The First Complete Chloroplast Genome of Campanula carpatica: Genome Characterization and Phylogenetic Diversity.

Campanula carpatica is an ornamental flowering plant belonging to the family Campanulaceae. The complete chloroplast genome of C. carpatica was obtained using Illumina HiSeq X and Oxford Nanopore (Nanopore GridION) platforms. The chloroplast genome exhibited a typical circular structure with a total length of 169,341 bp, comprising a large single-copy region of 102,323 bp, a small single-copy region of 7744 bp, and a pair of inverted repeats (IRa/IRb) of 29,637 bp each. Out of a total 120 genes, 76 were protein-coding genes, 36 were transfer RNA genes, and eight were ribosomal RNA genes. The genomic characteristics of C. carpatica are similar to those of other Campanula species in terms of repetitive sequences, sequence divergence, and contraction/expansion events in the inverted repeat regions. A phylogenetic analysis of 63 shared genes in 16 plant species revealed that Campanula zangezura is the closest relative of C. carpatica. Phylogenetic analysis indicated that C. carpatica was within the Campanula clade, and C. pallida occupied the outermost position of that clade.

The complete mitochondrial genome sequences of Bupleurum falcatum (Apiales: Apiaceae).

Bupleurum falcatum has a long history of use in traditional oriental medicine. The first complete mitochondrial genome sequences of B. falcatum were 463,792 bp based on 494,582 aligned reads. A total of 51 genes was annotated including 32 protein-coding genes, 16 tRNA genes, and three rRNA genes. In a comparison of B. falcatum and carrot (Daucus carota) revealed that the former species has four exclusive genes, but lacks six genes present in the latter. The compositional structure and phylogenetic relationships indicated that the mitochondrial genome of B. falcatum is similar to that of D. carota.

The multipartite mitochondrial genome of Cynanchum auriculatum (Gentianales: Apocynaceae).

Cynanchum auriculatum is a Chinese herbal medicine species in the family Apocynaceae. The mitochondrial genome of C. auriculatum has heteroplasmy and consists of two chromosomes (chromosomes I and II), the lengths of which are 614,836 and 426,495 nucleotides. The multipartite mitochondrial genome encodes 57 genes, including 37 protein-coding genes, 17 transfer RNA genes, and three ribosomal RNA genes. Including 44 overlapping genes, we identified 57 genes on chromosome I and 44 genes on chromosome II. A phylogenetic tree revealed that C. auriculatum is most closely related to Asclepias syriaca.

The multipartite mitochondrial genome of Fallopia multiflora (Caryophyllales: Polygonaceae).

Fallopia multiflora is an important Oriental herb belonging to the family Polygonaceae. The F. multiflora mitochondrial genome consists of two circular chromosomes that were 200,352- and 112,098-nucleotides long. The mitochondrial genome encodes 57 genes, including 34 protein-coding genes, 20 transfer RNA (tRNA) genes, and three ribosomal RNA (rRNA) genes. When accounting for 3 overlapping genes, 38 genes were found in chromosome I, and 22 in chromosome II. The phylogenetic analysis suggests that F. multiflora is closely related to Beta macrocarpa and Silene latifolia.

A Multilayered Screening Method for the Identification of Regulatory Genes in Rice by Agronomic Traits.

We developed a multilayered screening method that integrates both genome and transcriptome data to effectively identify regulatory genes in rice (Oryza sativa). We tested our method using eight rice accessions that differed in three important nutritional and agricultural traits, anthocyanin biosynthesis, amylose content, and heading date. In the genome resequencing of eight rice accessions with 24 RNA sequencing experiments, 98% of the preprocessed reads could be uniquely mapped to the reference genome, resulting in the identification of 42,699 unique transcripts. Comparison between black and white rice cultivars showed evidence of intensive selective sweeps in chromosomes 3, 10, and 12. A total of 131 genes were differentially expressed among the black rice cultivars and found to be associated with three Gene Ontology terms (secondary metabolic process, biosynthetic process, and response to stimulus). We identified nonsynonymous Single Nucleotide Polymorphism (SNP) that likely play an important role in determining the agronomic traits differences, two upregulated and three downregulated genes in the black cultivars, and two downregulated genes in the white cultivars. The three agronomic traits were clearly grouped together by the developmental stages, regardless of any other traits, suggesting that the developmental stage is the most important factor that triggers global changes in gene expression. Interestingly, glutinous and nonglutinous black rice cultivars were distinguished from one another by different heading dates.

