Genome-wide identification and functional exploration of the legume lectin genes in Brassica napus and their roles in Sclerotinia disease resistance

As one of the largest classes of lectins, legume lectins have a variety of desirable features such as antibacterial and insecticidal activities as well as anti-abiotic stress ability. The Sclerotinia disease (SD) caused by the soil-borne fungus Sclerotinia sclerotiorum is a devastating disease affecting most oil crops such as Brassica napus. Here, we identified 130 legume lectin (LegLu) genes in B. napus, which could be phylogenetically classified into seven clusters. The BnLegLu gene family has been significantly expanded since the whole-genome duplication (WGD) or segmental duplication. Gene structure and conserved motif analysis suggested that the BnLegLu genes were well conserved in each cluster. Moreover, relative to those genes only containing the legume lectin domain in cluster VI–VII, the genes in cluster I–V harbored a transmembrane domain and a kinase domain linked to the legume lectin domain in the C terminus. The expression of most BnLegLu genes was relatively low in various tissues. Thirty-five BnLegLu genes were responsive to abiotic stress, and 40 BnLegLu genes were strongly induced by S. sclerotiorum, with a most significant up-regulation of 715-fold, indicating their functional roles in SD resistance. Four BnLegLu genes were located in the candidate regions of genome-wide association analysis (GWAS) results which resulted from a worldwide rapeseed population consisting of 324 accessions associated with SD. Among them, the positive role of BnLegLus-16 in SD resistance was validated by transient expression in tobacco leaves. This study provides important information on BnLegLu genes, particularly about their roles in SD resistance, which may help targeted functional research and genetic improvement in the breeding of B. napus

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Whole-genome sequencing of <em>Oryza brachyantha</em> reveals mechanisms underlying <em>Oryza</em> genome evolution

The wild species of the genus Oryza contain a largely untapped reservoir of agronomically important genes for rice improvement. Here we report the 261-Mb de novo assembled genome sequence of Oryza brachyantha. Low activity of long-terminal repeat retrotransposons and massive internal deletions of ancient long-terminal repeat elements lead to the compact genome of Oryza brachyantha. We model 32,038 protein-coding genes in the Oryza brachyantha genome, of which only 70% are located in collinear positions in comparison with the rice genome. Analysing breakpoints of non-collinear genes suggests that double-strand break repair through non-homologous end joining has an important role in gene movement and erosion of collinearity in the Oryza genomes. Transition of euchromatin to heterochromatin in the rice genome is accompanied by segmental and tandem duplications, further expanded by transposable element insertions. The high-quality reference genome sequence of Oryza brachyantha provides an important resource for functional and evolutionary studies in the genus Oryza

The Characterization of the Phloem Protein 2 Gene Family Associated with Resistance to Sclerotinia sclerotiorum in Brassica napus

In plants, phloem is not only a vital structure that is used for nutrient transportation, but it is also the location of a response that defends against various stresses, named phloem-based defense (PBD). Phloem proteins (PP2s) are among the predominant proteins in phloem, indicating their potential functional role in PBD. Sclerotinia disease (SD), which is caused by the necrotrophic fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum), is a devastating disease that affects oil crops, especially Brassica napus (B. napus), mainly by blocking nutrition and water transportation through xylem and phloem. Presently, the role of PP2s in SD resistance is still largely estimated. Therefore, in this study, we identified 62 members of the PP2 gene family in the B. napus genome with an uneven distribution across the 19 chromosomes. A phylogenetic analysis classified the BnPP2s into four clusters (I&ndash;IV), with cluster I containing the most members (28 genes) as a consequence of its frequent genome segmental duplication. A comparison of the gene structures and conserved motifs suggested that BnPP2 genes were well conserved in clusters II to IV, but were variable in cluster I. Interestingly, the motifs in different clusters displayed unique features, such as motif 6 specifically existing in cluster III and motif 1 being excluded from cluster IV. These results indicated the possible functional specification of BnPP2s. A transcriptome data analysis showed that the genes in clusters II to IV exhibited dynamic expression alternation in tissues and the stimulation of S. sclerotiorum, suggesting that they could participate in SD resistance. A GWAS analysis of a rapeseed population comprising 324 accessions identified four BnPP2 genes that were potentially responsible for SD resistance and a transgenic study that was conducted by transiently expressing BnPP2-6 in tobacco (Nicotiana tabacum) leaves validated their positive role in regulating SD resistance in terms of reduced lesion size after inoculation with S. sclerotiorum hyphal plugs. This study provides useful information on PP2 gene functions in B. napus and could aid elaborated functional studies on the PP2 gene family

Collaborative biofluid analysis based multi-channel integrated wearable detection system for the monitoring of wound infection

