Rice Genome Research Articles

Genome-Wide Identification, Characterization, and Expression Profile Analysis of CONSTANS-like Genes in Woodland Strawberry (Fragaria vesca).

CONSTANS-like (CO-like) gene is one of the most important regulators in the flowering process of the plant, playing a core role in the photoperiodic flowering induction pathway. In this study, we identified 10 distinct CO-like genes (FveCOs) in woodland strawberry (Fragaria vesca). They were classified into three groups with specific gene structure characteristics or protein domains in each group. The effect of selection pressure on the FveCOs in the woodland strawberry was tested by Ka/Ks, and it was shown that the evolution rate of FveCOs was controlled by purification selection factors. Intraspecific synteny analysis of woodland strawberry FveCOs showed that at least one duplication event existed in the gene family members. Collinearity analysis of woodland strawberry genome with genomes of Arabidopsis, rice (Oryza sativa), and apple (Malus × domestica) showed that CO-like genes of F. vesca and Malus × domestica owned higher similarity for their similar genomes compared with those of other two species. The FveCOs showed different tissue-specific expression patterns. Moreover, real-time quantitative PCR results revealed that the expressions of the most FveCOs followed a 24-h rhythm oscillation under both long-day (LD) and short-day (SD) conditions. Further expression analysis showed that the individual expression changing profile of FveCO3 and FveCO5 was opposite to each other under both LD and SD conditions. Moreover, the expression of FveCO3 and FveCO5 was both negatively correlated with the flowering time variation of the woodland strawberry grown under LD and SD conditions, indicating their potential vital roles in the photoperiodic flowering regulation. Further protein interaction network analysis also showed that most of the candidate interaction proteins of FveCO3 and FveCO5 were predicted to be the flowering regulators. Finally, LUC assay indicated that both FveCO3 and FveCO5 could bind to the promoter of FveFT1, the key regulator of flowering regulation in the woodland strawberry, and thus activate its expression. Taken together, this study laid a foundation for understanding the exact roles of FveCOs in the reproductive development regulation of the woodland strawberry, especially in the photoperiodic flowering process.

An ORFeome of rice E3 ubiquitin ligases for global analysis of the ubiquitination interactome

BackgroundUbiquitination is essential for many cellular processes in eukaryotes, including 26S proteasome-dependent protein degradation, cell cycle progression, transcriptional regulation, and signal transduction. Although numerous ubiquitinated proteins have been empirically identified, their cognate ubiquitin E3 ligases remain largely unknown.ResultsHere, we generate a complete ubiquitin E3 ligase-encoding open reading frames (UbE3-ORFeome) library containing 98.94% of the 1515 E3 ligase genes in the rice (Oryza sativa L.) genome. In the test screens with four known ubiquitinated proteins, we identify both known and new E3s. The interaction and degradation between several E3s and their substrates are confirmed in vitro and in vivo. In addition, we identify the F-box E3 ligase OsFBK16 as a hub-interacting protein of the phenylalanine ammonia lyase family OsPAL1–OsPAL7. We demonstrate that OsFBK16 promotes the degradation of OsPAL1, OsPAL5, and OsPAL6. Remarkably, we find that overexpression of OsPAL1 or OsPAL6 as well as loss-of-function of OsFBK16 in rice displayed enhanced blast resistance, indicating that OsFBK16 degrades OsPALs to negatively regulate rice immunity.ConclusionsThe rice UbE3-ORFeome is the first complete E3 ligase library in plants and represents a powerful proteomic resource for rapid identification of the cognate E3 ligases of ubiquitinated proteins and establishment of functional E3–substrate interactome in plants.

Genome Size Variation and Evolution Driven by Transposable Elements in the Genus Oryza.

