Genetic Diversity and Relationship of Shanlan Upland Rice Were Revealed Based on 214 Upland Rice SSR Markers.

Shanlan upland rice (Oryza sativa L.) is a unique upland rice variety cultivated by the Li nationality for a long time, which has good drought resistance and high utilization value in drought resistance breeding. To explore the origin of Shanlan upland rice and its genetic relationship with upland rice from other geographical sources, 214 upland rice cultivars from Southeast Asia and five provinces (regions) in southern China were used to study genetic diversity by using SSR markers. Twelve SSR primers were screened and 164 alleles (Na) were detected, with the minimum number of alleles being 8 and the maximum number of alleles being 23, with an average of 13.667. The analysis of genetic diversity and analysis of molecular variance (AMOVA) showed that the differences among the materials mainly came from the individuals of upland rice. The results of gene flow and genetic differentiation revealed the relationship between the upland rice populations, and Hainan Shanlan upland rice presumably originated from upland rice in Guangdong province, and some of them were genetically differentiated from Hunan upland rice. It can be indirectly proved that the Li nationality in Hainan is a descendant of the ancient Baiyue ethnic group, which provides circumstantial evidence for the migration history of the Li nationality in Hainan, and also provides basic data for the advanced protection of Shanlan upland rice, and the innovative utilization of germplasm resources.

Genetically modified soybean lines exhibit less transcriptomic variation compared to natural varieties.

Genetically modified (GM) soybeans provide a huge amount of food for human consumption and animal feed. However, the possibility of unexpected effects of transgenesis has increased food safety concerns. High-throughput sequencing profiling provides a potential approach to directly evaluate unintended effects caused by foreign genes. In this study, we performed transcriptomic analyses to evaluate differentially expressed genes (DEGs) in individual soybean tissues, including cotyledon (C), germ (G), hypocotyl (H), and radicle (R), instead of using the whole seed, from four GM and three non-GM soybean lines. A total of 3,351 DEGs were identified among the three non-GM soybean lines. When the GM lines were compared with their non-GM parents, 1,836 to 4,551 DEGs were identified. Furthermore, Gene Ontology (GO) analysis of the DEGs showed more abundant categories of GO items (199) among non-GM lines than between GM lines and the non-GM natural varieties (166). Results of Kyoto Encyclopedia of Genes and Genomes (KEGG) analysis showed that most KEGG pathways were the same for the two types of comparisons. The study successfully employed RNA sequencing to assess the differences in gene expression among four tissues of seven soybean varieties, and the results suggest that transgenes do not induce massive transcriptomic alterations in transgenic soybeans compared with those that exist among natural varieties. This work offers empirical evidence to investigate the genomic-level disparities induced by genetic modification in soybeans, specifically focusing on seed tissues.

Identification of LncRNAs and Functional Analysis of ceRNA Related to Fatty Acid Synthesis during Flax Seed Development.

Flax is a flowering plant cultivated for its oil and contains various unsaturated fatty acids. Linseed oil is known as the "deep-sea fish oil" of plants, and is beneficial to brain and blood lipids, among other positive effects. Long non-coding RNAs (lncRNAs) play an important role in plant growth and development. There are not many studies assessing how lncRNAs are related to the fatty acid synthesis of flax. The relative oil contents of the seeds of the variety Heiya NO.14 (for fiber) and the variety Macbeth (for oil) were determined at 5 day, 10 day, 20 day, and 30 day after flowering. We found that 10-20 day is an important period for ALA accumulation in the Macbeth variety. The strand-specific transcriptome data were analyzed at these four time points, and a series of lncRNAs related to flax seed development were screened. A competing endogenous RNA (ceRNA) network was constructed and the accuracy of the network was verified using qRT-PCR. MSTRG.20631.1 could act with miR156 on the same target, squamosa promoter-binding-like protein (SPL), to influence fatty acid biosynthesis through a gluconeogenesis-related pathway during flax seed development. This study provides a theoretical basis for future studies assessing the potential functions of lncRNAs during seed development.

Overexpression of the Caragana korshinskii com58276 Gene Enhances Tolerance to Drought in Cotton (Gossypium hirsutum L.).

