Characterization and fine-mapping of a new Asian rice selfish genetic locus S58 in Asian-African rice hybrids.

KEY MESSAGE: We identified and fine-mapped S58, a selfish genetic locus from Asian rice that confers hybrid male sterility in crosses between Asian and African cultivated rice, and found a natural neutral allele in Asian rice lines that will be useful for overcoming S58-mediated hybrid sterility. Hybrids between Asian cultivated rice (Oryza sativa L.) and African cultivated rice (Oryza glaberrima Steud) display severe hybrid sterility (HS), hindering the utilization of strong heterosis in hybrids between these species. Several African rice selfish loci causing HS in Asian-African cultivated rice hybrids have been identified, but few such Asian rice selfish loci have been found. In this study, we identified an Asian rice selfish locus, S58, which causes hybrid male sterility (HMS) in hybrids between the Asian rice variety 02428 and the African rice line CG14. Genetic analysis confirmed that S58 causes a transmission advantage for the Asian rice S58 allele in the hybrid offspring. Genetic mapping with near-isogenic lines and DNA markers delimited S58 to 186 kb and 131 kb regions of chromosome 1 in 02428 and CG14, respectively, and revealed complex genomic structural variation over these mapped regions. Gene annotation analysis and expression profiling analyses identified eight anther-expressed candidate genes potentially responsible for S58-mediated HMS. Comparative genomic analysis determined that some Asian cultivated rice varieties harbor a 140 kb fragment deletion in this region. Hybrid compatibility analysis showed that this large deletion allele in some Asian cultivated rice varieties can serve as a natural neutral allele, S58-n, that can overcome S58-mediated interspecific HMS. Our study demonstrates that this selfish genetic element from Asian rice is important for HMS between Asian and African cultivated rice, broadening our understanding of interspecific HS. This study also provides an effective strategy for overcoming HS in future interspecific rice breeding.

All-in-one: a robust fluorescent fusion protein vector toolbox for protein localization and BiFC analyses in plants.

Fluorescent tagging protein localization (FTPL) and bimolecular fluorescence complementation (BiFC) are popular tools for in vivo analyses of the subcellular localizations of proteins and protein-protein interactions in plant cells. The efficiency of fluorescent fusion protein (FFP) expression analyses is typically impaired when the FFP genes are co-transformed on separate plasmids compared to when all are cloned and transformed in a single vector. Functional genomics applications using FFPs such as a gene family studies also often require the generation of multiple plasmids. Here, to address these needs, we developed an efficient, modular all-in-one (Aio) FFP (AioFFP) vector toolbox, including a set of fluorescently labelled organelle markers, FTPL and BiFC plasmids and associated binary vectors. This toolbox uses Gibson assembly (GA) and incorporates multiple unique nucleotide sequences (UNSs) to facilitate efficient gene cloning. In brief, this system enables convenient cloning of a target gene into various FFP vectors or the insertion of two or more target genes into the same FFP vector in a single-tube GA reaction. This system also enables integration of organelle marker genes or fluorescently fused target gene expression units into a single transient expression plasmid or binary vector. We validated the AioFFP system by testing genes encoding proteins known to be functional in FTPL and BiFC assays. In addition, we performed a high-throughput assessment of the accurate subcellular localizations of an uncharacterized rice CBSX protein subfamily. This modular UNS-guided GA-mediated AioFFP vector toolkit is cost-effective, easy to use and will promote functional genomics research in plants.

Research on Chinese medical named entity recognition based on collaborative cooperation of multiple neural network models.

Medical named entity recognition (NER) in Chinese electronic medical records (CEMRs) has drawn much research attention, and plays a vital prerequisite role for extracting high-value medical information. In 2018, China Health Information Processing Conference (CHIP2018) organized a medical NER academic competition aiming to extract three types of malignant tumor entity from CEMRs. Since the three types of entity are highly domain-specific and interdependency, extraction of them cannot be achieved with a single neural network model. Based on comprehensive study of the three types of entity and the entity interdependencies, we propose a collaborative cooperation of multiple neural network models based approach, which consists of two BiLSTM-CRF models and a CNN model. In order to tackle the problem that target scene dataset is small and entity distributions are sparse, we introduce non-target scene datasets and propose sentence-level neural network model transfer learning. Based on 30,000 real-world CEMRs, we pre-train medical domain-specific Chinese character embeddings with word2vec, GloVe and ELMo, and apply them to our approach respectively to validate effects of pre-trained language models in Chinese medical NER. Also, as control experiments, we apply Gated Recurrent Unit to our approach. Finally, our approach achieves an overall F1-score of 87.60%, which is the state-of-the-art performance to the best of our knowledge. In addition, our approach has won the champion of the medical NER academic competition organized by 2019 China Conference on Knowledge Graph and Semantic Computing, which proves the outstanding generalization ability of our approach.

Suppression or knockout of SaF/SaM overcomes the Sa-mediated hybrid male sterility in rice.

