selection pressure. In addition, we found that the duplications of the Juglandaceae family GRF genes were all segmental duplication events, and a total of 79 orthologous gene pairs and one paralogous homologous gene pair were identified in four Juglandaceae families. The Ka/KS ratios between these homologous gene pairs were further analyzed, and the Ka/KS values were all less than 1, indicating that purifying selection plays an important role in the evolution of the Juglandaceae family GRF genes. The codon bias of genes in the GRF family of Juglandaceae species is weak, and is affected by both natural selection pressure and base mutation, and translation selection plays a dominant role in the mutation pressure in codon usage. Finally, expression analysis showed that GRF genes play important roles in pecan embryo development and walnut male and female flower bud development, but with different expression patterns. In conclusion, this study will serve as a rich genetic resource for exploring the molecular mechanisms of flower bud differentiation and embryo development in Juglandaceae. In addition, this is the first study to report the GRF gene family in the Juglandaceae family; therefore, our study will provide guidance for future comparative and functional genomic studies of the GRF gene family in the Juglandaceae specie.

Identification and Expression Analysis of R2R3-MYB Family Genes Associated with Salt Tolerance in Cyclocarya paliurus.

R2R3-MYB transcription factors are most abundant in the MYB superfamily, while the R2R3-MYB genes play an important role in plant growth and development, especially in response to environmental stress. Cyclocarya paliurus is a multifunction tree species, and the existing resources cannot meet the requirement for its leaf production and medical use. Therefore, lands with some environmental stresses would be potential sites for developing C. paliurus plantations. However, the function of R2R3-MYB genes in C.paliurus in response to environmental stress remains unknown. In this study, to identify the roles of R2R3-MYB genes associated with salt stress response, 153 CpaMYB genes and their corresponding protein sequences were identified from the full-length transcriptome. Based on the comparison with MYB protein sequences of Arabidopsis thaliana, 69 R2R3-MYB proteins in C. paliurus were extracted for further screening combined with conserved functional domains. Furthermore, the MYB family members were analyzed from the aspects of protein sequences alignment, evolution, motif prediction, promoter cis-acting element analysis, and gene differential expression under different salt treatments using both a pot experiment and hydroponic experiment. The results showed that the R2R3-MYB genes of C.paliurus conserved functional domains, whereas four R2R3-MYB genes that might respond to salt stress via regulating plant hormone signals were identified in this study. This work provides a basis for further functional characterization of R2R3-MYB TFs in C. paliurus.

Transcriptome analysis of Cyclocarya paliurus flavonoids regulation of differently expressed genes in Enterococcus faecalis under low pH stress.

Enterococcus faecalis (E. faecalis) is an indigenous intestinal bacterium and has potential to be applied as probiotic supplement. Low pH is one of the main stresses that E. faecalis has to deal with to colonize in the gastrointestinal tract. Previous study indicated low concentration of flavonoids may enhance the tolerance of probiotic to environmental stress. In the present research, transcriptome analysis was employed to investigate the influence of Cyclocarya paliurus flavonoids (CPF) on E. faecalis exposed to low pH environment. The results revealed that under the stress of low pH, genes related to cell wall and membrane, transmembrane transport, metabolism process, energy production, and conversion stress proteins were significantly differentially expressed. And certain undesired changes of which (such as genes for MFS transporter were downregulated) could be partially mitigated by CPF intervention, indicating their capacity to improve the low pH tolerance of E. faecalis. Results from this study deepened our understanding of the beneficial role of CPF on the probiotic in the gastrointestinal environment.

Phylogeny and divergence time estimation of the walnut family (Juglandaceae) based on nuclear RAD-Seq and chloroplast genome data.

The walnut family Juglandaceae was widely distributed in the Northern Hemisphere while several extant genera now exhibit intercontinental disjunctions. Recent progress in the systematics of Juglandaceae has greatly broadened our knowledge about its origin and evolution. However, there are still uncertainties about the intergeneric relationships within Juglandaceae, and discrepancies between fossil records and inferred divergence times for certain lineages were observed. In this study, well-resolved phylogenies of the Juglandaceae are reconstructed based on both the nuclear RAD-Seq and the whole chloroplast genome data. Our results support the Juglandoideae topology of (Hicoreae, (Platycaryeae, Juglandeae)) at the tribal level. Within Juglandeae, a discordant position of Pterocarya was detected between nuclear and plastid genome data, and a more likely topology (nuclear), (Juglans, (Pterocarya, Cyclocarya)), was discussed based on evidence from molecular data and fossil records. Based on carefully selected fossil calibrations, the divergence times of extant lineages were estimated and they corroborated well with fossil records (especially concerning Juglans and Pterocarya). Four sections within Juglans were strongly supported by the nuclear data. Within Juglans, the incongruent position of J. hopeiensis was recovered between the nuclear and plastid genomes. Yet the origin and evolutionary history of J. cinerea and J. hopeiensis are supported to be complicated and need further clarification. Integrative evidence from the fossil records, phylogeny and lineage divergence times shows that Juglandoideae originated in North America, and migrated to Eurasia via both the Bering and the North Atlantic land bridges. Our study shows the potential of integrative biogeographic studies for illuminating the evolutionary history of Juglandaceae.