Whole-genome resequencing and transcriptomic analysis to identify genes involved in leaf-color diversity in ornamental rice plants.

Rice field art is a large-scale art form in which people design rice fields using various kinds of ornamental rice plants with different leaf colors. Leaf color-related genes play an important role in the study of chlorophyll biosynthesis, chloroplast structure and function, and anthocyanin biosynthesis. Despite the role of different metabolites in the traditional relationship between leaf and color, comprehensive color-specific metabolite studies of ornamental rice have been limited. We performed whole-genome resequencing and transcriptomic analysis of regulatory patterns and genetic diversity among different rice cultivars to discover new genetic mechanisms that promote enhanced levels of various leaf colors. We resequenced the genomes of 10 rice leaf-color accessions to an average of 40× reads depth and >95% coverage and performed 30 RNA-seq experiments using the 10 rice accessions sampled at three developmental stages. The sequencing results yielded a total of 1,814 × 106 reads and identified an average of 713,114 SNPs per rice accession. Based on our analysis of the DNA variation and gene expression, we selected 47 candidate genes. We used an integrated analysis of the whole-genome resequencing data and the RNA-seq data to divide the candidate genes into two groups: genes related to macronutrient (i.e., magnesium and sulfur) transport and genes related to flavonoid pathways, including anthocyanidin biosynthesis. We verified the candidate genes with quantitative real-time PCR using transgenic T-DNA insertion mutants. Our study demonstrates the potential of integrated screening methods combined with genetic-variation and transcriptomic data to isolate genes involved in complex biosynthetic networks and pathways.

A multistep screening method to identify genes using evolutionary transcriptome of plants.

We introduced a multistep screening method to identify the genes in plants using microarrays and ribonucleic acid (RNA)-seq transcriptome data. Our method describes the process for identifying genes using the salt-tolerance response pathways of the potato (Solanum tuberosum) plant. Gene expression was analyzed using microarrays and RNA-seq experiments that examined three potato lines (high, intermediate, and low salt tolerance) under conditions of salt stress. We screened the orthologous genes and pathway genes involved in salinity-related biosynthetic pathways, and identified nine potato genes that were candidates for salinity-tolerance pathways. The nine genes were selected to characterize their phylogenetic reconstruction with homologous genes of Arabidopsis thaliana, and a Circos diagram was generated to understand the relationships among the selected genes. The involvement of the selected genes in salt-tolerance pathways was verified by reverse transcription polymerase chain reaction analysis. One candidate potato gene was selected for physiological validation by generating dehydration-responsive element-binding 1 (DREB1)-overexpressing transgenic potato plants. The DREB1 overexpression lines exhibited increased salt tolerance and plant growth when compared to that of the control. Although the nine genes identified by our multistep screening method require further characterization and validation, this study demonstrates the power of our screening strategy after the initial identification of genes using microarrays and RNA-seq experiments.

A Multi-Layered Screening Method to Identify Plant Regulatory Genes.

We used a seven-step process to identify genes involved in glucosinolate biosynthesis and metabolism in the Chinese cabbage (Brassica rapa). We constructed an annotated data set with 34,570 unigenes from B. rapa and predicted 11,526 glucosinolate-related candidate genes using expression profiles generated across nine stages of development on a 47k-gene microarray. Using our multi-layered screening method, we screened 392 transcription factors, 843 pathway genes, and 4,162 ortholog genes associated with glucosinolate-related biosynthesis. Finally, we identified five genes by comparison of the pathway-network genes including the transcription-factor genes and the ortholog-ontology genes. The five genes were anchored to the chromosomes of B. rapa to characterize their genetic-map positions, and phylogenetic reconstruction with homologous genes was performed. These anchored genes were verified by reverse-transcription polymerase chain reaction. While the five genes identified by our multi-layered screen require further characterization and validation, our study demonstrates the power of multi-layered screening after initial identification of genes on microarrays.