Genome size variation and evolutionary forces behind have been long pursued in flowering plants. The genus Oryza, consisting of approximately 25 wild species and two cultivated rice, harbors eleven extant genome types, six of which are diploid (AA, BB, CC, EE, FF, and GG) and five of which are tetraploid (BBCC, CCDD, HHJJ, HHKK, and KKLL). To obtain the most comprehensive knowledge of genome size variation in the genus Oryza, we performed flow cytometry experiments and estimated genome sizes of 166 accessions belonging to 16 non-AA genome Oryza species. k-mer analyses were followed to verify the experimental results of the two accessions for each species. Our results showed that genome sizes largely varied fourfold in the genus Oryza, ranging from 279 Mb in Oryza brachyantha (FF) to 1,203 Mb in Oryza ridleyi (HHJJ). There was a 2-fold variation (ranging from 570 to 1,203 Mb) in genome size among the tetraploid species, while the diploid species had 3-fold variation, ranging from 279 Mb in Oryza brachyantha (FF) to 905 Mb in Oryza australiensis (EE). The genome sizes of the tetraploid species were not always two times larger than those of the diploid species, and some diploid species even had larger genome sizes than those of tetraploids. Nevertheless, we found that genome sizes of newly formed allotetraploids (BBCC-) were almost equal to totaling genome sizes of their parental progenitors. Our results showed that the species belonging to the same genome types had similar genome sizes, while genome sizes exhibited a gradually decreased trend during the evolutionary process in the clade with AA, BB, CC, and EE genome types. Comparative genomic analyses further showed that the species with different rice genome types may had experienced dissimilar amplification histories of retrotransposons, resulting in remarkably different genome sizes. On the other hand, the closely related rice species may have experienced similar amplification history. We observed that the contents of transposable elements, long terminal repeats (LTR) retrotransposons, and particularly LTR/Gypsy retrotransposons varied largely but were significantly correlated with genome sizes. Therefore, this study demonstrated that LTR retrotransposons act as an active driver of genome size variation in the genus Oryza.

A Koshihikari X Oryza rufipogon Introgression Line with a High Capacity to Take up Nitrogen to Maintain Growth and Panicle Development under Low Nitrogen Conditions.

Nitrogen (N) is an important macronutrient for plant growth and development. Currently, N fertilizers are required for the efficient production of modern crops such as rice due to their limited capacity to take up N when present at low concentrations. Wild rice represents a useful genetic resource for improving crop responses to low nutrient stress. Here, we describe the isolation and characterization of an introgression line, KRIL37, that carries a small region of the Oryza rufipogon genome in the Oryza sativa L. cv Koshihikari (KH) background. This line was found to grow better under low N conditions and have similar or lower C/N ratios in aerial portions compared to those in the parental KH cultivar, suggesting that KRIL37 has a higher capacity to take up and assimilate N when present at low concentrations. KRIL37 performance in the field was also better than that of KH cultivated without N and fertilizer (-F). Transcriptome analyses of 3-week-old seedlings based on RNA-sequencing revealed that KH induced a wider suite of genes than the tolerant line KRIL37 in response to low N conditions. Some ammonium transporters and N assimilation genes were found to be induced under low N in KRIL37, but not in KH. Our findings suggest that the superior growth performance of KRIL37 under limited N conditions could be due to the expression of wild alleles influencing N uptake and assimilation. Our study demonstrates the potential to use wild rice genomes to improve modern crops for low nutrient tolerance.

Genomic regions and candidate genes selected during the breeding of rice in Vietnam.

Vietnam harnesses a rich diversity of rice landraces adapted to a range of conditions, which constitute a largely untapped source of diversity for the continuous improvement of cultivars. We previously identified a strong population structure in Vietnamese rice, which is captured in five Indica and four Japonica subpopulations, including an outlying Indica‐5 group. Here, we leveraged that strong differentiation and 672 native rice genomes to identify genomic regions and genes putatively selected during the breeding of rice in Vietnam. We identified significant distorted patterns in allele frequency (XP‐CLR) and population differentiation scores (F ST) resulting from differential selective pressures between native subpopulations, and later annotated them with QTLs previously identified by GWAS in the same panel. We particularly focussed on the outlying Indica‐5 subpopulation because of its likely novelty and differential evolution, where we annotated 52 selected regions, which represented 8.1% of the rice genome. We annotated the 4576 genes in these regions and selected 65 candidate genes as promising breeding targets, several of which harboured alleles with nonsynonymous substitutions. Our results highlight genomic differences between traditional Vietnamese landraces, which are likely the product of adaption to multiple environmental conditions and regional culinary preferences in a very diverse country. We also verified the applicability of this genome scanning approach to identify potential regions harbouring novel loci and alleles to breed a new generation of sustainable and resilient rice.