Shining a light on species delimitation in the tree genus Engelhardia Leschenault ex Blume (Juglandaceae).

Enhanced efficacy in species delimitation is critically important in biology given the pending biodiversity crisis under global warming and anthropogenic activity. In particular, delineation of traditional classifications in view of the complexity of species requires an integrative approach to effectively define species boundaries, and this is a major focus of systematic biology. Here, we explored species delimitation of Engelhardia in tropical and subtropical Asia. In total, 716 individuals in 71 populations were genotyped using five chloroplast regions, one nuclear DNA region (nrITS), and 11 nuclear simple sequence repeats (nSSR). Phylogenetic trees were constructed and relationships among species were assessed. Molecular analyses were then combined with 14 morphological characteristics of 720 specimens to further explore the species boundaries of Engelhardia. Integrating phylogenetic and morphological clusters provided well-resolved relationships to delineate seven species. The results suggested that: first, that E. fenzelii, E. roxburghiana, E. hainanensis, E. apoensis, and E. serrata are distinct species; second, E. spicata var. spicata, E. spicata var. aceriflora, E. spicata var. colebrookeana, and E. rigida should be combined under E. spicata and treated as a species complex; third, E. serrata var. cambodica should be raised to species level and named E. villosa. We illuminated that bias thresholds determining the cluster number for delimiting species boundaries were substantially reduced when morphological data were incorporated. Our results urge caution when using the concepts of subspecies and varieties in order to prevent confusion, particularly with respect to species delimitation for tropical and subtropical species. In some cases, re-ranking or combining subspecies and/or varieties may enable more accurate species delimitation.

Combined Analysis of the Metabolome and Transcriptome Identified Candidate Genes Involved in Phenolic Acid Biosynthesis in the Leaves of Cyclocarya paliurus.

To assess changes of metabolite content and regulation mechanism of the phenolic acid biosynthesis pathway at different developmental stages of leaves, this study performed a combined metabolome and transcriptome analysis of Cyclocarya paliurus leaves at different developmental stages. Metabolite and transcript profiling were conducted by ultra-performance liquid chromatography quadrupole time-of-flight tandem mass spectrometer and high-throughput RNA sequencing, respectively. Transcriptome identification showed that 58 genes were involved in the biosynthesis of phenolic acid. Among them, 10 differentially expressed genes were detected between every two developmental stages. Identification and quantification of metabolites indicated that 14 metabolites were located in the phenolic acid biosynthetic pathway. Among them, eight differentially accumulated metabolites were detected between every two developmental stages. Association analysis between metabolome and transcriptome showed that six differentially expressed structural genes were significantly positively correlated with metabolite accumulation and showed similar expression trends. A total of 128 transcription factors were identified that may be involved in the regulation of phenolic acid biosynthesis; these include 12 MYBs and 10 basic helix-loop-helix (bHLH) transcription factors. A regulatory network of the phenolic acid biosynthesis was established to visualize differentially expressed candidate genes that are involved in the accumulation of metabolites with significant differences. The results of this study contribute to the further understanding of phenolic acid biosynthesis during the development of leaves of C. paliurus.

Selection pressure causes differentiation of the SPL gene family in the Juglandaceae.

The SQUAMOSA promoter-binding protein-like (SPL) is a plant-specific transcription factor that influences flowering and vegetative development. Although the SPL genes have been functionally analyzed in many species, studies on the evolutionary history of the whole gene family, and in the Juglandaceae specifically, have been limited. Here, we conducted a phylogenetic relationship analysis of the Juglandaceae SPL gene family compared with other land plant species. Our results showed that the SPL genes were divided into three major clades, all of which were further divided into ten small clades. Selection pressure analysis suggested that all SPL genes were exposed to purifying selection pressure during evolution. The purifying selection was smaller for the Juglandaceae SPL genes than for other angiosperms, indicating a greater susceptibility to functional differentiation in the Juglandaceae. The SPL proteins encoded by Clade 1 contained a branch-specific transmembrane structure and many conserved motif combinations at the C-terminal. We also detected many selection sites in these motif combinations. Expression analysis showed that Clade 1 genes had spatial and temporal differences and were highly expressed in various organs. The expression profile was closely related to the selection sites and motif combinations at the C-terminal. These observations represent essential entry points for revealing the functional differentiation of the SPL gene family. Our data presented here may provide a basis for future investigations of SPL genes in the Juglandaceae, especially for flower development and perhaps crop yield improvement.