The increasing water scarcity associated with environmental change brings significant negative impacts to the growth of cotton plants, whereby it is urgent to enhance plant tolerance to drought. Here, we overexpressed the com58276 gene isolated from the desert plant Caragana korshinskii in cotton plants. We obtained three OE plants and demonstrated that com58276 confers drought tolerance in cotton after subjecting transgenic seeds and plants to drought. RNA-seq revealed the mechanisms of the possible anti-stress response, and that the overexpression of com58276 does not affect growth and fiber content in OE cotton plants. The function of com58276 is conserved across species, improving the tolerance of cotton to salt and low temperature, and demonstrating its applicability to improve plant resistance to environmental change.

A LuALS Mutation with High Sulfonylurea Herbicide Resistance in Linum usitatissimum L.

The cultivation of herbicide-resistant crops is an effective tool for weed management in agriculture. Weed control in flax (Linum usitatissimum L.) remains challenging due to the lack of available herbicide-resistant cultivars. In this study, a mutant resistant to acetolactate synthase (ALS)-inhibiting herbicides was obtained by ethyl methanesulphonate (EMS) mutagenesis using an elite cultivar, Longya10. Whole-plant dose-response assays revealed that, compared to Longya10, the mutant was 11.57-fold more resistant to tribenuron-methyl (TBM) and slightly resistant to imazethapyr (resistance index (mutant/Longya10) < 3). In vitro acetolactate synthase assays showed that the relative resistance of the mutant was 12.63 times more than that of Longya10. A biochemical analysis indicated that there was a Pro197Ser (relative to the Arabidopsis thaliana ALS sequence) substitution within the LuALS1, conferring high resistance to sulfonylurea herbicides in the mutant. Additionally, two cleaved amplified polymorphic sequence (CAPS) markers, BsaI-LuALS1 and EcoO109I-LuALS1, were developed based on the mutation site for marker assistant selection in breeding. Moreover, the mutant did not cause losses in natural field conditions. We find a mutant with ALS-inhibiting herbicide resistance chemically induced by EMS mutagenesis, providing a valuable germplasm for breeding herbicide-resistant flax varieties.

Understanding the molecular mechanism of drought resistance in Shanlan upland rice by transcriptome and phenotype analyses.

Rice (Oryza sativa L.) is an important grain crop worldwide, and drought has become an important factor restricting rice yield. As a unique rice germplasm in Hainan (China), Shanlan upland rice has rich genetic diversity and certain advantage for breeding water-saving and drought-resistance rice. 48 varieties, including 41 Shanlan upland rice, 3 upland rice, and 4 irrigated rice varieties was cultivated in soil pots. The drought resistance was assessed at the seedling stage using the stress coefficients of seven indicators, as the D value calculating from five principal components to rank the varieties. Five cultivars with strong, medium, and low resistance, were selected for transcriptome sequencing. The results of the GSEA analysis showed that free amino acid content increased through the redistribution of energy in Shanlan upland rice to cope with drought stress. In addition, we found that Os03g0623100 was significantly up-regulated under drought stress conditions in varieties with high drought resistance, as compared with low resistance cultivars. The Os03g0623100 was predicted to interact with LEA protein in the STRING database, which may contribute to maintaining the energy metabolisms to under stress conditions. This study provides a view of Shanlan upland rice as a drought-resistant germplasm resource, and a deeper understanding of the molecular mechanism of crop drought resistance.

Research on lncRNA related to drought resistance of Shanlan upland rice.

BACKGROUND: Drought has become the major abiotic stress that causes losses in rice yields and consequently is one of the main environmental factors threatening food security. Long non-coding RNA (lncRNA) is known to play an important role in plant response to drought stress, while the mechanisms of competing endogenous RNA (ceRNA) in drought resistance in upland rice have been rarely reported. RESULTS: In our study, a total of 191 lncRNAs, 2115 mRNAs and 32 miRNAs (microRNAs) were found by strand-specific sequencing and small RNA sequencing to be differentially expressed in drought-stressed rice. Functional analysis of results indicate that they play important roles in hormone signal transduction, chlorophyll synthesis, protein synthesis and other pathways. Construction of a ceRNA network revealed that MSTRG.28732.3 may interact with miR171 in the chlorophyll biosynthesis pathway and affect the ability of plants to withstand drought stress by regulating Os02g0662700, Os02g0663100 and Os06g0105350. The accuracy of the regulatory network was verified by qRT-PCR. CONCLUSION: Our results provide a theoretical basis for future studies on the potential function of lncRNA in plant drought resistance, and they provide new genetic resources for drought-resistant rice breeding.

