Nadia Raboanatahiry

Genome-wide identification, structural analysis and new insights into late embryogenesis abundant (LEA) gene family formation pattern in Brassica napus

Late embryogenesis abundant (LEA) proteins are a diverse and large group of polypeptides that play important roles in desiccation and freezing tolerance in plants. The LEA family has been systematically characterized in some plants but not Brassica napus. In this study, 108 BnLEA genes were identified in the B. napus genome and classified into eight families based on their conserved domains. Protein sequence alignments revealed an abundance of alanine, lysine and glutamic acid residues in BnLEA proteins. The BnLEA gene structure has few introns (<3), and they are distributed unevenly across all 19 chromosomes in B. napus, occurring as gene clusters in chromosomes A9, C2, C4 and C5. More than two-thirds of the BnLEA genes are associated with segmental duplication. Synteny analysis revealed that most LEA genes are conserved, although gene losses or gains were also identified. These results suggest that segmental duplication and whole-genome duplication played a major role in the expansion of the BnLEA gene family. Expression profiles analysis indicated that expression of most BnLEAs was increased in leaves and late stage seeds. This study presents a comprehensive overview of the LEA gene family in B. napus and provides new insights into the formation of this family.

Progress in Genome Editing Technology and Its Application in Plants

Genome editing technology (GET) is a versatile approach that has progressed rapidly as a mechanism to alter the genotype and phenotype of organisms. However, conventional genome modification using GET cannot satisfy current demand for high-efficiency and site-directed mutagenesis, retrofitting of artificial nucleases has developed into a new avenue within this field. Based on mechanisms to recognize target genes, newly-developed GETs can generally be subdivided into three cleavage systems, protein-dependent DNA cleavage systems (i.e., zinc-finger nucleases, ZFN, and transcription activator-like effector nucleases, TALEN), RNA-dependent DNA cleavage systems (i.e., clustered regularly interspaced short palindromic repeats-CRISPR associated proteins, CRISPR-Cas9, CRISPR-Cpf1, and CRISPR-C2c1), and RNA-dependent RNA cleavage systems (i.e., RNA interference, RNAi, and CRISPR-C2c2). All these techniques can lead to double-stranded (DSB) or single-stranded breaks (SSB), and result in either random mutations via non-homologous end-joining (NHEJ) or targeted mutation via homologous recombination (HR). Thus, site-directed mutagenesis can be induced via targeted gene knock-out, knock-in, or replacement to modify specific characteristics including morphology-modification, resistance-enhancement, and physiological mechanism-improvement along with plant growth and development. In this paper, an non-comprehensive review on the development of different GETs as applied to plants is presented.

Genetic dissection of seed oil and protein content and identification of networks associated with oil content in Brassica napus

High-density linkage maps can improve the precision of QTL localization. A high-density SNP-based linkage map containing 3207 markers covering 3072.7 cM of the Brassica napus genome was constructed in the KenC-8 × N53-2 (KNDH) population. A total of 67 and 38 QTLs for seed oil and protein content were identified with an average confidence interval of 5.26 and 4.38 cM, which could explain up to 22.24% and 27.48% of the phenotypic variation, respectively. Thirty-eight associated genomic regions from BSA overlapped with and/or narrowed the SOC-QTLs, further confirming the QTL mapping results based on the high-density linkage map. Potential candidates related to acyl-lipid and seed storage underlying SOC and SPC, respectively, were identified and analyzed, among which six were checked and showed expression differences between the two parents during different embryonic developmental periods. A large primary carbohydrate pathway based on potential candidates underlying SOC- and SPC-QTLs, and interaction networks based on potential candidates underlying SOC-QTLs, was constructed to dissect the complex mechanism based on metabolic and gene regulatory features, respectively. Accurate QTL mapping and potential candidates identified based on high-density linkage map and BSA analyses provide new insights into the complex genetic mechanism of oil and protein accumulation in the seeds of rapeseed.

Computational Prediction of acyl-coA Binding Proteins Structure in Brassica napus

Acyl-coA binding proteins could transport acyl-coA esters from plastid to endoplasmic reticulum, prior to fatty acid biosynthesis, leading to the formation of triacylglycerol. The structure and the subcellular localization of acyl-coA binding proteins (ACBP) in Brassica napus were computationally predicted in this study. Earlier, the structure analysis of ACBPs was limited to the small ACBPs, the current study focused on all four classes of ACBPs. Physicochemical parameters including the size and the length, the intron-exon structure, the isoelectric point, the hydrophobicity, and the amino acid composition were studied. Furthermore, identification of conserved residues and conserved domains were carried out. Secondary structure and tertiary structure of ACBPs were also studied. Finally, subcellular localization of ACBPs was predicted. The findings indicated that the physicochemical parameters and subcellular localizations of ACBPs in Brassica napus were identical to Arabidopsis thaliana. Conserved domain analysis indicated that ACBPs contain two or three kelch domains that belong to different families. Identical residues in acyl-coA binding domains corresponded to eight amino acid residues in all ACBPs of B. napus. However, conserved residues of common ACBPs in all species of animal, plant, bacteria and fungi were only inclusive in small ACBPs. Alpha-helixes were displayed and conserved in all the acyl-coA binding domains, representing almost the half of the protein structure. The findings confirm high similarities in ACBPs between A. thaliana and B. napus, they might share the same functions but loss or gain might be possible.