Genetic basis underlying tiller angle in rice (Oryza sativa L.) by genome-wide association study.

Novel alleles of two reported tiller angle genes and eleven candidate genes for rice tiller angle were identified by combining GWAS with transcriptomic, qRT-PCR and haplotype analysis. Rice tiller angle is a key agronomic trait determining rice grain yield. Several quantitative trait loci (QTLs) affecting rice tiller angle have been mapped in the past decades. Little is known about the genetic base of tiller angle in rice, because rice tiller angle is a complex polygenic trait. In this study, we performed genome-wide association study (GWAS) on tiller angle in rice using a population of 164 japonica varieties derived from the 3K Rice Genomes Project (3K RGP). We detected a total of 18 QTLs using 1135519 single-nucleotide polymorphisms (SNP) based on three GWAS models (GLM, FastLMM and FarmCPU). Among them, two identified QTLs, qTA8.3 and qTA8.4, overlapped with PAY1 and TIG1, respectively, and additional 16 QTLs were identified for the first time. Combined with haplotype and expression analyses, we further revealed that PAY1 harbors one non-synonymous variation at its coding region, likely leading to variable tiller angle in the population, and that nature variations in the promoter of TIG1 significantly affect its expression, closely correlating with tiller angle phenotypes observed. Similarly, using qRT-PCR and haplotype analysis, we identified 1 and 7 candidate genes in qTA6.1 and qTA8.1 that were commonly detected by two GWAS models, respectively. In addition, we identified 3 more candidate genes in the remaining 14 novel QTLs after filtering by transcriptome analysis and qRT-PCR. In summary, this study provides new insights into the genetic architecture of rice tiller angle and candidate genes for rice breeding.

Genome-Wide Analysis of von Willebrand Factor A Gene Family in Rice for Its Role in Imparting Biotic Stress Resistance with Emphasis on Rice Blast Disease

von Willebrand factor A (vWA) genes are well characterized in humans except for few BONZAI genes, but the vWA genes are least explored in plants. Considering the novelty and vital role of vWA genes, this study aimed at characterization of vWA superfamily in rice. Rice genome was found to have 40 vWA genes distributed across all the 12 chromosomes, and 20 of the 40 vWA genes were unique while the remaining shared large fragment similarities with each other, indicating gene duplication. In addition to vWA domain, vWA proteins possess other different motifs or domains, such as ubiquitin interacting motif in protein degradation pathway, and RING finger in protein-protein interaction. Expression analysis of vWA genes in available expression data suggested that they probably function in biotic and abiotic stress responses including hormonal response and signaling. The frequency of transposon elements in the entire 3K rice germplasm was negligible except for 9 vWA genes, indicating the importance of these genes in rice. Structural and functional diversities showed that the vWA genes in a blast-resistant rice variety Tetep had huge variations compared to blast-susceptible rice varieties HP2216 and Nipponbare. qRT-PCR analysis of vWA genes in Magnaporthe oryzae infected rice tissues indicated OsvWA9, OsvWA36, OsvWA37 and OsvWA18 as the optimal candidate genes for disease resistance. This is the first attempt to characterize vWA gene family in plant species.

Genome-Wide Comparative Analysis of Transposable Elements by Matrix-TE Method Revealed Indica and Japonica Rice Evolution

Transposons (TEs) are known to change the gene expression and function, and subsequently cause plant speciation and evolution. Nevertheless, efficient and new approaches are required to investigate the role of TEs in the plant genome structural variations. Here, we reported the method named matrix-TE to investigate the differentiation of intact and truncated LTR/TEs comprehensively in Indica and Japonica rice throughout whole genomes with a special eye on centromeric regions. Six LTR/TE super-families were identified in both Indica and Japonica rice genomes, and the TE ORF references were extracted by phylogenetic analysis. Indica rice specific TE peak P-Gypsy and Japonica rice specific TE peak P-Copia were observed, and were further analyzed by Gaussian probability density function (GPDF) fit. The individual TE peak P-Gypsy was observed in centromeric regions of the Indica genome. By the matrix-TE method, the divergence of Indica and Japonica genomes, especially their centromeric regions, mainly resulted from the Ty3/Gypsy insertion events at 0.77 Mya. Our data indicate that the optimized matrix-TE approach may be used to specifically analyze the TE content, family evolution, and time of the TE insertions.