Application of the maximum threshold distances to reduce gene flow frequency in the coexistence between genetically modified (GM) and non-GM maize.

On the coexistence of genetically modified (GM) and non-GM maize, the isolation distance plays an important role in controlling the transgenic flow. In this study, maize gene flow model was used to quantify the MTD0.1% and MTD1% in the main maize-planting regions of China; those were the maximum threshold distance for the gene flow frequency equal to or lower than 1% and 0.1%. The model showed that the extreme MTD1% and MTD0.1% were 187 and 548 m, respectively. The regions of northern China and the coastal plain, including Hainan crop winter-season multiplication base, showed a significantly high risk for maize gene flow, while the west-south of China was the largest low-risk areas. Except for a few sites, the isolation distance of 500 m could yield a seed purity of better than 0.1% and meet the production needs of breeder seeds. The parameters of genetic competitiveness (cp) were introduced to assess the effects of hybrid compatibility between the donor and recipient. The results showed that hybrid incompatibility could minimize the risk. When cp = 0.05, MTD1% and MTD0.1% could be greatly reduced within 19 m and 75 m. These data were helpful to provide scientific data to set the isolation distance between GM and non-GM maize and select the right place to produce the hybrid maize seeds.

Development of a Duck Genomic Reference Material by Digital PCR Platforms for the Detection of Meat Adulteration.

Low-cost meat, such as duck, is frequently used to adulterate more expensive foods like lamb or beef in many countries. However, the lack of DNA-based reference materials has limited the quality control and detection of adulterants. Here, we report the development and validation of duck genomic DNA certified reference materials (CRMs) through the detection of the duck interleukin 2 (IL2) gene by digital PCR (dPCR) for the identification of duck meat in food products. The certified value of IL2 in CRMs was 5.78 ± 0.51 × 103 copies/µL with extended uncertainty (coverage factor k = 2) based on IL2 quantification by eight independent collaborating laboratories. Quantification of the mitochondrial gene cytb revealed a concentration of 2.0 × 106 copies/µL, as an information value. The CRMs were also used to determine the limit of detection (LOD) for six commercial testing kits, which confirmed that these kits meet or exceed their claimed sensitivity and are reliable for duck detection.

Genome-wide identification and functional characterization of LEA genes during seed development process in linseed flax (Linum usitatissimum L.).

BACKGROUND: LEA proteins are widely distributed in the plant and animal kingdoms, as well as in micro-organisms. LEA genes make up a large family and function in plant protection against a variety of adverse conditions. RESULTS: Bioinformatics approaches were adopted to identify LEA genes in the flax genome. In total, we found 50 LEA genes in the genome. We also conducted analyses of the physicochemical parameters and subcellular location of the genes and generated a phylogenetic tree. LuLEA genes were unevenly mapped among 15 flax chromosomes and 90% of the genes had less than two introns. Expression profiles of LuLEA showed that most LuLEA genes were expressed at a late stage of seed development. Functionally, the LuLEA1 gene reduced seed size and fatty acid contents in LuLEA1-overexpressed transgenic Arabidopsis lines. CONCLUSION: Our study adds valuable knowledge about LEA genes in flax which can be used to improve related genes of seed development.

Enhanced tolerance to drought stress resulting from Caragana korshinskii CkWRKY33 in transgenic Arabidopsis thaliana.

BACKGROUND: It is well known that WRKY transcription factors play important roles in plant growth and development, defense regulation and stress responses. RESULTS: In this study, a WRKY transcription factor, WRKY33, was cloned from Caragana korshinskii. A sequence structure analysis showed that it belonged to the Group-I type. Subcellular localization experiments in tobacco epidermal cells showed the presence of CkWRKY33 in the nucleus. Additionally, CkWRKY33 was overexpressed in Arabidopsis thaliana. A phenotypic investigation revealed that compared with wild-type plants, CkWRKY33-overexpressing transgenic plants had higher survival rates, as well as relative soluble sugar, proline and peroxidase contents, but lower malondialdehyde contents, following a drought stress treatment. CONCLUSIONS: This suggested that the overexpression of CkWRKY33 led to an enhanced drought-stress tolerance in transgenic A. thaliana. Thus, CkWRKY33 may act as a positive regulator involved in the drought-stress responses in Caragana korshinskii.