The infection monitoring of chronic wounds can effectively improve the quality of wound care. However, the widely used single variable intermittent monitoring of wound provides little available information, which leads to inaccurate diagnosis and untimely warnings. In this study, a collaborative biofluid analysis based multi-channel integrated wearable detection system was constructed for the continuous detection of analytes such as pH, uric acid (UA), and C-reactive protein (CRP) in wound exudates with time division multiplexing. Based on the functionally modification with nanomaterials, integrated screen-printed electrodes (iSPE) with three working electrodes were designed for the collaboratively analyzing of wound exudates. Through the development of integrated circuits, the multi-channel wearable detection printed circuit board was constructed. With a self-designed interface, this iSPE was stably connected to the printed circuit board and realized the detection of three targets in the range of pH 3–8, UA concentrations 5–500 μmol/L, and CRP concentrations 1–1000 ng/mL at the same time. Combined with a smartphone, these results were collaborated analyzed and transferred for health management. Therefore, this integrated wearable multi-channel detection system can provide reliable and continuous evaluations for early warning of infection and further treatment of chronic wounds

The Characterization of the <i>Phloem Protein 2</i> Gene Family Associated with Resistance to <i>Sclerotinia sclerotiorum</i> in <i>Brassica napus</i>

In plants, phloem is not only a vital structure that is used for nutrient transportation, but it is also the location of a response that defends against various stresses, named phloem-based defense (PBD). Phloem proteins (PP2s) are among the predominant proteins in phloem, indicating their potential functional role in PBD. Sclerotinia disease (SD), which is caused by the necrotrophic fungal pathogen S. sclerotiorum (Sclerotinia sclerotiorum), is a devastating disease that affects oil crops, especially Brassica napus (B. napus), mainly by blocking nutrition and water transportation through xylem and phloem. Presently, the role of PP2s in SD resistance is still largely estimated. Therefore, in this study, we identified 62 members of the PP2 gene family in the B. napus genome with an uneven distribution across the 19 chromosomes. A phylogenetic analysis classified the BnPP2s into four clusters (I–IV), with cluster I containing the most members (28 genes) as a consequence of its frequent genome segmental duplication. A comparison of the gene structures and conserved motifs suggested that BnPP2 genes were well conserved in clusters II to IV, but were variable in cluster I. Interestingly, the motifs in different clusters displayed unique features, such as motif 6 specifically existing in cluster III and motif 1 being excluded from cluster IV. These results indicated the possible functional specification of BnPP2s. A transcriptome data analysis showed that the genes in clusters II to IV exhibited dynamic expression alternation in tissues and the stimulation of S. sclerotiorum, suggesting that they could participate in SD resistance. A GWAS analysis of a rapeseed population comprising 324 accessions identified four BnPP2 genes that were potentially responsible for SD resistance and a transgenic study that was conducted by transiently expressing BnPP2-6 in tobacco (Nicotiana tabacum) leaves validated their positive role in regulating SD resistance in terms of reduced lesion size after inoculation with S. sclerotiorum hyphal plugs. This study provides useful information on PP2 gene functions in B. napus and could aid elaborated functional studies on the PP2 gene family

The impact and origin of copy number variations in the Oryza species

Background: Copy number variation (CNV), a complex genomic rearrangement, has been extensively studied in humans and other organisms. In plants, CNVs of several genes were found to be responsible for various important traits; however, the cause and consequence of CNVs remains largely unknown. Recently released next-generation sequencing (NGS) data provide an opportunity for a genome-wide study of CNVs in rice. Results: Here, by an NGS-based approach, we generated a CNV map comprising 9,196 deletions compared to the reference genome 'Nipponbare'. Using Oryza glaberrima as the outgroup, 80 % of the CNV events turned out to be insertions in Nipponbare. There were 2,806 annotated genes affected by these CNV events. We experimentally validated 28 functional CNV genes including OsMADS56, BPH14, OsDCL2b and OsMADS30, implying that CNVs might have contributed to phenotypic variations in rice. Most CNV genes were found to be located in non-co-linear positions by comparison to O. glaberrima. One of the origins of these non-co-linear genes was genomic duplications caused by transposon activity or double-strand break repair. Comprehensive analysis of mutation mechanisms suggested an abundance of CNVs formed by non-homologous end-joining and mobile element insertion. Conclusions: This study showed the impact and origin of copy number variations in rice on a genomic scale

Brassica GLABRA2 genes: analysis of function related to seed oil content and development of functional markers