Amplification of LTRs of extrachromosomal linear DNAs (ALE-seq) identifies two active Oryco LTR retrotransposons in the rice cultivar Dongjin

Long terminal repeat retrotransposons (LTR-RTs) make up a considerable portion of plant genomes. New insertions of these active LTR-RTs modify gene structures and functions and play an important role in genome evolution. Therefore, identifying active forms of LTR-RTs could uncover the effects of these elements in plants. Extrachromosomal linear DNA (eclDNA) forms during LTR-RT replication; therefore, amplification LTRs of eclDNAs followed by sequencing (ALE-seq) uncover the current transpositional potential of the LTR-RTs. The ALE-seq protocol was validated by identification of Tos17 in callus of Nipponbare cultivar. Here, we identified two active LTR-RTs belonging to the Oryco family on chromosomes 6 and 9 in rice cultivar Dongjin callus based on the ALE-seq technology. Each Oryco family member has paired LTRs with identical sequences and internal domain regions. Comparison of the two LTR-RTs revealed 97% sequence identity in their internal domains and 65% sequence identity in their LTRs. These two putatively active Oryco LTR-RT family members could be used to expand our knowledge of retrotransposition mechanisms and the effects of LTR-RTs on the rice genome.

The Long Read Transcriptome of Rice (Oryza sativa ssp. japonica var. Nipponbare) Reveals Novel Transcripts

BackgroundHigh-throughput next-generation sequencing technologies offer a powerful approach to characterizing the transcriptomes of plants. Long read sequencing has been shown to support the discovery of novel isoforms of transcripts. This approach enables the generation of full-length sequences revealing splice variants that may be important in regulating gene action. Investigation of the diversity of transcripts in the rice transcriptome including splice variants was conducted using PacBio long-read sequence data to improve the annotation of the rice genome.ResultsA cDNA library was prepared from RNA extracted from leaves, roots, seeds, inflorescences, and panicles of O. sativa ssp. japonica var Nipponbare and sequenced on a PacBio Sequel platform. This produced 346,190 non-redundant full-length non-chimeric reads (FLNC) resulting in 33,504 high-quality transcripts. Half of the transcripts were multi-exonic and entirely matched with the reference transcripts. However, 14,874 novel isoforms were also identified resulting predominantly from intron retention and at least one novel splice site. Intron retention was the prevalent alternative splicing event and exon skipping was the least observed. Of 73,659 splice junctions, 12,755 (17%) represented novel splice junctions with canonical and non-canonical intron boundaries. The complexity of the transcriptome was examined in detail for 19 starch synthesis-related genes, defining 276 spliced isoforms of which 94 splice variants were novel.ConclusionThe data reveal the great complexity of the rice transcriptome. The novel transcripts provide new insights that may be a key input in future research to improve the annotation of the rice genome.

Transcriptional and post-transcriptional regulation of young genes in plants.

BackgroundNew genes continuously emerge from non-coding DNA or by diverging from existing genes, but most of them are rapidly lost and only a few become fixed within the population. We hypothesized that young genes are subject to transcriptional and post-transcriptional regulation to limit their expression and minimize their exposure to purifying selection.ResultsWe performed a protein-based homology search across the tree of life to determine the evolutionary age of protein-coding genes present in the rice genome. We found that young genes in rice have relatively low expression levels, which can be attributed to distal enhancers, and closed chromatin conformation at their transcription start sites (TSS). The chromatin in TSS regions can be re-modeled in response to abiotic stress, indicating conditional expression of young genes. Furthermore, transcripts of young genes in Arabidopsis tend to be targeted by nonsense-mediated RNA decay, presenting another layer of regulation limiting their expression.ConclusionsThese data suggest that transcriptional and post-transcriptional mechanisms contribute to the conditional expression of young genes, which may alleviate purging selection while providing an opportunity for phenotypic exposure and functionalization.Supplementary InformationThe online version contains supplementary material available at 10.1186/s12915-022-01339-7.

