Dynamic changes in the plastid and mitochondrial genomes of the angiosperm Corydalis pauciovulata (Papaveraceae).

BACKGROUND: Corydalis DC., the largest genus in the family Papaveraceae, comprises > 465 species. Complete plastid genomes (plastomes) of Corydalis show evolutionary changes, including syntenic arrangements, gene losses and duplications, and IR boundary shifts. However, little is known about the evolution of the mitochondrial genome (mitogenome) in Corydalis. Both the organelle genomes and transcriptomes are needed to better understand the relationships between the patterns of evolution in mitochondrial and plastid genomes. RESULTS: We obtained complete plastid and mitochondrial genomes from Corydalis pauciovulata using a hybrid assembly of Illumina and Oxford Nanopore Technologies reads to assess the evolutionary parallels between the organelle genomes. The mitogenome and plastome of C. pauciovulata had sizes of 675,483 bp and 185,814 bp, respectively. Three ancestral gene clusters were missing from the mitogenome, and expanded IR (46,060 bp) and miniaturized SSC (202 bp) regions were identified in the plastome. The mitogenome and plastome of C. pauciovulata contained 41 and 67 protein-coding genes, respectively; the loss of genes was a plastid-specific event. We also generated a draft genome and transcriptome for C. pauciovulata. A combination of genomic and transcriptomic data supported the functional replacement of acetyl-CoA carboxylase subunit ß (accD) by intracellular transfer to the nucleus in C. pauciovulata. In contrast, our analyses suggested a concurrent loss of the NADH-plastoquinone oxidoreductase (ndh) complex in both the nuclear and plastid genomes. Finally, we performed genomic and transcriptomic analyses to characterize DNA replication, recombination, and repair (DNA-RRR) genes in C. pauciovulata as well as the transcriptomes of Liriodendron tulipifera and Nelumbo nuicifera. We obtained 25 DNA-RRR genes and identified their structure in C. pauciovulata. Pairwise comparisons of nonsynonymous (dN) and synonymous (dS) substitution rates revealed that several DNA-RRR genes in C. pauciovulata have higher dN and dS values than those in N. nuicifera. CONCLUSIONS: The C. pauciovulata genomic data generated here provide a valuable resource for understanding the evolution of Corydalis organelle genomes. The first mitogenome of Papaveraceae provides an example that can be explored by other researchers sequencing the mitogenomes of related plants. Our results also provide fundamental information about DNA-RRR genes in Corydalis and their related rate variation, which elucidates the relationships between DNA-RRR genes and organelle genome stability.

Characterization of CYP82 genes involved in the biosynthesis of structurally diverse benzylisoquinoline alkaloids in Corydalis yanhusuo.

Benzylisoquinoline alkaloids (BIAs) represent a significant class of secondary metabolites with crucial roles in plant physiology and substantial potential for clinical applications. CYP82 genes are involved in the formation and modification of various BIA skeletons, contributing to the structural diversity of compounds. In this study, Corydalis yanhusuo, a traditional Chinese medicine rich in BIAs, was investigated to identify the catalytic function of CYP82s during BIA formation. Specifically, 20 CyCYP82-encoding genes were cloned, and their functions were identified in vitro. Ten of these CyCYP82s were observed to catalyze hydroxylation, leading to the formation of protopine and benzophenanthridine scaffolds. Furthermore, the correlation between BIA accumulation and the expression of CyCYP82s in different tissues of C. yanhusuo was assessed their. The identification and characterization of CyCYP82s provide novel genetic elements that can advance the synthetic biology of BIA compounds such as protopine and benzophenanthridine, and offer insights into the biosynthesis of BIAs with diverse structures in C. yanhusuo.

Comparative analysis of the complete chloroplast genome of Papaveraceae to identify rearrangements within the Corydalis chloroplast genome.