CRISPR/Cas9 editing of wheat TaQ genes alters spike morphogenesis and grain threshability.

The TaQ alleles as one of the AP2-like transcription factors in common wheat (Triticum aestivum) play an important role in the evolution of spike characteristics from wild and domesticated emmer to modern wheat cultivars. Its loss-of-function mutant not only changed threshability and spike architecture but also affected plant height, flowering time, and floret structure. However, the comprehensive functions of TaAQ and TaDq genes in wheat have not been fully elucidated yet. Here, CRISPR/SpCas9 was used to edit wheat TaAQ and TaDq. We obtained homozygous plants in the T1 generation with loss of function of only TaAQ or TaDq and simultaneous loss of function of TaAQ and TaDq to analyze the effect of these genes on wheat spikes and floret shapes. The results demonstrated that the TaAQ-edited plants and the TaAQ and TaDq simultaneously-edited plants were nearly similar in spike architecture, whereas the TaDq-edited plants were different from the wild-type ones only in plant height. Moreover, the TaAQ-edited plants or the TaAQ and TaDq simultaneously-edited plants were more brittle than the wild-type and the TaDq-edited plants. Based on the expression profiling, we postulated that the VRN1, FUL2, SEP2, SEP5, and SEP6 genes might affect the number of spikelets and florets per spike in wheat by regulating the expression of TaQ. Combining the results of this report and previous reports, we conceived a regulatory network of wheat traits, including plant height, spike shape, and floral organs, which were influenced by AP2-like family genes. The results achieved in this study will help us to understand the regulating mechanisms of TaAQ and TaDq alleles on wheat floral organs and inflorescence development.

An electrochemical DNA biosensor based on nitrogen-doped graphene nanosheets decorated with gold nanoparticles for genetically modified maize detection.

A reliable electrochemical biosensor is reported based on nitrogen-doped graphene nanosheets and gold nanoparticle (Au/N-G) nanocomposites for the event-specific detection of GM maize MIR162. The differential pulse voltammetry response of methylene blue (MB) was chosen to monitor the target DNA hybridization event. Under the optimum conditions, the peak current increased linearly with the logarithm of the concentration of DNA in the range 1.0 × 10-14 to 1.0 × 10-8 M, and the detection limit was 2.52 × 10-15 M (S/N = 3). It is also demonstrated that the DNA biosensor has high selectivity, good stability, and fabrication reproducibility. The biosensor has been effectively applied to detect MIR162 in real samples, showing its potential as an effective tool for GM crop analysis. These results will contribute to the development of new portable transgenic detection systems. Graphical abstract .

Identification and Characterization of microRNAs in the Developing Seed of Linseed Flax (Linum usitatissimum L.).

Seed development plays an important role during the life cycle of plants. Linseed flax is an oil crop and the seed is a key organ for fatty acids synthesis and storage. So it is important to understand the molecular mechanism of fatty acid biosynthesis during seed development. In this study, four small RNA libraries from early seeds at 5, 10, 20 and 30 days after flowering (DAF) were constructed and used for high-throughput sequencing to identify microRNAs (miRNAs). A total of 235 miRNAs including 114 known conserved miRNAs and 121 novel miRNAs were identified. The expression patterns of these miRNAs in the four libraries were investigated by bioinformatics and quantitative real-time polymerase chain reaction (qPCR) analysis. It was found that several miRNAs, including Lus-miRNA156a was significantly correlated with seed development process. In order to confirm the actual biological function of Lus-miRNA156a, over-expression vector was constructed and transformed to Arabidopsis. The phenotypes of homozygous transgenic lines showed decreasing of oil content and most of the fatty acid content in seeds as well as late flowering time. The results provided a clue that miRNA156a participating the fatty acid biosynthesis pathway and the detailed molecular mechanism of how it regulates the pathway needs to be further investigated.