Distance-based Support Vector Machine to Predict DNA N6- methyladenine Modification

Background: DNA N6-methyladenine plays an important role in the restriction-modification system to isolate invasion from adventive DNA. The shortcomings of the high time consumption and high costs of experimental methods have been exposed, and some computational methods have emerged. The support vector machine theory has received extensive attention in the bioinformatics field due to its solid theoretical foundation and many good characteristics. Objective: General machine learning methods include an important step of extracting features. The research has omitted this step and replaced with easy-to-obtain sequence distances matrix to obtain better results. Method: First sequence alignment technology was used to achieve the similarity matrix. Then, a novel transformation turned the similarity matrix into a distance matrix. Next, the similarity-distance matrix was made positive semi-definite so that it can be used in the kernel matrix. Finally, the LIBSVM software was applied to solve the support vector machine. Results: The five-fold cross-validation of this model on rice and mouse data has achieved excellent accuracy rates of 92.04% and 96.51%, respectively. This shows that the DB-SVM method has obvious advantages over traditional machine learning methods. Meanwhile, this model achieved 0.943,0.982 and 0.818 accuracy; 0.944, 0.982, and 0.838 Matthews correlation coefficient; and 0.942, 0.982 and 0.840 F1 scores for the rice, M. musculus and cross-species genome datasets, respectively. Conclusion: These outcomes show that this model outperforms the iIM-CNN and csDMA in the prediction of DNA 6mA modification, which is the latest research finding on DNA 6mA.

Pup1 QTL Regulates Gene Expression Through Epigenetic Modification of DNA Under Phosphate Starvation Stress in Rice.

Cytosine methylation, epigenetic DNA modification, is well known to regulate gene expression. Among the epigenetic modifications, 5-methylcytosine (5-mC) has been one of the extensively studied epigenetic changes responsible for regulating gene expression in animals and plants. Though a dramatic change in 5-mC content is observed at the genome level, the variation in gene expression is generally less than that it is expected. Only less is understood about the significance of 5-mC in gene regulation under P-starvation stress in plants. Using whole-genome bisulfite sequencing of a pair of rice [Pusa-44 and its near-isogenic line (NIL)-23 harboring Pup1 QTL] genotypes, we could decipher the role of Pup1 on DNA (de)methylation-mediated regulation of gene expression under P-starvation stress. We observed 13–15% of total cytosines to be methylated in the rice genome, which increased significantly under the stress. The number of differentially methylated regions (DMRs) for hypomethylation (6,068) was higher than those (5,279) for hypermethylated DMRs under the stress, particularly in root of NIL-23. Hypomethylation in CHH context caused upregulated expression of 489 genes in shoot and 382 genes in root of NIL-23 under the stress, wherein 387 genes in shoot and 240 genes in root were upregulated exclusively in NIL-23. Many of the genes for DNA methylation, a few for DNA demethylation, and RNA-directed DNA methylation were upregulated in root of NIL-23 under the stress. Methylation or demethylation of DNA in genic regions differentially affected gene expression. Correlation analysis for the distribution of DMRs and gene expression indicated the regulation of gene mainly through (de)methylation of promoter. Many of the P-responsive genes were hypomethylated or upregulated in roots of NIL-23 under the stress. Hypermethylation of gene body in CG, CHG, and CHH contexts caused up- or downregulated expression of transcription factors (TFs), P transporters, phosphoesterases, retrotransposon proteins, and other proteins. Our integrated transcriptome and methylome analyses revealed an important role of the Pup1 QTL in epigenetic regulation of the genes for transporters, TFs, phosphatases, carbohydrate metabolism, hormone-signaling, and chromatin architecture or epigenetic modifications in P-starvation tolerance. This provides insights into the molecular function of Pup1 in modulating gene expression through DNA (de)methylation, which might be useful in improving P-use efficiency or productivity of rice in P-deficient soil.