Chloroplast genomes are valuable for inferring evolutionary relationships. We report the complete chloroplast genomes of 36 Corydalis spp. and one Fumaria species. We compared these genomes with 22 other taxa and investigated the genome structure, gene content, and evolutionary dynamics of the chloroplast genomes of 58 species, explored the structure, size, repeat sequences, and divergent hotspots of these genomes, conducted phylogenetic analysis, and identified nine types of chloroplast genome structures among Corydalis spp. The ndh gene family suffered inversion and rearrangement or was lost or pseudogenized throughout the chloroplast genomes of various Corydalis species. Analysis of five protein-coding genes revealed simple sequence repeats and repetitive sequences that can be potential molecular markers for species identification. Phylogenetic analysis revealed three subgenera in Corydalis. Subgenera Cremnocapnos and Sophorocapnos represented the Type 2 and 3 genome structures, respectively. Subgenus Corydalis included all types except type 3, suggesting that chloroplast genome structural diversity increased during its differentiation. Despite the explosive diversification of this subgenus, most endemic species collected from the Korean Peninsula shared only one type of genome structure, suggesting recent divergence. These findings will greatly improve our understanding of the chloroplast genome of Corydalis and may help develop effective molecular markers.

An updated classification for the hyper-diverse genus Corydalis (Papaveraceae: Fumarioideae) based on phylogenomic and morphological evidence.

The genus Corydalis, with ca. 530 species, has long been considered taxonomically challenging because of its great variability. Previous molecular analyses, based on a few molecular markers and incomplete taxonomic sampling, were clearly inadequate to delimit sections and subgenera. We have performed phylogenetic analyses of Corydalis and related taxa, using 65 shared protein-coding plastid genes from 313 accessions (including 280 samples of ca. 226 species of Corydalis) and 152 universal low-copy nuclear genes from 296 accessions (including 271 samples of Corydalis) covering all 42 previously recognized sections and five independent "series". Phylogenetic trees were inferred using Bayesian Inference and Maximum Likelihood. Eight selected morphological characters were estimated using ancestral state reconstructions. Results include: (i) of the three subgenera of Corydalis, two are fully supported by both the plastid and nuclear data; the third, subg. Cremnocapnos, is weakly supported by plastid DNA only, whereas in the nuclear data the two included sections form successive outgroups to the rest of the genus; (ii) among all 42 sections and five "series", 25 sections and one "series" are resolved as monophyletic in both data sets; (iii) the common ancestor of Corydalis is likely to be a perennial plant with a taproot, yellow flowers with a short saccate spur, linear fruits with recurved fruiting pedicels, and seeds with elaiosomes; (iv) we provide a new classification of Corydalis with four subgenera (of which subg. Bipapillatae is here newly described), 39 sections, 16 of which are consistent with the previous classification, 16 sections have been recircumscribed, one section has been reinstated and six new sections are established. Characters associated with lifespan, underground structures, floral spur, fruit and elaiosomes are important for the recognition of subgenera and sections. These new phylogenetic analyses combined with ancestral character reconstructions uncovered previously unrecognized relationships, and greatly improved our understanding of the evolution of the genus.

Phylogenetic analysis based on single-copy orthologous proteins in highly variable chloroplast genomes of Corydalis.

Corydalis is one of the few lineages that have been reported to have extensive large-scale chloroplast genome (cp-genome) rearrangements. In this study, novel cp-genome rearrangements of Corydalis pinnata, C. mucronate, and C. sheareri are described. C. pinnata is a narrow endemic species only distributed at Qingcheng Mountain in southwest China. Two independent relocations of the same four genes (trnM-CAU-rbcL) were found relocated from the typically posterior part of the large single-copy region to the front of it. A uniform inversion of an 11-14-kb segment (ndhB-trnR-ACG) was found in the inverted repeat region; and extensive losses of accD, clpP, and trnV-UAC genes were detected in all cp-genomes of all three species of Corydalis. In addition, a phylogenetic tree was reconstructed based on 31 single-copy orthologous proteins in 27 cp-genomes. This study provides insights into the evolution of cp-genomes throughout the genus Corydalis and also provides a reference for further studies on the taxonomy, identification, phylogeny, and genetic transformation of other lineages with extensive rearrangements in cp-genomes.

The genome of Corydalis reveals the evolution of benzylisoquinoline alkaloid biosynthesis in Ranunculales.