Increased pollen source area does not always enhance the risk of pollen dispersal and gene flow in Oryza sativa L.

Pollen dispersal is one of the main ways of gene flow. In the past years, rice pollen dispersal and gene flow have been well studies. However, there is much dispute whether the risk of pollen dispersal and gene flow continuously increases with the source area. A Lagrangian stochastic model was used to simulate the pollen depositions at different distances from different pollen source areas. The field experiments showed a good fit in the pollen depositions. The larger the source area, the more the pollen grains were deposited at each distance, with the pollen dispersal distance increasing accordingly. However, this effect gradually leveled off as the source area increased. In the large-area of pollen source, we found a significantly higher saturation point for the amount of pollen deposition. Once the source area exceeded 1000 × 1000 m2, the pollen deposition no longer increased, even if the source area continued to increase, indicating the "critical source area" of rice pollen dispersal. However, a 100 × 100 m2 critical source area for conventional rice and hybrid rice was sufficient, while the critical source area for the sterile line was about 230 × 230 m2.

Genomic Comparison and Population Diversity Analysis Provide Insights into the Domestication and Improvement of Flax.

Flax has been cultivated for its oil and fiber for thousands of years. However, it remains unclear how the modifications of agronomic traits occurred on the genetic level during flax cultivation. In this study, we conducted genome-wide variation analyses on multiple accessions of oil-use, fiber-use, landraces, and pale flax to identify the genomic variations during flax cultivation. Our findings indicate that, during flax domestication, genes relevant to flowering, dehiscence, oil production, and plant architecture were preferentially selected. Furthermore, regardless of origins, the improvement of the modern oil-use flax preceded that of the fiber-use flax, although the dual selection on oil-use and fiber-use characteristics might have occurred in the early flax domestication. We also found that the expansion of MYB46/MYB83 genes may have contributed to the unique secondary cell wall biosynthesis in flax and the directional selections on MYB46/MYB83 may have shaped the morphological profile of the current oil-use and fiber-use flax.

Identification of drought response genes by digital gene expression (DGE) analysis in Caragana korshinskii Kom.

Caragana korshinskii Kom. is a legume shrub that is widely distributed across desert habitats with gravely, sandy, and saline soils in Asia and Africa. C. korshinskii has highly developed roots and a strong tolerance to abiotic stress. At present, there are few genetic studies of C. korshinskii because of the limited availability of genomic resources. To understand the comprehensive mechanisms that are associated with drought tolerance, we used RNA-seq to survey the differentially expressed genes (DEGs) in comparisons of drought-treated and control plants. After analysing the sequencing results, we found 440 differentially expressed genes existing in drought-treated and control plants. Among the DEGs, 39 unigenes showed up-regulated expression after drought treatment, while 401 unigenes were down-regulated. We used the KEGG database to annotate these drought-induced genes; 126 unigenes were identified by KEGG pathway annotation, and approximately 28% of the unigenes with known function fell into categories related to fatty acid metabolism, starch, sucrose metabolism, and nitrogen metabolism, suggesting that these pathways or processes may be involved in the drought response. Finally, we confirmed that one gene has a potential function in drought tolerance. Our study is the first to provide transcriptomic resources for Caragana korshinskii and to determine its digital gene expression profile under conditions of drought stress using the assembled transcriptomic data for reference. These data provide a valuable resource for genetic and genomic studies of desert plants under abiotic stress conditions.

Efficient induction of haploid plants in wheat by editing of TaMTL using an optimized Agrobacterium-mediated CRISPR system.