Comprehensive Bioinformatics and Expression Analysis of the TLP Gene Family Revealed Its Role in Regulating the Response of Oryza sativa to Nilaparvata lugens, Laodelphax striatellus, and Jinggangmycin

Thaumatin-like proteins (TLPs), which belong to pathogenesis-related (PR) protein family 5 (PR5), are involved in the plant host defense and developmental processes. Detailed information regarding the TLP gene family in rice remains elusive. Here, we identified 43 OsTLP genes from the rice genome database. The in silico analysis comprised of the evolutionary tree, gene structure, conserved motifs, and chemical properties highlighted the key aspects of the OsTLP genes. By phylogenetic analysis, the OsTLP genes were classified into ten groups (OsTLPI-OsTLPX). Gene ontology (GO) analysis showed that OsTLP genes regulate plant growth and response to various environmental stimuli. Moreover, most of the OsTLP genes are expressed in almost all tissues. Microarray data showed differential expression trends of OsTLP genes under salinity stress and hormonal treatments, whereas under BPH, SSB, and RFL, only OsTLP1, OsTLP2 and OsTLP27 were expressed. The qRT-PCR analysis showed OsTLP27 as the prominent gene, displayed by its upregulated expression under JGM. Our results provide detailed knowledge of OsTLP gene resistance in rice plants, and we believe the current study will facilitate the development of cultivars resistant to biotic/abiotic stress, particularly stress caused by the chewing and sucking of insect pests.

Fine Mapping and Cloning of a Major QTL qph12, Which Simultaneously Affects the Plant Height, Panicle Length, Spikelet Number and Yield in Rice (Oryza sativa L.).

Plant height is one of the most important agronomical traits in rice (Oryza sativa L.). Introducing the semidwarf rice in the 1960s significantly enhanced the rice yield potential in Asia. Implementing near-isogenic lines (NILs) is the most powerful tool for the identification and fine mapping of quantitative trait loci (QTLs). In this study, 176 NILs were produced from the crossing and back-crossing of two rice cultivars. Specifically, the indica rice cultivar Jiafuzhan served as a recipient, and the restorer japonica cultivar Hui1586 served as a donor. Using the 176 NILs, we identified a novel major QTL for reduced plant height in the NIL36 line. The qph12 QTL was mapped to a 31 kb genomic region between the indel markers Indel12-29 and Indel12-31. The rice genome annotation indicated the presence of three candidate genes in this genomic region. Through gene prediction and cDNA sequencing, we confirmed that LOC_Os12g40890 (qPH12) is the target gene in the NIL36 line. Further analysis showed that the qph12 QTL is caused by a 1 bp deletion in the first exon that resulted in premature termination of the qPH12. Knockout experiments showed that the qph12 QTL is responsible for the reduced plant height phenotype of the NIL36 line. Although the qph12 gene from the NIL36 line showed a shorter panicle length, fewer spikelets per panicle and a lower plant grain yield, the plant also exhibited a lower plant height. Taken together, our results revealed that the qph12 have good specific application prospects in future rice breeding.

Genome-wide identification of Oryza sativa: A new insight for advanced analysis of ABC transporter genes associated with the degradation of four pesticides

ATP-binding cassette (ABC) transporter is a large genes superfamily. It involves transportation of diverse substrates (e.g., heavy metal, amino acids, pesticides, metabolites). The ABC transporters can be strongly induced by environmental stress and responsible for the phase III metabolic process of toxic compounds in plants. To investigate the potential molecular and biochemical function of ABC transporters in response to pesticides, we used bioinformatics and high-throughput sequencing to identify 107 loci from rice (Oryza sativa) exposed to different pesticides (ametryn, AME; bentazone, BNTZ; fomesafen, FSA; mesotrione, MTR) and annotated as ABC transporter genes. ABC transporter genes were categorized to eight subfamilies including ABCA-G and ABCI. ABCG subfamily was the largest group in rice genome followed by ABCC subfamily and ABCB subfamily. The distribution of each ABC transporter on twelve chromosomes was identified. The result showed that a large number of genes were scattered around chromosome. Differentially expressed genes (DEGs) were selected for cis-acting analysis under pesticide stress. Multiple cis-elements for biological functions such as hormone-sensitive elements and defense-related elements were found to involve the initiation and regulation of transcription. Comprehensive phylogenetic analysis and domain prediction of all ABC DEGs from rice and Arabidopsis were carried out. The docking analysis of ABC transporters and pesticides provided insights into the key amino acid residues involved in the binding of the pesticides. Consequently, the results provided applicable information and reference for a more functional analysis of ABC transporter genes on regulation of pesticide metabolism and transport in plants.

