Unraveling the complexity of faba bean (Vicia faba L.) transcriptome to reveal cold-stress-responsive genes using long-read isoform sequencing technology.

Faba bean (Vicia faba L.), a globally important grain legume providing a stable source of dietary protein, was one of the earliest plant cytogenetic models. However, the lack of draft genome annotations and unclear structural information on mRNA transcripts have impeded its genetic improvement. To address this, we sequenced faba bean leaf transcriptome using the PacBio single-molecule long-read isoform sequencing platform. We identified 28,569 nonredundant unigenes, ranging from 108 to 9669 bp, with a total length of 94.5 Mb. Many unigenes (3597, 12.5%) had 2-20 isoforms, indicating a highly complex transcriptome. Approximately 96.5% of the unigenes matched sequences in public databases. The predicted proteins and transcription factors included NB-ARC, Myb_domain, C3H, bHLH, and heat shock proteins, implying that this genome has an abundance of stress resistance genes. To validate our results, we selected WCOR413-15785, DHN2-12403, DHN2-14197, DHN2-14797, COR15-14478, and HVA22-15 unigenes from the ICE-CBF-COR pathway to analyze their expression patterns in cold-treated samples via qRT-PCR. The expression of dehydrin-related genes was induced by cold stress. The assembled data provide the first insights into the deep sequencing of full-length RNA from faba bean at the single-molecule level. This study provides an important foundation to improve gene modeling and protein prediction.

Analysis of genome variants in dwarf soybean lines obtained in F6 derived from cross of normal parents (cultivated and wild soybean).

Plant height is an important component of plant architecture and significantly affects crop breeding practices and yield. We studied DNA variations derived from F5 recombinant inbred lines (RILs) with 96.8% homozygous genotypes. Here, we report DNA variations between the normal and dwarf members of four lines harvested from a single seed parent in an F6 RIL population derived from a cross between Glycine max var. Peking and Glycine soja IT182936. Whole genome sequencing was carried out, and the DNA variations in the whole genome were compared between the normal and dwarf samples. We found a large number of DNA variations in both the dwarf and semi-dwarf lines, with one single nucleotide polymorphism (SNP) per at least 3.68 kb in the dwarf lines and 1 SNP per 11.13 kb of the whole genome. This value is 2.18 times higher than the expected DNA variation in the F6 population. A total of 186 SNPs and 241 SNPs were discovered in the coding regions of the dwarf lines 1282 and 1303, respectively, and we discovered 33 homogeneous nonsynonymous SNPs that occurred at the same loci in each set of dwarf and normal soybean. Of them, five SNPs were in the same positions between lines 1282 and 1303. Our results provide important information for improving our understanding of the genetics of soybean plant height and crop breeding. These polymorphisms could be useful genetic resources for plant breeders, geneticists, and biologists for future molecular biology and breeding projects.

Sequence-Specific Amplified Polymorphism (SSAP) and Sequence Characterized Amplified Region (SCAR) Markers in Zea mays.

Transposable elements (TEs) are mobile, recurring DNA sequences scattered throughout genome and have a large impact on genome structure and function. Several genetic marker techniques were developed to exploit their ubiquitous nature. Sequence-specific amplified polymorphism (SSAP) is a TE-based genetic marker system that has been used in various purposes such as measuring genetic relatedness between species, deciphering the population structures, molecular tagging for agronomic development in marker-assisted breeding (MAS). In addition to SSAP, sequence characterized amplified region (SCAR) from the SSAP markers provides an added advantage in identifying qualitative traits. Once developed SCAR markers are efficient, fast, and reliable method for genetic evaluations. These methods can be useful especially for the crops which have no genetic sequence information. With improved discriminatory ability they offer access to dynamic and polymorphic regions of genome. These techniques can be useful in breeding programs to improve or develop high yielding crops.

Characterization of transcription factor genes related to cold tolerance in Brassica napus.

Brassica napus is the third most important oilseed crop in the world; however, in Korea, it is greatly affected by cold stress, limiting seed growth and production. Plants have developed specific stress responses that are generally divided into three categories: cold-stress signaling, transcriptional/post-transcriptional regulation, and stress-response mechanisms. Large numbers of functional and regulatory proteins are involved in these processes when triggered by cold stress. Here, our objective was to investigate the different genetic factors involved in the cold-stress responses of B. napus. Consequently, we treated the Korean B. napus cultivar Naehan at the 4-week stage in cold chambers under different conditions, and RNA and cDNA were obtained. An in silico analysis included 80 cold-responsive genes downloaded from the National Center for Biotechnology Information (NCBI) database. Expression levels were assessed by reverse transcription polymerase chain reaction, and 14 cold-triggered genes were identified under cold-stress conditions. The most significant genes encoded zinc-finger proteins (33.7%), followed by MYB transcription factors (7.5%). In the future, we will select genes appropriate for improving the cold tolerance of B. napus.