Species belonging to the order Ranunculales have attracted much attention because of their phylogenetic position as a sister group to all other eudicot lineages and their ability to produce unique yet diverse benzylisoquinoline alkaloids (BIAs). The Papaveraceae family in Ranunculales is often used as a model system for studying BIA biosynthesis. Here, we report the chromosome-level genome assembly of Corydalis tomentella, a species of Fumarioideae, one of the two subfamilies of Papaveraceae. Based on comparisons of sequenced Ranunculalean species, we present clear evidence of a shared whole-genome duplication (WGD) event that has occurred before the divergence of Ranunculales but after its divergence from other eudicot lineages. The C. tomentella genome enabled us to integrate isotopic labeling and comparative genomics to reconstruct the BIA biosynthetic pathway for both sanguinarine biosynthesis shared by papaveraceous species and the cavidine biosynthesis that is specific to Corydalis. Also, our comparative analysis revealed that gene duplications, especially tandem gene duplications, underlie the diversification of BIA biosynthetic pathways in Ranunculales. In particular, tandemly duplicated berberine bridge enzyme-like genes appear to be involved in cavidine biosynthesis. In conclusion, our study of the C. tomentella genome provides important insights into the occurrence of WGDs during the early evolution of eudicots, as well as into the evolution of BIA biosynthesis in Ranunculales.

Characterization and expression of a novel thaumatin-like protein (CcTLP1) from papaveraceous plant Corydalis cava.

Thaumatin-like proteins (TLPs, osmotins) form a protein family which shares a significant sequence homology to the sweet-tasting thaumatin from the plant Thaumatococcus daniellii. TLPs are not sweet-tasting and are involved in response to biotic stresses and developmental processes. Recently it has been shown using a proteomic approach that the tuber extract from Corydalis cava (Papaveraceae) contains a TLP protein. The aim of this work was to characterize the structure and expression of TLP from C. cava tubers. The results obtained using a PCR approach with degenerate primers demonstrated a coding sequence of a novel protein, named CcTLP1. It consists of 225 aa, has a predicted molecular weight of 24.2 kDa (NCBI GenBank accession no. KJ513303) and has 16 strictly conserved cysteine residues, which form 8 disulfide bridges and stabilize the 3D structure. CcTLP1 may be classified into class IX of plant TLPs. The highest CcTLP1 expression levels were shown by qPCR in the stem of the plant compared to other organs and in the medium-size plants compared to other growth phases. The results confirm that CcTLP1 is expressed during plant growth and development until flowering, with a possible defensive function against different stress conditions.

Quality Evaluation of Corydalis yanhusuo by High-Performance Liquid Chromatography Fingerprinting Coupled with Multicomponent Quantitative Analysis.

Corydalis Rhizoma is the tuber of Corydalis yanhusuo W. T. Wang, which has been long used in traditional Chinese medicine. Herein, the quality of C. yanhusuo samples collected from 23 regions of three provinces in China is evaluated through high-performance liquid chromatography fingerprinting coupled with similarity, hierarchical clustering, and principal component analyses. Sample similarities are evaluated according to the State Food and Drug Administration requirements by selection of 18 characteristic chromatographic fingerprint peaks and are found to vary between 0.455 and 0.999. Moreover, common patterns of a typical local variety of C. yanhusuo sourced in the Panan County are established. The obtained results show that the combination of quantitative analysis and chromatographic fingerprint analysis can be readily utilized for quality control purposes, offering a comprehensive strategy for quality evaluation of C. yanhusuo and related products.

Identification and Selection of Reference Genes for Quantitative Transcript Analysis in Corydalis yanhusuo.

Key word: qPCR; Corydalisyanhusuo; reference genes; geNorm; NormFinder; BestKeeper.

[Identification of Corydalis yanhusuo,C. turtschaninovii,C. decumbens by allele-specific PCR].

