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.

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.

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.

The comparative gene expression concern to the seed pigmentation in maize (Zea mays L.)

Maize seed pigmentation is one of the important issue to develop maize seed breeding. The differently gene expression was characterized and compared for three inbred lines, such as the pigment accumulated seed (CM22) and non-pigmented seed (CM5 and CM19) at 10 days after pollination. We obtained a total of 63,870, 82,496 and 54,555 contigs by de novo assembly to identify gene expression in the CM22, CM5, and CM19, respectably. In differentially expressed gene analysis, it was revealed that 7,044 genes were differentially expressed by at least two-fold, with 4,067 upregulated in colored maize inbred lines and 2,977 upregulated in colorless maize inbred lines. Of them,18 genes were included to the anthocyanin biosynthesis pathways, while 15 genes were upregulated in both CM22/5 and CM22/19. Additionally, 37 genes were detected in the metabolic pathway concern to the seed pigmentation by BINs analysis using MAPMAN software. Finally, these differently expressed genes may aid in the research on seed pigmentation in maize breeding programs.

The comparative gene expression concern to the seed pigmentation in maize (Zea mays L.)

Maize seed pigmentation is one of the important issue to develop maize seed breeding. The differently gene expression was characterized and compared for three inbred lines, such as the pigment accumulated seed (CM22) and non-pigmented seed (CM5 and CM19) at 10 days after pollination. We obtained a total of 63,870, 82,496 and 54,555 contigs by de novo assembly to identify gene expression in the CM22, CM5, and CM19, respectably. In differentially expressed gene analysis, it was revealed that 7,044 genes were differentially expressed by at least two-fold, with 4,067 upregulated in colored maize inbred lines and 2,977 upregulated in colorless maize inbred lines. Of them,18 genes were included to the anthocyanin biosynthesis pathways, while 15 genes were upregulated in both CM22/5 and CM22/19. Additionally, 37 genes were detected in the metabolic pathway concern to the seed pigmentation by BINs analysis using MAPMAN software. Finally, these differently expressed genes may aid in the research on seed pigmentation in maize breeding programs.

RNA-Seq De Novo Assembly and Differential Transcriptome Analysis of Korean Medicinal Herb Cirsium japonicum var. spinossimum

Cirsium japonicum belongs to the Asteraceae or Compositae family and is a medicinal plant in Asia that has a variety of effects, including tumour inhibition, improved immunity with flavones, and antidiabetic and hepatoprotective effects. Silymarin is synthesized by 4-coumaroyl-CoA via both the flavonoid and phenylpropanoid pathways to produce the immediate precursors taxifolin and coniferyl alcohol. Then, the oxidative radicalization of taxifolin and coniferyl alcohol produces silymarin. We identified the expression of genes related to the synthesis of silymarin in C. japonicum in three different tissues, namely, flowers, leaves, and roots, through RNA sequencing. We obtained 51,133 unigenes from transcriptome sequencing by de novo assembly using Trinity v2.1.1, TransDecoder v2.0.1, and CD-HIT v4.6 software. The differentially expressed gene analysis revealed that the expression of genes related to the flavonoid pathway was higher in the flowers, whereas the phenylpropanoid pathway was more highly expressed in the roots. In this study, we established a global transcriptome dataset for C. japonicum. The data shall not only be useful to focus more deeply on the genes related to product medicinal metabolite including flavolignan but also to study the functional genomics for genetic engineering of C. japonicum.

Efficiency to Discovery Transgenic Loci in GM Rice Using Next Generation Sequencing Whole Genome Re-sequencing

Molecular characterization technology in genetically modified organisms, in addition to how transgenic biotechnologies are developed now require full transparency to assess the risk to living modified and non-modified organisms. Next generation sequencing (NGS) methodology is suggested as an effective means in genome characterization and detection of transgenic insertion locations. In the present study, we applied NGS to insert transgenic loci, specifically the epidermal growth factor (EGF) in genetically modified rice cells. A total of 29.3 Gb (~72x coverage) was sequenced with a 2 x 150 bp paired end method by Illumina HiSeq2500, which was consecutively mapped to the rice genome and T-vector sequence. The compatible pairs of reads were successfully mapped to 10 loci on the rice chromosome and vector sequences were validated to the insertion location by polymerase chain reaction (PCR) amplification. The EGF transgenic site was confirmed only on chromosome 4 by PCR. Results of this study demonstrated the success of NGS data to characterize the rice genome. Bioinformatics analyses must be developed in association with NGS data to identify highly accurate transgenic sites.

Survey of the Applications of NGS to Whole-Genome Sequencing and Expression Profiling

Recently, the technologies of DNA sequence variation and gene expression profiling have been used widely as approaches in the expertise of genome biology and genetics. The application to genome study has been particularly developed with the introduction of the next-generation DNA sequencer (NGS) Roche/454 and Illumina/Solexa systems, along with bioinformation analysis technologies of whole-genome de novo assembly, expression profiling, DNA variation discovery, and genotyping. Both massive whole-genome shotgun paired-end sequencing and mate paired-end sequencing data are important steps for constructing de novo assembly of novel genome sequencing data. It is necessary to have DNA sequence information from a multiplatform NGS with at least 2x and 30x depth sequence of genome coverage using Roche/454 and Illumina/Solexa, respectively, for effective an way of de novo assembly. Massive short-length reading data from the Illumina/Solexa system is enough to discover DNA variation, resulting in reducing the cost of DNA sequencing. Whole-genome expression profile data are useful to approach genome system biology with quantification of expressed RNAs from a whole-genome transcriptome, depending on the tissue samples. The hybrid mRNA sequences from Rohce/454 and Illumina/Solexa are more powerful to find novel genes through de novo assembly in any whole-genome sequenced species. The 20x and 50x coverage of the estimated transcriptome sequences using Roche/454 and Illumina/Solexa, respectively, is effective to create novel expressed reference sequences. However, only an average 30x coverage of a transcriptome with short read sequences of Illumina/Solexa is enough to check expression quantification, compared to the reference expressed sequence tag sequence.