Adaptive genetic differentiation in Pterocarya stenoptera (Juglandaceae) driven by multiple environmental variables were revealed by landscape genomics.

BACKGROUND: The investigation of the genetic basis of local adaptation in non-model species is an interesting focus of evolutionary biologists and molecular ecologists. Identifying these adaptive genetic variabilities on the genome responsible can provide insight into the genetic mechanism of local adaptation. RESULTS: We investigated the spatial distribution of genetic variation in 22 natural populations of Pterocarya stenoptera across its distribution area in China to provide insights into the complex interplay between multiple environmental variables and adaptive genetic differentiation. The Bayesian analysis of population structure showed that the 22 populations of P. stenoptera were subdivided into two groups. Redundancy analysis demonstrated that this genetic differentiation was caused by the divergent selection of environmental difference. A total of 44 outlier loci were mutually identified by Arlequin and BayeScan, 43 of which were environment-associated loci (EAL). The results of latent factor mixed model analysis showed that solar radiation in June (Sr6), minimum temperature of the coldest month (Bio6), temperature seasonality (Bio4), and water vapor pressure in January (Wvp1) were associated with the highest numbers of EAL. Sr6 was associated with the ecological habitat of "prefered light", and Bio6 and Wvp1 were associated with the ecological habitat of "warm and humid environment". CONCLUSIONS: Our results provided empirical evidence that environmental variables related to the ecological habitats of species play key roles in driving adaptive differentiation of species genome.

Chromosome-level genome assembly of Platycarya strobilacea.

Platycarya strobilacea belongs to the walnut family (Juglandaceae), is commonly known as species endemic to East Asia, and is an ecologically important, wind pollinated, woody deciduous tree. To facilitate this ancient tree for the ecological value and conservation of this ancient tree, we report a new high-quality genome assembly of P. strobilacea. The genome size was 677.30 Mb, with a scaffold N50 size of 45,791,698 bp, and 98.43% of the assembly was anchored to 15 chromosomes. We annotated 32,246 protein-coding genes in the genome, of which 96.30% were functionally annotated in six databases. This new high-quality assembly of P. strobilacea provide valuable resource for the phylogenetic and evolutionary analysis of the walnut family and angiosperm.

The impact of genetic similarity and environment on the flavonoids variation pattern of Cyclocarya paliurus.

The leaves of Cyclocarya paliurus (Batalin) Iljinskaja, an endemic tree with a scattered distribution in subtropical China, are rich in flavonoids with beneficial, health-promoting properties. To understand the impact of environment and genetic similarity on the variation pattern of flavonoids in this species, we analyzed C. paliurus germplasm resources from 26 different populations previously sampled from the main distribution area. Environmental, genetic and biochemical data was associated by genetic structure analysis, non-parametric tests, correlation analysis and principal component analysis. We found that populations with higher flavonoid contents were distributed at higher elevations and latitudes and fell into two groups with similar genetic diversities. Significant accumulations of isoquercitrin and kaempferol 3-O-glucoside were detected in the higher flavonoid-content resources. In addition, the genetic clusters with higher flavonoid contents exhibited broader environmental-adaptive capacities. Even in the presence of environmental factors promoting C. paliurus flavonoid accumulation, only those populations having a specific level of genetic similarity were able to exploit such environments.

Conservation genetics of Annamocarya sinensis (Dode) Leroy, an endangered endemic species.

The endangered perennial plant Annamocarya sinensis (Dode) Leroy is a tertiary relict tree restricted to southeastern China and northern Vietnam. To explore endangerment mechanisms, develop protection strategies, and guide reintroduction efforts for this species, we investigated genetic diversity and population structure by surveying 70 individuals from three distinct populations using 12 polymorphic microsatellite markers. We found high genetic diversity for A. sinensis as indicated by high allelic diversity (allelic number = 4.667 ± 0.436, effective number of alleles = 2.913 ± 0.249), excess heterozygosity (observed heterozygosity = 0.586 ± 0.039, expected heterozygosity = 0.582 ± 0.029), and low fixation index (-0.028 ± 0.057). Our research revealed low genetic differentiation (FST = 0.066 ± 0.011) and no correlation between genetic distance and geographic distance. Analysis of molecular variance attributed 87% of the variance to differences within the population, whereas 13% was distributed among populations. The protection strategy should aim to protect as many populations as possible. Promoting sexual reproduction among various genotypes and establishing an outcrossing program are advisable for A. sinensis.