The complete chloroplast genome of a fern genus Thelypteris interrupta.

In this study, we report the complete chloroplast (cp) genome of Thelypteris interrupta, a fern member, and comparative analysis with its related family members. The cp genome was 155,983 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (25,614 bp) separated by a large (82,769 bp) and small (21,986 bp) single-copy (SC) region. The genome encodes a total of 88 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Additionally, we identified 86 RNA editing sites in 52 genes; most of the substitution was U to C (52 sites), while C to U conversion occurred in 34 positions. The phylogenetic analysis strongly supported the relationship of T. interrupta with Ampelopteris prolifera and Christella appendiculata of Thelypteridoideae family.

De novo assembly and characterization of transcriptome in the medicinal plant Euphorbia jolkini.

BACKGROUND: Euphorbia jolkini, a medicinal herb that grows on the warm beaches in Japan and South Korea, is known to be used for traditional medicines to treat a variety of ailments, including bruises, stiffness, indigestion, toothache, and diabetes. OBJECTIVE: It is to analyze the whole transcriptome and identify the genes related to the phenylpropanoid biosynthesis in the medicinally important herb E jolkini. METHODS: Paired-end Illumina HiSeq™ 2500 sequencing technology was employed for cDNA library construction and Illumina sequencing. Public databases like TAIR (The Arabidopsis Information Resource), Swissprot and KEGG (Kyoto Encyclopedia of Genes and Genomes) were used for annotations of unigenes obtained. RESULTS: The transcriptome of E. jolkini generated 139,215 assembled transcripts with an average length of 868 bp and an N50 value of 1460 bp that were further clustered using CD-HIT into 93,801 unigenes with an average length of 847 bp (N50-1410 bp). Sixty-three percent of the coding sequences (CDS) were annotated from the longest open reading frame (ORF). A remarkable percentage of unigenes were annotated against various databases. The differentially expressed gene analysis revealed that the expression of genes related to the terpenoid backbone biosynthesis pathway was higher in the flowers, whereas that of genes related to the phenylpropanoid biosynthesis pathway was both up- and downregulated in flowers and leaves. A search of against the transcription factor domain found 1023 transcription factors (TFs) that were from 54 TF families. CONCLUSION: Assembled sequences of the E. jolkini transcriptome are made available for the first time in this study E. jolkini and lay a foundation for the investigation of secondary metabolite biosynthesis.

Characterization of Gene Isoforms Related to Cellulose and Lignin Biosynthesis in Kenaf (Hibiscus cannabinus L.) Mutant.

Kenaf is a source of fiber and a bioenergy crop that is considered to be a third world crop. Recently, a new kenaf cultivar, "Jangdae," was developed by gamma irradiation. It exhibited distinguishable characteristics such as higher biomass, higher seed yield, and earlier flowering than the wild type. We sequenced and analyzed the transcriptome of apical leaf and stem using Pacific Biosciences single-molecule long-read isoform sequencing platform. De novo assembly yielded 26,822 full-length transcripts with a total length of 59 Mbp. Sequence similarity against protein sequence allowed the functional annotation of 11,370 unigenes. Among them, 10,100 unigenes were assigned gene ontology terms, the majority of which were associated with the metabolic and cellular process. The Kyoto encyclopedia of genes and genomes (KEGG) analysis mapped 8875 of the annotated unigenes to 149 metabolic pathways. We also identified the majority of putative genes involved in cellulose and lignin-biosynthesis. We further evaluated the expression pattern in eight gene families involved in lignin-biosynthesis at different growth stages. In this study, appropriate biotechnological approaches using the information obtained for these putative genes will help to modify the desirable content traits in mutants. The transcriptome data can be used as a reference dataset and provide a resource for molecular genetic studies in kenaf.

Genetic differentiation of Mutator insertion polymorphisms and association with agronomic traits in waxy and common maize.