Hybrids between the indica and japonica subspecies of rice (Oryza sativa) are usually sterile, which hinders utilization of heterosis in the inter-subspecific hybrid breeding. The complex locus Sa comprises two adjacently located genes, SaF and SaM, which interact to cause abortion of pollen grains carrying the japonica allele in japonica-indica hybrids. Here we showed that silencing of SaF or SaM by RNA interference restored male fertility in indica-japonica hybrids with heterozygous Sa. We further used clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9-based genome editing to knockout the SaF and SaM alleles, respectively, of an indica rice line to create hybrid-compatible lines. The resultant artificial neutral alleles did not affect pollen viability and other agricultural traits, but did break down the reproductive barrier in the hybrids. We found that some rice lines have natural neutral allele Sa-n, which was compatible with the typical japonica or indica Sa alleles in hybrids. Our results demonstrate that SaF and SaM are required for hybrid male sterility, but are not essential for pollen development. This study provides effective approaches for the generation of hybrid-compatible lines by knocking out the Sa locus or using the natural Sa-n allele to overcome hybrid male sterility in rice breeding. © 2017 The Authors. Bioelectromagnetics published by Wiley Periodicals, Inc.

STI PCR: An efficient method for amplification and de novo synthesis of long DNA sequences.

Despite continuous improvements, it is difficult to efficiently amplify large sequences from complex templates using current PCR methods. Here, we developed a suppression thermo-interlaced (STI) PCR method for the efficient and specific amplification of long DNA sequences from genomes and synthetic DNA pools. This method uses site-specific primers containing a common 5' tag to generate a stem-loop structure, thereby repressing the amplification of smaller non-specific products through PCR suppression (PS). However, large target products are less affected by PS and show enhanced amplification when the competitive amplification of non-specific products is suppressed. Furthermore, this method uses nested thermo-interlaced cycling with varied temperatures to optimize strand extension of long sequences with an uneven GC distribution. The combination of these two factors in STI PCR produces a multiplier effect, markedly increasing specificity and amplification capacity. We also developed a webtool, calGC, for analyzing the GC distribution of target DNA sequences and selecting suitable thermo-cycling programs for STI PCR. Using this method, we stably amplified very long genomic fragments (up to 38 kb) from plants and human and greatly increased the length of de novo DNA synthesis, which has many applications such as cloning, expression, and targeted genomic sequencing. Our method greatly extends PCR capacity and has great potential for use in biological fields.

Di[12]aneN3-Functionalized Green Fluorescent Protein Chromophore for GFP Luminescence Simulation and Efficient Gene Transfection In Vitro and In Vivo.

Although a plethora of gene carriers have been developed for potential gene therapy, imageable stimuli-responsive gene vectors with fast access to the nucleus, high biocompatibility, and transfection efficiency are still scarce. Herein, we report the design and synthesis of four dendrite-shaped cationic liposomes, MPA-HBI-R/DOPE (R: n-butyl, 1; n-octyl, 2; n-dodecyl, 3; palmyl, 4), prepared via esterification of 4-alkoxybenzylideneimidazolinone containing aliphatic chains of different lengths (HBI-R), the green fluorescent protein (GFP) chromophore, with a di[12]aneN3 unit. Liposomes were fabricated via the self-assembly of MPA-HBI-R, assisted with 1,2-dioleoyl-sn-glycerol-3-phosphorylethanolamine (DOPE). These liposomes (MPA-HBI-R/DOPE) exhibited efficient DNA condensation, pH-responsive degradation, excellent cellular biocompatibility (up to 150 µM), and high transfection efficiency. Molecular docking experiments were also used to verify the optimal interaction between MPA-HBI-R and DNA, as well as the fluorescence enhancements. In particular, MPA-HBI-2/DOPE delivered DNA into the nucleus in less than an hour, and its luciferase transfection activity was more than 10 times that by Lipo2000, across multiple cell lines. The GFP chromophore conjugation allowed trackable intracellular delivery and release of DNA in real time via fluorescence imaging. Furthermore, efficient red fluorescent protein (RFP) transfection in zebrafish, with an efficiency of more than 6 times that by Lipo2000, was also achieved. The results not only realized, for the first time, the combination of gene delivery and GFP-simulated light emission, allowing fluorescent tracking and highly efficient gene transfection, but also offered valuable insights into the use of biomimetic chromophore for the development of the next-generation nonviral vectors.

An asymmetric allelic interaction drives allele transmission bias in interspecific rice hybrids.

Hybrid sterility (HS) between Oryza sativa (Asian rice) and O. glaberrima (African rice) is mainly controlled by the S1 locus. However, our limited understanding of the HS mechanism hampers utilization of the strong interspecific heterosis. Here, we show that three closely linked genes (S1A4, S1TPR, and S1A6) in the African S1 allele (S1-g) constitute a killer-protector system that eliminates gametes carrying the Asian allele (S1-s). In Asian-African rice hybrids (S1-gS1-s), the S1TPR-S1A4-S1A6 interaction in sporophytic tissues generates an abortion signal to male and female gametes. However, S1TPR can rescue S1-g gametes, while the S1-s gametes selectively abort for lacking S1TPR. Knockout of any of the S1-g genes eliminates the HS. Evolutionary analysis suggests that S1 may have arisen from newly evolved genes, multi-step recombination, and nucleotide variations. Our findings will help to overcome the interspecific reproductive barrier and use Asian-African hybrids for increasing rice production.