Expression of NDV HN protein in rice and development of a semi-quantitative rapid method for detection of antibodies

The aim of this study was to develop a semi-quantitative immunochromatographic method for rapid detection of Newcastle disease virus (NDV) antibodies by expressing HN protein in rice endosperm bioreactor. The recombinant plasmid pUC57-HN was digested by MlyⅠ and XhoⅠ to retrieve the HN gene, while the intermediate vector pMP3 containing promoter, signal peptide and terminator was digested by NaeⅠ and XhoⅠ. The HN gene and the linearized pMP3 were purified and ligated to form a recombinant plasmid pMP3-HN1. Subsequently, pMP3-HN1 and plant vector pCAMBIA1300 were digested by EcoRⅠ and Hind Ⅲ, and the HN1 gene was cloned into pCAMBIA1300. The recombinant plasmid pCAMBIA1300-HN1 was introduced into Agrobacterium tumefaciens EHA105 by electrotransformation, and the pCAMBIA1300-HN1 was transferred into rice callus by agrobacterium-mediated method. After dark culture, callus screening, differentiation, rooting and transplanting, transgenic rice seeds were obtained 4 months later. PCR identified that the HN gene has been inserted into the rice genome. SDS-PAGE and Western blotting indicated that the HN protein was successfully expressed in the positive rice endosperm. The purity of the HN protein was more than 90% by SP cation exchange chromatography and gel filtration chromatography. According to the national standards for the diagnostic techniques of Newcastle disease HI test (HI≥4log2, positive antibody reaction), a colloidal gold labeled purified HN protein was used to prepare a semi-quantitative test strip by double-antibody sandwich method for rapid detection of NDV antibody. The results showed that the test strip did not cross-react with positive sera against other viruses, and the sensitivity of the test strip reached 1:102 400 for standard positive sera of Newcastle disease. Testing of a total of 308 clinical sera showed that the compliance rate of the test strip with HI test was 97.08%, and the Kappa value was 0.942. In conclusion, high purity recombinant HN protein was obtained from rice endosperm, and a simple, rapid, highly sensitive and highly specific semi-quantitative immunochromatographic strip was developed. The test strip could be used for immune evaluation of the Newcastle disease vaccine.

Phenotypic variation and genome-wide association studies of main culm panicle node number, maximum node production rate, and degree-days to heading in rice

BackgroundGrain yield is a complex trait that results from interaction between underlying phenotypic traits and climatic, edaphic, and biotic variables. In rice, main culm panicle node number (MCPNN; the node number on which the panicle is borne) and maximum node production rate (MNPR; the number of leaves that emerge per degree-day > 10°C) are primary phenotypic plant traits that have significant positive direct effects on yield-related traits. Degree-days to heading (DDTH), which has a significant positive effect on grain yield, is influenced by the interaction between MCPNN and MNPR. The objective of this research is to assess the phenotypic variation of MCPNN, MNPR, and DDTH in a panel of diverse rice accessions, determine regions in the rice genome associated with these traits using genome-wide association studies (GWAS), and identify putative candidate genes that control these traits.ResultsConsiderable variation was observed for the three traits in a 220-genotype diverse rice population. MCPNN ranged from 8.1 to 20.9 nodes in 2018 and from 9.9 to 21.0 nodes in 2019. MNPR ranged from 0.0097 to 0.0214 nodes/degree day > 10°C in 2018 and from 0.0108 to 0.0193 nodes/degree-day > 10°C in 2019. DDTH ranged from 713 to 2,345 degree-days > 10°C in 2018 and from 778 to 2,404 degree-days > 10°C in 2019.Thirteen significant (P < 2.91 x 10-7) trait-single nucleotide polymorphism (SNP) associations were identified using the multilocus mixed linear model for GWAS. Significant associations between MCPNN and three SNPs in chromosome 2 (S02_12032235, S02_11971745, and S02_12030176) were detected with both the 2018 and best linear unbiased prediction (BLUP) datasets. Nine SNPs in chromosome 6 (S06_1970442, S06_2310856, S06_2550351, S06_1968653, S06_2296852, S06_1968680, S06_1968681, S06_1970597, and S06_1970602) were significantly associated with MNPR in the 2019 dataset. One SNP in chromosome 11 (S11_29358169) was significantly associated with the DDTH in the BLUP dataset.ConclusionsThis study identifies SNP markers that are putatively associated with MCPNN, MNPR, and DDTH. Some of these SNPs were located within or near gene models, which identify possible candidate genes involved in these traits. Validation of the putative candidate genes through expression and gene editing analyses are necessary to confirm their roles in regulating MCPNN, MNPR, and DDTH. Identifying the underlying genetic basis for primary phenotypic traits MCPNN and MNPR could lead to the development of fast and efficient approaches for their estimation, such as marker-assisted selection and gene editing, which is essential in increasing breeding efficiency and enhancing grain yield in rice. On the other hand, DDTH is a resultant variable that is highly affected by nitrogen and water management, plant density, and several other factors.