Proteomic Dissection of Seed Germination and Seedling Establishment in Brassica napus.

The success of seed germination and establishment of a normal seedling are key determinants of plant species propagation. At present, only a few studies have focused on the genetic control of seed germination by using a proteomic approach in Brassica napus. In the present study, the protein expression pattern of seed germination was investigated using differential fluorescence two-dimensional gel electrophoresis in B. napus. One hundred and thirteen differentially expressed proteins (DEPs) that were mainly involved in storage (23.4%), energy metabolism (18.9%), protein metabolism (16.2%), defense/disease (12.6%), seed maturation (11.7%), carbohydrate metabolism (4.5%), lipid metabolism (4.5%), amino acids metabolism (3.6%), cell growth/division (3.6%), and some unclear functions (2.7%) were observed by proteomic analysis. Seventeen genes corresponding to 11 DEPs were identified within or near the associated linkage disequilibrium regions related to seed germination and vigor quantitative traits reported in B. napus in previous studies. The expression pattern of proteins showed that heterotrophic metabolism could be activated in the process of seed germination and that the onset of defense mechanisms might start during seed germination. These findings will help generate a more in-depth understanding of the mobilization of seed storage reserves and regulation mechanisms of the germination process in B. napus.

Transcriptome Analysis of Stem and Globally Comparison with Other Tissues in Brassica napus

Brassica napus is one of the most important oilseed crops in the world. However, there is currently no enough stem transcriptome information and comparative transcriptome analysis of different tissues, which impedes further functional genomics research on B. napus. In this study, the stem transcriptome of B. napus was characterized by RNA-seq technology. Approximately 13.4 Gb high-quality clean reads with an average length of 100 bp were generated and used for comparative transcriptome analysis with the existing transcriptome sequencing data of roots, leaves, flower buds, and immature embryos of B. napus. All the transcripts were annotated against GO and KEGG databases. The common genes in five tissues, differentially expressed genes (DEGs) of the common genes between stems and other tissues, and tissue-specific genes were detected, and the main biochemical activities and pathways implying the common genes, DEGs and tissue-specific genes were investigated. Accordingly, the common transcription factors (TFs) in the five tissues and tissue-specific TFs were identified, and a TFs-based regulation network between TFs and the target genes involved in ‘Phenylpropanoid biosynthesis’ pathway were constructed to show several important TFs and key nodes in the regulation process. Collectively, this study not only provided an available stem transcriptome resource in B. napus, but also revealed valuable comparative transcriptome information of five tissues of B. napus for future investigation on specific processes, functions and pathways.

Synteny analysis of genes and distribution of loci controlling oil content and fatty acid profile based on QTL alignment map in Brassica napus

BackgroundDeciphering the genetic architecture of a species is a good way to understand its evolutionary history, but also to tailor its profile for breeding elite cultivars with desirable traits. Aligning QTLs from diverse population in one map and utilizing it for comparison, but also as a basis for multiple analyses assure a stronger evidence to understand the genetic system related to a given phenotype.ResultsIn this study, 439 genes involved in fatty acid (FA) and triacylglycerol (TAG) biosyntheses were identified in Brassica napus. B. napus genome showed mixed gene loss and insertion compared to B. rapa and B. oleracea, and C genome had more inserted genes. Identified QTLs for oil (OC-QTLs) and fatty acids (FA-QTLs) from nine reported populations were projected on the physical map of the reference genome “Darmor-bzh” to generate a map. Thus, 335 FA-QTLs and OC-QTLs could be highlighted and 82 QTLs were overlapping. Chromosome C3 contained 22 overlapping QTLs with all trait studied except for C18:3. In total, 218 candidate genes which were potentially involved in FA and TAG were identified in 162 QTLs confidence intervals and some of them might affect many traits. Also, 76 among these candidate genes were found inside 57 overlapping QTLs, and candidate genes for oil content were in majority (61/76 genes). Then, sixteen genes were found in overlapping QTLs involving three populations, and the remaining 60 genes were found in overlapping QTLs of two populations. Interaction network and pathway analysis of these candidate genes indicated ten genes that might have strong influence over the other genes that control fatty acids and oil formation.ConclusionThe present results provided new information for genetic basis of FA and TAG formation in B. napus. A map including QTLs from numerous populations was built, which could serve as reference to study the genome profile of B. napus, and new potential genes emerged which might affect seed oil. New useful tracks were showed for the selection of population or/and selection of interesting genes for breeding improvement purpose.