To establish a DNA molecular markers method for identification of Corydalis yanhusuo,C. turtschaninovii and C. decumbens,the mat K,trn G and psb A-trn H sequences of 56 samples from 14 species of C. yanhusuo,C. turtschaninovii,C. decumbens and their related species were obtained by sequencing. The SNP loci were obtained by Bio Edit 7. 2. 2 software. The primers for AS-PCR identification were designed based on the mutation sites,and the conditions of PCR were optimized to identify C. yanhusuo,C. turtschaninovii,and C. decumbens according to the specific bands. The results showed that the amount of template( 0. 6-1 200 ng)and annealing temperature( 42-60 ℃) had little influence on the amplification results,and the number of cycles had much influence on the amplification results. When the number of cycles was 20,the specific bands of 297 bp( mat K),353 bp( trn G) and 544 bp( mat K) were amplified from C. yanhusuo,C. turtschaninovii and C. decumbens,respectively. The method established in this study had a minimum detection limit of 6 ng for C. yanhusuo,60 ng for C. decumbens and less than 0. 6 ng for C. turtschaninovii. Thus,the allelespecific PCR method established in the research can specifically identify C. yanhusuo,C. turtschaninovii,and C. decumbens.

Identification and developmental expression profiling of putative alkaloid biosynthetic genes in Corydalis yanhusuo bulbs.

Alkaloids in bulbs of Corydalis (C.) yanhusuo are the major pharmacologically active compounds in treatment of blood vessel diseases, tumors and various pains. However, due to the absence of gene sequences in C. yanhusuo, the genes involved in alkaloid biosynthesis and their expression during bulb development remain unknown. We therefore established the first transcriptome database of C. yanhusuo via Illumina mRNA-Sequencing of a RNA composite sample collected at Bulb initiation (Day 0), early enlargement (Day 10) and maturation (Day 30). 25,013,630 clean 90 bp paired-end reads were de novo assembled into 47,081 unigenes with an average length of 489 bp, among which 30,868 unigenes (65.56%) were annotated in four protein databases. Of 526 putative unigenes involved in biosynthesis o f various alkaloids, 187 were identified as the candidate genes involved in the biosynthesis of benzylisoquinoline alkaloids (BIAs), the only alkaloid type reported in C. yanhusuo untill now. BIAs biosynthetic genes were highly upregulated in the overall pathway during bulb development. Identification of alkaloid biosynthetic genes in C. yanhusuo provide insights on pathways and molecular regulation of alkaloid biosynthesis, to initiate metabolic engineering in order to improve the yield of interesting alkaloids and to identify potentially new alkaloids predicted from the transcriptomic information.

[Identification and analysis of Corydalis boweri, Meconopsis horridula and their close related species of the same genus by using ITS2 DNA barcode].

The study is aimed to ensure the quality and safety of medicinal plants by using ITS2 DNA barcode technology to identify Corydalis boweri, Meconopsis horridula and their close related species. The DNA of 13 herb samples including C. boweri and M. horridula from Lhasa of Tibet was extracted, ITS PCR were amplified and sequenced. Both assembled and web downloaded 71 ITS2 sequences were removed of 5. 8S and 28S. Multiple sequence alignment was completed and the intraspecific and interspecific genetic distances were calculated by MEGA 5.0, while the neighbor-joining phylogenetic trees were constructed. We also predicted the ITS2 secondary structure of C. boweri, M. horridula and their close related species. The results showed that ITS2 as DNA barcode was able to identify C. boweri, M. horridula as well as well as their close related species effectively. The established based on ITS2 barcode method provides the regular and safe detection technology for identification of C. boweri, M. horridula and their close related species, adulterants and counterfeits, in order to ensure their quality control, safe medication, reasonable development and utilization.

Genetic variation in wild and cultivated rhizoma corydalis revealed by ISSRs markers.

Rhizoma Corydalis is an important Chinese medicinal herb. In this paper, we employed ISSR data to explore the genetic variation in domesticated populations and wild populations of the species. The average of within-population ISSR diversity in cultivated populations (PPF=25.32%, Hpop=0.094) was lower than that in wild populations (PPF=47.70%, Hpop=0.144). Cultivated populations (PhiST=0.515, GST=0.429) have a greater proportion of their genetic variability distributed among populations than wild populations (PhiST=0.277, GST=0.226). Based on hierarchical estimates of variance components, significant statistical differences (57.77%, P<0.001) were found between the wild and cultivated groups. The low levels of genetic diversity within cultivated populations and high levels of genetic differentiation among populations/groups may result from artificial selection, the mode of clonal propagation, and only limited exchange of material among localities. Finally, some suggestions for conservation and efficient management of the genetic resources of this important medicinal herb are proposed.