Terpene Synthases in Rice Pan-Genome and Their Responses to Chilo suppressalis Larvae Infesting.

Terpene synthase (TPS) catalyzes the synthesis of terpenes and plays an important role in plant defense. This study identified 45 OsTPS genes (32 core genes and 13 variable genes) based on the high-quality rice gene-based pan-genome. This indicates limitations in OsTPS gene studies based on a single reference genome. In the present study, through collinearity between multiple rice genomes, one OsTPS gene absent in the reference (Nipponbare) genome was found and two TPS genes in the reference genome were found to have atypical structures, which would have been ignored in single genome analysis. OsTPS genes were divided into five groups and TPS-b was lost according to the phylogenetic tree. OsTPSs in TPS-c and TPS-g were all core genes indicating these two groups were stable during domestication. In addition, through the analysis of transcriptome data, some structural variations were found to affect the expression of OsTPS genes. Through the Ka/Ks calculation of OsTPS genes, we found that different OsTPS genes were under different selection pressure during domestication; for example, OsTPS22 and OsTPS29 experienced stronger positive selection than the other OsTPS genes. After Chilo suppressalis larvae infesting, 25 differentially expressed OsTPS genes were identified, which are involved in the diterpene phytoalexins precursors biosynthesis and ent-kaurene biosynthesis pathways. Overall, the present study conducted a bioinformatics analysis of OsTPS genes using a high-quality rice pan-genome, which provided a basis for further study of OsTPS genes.

Genome-wide identification and expression analysis of CPP-like gene family in Triticum aestivum L. under different hormone and stress conditions.

The CPP-like plant‐specific transcription factor has a prominent role in plant development and growth through cell division and differential activities. However, little information is available about the CPP gene family in Triticum aestivum L. Herein, we identified 37 and 11 CPP genes in the wheat and rice genome databases, respectively. The phylogeny of the CPP protein-like family members was further divided into five subfamilies based on structural similarities and phenotypic functional diversities. The in silico expression analysis showed that CPP genes are highly expressed in some tissues, such as shoot apex, shoot, leaf, leaf sheath, and microspore. Furthermore, the qRT-PCR found higher expression for TaCPP gene family members in leaf, leaf blade, young spike, mature spike, and differential expression patterns under abiotic stresses, including heat, drought, salt, and hormonal treatment, such as indole acetic acid and 1-aminocyclopropane-1 carboxylic acid. We found that CPP gene family members are mostly located in the nucleus after infiltrating the CPP5-1B-GFP and TaCPP11-3B-GFP into tobacco leaves. The overexpression of the TaCPP5-1D gene revealed that the CPP gene positively regulates the germanium, shoot, and root activities in Arabidopsis. The TaCPP5-1D-overexpressed plants showed less anti-oxidative sensitivity under drought stress conditions. These results demonstrated that TaCPP5-1D protein has a crucial contribution by interacting with TaCPP11-3B protein in maintaining stress homeostasis under the natural and unfavorable environmental conditions for growth, development, and stress resistance activities. Therefore, this study could be used as pioneer knowledge to further investigate the function of CPP genes in plant growth and development.