BACKGROUND: As waxy maize is considered a key economic crop in Korea, an understanding of its genetic variation and differentiation is fundamental for the selective plant breeding. The maize genome is primarily composed of transposable elements, for which large and stable insertions generate variations that reflect selection during evolution. OBJECTIVES: This study was to elucidate the genetic diversity based on the contribution of TEs and to investigate the effect of Mu transposition on the genetic divergence of waxy and common maize. We also performed an association analysis on these inbred lines to determine the Mu insertions associated with agronomic traits. METHODS: In this study, we utilized a Mutator-based transposon display method to study the genetic diversity and population structure of 40 waxy and 40 common inbred lines of maize in the Gangwon Agricultural Research and Extension Services collection at the Maize Research Institute. RESULTS: We detected polymorphisms in 86.33% of 278 Mutator (Mu) anchored loci, reflecting the activity of the Mu element and its contribution to genetic variation. Common maize showed a substantial amount of genetic diversity, which was greater than that observed in waxy maize. Principal-coordinate and neighbor-joining cluster analyzes consistently supported the presence of two genetically distinct groups. However, the distribution of genetic variation within the populations was much higher than the genetic differentiation among the populations. To explore the contribution of the Mu element to phenotypic variation, we analyzed the associations with ten important agronomical traits. On the basis of the combined results from two models (QGLM and Q + KLM), we found significant associations between seven Mu loci and four different traits. CONCLUSIONS: These results will assist waxy maize breeders in choosing parental lines and be useful for marker-assisted selection.

The complete chloroplast genome of a Korean endemic species Sophora koreensis, Nakai.

In this study, we report the complete chloroplast (cp) genome of Sophora koreensis and its relation with other species within the Fabaceae family. The cp genome was 154,870 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (25,866 bp) separated by a large (85,037 bp) and small (18,101 bp) single-copy (SC) region. The genome encodes a total of 84 protein-coding genes, 35 tRNA genes, and 8 rRNA genes. Phylogenetic analysis suggested that S. koreensis is closely related to genus Sophora alopecuroides var. alopecuroides within Fabaceae.

The complete chloroplast genome of a rare species in Korea, Lilium Dauricum Ker Gawl.

Lilium dauricum Ker Gawler is a wild lily species that belongs to section Sinomartagon and is one of the ancestors of the Asiatic hybrid lilies. Unique traits such as disease resistance and early flowering make L. dauricum a desirable resource for interspecific hybridization. However, in Korea, the natural resources of L. dauricum are being exhausted by excessive exploitation and require urgent conservation. In this study, the complete chloroplast genome of L. dauricum was generated using Illumina paired-end sequencing technology, and its structure was compared with that of other Lilium species. The chloroplast genome was 152,063 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (26,492 bp) separated by a large (81,485 bp) and small (17,584 bp) single-copy (SC) region. The genome encodes 131 different genes, including 85 protein-coding genes, 38 tRNA genes, and 8 rRNA genes. A phylogenetic analysis strongly supported the relationship of L. dauricum with other members of Sinomartagon and Martagon lilies.

The complete chloroplast genome of a Woodwardia japonica.

Woodwardia japonica is one of the diverse members of the fern group and medicinally important genus. In Korea, the natural resources of W. japonica are being exhausted by excessive exploitation and require urgent conservation. In this study, the complete chloroplast genome of W. japonica was generated, and its structure was compared with that of other members of same family. The chloroplast genome was 153224 bp long, with a typical quadripartite structure including a pair of inverted repeat regions (24591 bp) separated by a large (82480 bp) and small (21562 bp) single-copy (SC) region. The genome encodes a total of 88 protein-coding genes, 35 tRNA genes, and eight rRNA genes. Additionally we identified 87 RNA editing sites in 52 genes; most of the substitution was U to C (50 sites), while C to U conversion occurred in 37 positions. The phylogenetic analysis strongly supported the relationship of W. japonica with W. unigemmata and. A. melanocaulon (Blechnoideae).

Construction of genetic linkage map and identification of QTLs related to agronomic traits in maize using DNA transposon-based markers.