Squalene epoxidase (SE) gene related to triterpenoid biosynthesis assists to select elite genotypes in medicinal plant: Cyclocarya paliurus (Batal.) Iljinskaja.

Triterpenoids, known for their anti-inflammatory, anticancer, and hypoglycemic properties, are the major bioactive components in Cyclocarya paliurus (Batal.) Iljinskaja. Selecting elite individuals with high triterpenoids content is the basis of C. paliurus industry for medicinal use. In this study, seasonal variation patterns of total triterpenoids and five triterpene monomers accumulation for three groups with different total triterpenoid contents (TTC; H: 59.74-64.03 mg g-1; M: 47.66-57.08 mg g-1, and L: 35.26-42.22 mg g-1) were surveyed. Seasonal expression dynamics of 6 key genes relevant to triterpenoids biosynthesis, including HMGR, DXR, SQS, SE, LUS, and ß-AS, were described by quantitative real-time PCR (qRT-PCR) for three groups. The expression levels of HMGR, SE, LUS, and ß-AS genes in group H were higher than in groups M and L. In addition, Pearson correlation analysis showed that they were significantly positively correlated with triterpene accumulation, and the expression level of SE gene not only was significantly correlated with downstream genes, but also exhibited a linear relationship with TTC, especially in September. These results suggest that SE gene could serve as an effective make for screening elite individuals with high TTC from the germplasm of C. paliurus for medicinal use. Further testing on randomly selected individuals in next September proved the feasibility and reliability of SE gene in assisted selection. Also, we successfully cloned the full-length cDNA of SE. Thus, our work provides an efficient way to attain superior genotypes to develop medicinal industry of C. paliurus in practice.

Genome assembly of two diploid and one auto-tetraploid Cyclocarya paliurus genomes.

Cyclocarya paliurus, an endemic species in the genus Juglandaceae with the character of heterodichogamy, is one of triterpene-rich medicinal plants in China. To uncover the genetic mechanisms behind the special characteristics, we sequenced the genomes of two diploid (protandry, PA-dip and protogyny, PG-dip) and one auto-tetraploid (PA-tetra) C. paliurus genomes. Based on 134.9 (~225x), 75.5 (~125x) and 271.8 Gb (~226x) subreads of PacBio platform sequencing data, we assembled 586.62 Mb (contig N50 = 1.9 Mb), 583.45 Mb (contig N50 = 1.4 Mb), and 2.38 Gb (contig N50 = 430.9 kb) for PA-dip, PG-dip and PA-tetra genome, respectively. Furthermore, 543.53, 553.87, and 2168.65 Mb in PA-dip, PG-dip, and PA-tetra, were respectively anchored to 16, 16, and 64 pseudo-chromosomes using over 65.4 Gb (~109x), 68 Gb (~113x), and 264 (~220x) Hi-C sequencing data. Annotation of PA-dip, PG-dip, and PA-tetra genome assembly identified 34,699, 35,221, and 34,633 protein-coding genes (90,752 gene models) or allele-defined genes, respectively. In addition, 45 accessions from nine locations were re-sequenced, and more than 10 × coverage reads were generated.

Genomic Analysis of Plastid-Nuclear Interactions and Differential Evolution Rates in Coevolved Genes across Juglandaceae Species.

The interaction between the nuclear and chloroplast genomes in plants is crucial for preserving essential cellular functions in the face of varying rates of mutation, levels of selection, and modes of transmission. Despite this, identifying nuclear genes that coevolve with chloroplast genomes at a genome-wide level has remained a challenge. In this study, we conducted an evolutionary rate covariation analysis to identify candidate nuclear genes coevolving with chloroplast genomes in Juglandaceae. Our analysis was based on 4,894 orthologous nuclear genes and 76 genes across seven chloroplast partitions in nine Juglandaceae species. Our results indicated that 1,369 (27.97%) of the nuclear genes demonstrated signatures of coevolution, with the Ycf1/2 partition yielding the largest number of hits (765) and the ClpP1 partition yielding the fewest (13). These hits were found to be significantly enriched in biological processes related to leaf development, photoperiodism, and response to abiotic stress. Among the seven partitions, AccD, ClpP1, MatK, and RNA polymerase partitions and their respective hits exhibited a narrow range, characterized by dN/dS values below 1. In contrast, the Ribosomal, Photosynthesis, Ycf1/2 partitions and their corresponding hits, displayed a broader range of dN/dS values, with certain values exceeding 1. Our findings highlight the differences in the number of candidate nuclear genes coevolving with the seven chloroplast partitions in Juglandaceae species and the correlation between the evolution rates of these genes and their corresponding chloroplast partitions.