The use of CRISPR/LbCpf1 and CRISPR/xCas9 systems in wheat have not yet been reported. In this study, we compared the efficiencies of three CRISPR editing systems (SpCas9, LbCpf1, and xCas9), and three different promoters (OsU6a, TaU3, and TaU6) that drive single-guide (sg)RNA, which were introduced into wheat via Agrobacterium-mediated transformation. The results indicated that TaU3 was a better choice than OsU6a or TaU6. The editing efficiency was higher using two sgRNAs than one sgRNA, and mutants with a large fragment deletion between the two sgRNAs were produced. The LbCpf1 and xCas9 systems could both be used successfully. Two endogenous genes, TaWaxy and TaMTL, were edited with high efficiency by the optimized SpCas9 system, with the highest efficiency (80.5%) being achieved when using TaU3 and two sgRNAs to target TaWaxy. Rates of seed set in the TaMTL-edited T0 transgenic plants were much lower than that of the wild-type. A haploid induction rate of 18.9% was found in the TaMTL-edited T1 plants using the CRISPR/SpCas9 system. Mutants with reverse insertion of the deleted sequences of TaMTL and TaWaxy between the two sgRNAs were identified in the edited T0 plants. In addition, wheat grains lacking embryos or endosperms were observed in the TaMTL-edited T1 generation.

High-throughput sequencing analysis of microbial community diversity in response to indica and japonica bar-transgenic rice paddy soils.

Potential environmental risks of genetically modified (GM) crops have raised concerns. To better understand the effect of transgenic rice on the bacterial community in paddy soil, a field experiment was carried out using pairs of rice varieties from two subspecies (indica and japonica) containing bar transgene with herbicide resistance and their parental conventional rice. The 16S rRNA gene of soil genomic DNA from different soil layers at the maturity stage was sequenced using high-throughput sequencing on the Illumina MiSeq platform to explore the microbial community diversity among different rice soils. There were no significant differences in diversity indices between transgenic japonica rice and its sister conventional rice (japonica pair) among different soil layers, but, significant differences was observed between transgenic indica rice and its conventional rice (indica pair) in the topsoil layer around concentrated rice roots according to the ace diversity index. Though the japonica rice soil and indica rice soil were shared several key genera, including Rivibacter, Anaeromyxobacter, Roseomonas, Geobacter, Thiobacillus, Clostridium, and Desulfobulbus, the primary bacterial genera in indica rice soil were different from those in japonica rice. Synechococcus and Dechloromonas were present in japonica rice samples, while Chloronema, Flexibacter, and Blastocatella were observed in indica rice soil. Moreover, the abundance of genera between GM and non-GM varieties in japonica rice was significantly different from indica rice, and several bacterial communities influenced these differences. Anaerovorax was more abundant in transgenic japonica rice soil than conventional rice soil, while it was deficient in transgenic indica rice soil compared to conventional rice soil, and opposite responses to Deferrisoma were in that of indica rice. Thus, we concluded that transgenic indica and japonica rice had different effects on soil bacteria compared with their corresponding sister conventional rice. However, these composition and abundance difference only occurred for a few genera but had no effect on the primary genera and soil characteristics were mainly contributed to these differences. Thus, differences in bacterial community structure can be ignored when evaluating the impacts of transgenic rice in the complex soil microenvironment.

The Novel Cerato-Platanin-Like Protein FocCP1 from Fusarium oxysporum Triggers an Immune Response in Plants.

Panama disease, or Fusarium wilt, the most serious disease in banana cultivation, is caused by Fusarium oxysporum f. sp. cubense (FOC) and has led to great economic losses worldwide. One effective way to combat this disease is by enhancing host plant resistance. The cerato-platanin protein (CPP) family is a group of small secreted cysteine-rich proteins in filamentous fungi. CPPs as elicitors can trigger the immune system resulting in defense responses in plants. In this study, we characterized a novel cerato-platanin-like protein in the secretome of Fusarium oxysporum f. sp. cubense race 4 (FOC4), named FocCP1. In tobacco, the purified recombinant FocCP1 protein caused accumulation of reactive oxygen species (ROS), formation of necrotic reaction, deposition of callose, expression of defense-related genes, and accumulation of salicylic acid (SA) and jasmonic acid (JA) in tobacco. These results indicated that FocCP1 triggered a hypersensitive response (HR) and systemic acquired resistance (SAR) in tobacco. Furthermore, FocCP1 enhanced resistance tobacco mosaic virus (TMV) disease and Pseudomonas syringae pv. tabaci 6605 (Pst. 6605) infection in tobacco and improved banana seedling resistance to FOC4. All results provide the possibility of further research on immune mechanisms of plant and pathogen interactions, and lay a foundation for a new biological strategy of banana wilt control in the future.