Regulation of seed oil accumulation in oilseed rape (Brassica napus) has important economic signiWcance. However, few genes have been characterized that aVect Wnal seed oil content. Through a mutant identiWcation, the class IV homeodomain-ZIP transcription factor GLABRA2 (GL2) has been found to regulate seed oil accumulation in Arabidopsis, in addition to its role in trichome development. In this study, we isolated four distinct orthologues of GL2 from B. napus (AC-genome), B. rapa (A) and B. oleracea (C), using an overlapping-PCR strategy. The four GL2 orthologues were very similar, with 96.10– 99.69% identity in exon regions, 75.45–93.84% in intron regions, 97.34–99.87% in amino acid sequences. Alignments of the four genes revealed that the A-genome sequences of BnaA.GL2.a from B. napus and BraA.GL2.a from B. rapa are more similar than the others, and likewise the C-genome sequences of BnaC.GL2.b from B. napus and BolC.GL2.a from B. oleracea are more similar. BnaA.GL2.a and BraA.GL2.a from the A-genome are highly expressed in roots, whilst BnaC.GL2.b and BolC.GL2.a from the C-genome are preferentially expressed in seeds. Transgenic ectopic overexpression and suppression of BnaC.GL2.b in Arabidopsis allowed further investigation of the eVect on seed oil content. Overexpression generated two phenotypes: the wild-type-like and the gl2-mutant-like (an Arabidopsis glabrous mutant of gl2-2), with increases in seed oil content of 3.5–5.0% in the gl2-mutant-like transgenic plants. Suppression resulted in increases of 2.5–6.1% in seed oil content, and reduced trichome number at the leaf margins. These results suggest that BnaC.GL2.b can negatively regulate oil accumulation in Arabidopsis seeds. As a result of comparing the four GL2 genes, three A/C-genomespeciWc primer sets were developed and a C-genome-speciWc EcoRV cleavage site was identiWed, which can be used as functional markers to distinguish these orthologues within Brassica species. The genes identiWed and their molecular markers developed in this study will be valuable both for oilseed rape breeding focusing on improvement of seed oil content, and for detecting gene Xow between populations

Genome-Wide Identification and Analysis of Ariadne Gene Family Reveal Its Genetic Effects on Agronomic Traits of <i>Brassica napus</i>

E3 ligases promote protein ubiquitination and degradation, which regulate every aspect of eukaryotic life. The Ariadne (ARI) proteins of RBR (ring between ring fingers) protein subfamily has been discovered as a group of potential E3 ubiquitin ligases. Only a few available research studies show their role in plant adaptations processes against the external environment. Presently, the functions of ARI proteins are largely unknown in plants. Therefore, in this study, we performed genome-wide analysis to identify the ARI gene family and explore their potential importance in B. napus. A total of 39 ARI genes were identified in the B. napus genome and were classified into three subfamilies (A, B and C) based on phylogenetic analysis. The protein–protein interaction networks and enrichment analysis indicated that BnARI genes could be involved in endoreduplication, DNA repair, proteasome assembly, ubiquitination, protein kinase activity and stress adaptation. The transcriptome data analysis in various tissues provided us an indication of some BnARI genes’ functional importance in tissue development. We also identified potential BnARI genes that were significantly responsive towards the abiotic stresses. Furthermore, eight BnARI genes were identified as candidate genes for multiple agronomic traits through association mapping analysis in B. napus; among them, BnaA02g12100D, which is the ortholog of AtARI8, was significantly associated with ten agronomic traits. This study provided useful information on BnARI genes, which could aid targeted functional research and genetic improvement for breeding in B. napus

Identification of Flower-Specific Promoters through Comparative Transcriptome Analysis in Brassica napus

Brassica napus (oilseed rape) is an economically important oil crop worldwide. Sclerotinia stem rot (SSR) caused by Sclerotinia sclerotiorum is a threat to oilseed rape production. Because the flower petals play pivotal roles in the SSR disease cycle, it is useful to express the resistance-related genes specifically in flowers to hinder further infection with S. sclerotiorum. To screen flower-specific promoters, we first analyzed the transcriptome data from 12 different tissues of the B. napus line ZS11. In total, 249 flower-specific candidate genes with high expression in petals were identified, and the expression patterns of 30 candidate genes were verified by quantitative real-time transcription-PCR (qRT-PCR) analysis. Furthermore, two novel flower-specific promoters (FSP046 and FSP061 promoter) were identified, and the tissue specificity and continuous expression in petals were determined in transgenic Arabidopsis thaliana with fusing the promoters to &beta;-glucuronidase (GUS)-reporter gene. GUS staining, transcript expression pattern, and GUS activity analysis indicated that FSP046 and FSP061 promoter were strictly flower-specific promoters, and FSP046 promoter had a stronger activity. The two promoters were further confirmed to be able to direct GUS expression in B. napus flowers using transient expression system. The transcriptome data and the flower-specific promoters screened in the present study will benefit fundamental research for improving the agronomic traits as well as disease and pest control in a tissue-specific manner