Whole-Genome Identification and Expression Pattern of the Vicinal Oxygen Chelate Family in Rapeseed (Brassica napus L.)

Vicinal oxygen chelate proteins (VOC) are members of the metalloenzyme superfamily, which plays roles in many biological reactions. Some members of the VOC superfamily have been systematically characterized but not in Brassica napus. In this study, 38 VOC genes were identified based on their conserved domains. The present results revealed that most of the BnaVOC genes have few introns, and all contained the typical VOC structure of βαβββ modules. The BnaVOC genes are distributed unevenly across 15 chromosomes in B. napus and occur as gene clusters on chromosomes C5 and A6. The synteny and phylogenetic analyses revealed that the VOC gene family is a consequence of mesopolyploidy events that occurred in Brassica evolution, and whole-genome duplication and segmental duplication played a major role in the expansion of the BnaVOC gene family. The expression profile analysis indicated that the expression of most BnaVOCs was increased in the leaves and late stage seeds. Further results indicated that seeds of B. napus with a high oil content show higher expression levels under drought stress conditions, suggesting that BnaVOCs not only respond to abiotic stress but may also affect lipid metabolism in drought stress. This present study provides a comprehensive overview of the VOC gene family and provides new insights into their biological function in B. napus evolution.

QTL Alignment for Seed Yield and Yield Related Traits in Brassica napus

Worldwide consumption of oil is increasing with the growing population in need for edible oil and the expansion of industry using biofuels. Then, demand for high yielding varieties of oil crops is always increasing. Brassica napus (rapeseed) is one of the most important oil crop in the world, therefore, increasing rapeseed yield through breeding is inevitable in order to cater for the high demand of vegetable oil and high-quality protein for live stocks. Quantitative trait loci (QTL) analysis is a powerful tool to identify important loci and which is also valuable for molecular marker assisted breeding. Seed-yield (SY) is a complex trait that is controlled by multiple loci and is affected directly by seed weight, seeds per silique and silique number. Some yield-related traits, such as plant height, biomass yield, flowering time, and so on, also affect the SY indirectly. This study reports the assembly of QTLs identified for seed-yield and yield-related traits in rapeseed, in one unique map. A total of 972 QTLs for seed-yield and yield-related were aligned into the physical map of B. napus Darmor-bzh and 92 regions where 198 QTLs overlapped, could be discovered on 16 chromosomes. Also, 147 potential candidate genes were discovered in 65 regions where 131 QTLs overlapped, and might affect nine different traits. At the end, interaction network of candidate genes was studied, and showed nine genes that could highly interact with the other genes, and might have more influence on them. The present results would be helpful to develop molecular markers for yield associated traits and could be used for breeding improvement in B. napus.

Stable, Environmental Specific and Novel QTL Identification as Well as Genetic Dissection of Fatty Acid Metabolism in Brassica napus

Fatty acid (FA) composition is the typical quantitative trait in oil seed crops, of which study is not only closely related to oil content, but is also more critical for the quality improvement of seed oil. The double haploid (DH) population named KN with a high density SNP linkage map was applied for quantitative trait loci (QTL) analysis of FA composition in this study. A total of 406 identified QTL were detected for eight FA components with an average confidence interval (CI) of 2.92 cM, the explained phenotypic variation (PV) value ranged from 1.49 to 45.05%. Totally, 204 consensus and 91 unique QTL were further obtained via meta-analysis method for the purpose of detecting multiple environment expressed and pleiotropic QTL, respectively. Of which, 74 stable expressed and 22 environmental specific QTL were also revealed, respectively. In order to make clear the genetic mechanism of FA metabolism at individual QTL level, conditional QTL analysis was also conducted and more than two thousand conditional QTL which could not be detected under the unconditional mapping were detected, which indicated the complex interrelationship of the QTL controlling FA content in rapeseed. Through comparative genomic analysis and homologous gene annotation, 61 candidates related to acyl lipid metabolism were identified underlying the CI of FA QTL. To further visualize the genetic mechanism of FA metabolism, an intuitive and meticulous network about acyl lipid metabolism was constructed and some closely related candidates were positioned. This study provided a more accurate localization for stable and pleiotropic QTL, and a deeper dissection of the molecular regulatory mechanism of FA metabolism in rapeseed.