Transposable elements (TEs), are a rich source for molecular marker development as they constitute a significant fraction of the eukaryotic genome and impact the overall genome structure. Here, we utilize Mutator-based transposon display (Mu-TD), and CACTA-derived sequence-characterized amplified regions (SCAR) anchored by simple sequence repeats and single nucleotide polymorphisms to locate quantitative trait loci (QTLs) linked to agriculturally important traits on a genetic map. Specifically, we studied recombinant inbred line populations derived from a cross between dent corn and waxy corn. The resulting linkage map included 259 Mu-anchored fragments, 34 SCARs, and 614 SSR markers distributed throughout the ten maize chromosomes. Linkage analysis revealed three SNP loci associated with kernel starch synthesis genes (sh2, su1, wx1) linked to either Mu-TD loci or SSR markers, which may be useful for maize breeding programs. In addition, we used QTL analysis to determine the chromosomal location of traits related to grain yield and kernel quality. We identified 24 QTLs associated with nine traits located on nine out of ten maize chromosomes. Among these, 13 QTLs involved Mu loci and two involved SCARs. This study demonstrates the potential use of DNA transposon-based markers to construct linkage maps and identify QTLs linked to agronomic traits.

Mutator-Based Transposon Display: A Genetic Tool for Evolutionary and Crop-Improvement Studies in Maize.

Transposable elements account for up to 85% of the maize genome and have significant implications in crop-improvement and evolutionary analyses. The Mutator (Mu) transposon superfamily, a class of DNA transposons, comprises the most complex and active elements in the maize genome, suggesting a special role in plant evolution. Here, we designed a set of Mu-specific primers based on terminal invert repeats and used a transposon display (TD) method for genotyping. We analyzed the distribution pattern of Mu insertions in teosinte (wild relative), sorghum (distant relative), and domesticated maize accessions (dent, sweet, and waxy). The MU-TD analysis suggested the presence of high polymorphic insertions among the species and subspecies, indicating the utility of the method in studying genetic variation and species relationships. Furthermore, we analyzed 80 maize recombinant inbred line populations. Mu-TD generated an average of 60% Mu-anchored polymorphic fragments in which insertions appeared to be segregating in significantly high numbers. The amplification profile was highly reproducible, confirming the utility of Mu elements as a new set of TD markers for developing high-density genetic maps.

Enrichment of genomic resources and identification of simple sequence repeats from medicinally important Clausena excavata.

To broaden and delve into the genomic information of Clausena excavata, an important medicinal plant in many Asian countries, RNA sequencing (RNA-seq) analysis was performed and a total of 16,638 non-redundant unigenes (≥ 300 bp) with an average length of 755 bp were generated by de novo assembly from 17,580,456 trimmed clear reads. The functional categorization of the identified unigenes by a gene ontology (GO) term resulted in 2305 genes in the cellular component, 5577 in the biological processes, and 8056 in the molecular functions, respectively. The top sub-category in biological processes was the metabolic process with 4374 genes. Among annotated genes, 3006 were mapped to 123 metabolic pathways by the Kyoto Encyclopedia of Genes and Genomes (KEGG) metabolic pathway analysis tool. The search for simple sequence repeats (SSRs) resulted in 845 SSRs from 749 SSR-containing unigenes and the most abundant SSR motifs was AAG/CTT with 179 occurrences. Twelve SSR markers were tested for cross transferability among five Clausena species; eight of them exhibited polymorphism. Taken together, these data provide valuable resources for genomic or genetic studies of Clausena species and other relative studies. The transcriptome shotgun assembly data have been deposited at DDBJ/EMBL/GenBank under the accession GGEM00000000.

Development of CACTA transposon derived SCAR markers and their use in population structure analysis in Zea mays.

Molecular marker technologies have proven to be an important breakthrough for genetic studies, construction of linkage maps and population genetics analysis. Transposable elements (TEs) constitute major fractions of repetitive sequences in plants and offer a wide range of possible areas to be explored as molecular markers. Sequence characterized amplified region (SCAR) marker development provides us with a simple and time saving alternative approach for marker development. We employed the CACTA-TD to develop SCARs and then integrated them into linkage map and used them for population structure and genetic diversity analysis of corn inbred population. A total of 108 dominant SCAR markers were designed out of which, 32 were successfully integrated in to the linkage map of maize RIL population and the remaining were added to a physical map for references to check the distribution throughout all chromosomes. Moreover, 76 polymorphic SCARs were used for diversity analysis of corn accessions being used in Korean corn breeding program. The overall average polymorphic information content (PIC) was 0.34, expected heterozygosity was 0.324 and Shannon's information index was 0.491 with a percentage of polymorphism of 98.67%. Further analysis by associating with desirable traits may also provide some accurate trait specific tagged SCAR markers. TE linked SCARs can provide an added level of polymorphism as well as improved discriminating ability and therefore can be useful in further breeding programs to develop high yielding germplasm.